Hyaluronic acid-modified beauty peptide, preparation method and use thereof

Abstract

The present invention discloses a hyaluronic acid-modified beauty peptide, a preparation method thereof and uses thereof, relating to the field of beauty peptides. The structural formula of the hyaluronic acid-modified beauty peptide is shown as formula (I): M-C (I); wherein, M represents sodium hyaluronate, and its structure is shown as formula (II): #imgabs0# (II), where y is a natural number greater than or equal to 1; C represents a beauty peptide, and the beauty peptide includes a polypeptide or a derivative thereof having beauty and / or skin care effects, and the polypeptide includes a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide or a nonapeptide; wherein, the open-ring glucuronic acid at the M terminal is linked to the amino group in the C structure. The hyaluronic acid-modified beauty peptide provided by the present invention can not only maintain the properties of sodium hyaluronate but also contain the functionality of the polypeptide, and the stability of the prepared novel compound can be significantly improved, having broad application prospects in the field of cosmetics / skin care products.

Description

Technical Field

[0001] The present invention belongs to the field of beauty peptides, and particularly relates to a beauty peptide modified with hyaluronic acid, a preparation method thereof, and uses thereof. Background Art

[0002] Hyaluronic acid is a macromolecular polysaccharide first isolated from bovine vitreous humor by Professor Meyer and Palmer of Columbia University in the United States in 1934. Its aqueous solution is in a transparent glassy state, so it is also known as hyaluronic acid. Hyaluronic acid is a natural biological substance widely present in tissues such as the vitreous humor of the eye, skin, umbilical cord, and synovial fluid of joints. The early raw materials of hyaluronic acid used were mainly extracted from chicken combs. Due to cost and raw material limitations, it was not further promoted. Now, cosmetic and medicinal hyaluronic acid mainly comes from microbial fermentation. Fermentation is not restricted by animal raw materials, has a lower cost, is easy to scale up production, and the product has a higher purity and is convenient for controlling the relative molecular mass. Research in this field started in China in the 1980s, and significant achievements have now been made. The hyaluronic acid produced by fermentation in China is in an internationally advanced position both in terms of quality and quantity. Hyaluronic acid is one of the main components constituting the extracellular matrix and interstitial substance of cells and is the filler between cells, playing an important role in the form, structure, and function of the skin. Due to the functions of hyaluronic acid such as moisturizing, repairing, and nourishing, having good affinity for the skin and being safe to use, its application in beauty cosmetics is becoming more and more extensive. Due to the characteristics of hyaluronic acid such as biodegradability, biocompatibility, chemical modification ability, and in vivo targeting, it has attracted much attention in the field of protein polypeptide modification. The present invention uses a beauty peptide modified with hyaluronic acid and further studies its synthesis method and application. Summary of the Invention

[0003] The purpose of the present invention is to provide a beauty peptide modified with hyaluronic acid, a preparation method thereof, and uses thereof. The beauty peptide modified with hyaluronic acid can not only maintain the properties of sodium hyaluronate but also contain the functionality of polypeptides, and the stability of the prepared new compound can be significantly improved, having broad application prospects in the field of cosmetics / skin care products.

[0004] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0005] A beauty peptide modified with hyaluronic acid, the structural formula of the beauty peptide modified with hyaluronic acid is shown in formula (I):

[0006] M - C (I);

[0007] Wherein, M represents sodium hyaluronate, and its structure is shown in formula (II):

[0008] (II), where y is a natural number greater than or equal to 1;

[0009] C represents a beauty peptide, and the above-mentioned beauty peptide includes a polypeptide or its derivative having beauty and / or skin care effects. The above-mentioned polypeptide includes dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or other polypeptides;

[0010] Among them, the M-terminal glucuronic acid links to the amino group in the C structure. In the present invention, hyaluronic acid is modified with a beauty polypeptide. Through a substitution reaction, sodium hyaluronate and the polypeptide are combined by a covalent bond to form a new chemical substance. The new chemical substance can not only maintain the properties of sodium hyaluronate but also contain the functionality of the polypeptide; and this compound has multiple effects, and its stability effects are significantly enhanced compared with the original effects. For example, the compound has more excellent moisturizing performance; the anti-wrinkle and firming performance, whitening performance, antioxidant capacity and anti-glycation performance of some compounds are significantly improved, and the soothing ability is also effectively improved. In the present invention, hyaluronic acid and a beauty polypeptide are covalently coupled to generate a beauty peptide modified with hyaluronic acid, and a new chemical structure is formed directly through chemical modification, which can fundamentally endow the compound with more excellent properties, and the preparation method has simple steps; at the same time, the present invention also provides its application in cosmetics and beauty products, significantly enhancing the use effect of functional cosmetics, such as moisturizing, anti-aging and firming, whitening, soothing, antioxidant and anti-glycation effects, and thus can improve the user's satisfaction.

[0011] Preferably, y is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; more preferably, y = 1 or 2 or 3.

[0012] Specifically, the compound shown in formula (I) includes the structure shown in the following formula (III):

[0013] (III);

[0014] Among them,

[0015] The above-mentioned n is a natural number;

[0016] The above-mentioned R is the remaining part of the beauty peptide structure after removing the reactive group amino group;

[0017] The above-mentioned beauty peptide includes a polypeptide or its derivative having beauty and / or skin care effects. The above-mentioned polypeptide includes dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or other polypeptides.

[0018] Preferably, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. More preferably, n = 0 or 1 or 2 or 3.

[0019] Specifically, the dipeptide or its derivative includes one of dipeptide-2 and carnosine; preferably carnosine.

[0020] Specifically, the tripeptide or its derivative includes one of serpentin, tripeptide-1, tripeptide-5, tripeptide-8, tripeptide-38, and palmitoyl tripeptide-38; preferably tripeptide-1 or tripeptide-8.

[0021] Specifically, the tetrapeptide or its derivative includes one of tetrapeptide-5, tetrapeptide-7, tetrapeptide-9, tetrapeptide-11, tetrapeptide-30, and tetrapeptide-15, or includes a tetrapeptide with the amino acid sequence H-Asp-Val-Lys-Tyr-OH; preferably tetrapeptide-7 and tetrapeptide-15.

[0022] Specifically, the pentapeptide or its derivative includes one of pentapeptide-4 and myristoyl pentapeptide-4; preferably pentapeptide-4.

[0023] Specifically, the hexapeptide or its derivative includes one of hexapeptide-1, hexapeptide-8, hexapeptide-9, hexapeptide-11, and hexapeptide-38, or includes one of the hexapeptides with the amino acid sequences H-Arg-Arg-Gln-Met-Glu-Glu-NH2, H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2, H-Trp-Phe-Arg-Leu-Ala-His-NH2, and H-Trp-Phe-Arg-D-Leu-Ala-His-NH2.

[0024] Preferably, the hexapeptide or its derivative includes hexapeptide-1, hexapeptide-8, hexapeptide-9, hexapeptide-11; more preferably, it includes one of the hexapeptides with the amino acid sequences H-Arg-Arg-Gln-Met-Glu-Glu-NH2, H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2, H-Trp-Phe-Arg-Leu-Ala-His-NH2, and H-Trp-Phe-Arg-D-Leu-Ala-His-NH2.

[0025] Specifically, the heptapeptide or its derivative includes a heptapeptide with the amino acid sequence H-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH or H-Glu-Glu-Met-Gln-Arg-Arg-Ala-NH2.

[0026] Specifically, the octapeptide or its derivative includes an octapeptide with the amino acid sequence H-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2.

[0027] Specifically, the nonapeptide or its derivative includes nonapeptide-1.

[0028] Specifically, the compounds shown in the above (I) include one or more of H-Glu-Glu-Met-Gln-Arg-Arg-NH-hyaluronic acid (sodium), hyaluronic acid (sodium)-NH-Phe-Val-Ala-Pro-Phe-Pro-OH, H-His-D-Phe-Arg-NH-hyaluronic acid (sodium), H-His-Ala-Leu-Arg-Phe-Trp-NH-hyaluronic acid (sodium), H-Trp-Phe-Arg-D-Leu-Ala-His-NH-hyaluronic acid (sodium), H-Trp-Phe-Arg-Leu-Ala-His-NH-hyaluronic acid (sodium), H-Arg-Arg-Gln-D-Met-Glu-Glu-NH-hyaluronic acid (sodium) or H-Arg-Arg-Gln-Met-Glu-Glu-NH-hyaluronic acid (sodium).

[0029] Furthermore, the compounds shown in the above (I) may also include one or more of hyaluronic acid (sodium)-HN-Gly-Gln-Pro-Arg-OH, hyaluronic acid (sodium)-HN-Lys-Thr-Thr-Lys-Ser-OH, H-Lys-Leu-Ala-Lys-Lys-NH-hyaluronic acid (sodium), H-Gly-Pro-Gln-Gly-Pro-Gln-NH-hyaluronic acid (sodium), H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH-hyaluronic acid (sodium), H-Tyr-Pro-Phe-Phe-NH-hyaluronic acid (sodium), hyaluronic acid (sodium)-NH-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH or hyaluronic acid (sodium)-NH-Asp-Val-Lys-Tyr-OH.

[0030] It should be noted that the above-mentioned beauty peptides modified with hyaluronic acid may represent the case where there is only one reactive site for the bonding between hyaluronic acid (sodium) and the beauty peptide in the structure, or the case where there are multiple reactive sites. Further, it should be noted that the bonding part is formed by the reaction of the reactive amino group in the beauty polypeptide structure with the reactive hydroxyl site in the endocyclic group of the hyaluronic acid (sodium) structure that can be further polymerized.

[0031] More preferably, the compounds shown in the above formula (I) include one or more of the following: A2;

[0032] A4;

[0033] A6;

[0034] B2;

[0035] B4;

[0036] B6;

[0037] D2;

[0038] D4;

[0039] D6;

[0040] E2;

[0041] E4;

[0042] E6; F2; F4;

[0043] F6; G2; G4;

[0044] G6.

[0045] The present invention also discloses a preparation method of a hyaluronic acid-modified beauty peptide, comprising: subjecting sodium hyaluronate and the beauty peptide to a condensation reaction to obtain the hyaluronic acid-modified beauty peptide.

[0046] Further, the synthesis route of the above-mentioned hyaluronic acid-modified beauty peptide is as follows:

[0047] ;

[0048] Wherein, n is a natural number;

[0049] Y is Na or H;

[0050] Peptide = R; R is the remaining part of the beauty peptide structure after removing the reactive group amino;

[0051] X includes H, sodium or potassium;

[0052] The above-mentioned beauty peptides include polypeptides or their derivatives having beauty and / or skin care effects, and the polypeptides include dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides or other polypeptides.

[0053] It should be noted that the final form of the hyaluronic acid-modified beauty peptide provided by the present invention can be the hyaluronic acid-modified beauty peptide, or can be hydrochloride, sulfate, phosphate, acetate, sodium salt, potassium salt, trifluoroacetate, maleate or fumarate, etc.

[0054] The preparation method of the above-mentioned hyaluronic acid-modified beauty peptide includes: subjecting sodium hyaluronate and a beauty peptide to a ring-opening reaction to obtain the hyaluronic acid-modified beauty peptide.

[0055] Specifically, the preparation method of the above-mentioned hyaluronic acid-modified beauty peptide includes the following steps:

[0056] Add the beauty peptide to an organic solvent, stir to dissolve, then add TFA, stir for 0.5 - 1 h, then add DIEA, and then weigh and add sodium hyaluronate; at 40 - 50 °C, stir and react overnight, take a sample to detect LC-MS, when the reaction is basically complete, add acetic acid, control the temperature of the water bath at 30 - 45 °C, carry out a rearrangement reaction for 2 - 4 h, take a sample to detect LC-MS, when the reaction is basically complete; then carry out reverse chromatography purification to obtain the hyaluronic acid-modified beauty peptide.

[0057] Specifically, the above-mentioned organic solvent is selected from single solvents or mixed solvents such as DMF, N,N-diethylacetamide, DMSO, acetonitrile, N-methylpyrrolidone (NMP), methanol, ethanol, acetone, THF, dichloromethane, ethyl acetate, dioxane and water; preferably DMSO.

[0058] Specifically, the mass-volume ratio of the above-mentioned beauty peptide to DMSO is 1 g:8 - 25 mL; preferably 1 g:10 mL.

[0059] Specifically, the mass-volume ratio of the above-mentioned beauty peptide to TFA is 1 g:0 - 1.2 mL.

[0060] Specifically, the mass ratio of the above-mentioned beauty peptide to DIEA is 1:0.5 - 5.5; preferably 1:0.8 - 2.5.

[0061] Specifically, the molar ratio of the above-mentioned beauty peptide to hyaluronic acid is 1:2 - 8; preferably 1:3 - 5; more preferably 1:4.

[0062] Specifically, the mass-volume ratio of the above-mentioned beauty peptide to acetic acid is 1 g:4 - 10 mL; preferably 1 g:5 - 9 mL; more preferably 1 g:8 mL.

[0063] Another object of the present invention is to disclose the use of the above-mentioned hyaluronic acid-modified beauty peptide in the preparation of cosmetics and / or skin care products.

[0064] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the moisturizing performance of cosmetics and / or skin care products.

[0065] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the soothing performance of cosmetics and / or skin care products.

[0066] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the anti-wrinkle and firming performance of cosmetics and / or skin care products.

[0067] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the whitening performance of cosmetics and / or skin care products.

[0068] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the anti-glycation performance of cosmetics and / or skin care products.

[0069] The present invention also discloses the use of the above-mentioned hyaluronic acid-modified beauty peptide in enhancing the antioxidant performance of cosmetics and / or skin care products.

[0070] A cosmetic comprising the above-mentioned hyaluronic acid-modified beauty peptide.

[0071] A skin care product comprising the above-mentioned hyaluronic acid-modified beauty peptide.

[0072] The beneficial effects of the present invention include:

[0073] The present invention uses beauty polypeptides to modify hyaluronic acid to form a new compound through the Maillard reaction. This compound can not only maintain the properties of sodium hyaluronate but also contain the functionality of polypeptides; and its stability and efficacy are significantly enhanced compared to the original efficacy. For example, the compound has more excellent moisturizing performance; the anti-wrinkle and firming performance, whitening performance, antioxidant capacity, and anti-glycation performance of some compounds are significantly improved, and the soothing ability is also effectively improved. The hyaluronic acid-modified beauty peptide provided by the present invention directly forms a new chemical structure through chemical modification, which can fundamentally endow the compound with more excellent properties, and the preparation method has simple steps; at the same time, the present invention also provides its application in cosmetics and beauty products, significantly enhancing the use effect of functional cosmetics, such as moisturizing, anti-aging and firming, whitening, soothing, antioxidant, and anti-glycation effects, thereby improving the user's satisfaction.

[0074] Therefore, the present invention provides a hyaluronic acid-modified beauty peptide, a preparation method thereof, and uses thereof. The hyaluronic acid-modified beauty peptide can not only maintain the properties of sodium hyaluronate but also contain the functionality of polypeptides, and the stability of the prepared novel compound can be significantly improved, having broad application prospects in the field of cosmetics / skin care products. Description of the Drawings

[0075] Figure 1 Mass spectrometry test results of H-Gly-Gln-Pro-Arg-OH prepared in Example 1;

[0076] Figure 2 Liquid chromatography test results of H-Gly-Gln-Pro-Arg-OH prepared in Example 1;

[0077] Figure 3 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide prepared in Example 1;

[0078] Figure 4 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide prepared in Example 1;

[0079] Figure 5 Mass spectrometry test results of H-Lys-Thr-Thr-Lys-Ser-OH prepared in Example 2;

[0080] Figure 6 Liquid chromatography test results of H-Lys-Thr-Thr-Lys-Ser-OH prepared in Example 2;

[0081] Figure 7 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide prepared in Example 2;

[0082] Figure 8 Mass spectrometry test results of the sample at 5.801 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 2;

[0083] Figure 9 Mass spectrometry test results of the sample at 6.102 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 2;

[0084] Figure 10 Mass spectrometry test results of the sample at 6.558 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 2;

[0085] Figure 11 Mass spectrometry test results of H-Lys-Leu-Ala-Lys-Lys-NH2 prepared in Example 3;

[0086] Figure 12 LC-MS test results of H-Lys-Leu-Ala-Lys-Lys-NH2 prepared in Example 3;

[0087] Figure 13 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide prepared in Example 3;

[0088] Figure 14 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide prepared in Example 3;

[0089] Figure 15 Mass spectrometry test results of H-Glu-Glu-Met-Gln-Arg-Arg-NH2 prepared in Example 4;

[0090] Figure 16 LC-MS test results of H-Glu-Glu-Met-Gln-Arg-Arg-NH2 prepared in Example 4;

[0091] Figure 17 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide D2 prepared in Example 4;

[0092] Figure 18 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide D2 prepared in Example 4;

[0093] Figure 19 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide D4 prepared in Example 4;

[0094] Figure 20 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide D4 prepared in Example 4;

[0095] Figure 21 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide D6 prepared in Example 4;

[0096] Figure 22 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide D6 prepared in Example 4;

[0097] Figure 23 Mass spectrometry test results of the hyaluronic acid-modified beauty peptide prepared in Example 5;

[0098] Figure 24 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide prepared in Example 5;

[0099] Figure 25 Mass spectrometry test results of H-Gly-Pro-Gln-Gly-Pro-Gln-NH2 prepared in Example 6;

[0100] Figure 26 Test results of the liquid chromatography of H-Gly-Pro-Gln-Gly-Pro-Gln-NH2 prepared in Example 6;

[0101] Figure 27 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide prepared in Example 6;

[0102] Figure 28 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 6;

[0103] Figure 29 Test results of the mass spectrometry of H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH2 prepared in Example 7;

[0104] Figure 30 Test results of the liquid chromatography of H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH2 prepared in Example 7;

[0105] Figure 31 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide prepared in Example 7;

[0106] Figure 32 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 7;

[0107] Figure 33 Test results of the mass spectrometry of H-Phe-Val-Ala-Pro-Phe-Pro-OH prepared in Example 8;

[0108] Figure 34 Test results of the LC-MS of H-Phe-Val-Ala-Pro-Phe-Pro-OH prepared in Example 8;

[0109] Figure 35 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide H2 prepared in Example 8;

[0110] Figure 36 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide H2 prepared in Example 8;

[0111] Figure 37 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide H4 prepared in Example 8;

[0112] Figure 38Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide H4 prepared in Example 8;

[0113] Figure 39 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide H6 prepared in Example 8;

[0114] Figure 40 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide H6 prepared in Example 8;

[0115] Figure 41 Test results of the mass spectrometry of H-His-D-Phe-Arg-NH2 prepared in Example 9;

[0116] Figure 42 Test results of the LC-MS of H-His-D-Phe-Arg-NH2 prepared in Example 9;

[0117] Figure 43 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide L2 prepared in Example 9;

[0118] Figure 44 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide L2 prepared in Example 9;

[0119] Figure 45 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide L4 prepared in Example 9;

[0120] Figure 46 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide L4 prepared in Example 9;

[0121] Figure 47 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide L6 prepared in Example 9;

[0122] Figure 48 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide L6 prepared in Example 9;

[0123] Figure 49 Test results of the mass spectrometry of H-Tyr-Pro-Phe-Phe-NH2 prepared in Example 10;

[0124] Figure 50 Test results of the liquid chromatography of H-Tyr-Pro-Phe-Phe-NH2 prepared in Example 10;

[0125] Figure 51 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide prepared in Example 10;

[0126] Figure 52Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 10;

[0127] Figure 53 Test results of the mass spectrometry of some products (reaction products of sodium hyaluronate with beauty peptide where y = 1, y = 2, and y = 3) in the hyaluronic acid-modified beauty peptide prepared in Example 11;

[0128] Figure 54 Test results of the high-performance liquid chromatography of some products (reaction products of sodium hyaluronate with beauty peptide where y = 1, y = 2, and y = 3) in the hyaluronic acid-modified beauty peptide prepared in Example 11;

[0129] Figure 55 Test results of the mass spectrometry of some products (reaction products of sodium hyaluronate with beauty peptide where y = 2 and y = 3) in the hyaluronic acid-modified beauty peptide prepared in Example 11;

[0130] Figure 56 Test results of the liquid chromatography of some products (reaction products of sodium hyaluronate with beauty peptide where y = 2 and y = 3) in the hyaluronic acid-modified beauty peptide prepared in Example 11;

[0131] Figure 57 Test results of the mass spectrometry of H-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH prepared in Example 12;

[0132] Figure 58 Test results of the liquid chromatography of H-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH prepared in Example 12;

[0133] Figure 59 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 12;

[0134] Figure 60 Test results of the mass spectrometry of the sample at 11.577 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 12;

[0135] Figure 61 Test results of the mass spectrometry of the sample at 12.223 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 12;

[0136] Figure 62 Test results of the mass spectrometry of H-Asp-Val-Lys-Tyr-OH prepared in Example 13;

[0137] Figure 63Test results of the liquid chromatography of H-Asp-Val-Lys-Tyr-OH prepared in Example 13;

[0138] Figure 64 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 13;

[0139] Figure 65 Test results of the mass spectrometry of the sample at 8.226 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 13;

[0140] Figure 66 Test results of the mass spectrometry of the sample at 12.711 min in the liquid chromatography of the hyaluronic acid-modified beauty peptide prepared in Example 13;

[0141] Figure 67 Test results of the mass spectrometry of H-His-Ala-Leu-Arg-Phe-Trp-NH2 prepared in Example 14;

[0142] Figure 68 Test results of the liquid chromatography of H-His-Ala-Leu-Arg-Phe-Trp-NH2 prepared in Example 14;

[0143] Figure 69 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide A2 prepared in Example 14;

[0144] Figure 70 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide A2 prepared in Example 14;

[0145] Figure 71 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0146] Figure 72 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0147] Figure 73-1 Partial 1H NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0148] Figure 73-2 Partial 1H NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0149] Figure 73-3 Partial 1H NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0150] Figure 73-4Partial 1H NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0151] Figure 73-5 Partial 1H NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0152] Figure 74-1 Partial 13C NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0153] Figure 74-2 Partial 13C NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0154] Figure 74-3 Partial 13C NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0155] Figure 74-4 Partial 13C NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0156] Figure 74-5 Partial 13C NMR test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0157] Figure 75 2D cosy spectrum test results of the hyaluronic acid-modified beauty peptide A4 prepared in Example 14;

[0158] Figure 76 Mass spectrum test results of the hyaluronic acid-modified beauty peptide A6 prepared in Example 14;

[0159] Figure 77 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide A6 prepared in Example 14;

[0160] Figure 78 Mass spectrum test results of H-Trp-Phe-Arg-D-Leu-Ala-His-NH2 prepared in Example 27;

[0161] Figure 79 Liquid chromatography test results of H-Trp-Phe-Arg-D-Leu-Ala-His-NH2 prepared in Example 27;

[0162] Figure 80 Liquid chromatography test results of the hyaluronic acid-modified beauty peptide E2 prepared in Example 27;

[0163] Figure 81 Mass spectrum test results of the hyaluronic acid-modified beauty peptide E2 prepared in Example 27;

[0164] Figure 82 Mass spectrometry test results of hyaluronic acid-modified beauty peptide E4 prepared in Example 27;

[0165] Figure 83 Liquid chromatography test results of hyaluronic acid-modified beauty peptide E4 prepared in Example 27;

[0166] Figure 84 Mass spectrometry test results of hyaluronic acid-modified beauty peptide E6 prepared in Example 27;

[0167] Figure 85 Liquid chromatography test results of hyaluronic acid-modified beauty peptide E6 prepared in Example 27;

[0168] Figure 86 Mass spectrometry test results of H-Trp-Phe-Arg-Leu-Ala-His-NH2 prepared in Example 28;

[0169] Figure 87 Liquid chromatography test results of H-Trp-Phe-Arg-Leu-Ala-His-NH2 prepared in Example 28;

[0170] Figure 88 Mass spectrometry test results of hyaluronic acid-modified beauty peptide B2 prepared in Example 28;

[0171] Figure 89 Liquid chromatography test results of hyaluronic acid-modified beauty peptide B2 prepared in Example 28;

[0172] Figure 90 Liquid chromatography test results of hyaluronic acid-modified beauty peptide B4 prepared in Example 28;

[0173] Figure 91 Liquid chromatography test results of hyaluronic acid-modified beauty peptide B4 prepared in Example 28;

[0174] Figure 92 Mass spectrometry test results of hyaluronic acid-modified beauty peptide B6 prepared in Example 28;

[0175] Figure 93 Liquid chromatography test results of hyaluronic acid-modified beauty peptide B6 prepared in Example 28;

[0176] Figure 94 Mass spectrometry test results of H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2 prepared in Example 29;

[0177] Figure 95Test results of the liquid chromatography of H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2 prepared in Example 29;

[0178] Figure 96 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide G2 prepared in Example 29;

[0179] Figure 97 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide G2 prepared in Example 29;

[0180] Figure 98 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide G4 prepared in Example 29;

[0181] Figure 99 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide G4 prepared in Example 29;

[0182] Figure 100 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide G6 prepared in Example 29;

[0183] Figure 101 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide G6 prepared in Example 29;

[0184] Figure 102 Test results of the mass spectrometry of H-Arg-Arg-Gln-Met-Glu-Glu-NH2 prepared in Example 30;

[0185] Figure 103 Test results of the liquid chromatography of H-Arg-Arg-Gln-Met-Glu-Glu-NH2 prepared in Example 30;

[0186] Figure 104 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide F2 prepared in Example 30;

[0187] Figure 105 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide F2 prepared in Example 30;

[0188] Figure 106 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide F4 prepared in Example 30;

[0189] Figure 107 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide F4 prepared in Example 30;

[0190] Figure 108 Test results of the mass spectrometry of the hyaluronic acid-modified beauty peptide F6 prepared in Example 30;

[0191] Figure 109 Test results of the liquid chromatography of the hyaluronic acid-modified beauty peptide F6 prepared in Example 30. Detailed implementation manners

[0192] To make the objectives, technical solutions and advantages of the present invention more clear and definite, the technical solutions of the present invention will be further described in detail below in conjunction with the specific implementation manners:

[0193] It should be noted that the hyaluronic acid used in Examples 1 - 30 of the present invention is sodium hyaluronate hydrolyzate, purchased from Shandong Baolijia Biotechnology Co., Ltd. It is a mixture, and its main components include disaccharide sodium hyaluronate, tetrasaccharide sodium hyaluronate, and hexasaccharide sodium hyaluronate, with a mass ratio of 1:5:3.

[0194] Example 1:

[0195] A synthesis method of H-Gly-Gln-Pro-Arg-OH, comprising:

[0196] Place 3.13 mmol of Wang resin in a 125 mL solid-phase synthesis reactor, add 7.5 mmol of the amino acid Fmoc-Arg(Pbf)-OH, add 15 mL of dichloromethane (DCM), then add 2.01 mL of pyridine and 1.78 mL of DBU, react at 25 °C for 3 h, filter by suction, wash with DMF solution 3 times, 15 mL each time, add 15 mL of capping solution (the capping solution contains Ac2O, DMF, and DIEA, and the mass ratio of Ac2O:DMF:DIEA is 10:84:6) and react for 15 min; filter, wash the resin with dichloromethane 2 times, 15 mL each time, wash with methanol 2 times, 15 mL each time, and wash with DMF 2 times, 15 mL each time; add 15 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF 6 times, 15 mL each time, and drain for later use.

[0197] Take 5 mmol of Fmoc-Pro-OH·H2O and 5 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF and 0.8 mL of DIC, let stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 15 mL each time; after the washing is completed, proceed to the next step of the reaction; add 15 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 15 mL each time, and drain for later use.

[0198] Take 7.5 mmol of Fmoc-Gln(Trt)-OH and 7.5 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF, 1.2 mL of DIC, let it stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 15 mL each time; after washing, proceed to the next step of the reaction; add 15 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with 15 mL of DMF solution 6 times, and suction dry for later use.

[0199] Take 7.5 mmol of Fmoc-Gly-OH and 7.5 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF, 1.2 mL of DIC, let it stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 15 mL each time; after washing, proceed to the next step of the reaction; add 15 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 15 mL each time; wash with methanol 2 times, 15 mL each time; wash with DCM solution 2 times, 15 mL each time; wash with methanol 2 times, 15 mL each time; dry under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-Wang-resin, where AA1 is Gly; AA2 is Gln(Trt); AA3 is Pro; AA4 is Arg(Pbf). Cut the above peptide resin with TFA / Tis / H2O (the volume ratio of TFA, Tis and H2O is 90:5:5), the dosage is 30 mL, and the time is 2.5 h. Add the cutting solution to 300 mL of ether (5 °C) solution, precipitate white solid, centrifuge to obtain H-Gly-Gln-Pro-Arg-OH, and its mass spectrum ( Figure 1 ) and high performance liquid chromatography ( Figure 2 ) are as Figure 1-2 shown.

[0200] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0201] Weigh 0.46 g of H-Gly-Gln-Pro-Arg-OH·2TFA, add 5 mL of DMSO and stir to dissolve. Add 0.785 g of DIEA, and the tetrapeptide precipitates. Then weigh 2.1 g of hyaluronic acid, control the temperature of the water bath at 45 °C, stir for 15 min until the reaction solution becomes clear, continue to react overnight at 45 °C, take a sample for LC-MS detection, and the reaction is basically complete. Add 4 mL of acetic acid, control the temperature of the water bath at 35 °C, and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection, and the reaction is basically complete; then carry out reverse-phase chromatography purification. The purification conditions are as follows:

[0202] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0203] Packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0204] Mobile phase: A: 1% HAc; B: ACN;

[0205] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0206] Sample loading: Flow rate: 10 mL / min;

[0207] Elution: 0 - 10% B for 60 min;

[0208] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0209] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide. The mass spectrometry and high-performance liquid chromatography characterization results are as Figure 3 and Figure 4 shown.

[0210] Example 2:

[0211] A synthesis method of H-Lys-Thr-Thr-Lys-Ser-OH, comprising:

[0212] Place 8.75 mmol of CTC resin in a 250 mL solid-phase synthesis reactor, add 17.5 mmol of the amino acid Fmoc-Ser(tBu)-OH, add 75 mL of dichloromethane, then add 7.0 mL of DIEA, and react at 25 °C for 3 h. Add 10 mL of methanol and react for 5 min; filter, wash the resin with dichloromethane 2 times, 75 mL each time; wash with methanol 2 times, 75 mL each time; wash with DMF 2 times, 75 mL each time; add 75 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter, remove the deprotection solution, and then wash with DMF solution 6 times, 75 mL each time, and drain for use.

[0213] Take 21 mmol of Fmoc-Lys(Boc)-OH and 21 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 50 mL of DMF solution, let it stand for 15 min after adding 3.2 mL of DIC, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed; wash the resin with DMF solution three times, 75 mL each time; after washing, proceed to the next step of the reaction; add 75 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 65 mL each time, and dry by suction for later use.

[0214] Take 21 mmol of Fmoc-Thr(tBu)-OH and 21 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 50 mL of DMF solution, let it stand for 15 min after adding 3.2 mL of DIC, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed; wash the resin with DMF solution three times, 75 mL each time; after washing, proceed to the next step of the reaction; add 75 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 65 mL each time, and dry by suction for later use.

[0215] Take 21 mmol of Fmoc-Thr(tBu)-OH and 21 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 50 mL of DMF solution, let it stand for 15 min after adding 3.2 mL of DIC, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed; wash the resin with DMF solution three times, 75 mL each time; after washing, proceed to the next step of the reaction; add 75 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 65 mL each time, and dry by suction for later use.

[0216] Take 21 mmol of Fmoc-Lys(Boc)-OH and 21 mmol of HOBt in a 100 mL beaker. Cool down to 2 - 8 °C, add 50 mL of DMF solution, and 3.2 mL of DIC. Let it react statically for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with DMF solution, 75 mL each time. After washing, proceed to the next step. Add 75 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter to remove the deprotection solution, then wash 6 times with DMF solution, 65 mL each time, then wash 2 times with methanol, 75 mL each time, wash 2 times with DCM solution, 75 mL each time, and wash 2 times with methanol, 75 mL each time. Dry it under vacuum to obtain H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-CTC-resin;

[0217] Take 4.14 g of the above peptide resin, add 40 mL of cleavage solution TFA / Tis / H2O (the volume ratio of TFA, Tis, and H2O is 90:5:5), stir and react at 30 °C for 2.5 h, filter to remove the resin to obtain the filtrate; Dry the filtrate to obtain H-Lys-Thr-Thr-Lys-Ser-OH, and its mass spectrometry and high-performance liquid chromatography characterization results are as Figure 5 and Figure 6 shown.

[0218] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0219] Weigh 0.5 g of H-Lys-Thr-Thr-Lys-Ser-OH·3TFA, add 5 mL of DMSO and stir to dissolve. Add 82 μL of TFA, stir for 0.5 h, then add 0.641 g of DIEA. Weigh 1.71 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take samples for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take samples for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0220] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0221] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0222] Mobile phase: A: 1% HAc; B: ACN;

[0223] Equilibrium: A:B = 100:0, equilibrium for 10 min, flow rate: 10 mL / min;

[0224] Sample loading: flow rate: 10 mL / min;

[0225] Elution: 0 - 10%B for 60 min;

[0226] Column cleaning: clean with 80% ACN until baseline equilibrium;

[0227] Collect qualified products to obtain hyaluronic acid - modified beauty peptides, and their high - performance liquid chromatography characterization is as Figure 7 shown; among them, collect the samples at 5.801 min, 6.102 min, and 6.558 min respectively for mass spectrometry characterization, and the results are as Figure 8 、 Figure 9 、 Figure 10 shown.

[0228] Example 3:

[0229] A synthesis method of H - Lys - Leu - Ala - Lys - Lys - NH2, comprising:

[0230] Place 10 mmol of AM resin in a 250 - mL solid - phase synthesis reactor, add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and dry by suction for later use.

[0231] Take 20 mmol of Fmoc - Linker and 20 mmol of HOBt in a 100 - mL beaker, cool down to 5°C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 - mL beaker to the above - mentioned 250 - mL solid - phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and dry by suction for later use.

[0232] Take 20 mmol of Fmoc-Lys(Boc)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 70 mL each time; after the washing is completed, proceed to the next reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 70 mL each time, and filter to dryness for use.

[0233] Take 20 mmol of Fmoc-Lys(Boc)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 70 mL each time; after the washing is completed, proceed to the next reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 70 mL each time, and filter to dryness for use.

[0234] Take 20 mmol of Fmoc-Ala-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 70 mL each time; after the washing is completed, proceed to the next reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 70 mL each time, and filter to dryness for use.

[0235] Take 20 mmol of Fmoc-Leu-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 40 mL each time; after the washing is completed, proceed to the next reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 40 mL each time, and filter to dryness for use.

[0236] Take 20 mmol of Fmoc-Lys(Boc)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it react statically for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside. Then wash with methanol 2 times, 70 mL each time, wash with DCM solution 2 times, 70 mL each time, and wash with methanol 2 times, 70 mL each time. Dry under vacuum to obtain the peptide resin of H-Lys(Boc)-Leu-Ala-Lys(Boc)-Lys(Boc)-Linker-AM resin,

[0237] In the above peptide resin, add 100 mL of cleavage solution TFA / Tis / H2O (the volume ratio of TFA, Tis and H2O is 90:5:5), cleave for 2.5 hours, add the cleavage solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, dry under vacuum to obtain H-Lys-Leu-Ala-Lys-Lys-NH2, and its mass spectrometry and LC-MS characterization results are as Figure 11 and Figure 12 shown.

[0238] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0239] Weigh 0.5 g of H-Lys-Leu-Ala-Lys-Lys-NH2·4TFA, add 10 mL of DMSO and stir to dissolve, add 107 μL of TFA, stir at room temperature for 0.5 h, then add 0.557 g of DIEA, and then add 1.49 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take samples to detect LC-MS, the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples to detect LC-MS, the reaction is basically complete; then carry out reverse-phase chromatography purification, purification conditions:

[0240] Dissolution: Take 0.5 g of crude product and dilute it with 100 mL of H2O;

[0241] Filler: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0242] Mobile phase: A: 1% HAc; B: ACN;

[0243] Equilibrium: A:B = 100:0, equilibrium for 10 min, flow rate: 10 mL / min;

[0244] Sample loading: flow rate: 10 mL / min;

[0245] Elution: 0 - 10% B for 60 min;

[0246] Column cleaning: cleaning with 80% ACN until baseline equilibrium;

[0247] Collect the qualified products to obtain the hyaluronic acid - modified beauty peptide, and its mass spectrometry and high - performance liquid chromatography characterization results are as Figure 13 and Figure 14 shown.

[0248] Example 4:

[0249] A synthesis method of H - Glu - Glu - Met - Gln - Arg - Arg - NH2, comprising:

[0250] Place 5 mmol of AM resin in a 100 mL solid - phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for use;

[0251] Take 10 mmol of Fmoc - Linker and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid - phase synthesis reactor, stir and react for 1.5 h until the reaction is complete; wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for use.

[0252] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, then add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 30 mL of DMF solution each time, and filter to dryness for use.

[0253] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, then add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with 20 mL of DMF solution each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 30 mL of DMF solution each time, and filter to dryness for use.

[0254] Take 10 mmol of Fmoc-Gln(Trt)-OH, 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, then add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with 20 mL of DMF solution each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 30 mL of DMF solution each time, and filter to dryness for use.

[0255] Take 10 mmol of Fmoc-Met-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, then add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with 20 mL of DMF solution each time; after washing, proceed to the next step of the reaction; add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 40 mL of DMF solution each time, and filter to dryness for use.

[0256] Take 10 mmol of Fmoc-Glu(otBu)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it react statically for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 20 mL each time; after washing, proceed to the next step of the reaction; add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time, and drain and set aside.

[0257] Take 15 mmol of Fmoc-Glu(otBu)-OH and 1 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 2.3 mL (the content here seems incomplete, assuming it's a certain reagent), let it react statically for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin 3 times with DMF solution, 30 mL each time; after washing, proceed to the next step of the reaction; add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time, and drain and set aside; then wash with methanol 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, wash with methanol 2 times, 40 mL each time, and dry in vacuum to obtain the peptide resin of H-Glu(otBu)-Glu(otBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Linker-AM resin;

[0258] Cut the above peptide resin with 100 mL of TFA / anisole / phenol / H2O / EDT (the mass ratio of TFA, anisole, phenol, H2O and EDT is: 87.5:5:2.5:2.5:2.5) for 2.5 h, add the cutting solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry in vacuum to obtain H-Glu-Glu-Met-Gln-Arg-Arg-NH2, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 15 and Figure 16 shown.

[0259] The synthetic route of a hyaluronic acid-modified beauty peptide is: ;

[0260] Among them, -COOX is -COONa;

[0261] Preparation method of hyaluronic acid-modified beauty peptide, comprising the following steps:

[0262] Weigh 1 g of H-Glu-Glu-Met-Gln-Arg-Arg-NH2·3TFA, add 10 mL of DMSO and stir to dissolve, add 889 mg of DIEA, then add 3.67 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take a sample for LC-MS detection, the reaction is basically complete, add 8 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take a sample for LC-MS detection, the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0263] Dissolution: Take 1 g of the crude product and dilute it with 200 mL of H2O;

[0264] Filler: 50 DAC, C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0265] Mobile phase: A: 1% HAc; B: ACN;

[0266] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0267] Sample loading: Flow rate: 60 mL / min;

[0268] Elution: 0 - 20% B for 60 min;

[0269] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0270] Collect qualified products to obtain hyaluronic acid-modified beauty peptides with three structures, as follows:

[0271] D2, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 17 and Figure 18 shown;

[0272] D4, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 19 and Figure 20 shown;

[0273] D6, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 21 and Figure 22 shown.

[0274] Example 5:

[0275] Preparation method of hyaluronic acid-modified beauty peptide, comprising the following steps:

[0276] Weigh 0.5 g of H-Gly-His-Lys-OH·2AcOH, add 10 mL of DMSO and stir for 5 min to dissolve. Add 286 μL of TFA, and after stirring for 1 min, the solution becomes clear. Then stir for another 0.5 h, add 3 g of hyaluronic acid, stir to dissolve, add 1.11 g of DIEA, stir for 1 min, control the temperature of the water bath at 45 °C, stir and react overnight, take a sample to detect LC-MS, the reaction is basically complete, add 4 mL of acetic acid, control the temperature of the water bath at 35 °C, carry out the rearrangement reaction for 3 h, take a sample to detect LC-MS, the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0277] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0278] Packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0279] Mobile phase: A: 1% HAc; B: ACN;

[0280] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0281] Sample loading: Flow rate: 10 mL / min;

[0282] Elution: 0 - 20% B for 60 min;

[0283] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0284] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide, and its mass spectrometry and high performance liquid chromatography test results are as Figure 23 and Figure 24 shown.

[0285] Example 6:

[0286] A synthesis method of H-Gly-Pro-Gln-Gly-Pro-Gln-OH, comprising:

[0287] Place 10 mmol of AM resin in a 100 mL solid-phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain for use.

[0288] Take 20 mmol of Fmoc-Linker and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 3.1 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 30 mL each time, and filter until dry for use.

[0289] Take 20 mmol of Fmoc-Glu-OtBu and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 4.6 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 30 mL each time, and filter until dry for use.

[0290] Take 20 mmol of Fmoc-Pro-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 4.6 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 30 mL each time, and filter until dry for use.

[0291] Take 20 mmo of Fmoc-Gly-OH and 20 mmo of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 4.6 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 30 mL each time, and filter until dry for use.

[0292] Take 20 mmol of Fmoc-Gln(Trt)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, let 4.6 mL of DIC stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and dry by suction for use.

[0293] Take 20 mmol of Fmoc-Pro-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, let 4.6 mL of DIC stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and dry by suction for use.

[0294] Take 20 mmol of Fmoc-Gly-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, let 4.6 mL of DIC stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 30 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and dry by suction for use. Then wash with methanol 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, and wash with methanol 2 times, 40 mL each time. Dry in vacuum to obtain H-BB1-BB2-BB3-BB4-BB5-BB6-Linker-AM resin, where AA1 is Gly; AA2 is Pro; AA3 is Gln; AA4 is Gly; AA5 is Pro; and AA6 is Gln.

[0295] Take 8.3 g of the above peptide resin and cut it with 50 mL of a cleavage solution (the volume ratio of TFA, Tis, and H2O is 90:5:5) for 2.5 h. Add the cleavage solution to 500 mL of an ether (5 °C) solution to precipitate a white solid. Centrifuge and dry it under vacuum to obtain H-Gly-Pro-Gln-Gly-Pro-Gln-OH. The results of its mass spectrometry and high-performance liquid chromatography characterization are as Figure 25 and Figure 26 shown.

[0296] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0297] Weigh 0.5 g of H-Gly-Pro-Gln-Gly-Pro-Gln-OH·TFA, add 5 mL of DMSO and stir to dissolve. Add 0.834 g of DIEA, then add 2.23 g of hyaluronic acid. Control the temperature in a water bath at 45 °C and stir to react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid, control the temperature in the water bath at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then perform reverse-phase chromatography purification. The purification conditions are:

[0298] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0299] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0300] Mobile phase: A: 1% HAc; B: ACN;

[0301] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0302] Sample loading: Flow rate: 10 mL / min;

[0303] Elution: 0 - 20% B for 60 min;

[0304] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0305] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide. The results of its mass spectrometry and high-performance liquid chromatography characterization are as Figure 27 and Figure 28 shown.

[0306] Example 7:

[0307] A synthesis method of H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH2, comprising:

[0308] Place 5 mmol of AM resin in a 100 mL solid-phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for later use.

[0309] Take 10 mmol of Fmoc-Linker and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for later use.

[0310] Take 15 mmol of Fmoc-Val-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 2.3 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for later use.

[0311] Take 15 mmol of Fmoc-Pro-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 2.3 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 20 mL each time; after washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for later use.

[0312] Take 15 mmol of Fmoc-Lys(Boc)-OH and 15 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution, and 2.3 mL of DIC. Let it stand and react for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 30 mL of DMF solution each time, and suction dry for use.

[0313] Take 15 mmol of Fmoc-Phe-OH and 15 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution, and 2.3 mL of DIC. Let it stand and react for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 40 mL of DMF solution each time, and suction dry for use.

[0314] Take 10 mmol of Fmoc-D-Trp(Boc)-OH and 10 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution, and 1.5 mL of DIC. Let it stand and react for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 40 mL of DMF solution each time, and suction dry for use.

[0315] Take 15 mmol of Fmoc-Arg(Pbf)-OH and 15 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution, and 2.3 mL of DIC. Let it stand and react for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with 30 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 40 mL of DMF solution each time, and suction dry for use.

[0316] Take 10 mmol of Fmoc-D-Phe-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 1.5 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 30 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside for use.

[0317] Take 15 mmol of Fmoc-Pro-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 2.3 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 30 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside for use.

[0318] Take 15 mmol of Fmoc-Met-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution, and let 2.3 mL of DIC stand for reaction for 15 min. Then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 30 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside for use. Then wash with methanol 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, and wash with methanol 2 times, 40 mL each time; dry under vacuum to obtain the peptide resin of H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-Linker-AM resin.

[0319] Take 5.7 g of the above peptide resin and cleave it with 50 mL of cleavage solution TFA / anisole / EDT / phenol / H2O (mass ratio of TFA:anisole:EDT:phenol:H2O is 87.5:5:2.5:2.5:2.5) for 2.5 h. Add the cleavage solution to 500 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry in vacuum to obtain H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH2, whose mass spectrum and high performance liquid chromatography are as Figure 29 and Figure 30 shown.

[0320] Preparation method of hyaluronic acid-modified beauty peptide, comprising the following steps:

[0321] Weigh 0.5 g of H-Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val-NH2·3TFA, add 5 mL of DMSO and stir to dissolve, add 0.375 g of DIEA, then add 1 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take samples to detect LC-MS, the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples to detect LC-MS, the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0322] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0323] Filler: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0324] Mobile phase: A: 1% HAc; B: ACN;

[0325] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0326] Sample loading: Flow rate: 10 mL / min;

[0327] Elution: 15 - 35% B for 60 min;

[0328] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0329] Collect qualified products to obtain hyaluronic acid-modified beauty peptide, whose mass spectrum and high performance liquid chromatography characterization results are as Figure 31 and Figure 32 shown.

[0330] Example 8:

[0331] A method for synthesizing H-Phe-Val-Ala-Pro-Phe-Pro-OH, comprising:

[0332] Place 12.5 mmol of CTC resin in a 125 mL solid-phase synthesis reactor, add 25 mmol of the amino acid Fmoc-Pro-OH, add 100 mL of dichloromethane, then add 10.9 mL of DIEA, react at 25 °C for 3 h, add 15 mL of methanol, and react for 5 min; filter, wash the resin with dichloromethane twice, 100 mL each time, wash with methanol twice, 100 mL each time, and wash with DMF twice, 100 mL each time; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 20 mL each time, and drain to dryness for use.

[0333] Take 20 mmol of Fmoc-Phe-OH and 20 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 30 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution three times, 100 mL each time; after the washing is completed, proceed to the next step of the reaction; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 20 mL each time, and drain to dryness for use.

[0334] Take 20 mmol of Fmoc-Pro-OH and 20 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 30 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution three times, 100 mL each time; after the washing is completed, proceed to the next step of the reaction; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 20 mL each time, and drain to dryness for use.

[0335] Take 20 mmol of Fmoc-Ala-OH and 20 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 8 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 100 mL each time; after washing, proceed to the next step of the reaction; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 20 mL each time, and filter dry for later use.

[0336] Take 20 mmol of Fmoc-Val-OH and 20 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 8 mL of DMF solution and 20 mmol of DIC, let it stand and react for 5 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 100 mL each time; after washing, proceed to the next step of the reaction; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 20 mL each time, and filter dry for later use.

[0337] Take 20 mmol of Fmoc-Phe-OH and 20 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 30 mL of DMF solution and 3.1 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 100 mL each time; after washing, proceed to the next step of the reaction; add 100 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 20 mL each time, then wash with methanol 2 times, 100 mL each time, wash with DCM solution 2 times, 100 mL each time, wash with methanol 2 times, 100 mL each time, and dry in vacuum to obtain H-Phe-Val-Ala-Pro-Phe-Pro-CTC-resin;

[0338] Cut 2 g of the above peptide resin with 20 mL of cleavage solution TFA / DCM (the volume ratio of TFA to DCM is 2:98) at 30 °C for 1 hour, pull dry the cleavage solution to obtain H-Phe-Val-Ala-Pro-Phe-Pro-OH, and its mass spectrometry and LC-MS characterization results are as Figure 33 and Figure 34 shown. The synthesis route of a hyaluronic acid-modified beauty peptide is as follows:

[0339] ;

[0340] wherein, -COOX is -COONa;

[0341] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0342] Weigh 0.56 g of H-Phe-Val-Ala-Pro-Phe-Pro-OH.TFA, add 6 mL of DMSO and stir to dissolve, add 0.75 g of DIEA, then add 2.2 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out a rearrangement reaction for 3 h, take a sample for LC-MS detection, and the reaction is basically complete; then perform reverse-phase chromatography purification, and the purification conditions are as follows:

[0343] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0344] Packing: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0345] Mobile phase: A: 1% HAc; B: ACN;

[0346] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0347] Sample loading: Flow rate: 10 mL / min;

[0348] Elution: 12 - 32% B for 60 min;

[0349] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0350] Collect the qualified products to obtain hyaluronic acid-modified beauty peptides with three structures, namely:

[0351] Hyaluronic acid-modified beauty peptide H2, in which n = 0 in its structure, that is, y = 1 in the structure of the raw material hyaluronic acid, and the product prepared with the beauty peptide, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 35 and Figure 36 shown;

[0352] Hyaluronic acid-modified beauty peptide H4, in which n = 1 in its structure, that is, y = 2 in the structure of the raw material hyaluronic acid, and the product prepared with the beauty peptide, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 37 and Figure 38 shown;

[0353] The beauty peptide H6 modified with hyaluronic acid, where n = 2 in its structure, that is, y = 3 in the structure of the raw material hyaluronic acid, and the product prepared with the beauty peptide, its mass spectrometry and high performance liquid chromatography characterization results are as Figure 39 and Figure 40 shown.

[0354] Example 9:

[0355] A synthesis method of H-His-D-Phe-Arg-NH2, comprising:

[0356] Place 10 mmol of AM resin in a 250 mL solid-phase synthesis reactor, add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain and set aside.

[0357] Take 20 mmol of Fmoc-Linker and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after the washing is completed, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain and set aside.

[0358] Take 20 mmol of Fmoc-Arg(Pbf)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after the washing is completed, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain and set aside.

[0359] Take 20 mmol of Fmoc-D-Phe-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing is completed, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 70 mL each time, and drain and set aside for use.

[0360] Take 20 mmol of Fmoc-His(Trt)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 20 mmol of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing is completed, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time, and drain and set aside for use. Then add methanol to wash 2 times, 70 mL each time, wash with DCM solution 2 times, 70 mL each time, and wash with methanol 2 times, 70 mL each time. Dry under vacuum to obtain the peptide resin of H-His(Trt)-D-Phe-Arg(Pbf)-Linker-AM resin;

[0361] Cut the above peptide resin with TFA / Tis / H2O (the volume ratio of TFA, Tis and H2O is 90:5:5), the dosage is 100 mL, and the time is 2.5 h. Add the cutting solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry under vacuum to obtain H-His-D-Phe-Arg-NH2, and its mass spectrometry and LC-MS characterization results are as Figure 41 and Figure 42 shown. The synthesis route of a hyaluronic acid-modified beauty peptide is:

[0362] ;

[0363] wherein, n = 2, -COOX is -COONa;

[0364] The preparation method of the hyaluronic acid-modified beauty peptide includes the following steps:

[0365] Weigh 0.5 g of H-His-D-Phe-Arg-NH2·3TFA, add 5 mL of DMSO and stir to dissolve. Add 661 mg of DIEA, then add 1.94 g of hyaluronic acid. Control the temperature in a water bath at 45 °C and stir the reaction overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid. Control the temperature in a water bath at 35 °C and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse-phase chromatography purification. The purification conditions are as follows:

[0366] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0367] Packing material: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0368] Mobile phase: A: 1% HAc; B: ACN;

[0369] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0370] Sample loading: Flow rate: 10 mL / min;

[0371] Elution: 0 - 20% B for 60 min;

[0372] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0373] Collect the qualified products to obtain three kinds of hyaluronic acid-modified beauty peptides with different structures, namely:

[0374] Hyaluronic acid-modified beauty peptide L2, in which n = 0 in its structure, that is, y = 1 in the structure of the raw material hyaluronic acid. The product prepared with the beauty peptide, its mass spectrometry and high-performance liquid chromatography characterization results are as Figure 43 and Figure 44 shown;

[0375] Hyaluronic acid-modified beauty peptide L4, in which n = 1 in its structure, that is, y = 2 in the structure of the raw material hyaluronic acid. The product prepared with the beauty peptide, its mass spectrometry and high-performance liquid chromatography characterization results are as Figure 45 and Figure 46 shown;

[0376] Hyaluronic acid-modified beauty peptide L6, in which n = 2 in its structure, that is, y = 3 in the structure of the raw material hyaluronic acid. The product prepared with the beauty peptide, its mass spectrometry and high-performance liquid chromatography characterization results are as Figure 47 and Figure 48 shown.

[0377] Example 10:

[0378] A synthesis method of H-Tyr-Pro-Phe-Phe-NH2, comprising:

[0379] Place 10 mmol of AM resin in a 250 mL solid-phase synthesis reactor, add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain to dryness for later use.

[0380] Take 20 mmol of Fmoc-Linker and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain to dryness for later use.

[0381] Take 20 mmol of Fmoc-Phe-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed; wash the resin with DMF solution 3 times, 70 mL each time; after washing, proceed to the next step of the reaction; add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution L 6 times, 70 mL each time, and drain to dryness for later use.

[0382] Take 20 mmol of Fmoc-Phe-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 70 mL each time. After washing, proceed to the next step of the reaction. Add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and drain to dryness for later use.

[0383] Take 20 mmol of Fmoc-Pro-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 70 mL each time. After washing, proceed to the next reaction. Add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 70 mL each time, and filter to dryness for use.

[0384] Take 20 mmol of Fmoc-Tyr(tBu)-OH and 20 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 35 mL of DMF solution and 3.1 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 70 mL each time. After washing, proceed to the next reaction. Add 70 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and filter to dryness for use. Then add methanol to wash 2 times, 70 mL each time, wash with DCM solution 2 times, 70 mL each time, and wash with methanol 2 times, 70 mL each time. Dry under vacuum to obtain the peptide resin of H-Tyr(tBu)-Pro-Phe-Phe-Linker-AM resin;

[0385] Cut the above peptide resin with TFA / Tis / H2O (the volume ratio of TFA, Tis and H2O is 90:5:5), the dosage is 100 mL, and the time is 2.5 h. Add the cutting solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry under vacuum to obtain H-Tyr-Pro-Phe-Phe-NH2, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 49 and Figure 50 shown.

[0386] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0387] Weigh 0.5 g of H-Tyr-Pro-Phe-Phe-NH2.TFA, add 5 mL of DMSO and stir to dissolve. Add 0.771 g of DIEA, then add 2.27 g of hyaluronic acid. Control the water bath temperature at 45 °C and stir the reaction overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid. Control the water bath temperature at 35 °C and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification. The purification conditions are as follows:

[0388] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0389] Column packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0390] Mobile phase: A: 1% HAc; B: ACN;

[0391] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0392] Sample loading: Flow rate: 10 mL / min;

[0393] Elution: 12 - 32% B for 60 min;

[0394] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0395] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide. Its mass spectrometry and high-performance liquid chromatography characterization results are as Figure 51 and Figure 52 shown.

[0396] Example 11:

[0397] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0398] Weigh 0.5 g of H-β-Ala-His-OH.TFA, add 10 mL of DMSO and stir to dissolve. Add 0.5 g of TFA and stir for 10 min. Then add 2.5 g of DIEA, and then add 6.85 g of hyaluronic acid. Control the water bath temperature at 45 °C and stir the reaction overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid. Control the water bath temperature at 35 °C and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification. The purification conditions are as follows:

[0399] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0400] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0401] Mobile phase: A: 1% HAc; B: ACN;

[0402] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0403] Sample loading: Flow rate: 10 mL / min;

[0404] Elution: 0 - 10% B for 60 min;

[0405] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0406] Collect qualified products to obtain hyaluronic acid - modified beauty peptides. Among them, the mass spectrometry and high - performance liquid chromatography characterization results of the product where the beauty peptide binds to two tetrasaccharide sodium hyaluronates or the product where the beauty peptide binds to one hexasaccharide sodium hyaluronate and one disaccharide sodium hyaluronate are as Figure 53 and Figure 54 shown. The peak position in high - performance liquid chromatography is 6.460 min, and the product molecular weights are 872.3 / 582.0; The mass spectrometry and high - performance liquid chromatography characterization results of the product where the beauty peptide binds to one hexasaccharide sodium hyaluronate and one tetrasaccharide sodium hyaluronate are as Figure 55 and Figure 56 shown. The peak position in high - performance liquid chromatography is 7.202 min, and the product molecular weights are 708.4 / 1061.8 / 1415.8.

[0407] Example 12:

[0408] A synthesis method of H - Glu - Glu - Met - Gln - Arg - Arg - Ala - OH, comprising:

[0409] Place 7.5 mmol of Wang resin in a 125 mL solid - phase synthesis reactor, add 15 mmol of amino acid Fmoc - Ala - OH, add 50 mL of dichloromethane, then add 4.8 mL of pyridine and 4.2 mL of DBU, react at 25 °C for 3 h, filter by suction, wash 3 times with 50 mL of DMF solution each time, add 50 mL of capping solution Ac2O / DMF / DIEA (volume ratio of Ac2O, DMF to DIEA is 10:84:6) and react for 15 min. Filter, wash the resin 2 times with 50 mL of dichloromethane each time, 2 times with 50 mL of methanol each time, and 2 times with 50 mL of DMF each time; Add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with 50 mL of DMF solution each time, and dry by suction for later use.

[0410] Take 15 mmol of Fmoc-Arg(Pbf)-OH and 15 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 15 mL each time. After washing, proceed to the next step of the reaction. Add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 50 mL each time, and filter dry for use.

[0411] Take 15 mmol of Fmoc-Arg(Pbf)-OH and 15 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 15 mL each time. After washing, proceed to the next step of the reaction. Add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 50 mL each time, and filter dry for use.

[0412] Take 15 mmol of Fmoc-Gln(Trt)-OH and 15 mmol of HOBt in a 50 mL beaker, cool down to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand and react for 15 min, and add the solution in the 50 mL beaker to the above 125 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 15 mL each time. After washing, proceed to the next step of the reaction. Add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 50 mL each time, and filter dry for use.

[0413] Take 15 mmol of Fmoc-Met-OH and 15 mmol of HOBt in a 50 mL beaker. Cool the temperature to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 50 mL beaker to the above-mentioned 125 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 15 mL each time. After washing, proceed to the next step of the reaction. Add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 50 mL each time, and drain to dry for use.

[0414] Take 15 mmol of Fmoc-Glu(OtBu)-OH and 15 mmol of HOBt in a 50 mL beaker. Cool the temperature to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 50 mL beaker to the above-mentioned 125 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 15 mL each time. After washing, proceed to the next step of the reaction; add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 50 mL each time, and drain to dry for use.

[0415] Take 15 mmol of Fmoc-Glu(OtBu)-OH and 15 mmol of HOBt in a 50 mL beaker. Cool the temperature to 5 °C, add 20 mL of DMF solution and 2.32 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 50 mL beaker to the above-mentioned 125 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution three times, 15 mL each time. After washing, proceed to the next step of the reaction; add 50 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 50 mL each time, wash with methanol two times, 50 mL each time, wash with DCM solution two times, 50 mL each time, and wash with methanol two times, 50 mL each time. Dry under vacuum to obtain H-AA1-AA2-AA3-AA4-AA5-AA6-AA7-Wang-resin, where AA1 is Glu(OtBu); AA2 is Glu(OtBu); AA3 is Met; AA4 is Gln(Trt); AA5 is Arg(Pbf); AA6 is Arg(Pbf); AA7 is Ala.

[0416] Take 4.22 g of the above peptide resin and cleave it with 40 mL of TFA / anisole / phenol / H2O / EDT (the mass ratio of TFA, anisole, phenol, H2O and EDT is: 87.5:5:2.5:2.5:2.5) for 2.5 h. Add the cleavage solution to 400 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry in vacuum to obtain H-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH. The results of its mass spectrometry and high performance liquid chromatography characterization are as Figure 57 and Figure 58 shown.

[0417] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0418] Weigh 0.5 g of H-Glu-Glu-Met-Gln-Arg-Arg-Ala-OH·3TFA, add 5 mL of DMSO and stir to dissolve, then add 0.46 g of DIEA, then add 1.24 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take samples for LC-MS detection, the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples for LC-MS detection, the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0419] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0420] Filler: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0421] Mobile phase: A: 1% HAc; B: ACN;

[0422] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0423] Sample loading: Flow rate: 10 mL / min;

[0424] Elution: 0 - 10% B for 60 min;

[0425] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0426] Collect qualified products to obtain hyaluronic acid-modified beauty peptides. The results of its high performance liquid chromatography test are as Figure 59 shown. Among them, the sample at 11.577 min is the product of the beauty peptide combined with a tetrasaccharide sodium hyaluronate, and the results of its mass spectrometry characterization are as Figure 60 shown; the sample at 12.223 min is the product of the beauty peptide combined with a hexasaccharide sodium hyaluronate, and the results of its mass spectrometry characterization are as Figure 61 shown.

[0427] Example 13:

[0428] A method for synthesizing H-Asp-Val-Lys-Tyr-OH, comprising:

[0429] Place 6.25 mmol of CTC resin in a 250 mL solid-phase synthesis reactor, add 12.5 mmol of the amino acid Fmoc-Tyr(tBu)-OH, add 120 mL of dichloromethane, add 8.7 mL of DIEA, react at 25 °C for 3 h, add 12.5 mL of methanol, and react for 5 min. Filter, wash the resin with dichloromethane twice, 150 mL each time, wash with methanol twice, 150 mL each time, and wash with DMF twice, 390 mL each time. Add 65 mL of a 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with a DMF solution 6 times, 120 mL each time, and drain and set aside.

[0430] Take 15 mmol of Fmoc-Lys(Boc)-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 14 mL of a DMF solution and 1.89 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with a DMF solution 3 times, 120 mL each time. After washing, proceed to the next reaction. Add 65 mL of a 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with a DMF solution 6 times, 120 mL each time, and drain and set aside.

[0431] Take 15 mmol of Fmoc-Val-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 10 mL of a DMF solution and 2.32 mL of DIC, let stand and react for 15 min, and add the solution in the 100 mL beaker to the above 250 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with a DMF solution 3 times, 120 mL each time. After washing, proceed to the next reaction. Add 65 mL of a 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with a DMF solution 6 times, 120 mL each time, and drain and set aside.

[0432] Take 15 mmol of Fmoc-Asp(OtBu)-OH and 15 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 13 mL of DMF solution and 2.32 mL of DIC. Let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 250 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin 3 times with DMF solution, 120 mL each time. After washing, proceed to the next reaction. Add 65 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter to remove the deprotection solution, and then wash 6 times with DMF solution, 120 mL each time. Then wash 2 times with methanol, 125 mL each time, wash 2 times with DCM solution, 125 mL each time, and wash 2 times with methanol, 125 mL each time. Dry in vacuum to obtain H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-CTC-resin;

[0433] Cut 4.89 g of the above peptide resin with TFA / Tis / H2O (the volume ratio of TFA, Tis and H2O is 90:5:5), with a dosage of 40 mL. Stir and react at 30 °C for 2.5 h, filter to remove the resin, and obtain the filtrate. Dry the filtrate to obtain the crude peptide H-Asp-Val-Lys-Tyr-OH, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 62 and Figure 63 shown.

[0434] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0435] Weigh 0.5 g of H-Asp-Val-Lys-Tyr-OH.2TFA, add 7 mL of DMSO and stir to dissolve. Then add 0.773 g of DIEA, and then add 2.1 g of hyaluronic acid. Control the water bath temperature at 45 °C, stir and dissolve for 15 min, and then stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample to detect LC-MS. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0436] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0437] Filler: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0438] Mobile phase: A: 1% HAc; B: ACN;

[0439] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0440] Sample loading: Flow rate: 10 mL / min;

[0441] Elution: 0 - 20% B for 60 min;

[0442] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0443] Collect qualified products to obtain hyaluronic acid - modified beauty peptides, and the results of their high - performance liquid chromatography tests are as Figure 64 shown. Among them, the sample at 8.226 min is the product of the binding of a beauty peptide with two sodium hyaluronate hexasaccharides, and the results of its mass spectrometry characterization are as Figure 65 shown; the sample at 12.711 min is the product of the binding of a beauty peptide with one sodium hyaluronate hexasaccharide and one sodium hyaluronate disaccharide or the binding of a beauty polypeptide with two sodium hyaluronate tetrasaccharides, and the results of its mass spectrometry characterization are as Figure 66 shown.

[0444] Example 14:

[0445] A synthesis method of H - His - Ala - Leu - Arg - Phe - Trp - NH2, comprising:

[0446] Place 30 mmol of AM resin in a 500 mL solid - phase synthesis reactor, add 200 mL of 20% Pip / DMF (v / v) solution, stir for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 200 mL each time, and dry by suction for later use.

[0447] Take 10 mmol of Fmoc - Linker and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let stand for 15 min, and add the solution in the 100 mL beaker to the above 500 mL solid - phase synthesis reactor, stir for 1.5 h to complete the reaction. Wash the resin with DMF solution 3 times, 100 mL each time. After washing, proceed to the next step; add 200 mL of 20% Pip / DMF (v / v) solution, stir for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 200 mL each time, and dry by suction for later use.

[0448] Take 60 mmol of Fmoc-Trp(Boc)-OH and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution three times, 100 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 200 mL each time, and filter to dryness for use.

[0449] Take 60 mmol of Fmoc-Phe-OH and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution three times, 100 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 200 mL each time, and filter to dryness for use.

[0450] Take 60 mmol of Fmoc-Arg(Pbf)-OH and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution three times, 100 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution six times, 200 mL each time, and filter to dryness for use.

[0451] Take 60 mmol of Fmoc-Leu-OH and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with DMF solution, 100 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 200 mL each time, and filter to dryness for standby.

[0452] Take 60 mmol of Fmoc-Ala-OH and 60 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 60 mL of DMF solution and 9.3 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with DMF solution, 100 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 200 mL each time, and filter to dryness for standby.

[0453] Take 90 mmol of Fmoc-His(Trt)-OH and 90 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 100 mL of DMF solution and 13.9 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the 500 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with DMF solution, 150 mL each time. After washing, proceed to the next step of the reaction. Add 200 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash 6 times with DMF solution, 200 mL each time, and filter to dryness for standby. Then add methanol to wash 2 times, 200 mL each time, DCM solution to wash 2 times, 200 mL each time, and methanol to wash 2 times, 200 mL each time. Dry under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-AA5-AA6-Linker-AM resin, where AA1 is His(Trt); AA2 is Ala; AA3 is Leu; AA4 is Arg(Pbf); AA5 is Phe; and AA6 is Trp.

[0454] The above peptide resin was cleaved with 690 mL of TFA / anisole / phenol / H2O / EDT (mass ratio of TFA, anisole, phenol, H2O and EDT: 87.5:5:2.5:2.5:2.5) for 2.5 h. The cleavage solution was added to 7000 mL of diethyl ether (5 °C) solution, and a white solid was precipitated. After centrifugation and vacuum drying, H-His-Ala-Leu-Arg-Phe-Trp-NH2 was obtained. The characterization results of its mass spectrometry and high performance liquid chromatography are as Figure 67 and Figure 68 shown.

[0455] The synthesis route of a hyaluronic acid-modified beauty peptide is as follows: ;

[0456] wherein, -COOX is -COONa;

[0457] The preparation method of the hyaluronic acid-modified beauty peptide includes the following steps:

[0458] Weigh 1 g of H-His-Ala-Leu-Arg-Phe-Trp-NH2.TFA, add 10 mL of DMSO and stir to dissolve. Add 1.28 g of DIEA, stir for 5 min, then add 3.75 g of hyaluronic acid, control the water bath temperature at 45 °C, and stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 8 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample to detect LC-MS. When the reaction is basically complete; then carry out reverse chromatography purification. The purification conditions are:

[0459] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0460] Packing: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0461] Mobile phase: A: 1% HAc; B: ACN;

[0462] Equilibration: A:B = 95:5, equilibration for 10 min, flow rate: 10 mL / min;

[0463] Sample loading: Flow rate: 10 mL / min;

[0464] Elution: 12 - 32% B for 60 min;

[0465] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0466] Collect qualified products to obtain three structures of hyaluronic acid-modified beauty peptides as follows:

[0467] A2, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 69 and Figure 70 shown;

[0468] A4, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 71 and Figure 72 shown, its 1H NMR spectrum is as Figures 73-1 to 73-5 shown, and its 13C NMR spectrum is as Figures 74-1 to 74-5 shown, and its two-dimensional cosy spectrum is as Figure 75 shown;

[0469] A6, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 76 and Figure 77 shown.

[0470] Example 15:

[0471] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0472] Weigh 0.5 g of H-β-Ala-Pro-Dab-NH-Bzl.TFA, add 5 mL of DMSO and stir to dissolve. Add 704.32 mg of DIEA, stir for 5 min, then add 2064.08 mg of hyaluronic acid, control the water bath temperature at 45 °C, and stir and react overnight. Take samples for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take samples for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0473] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0474] Packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0475] Mobile phase: A: 1% HAc; B: ACN;

[0476] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0477] Sample loading: Flow rate: 10 mL / min;

[0478] Elution: 12 - 32% B for 60 min;

[0479] Column cleaning: Wash with 80% ACN until the baseline is balanced;

[0480] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide.

[0481] Example 16:

[0482] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0483] Weigh 0.5 g of H-Dab-Val-Dab-OH·2TFA, add 7 mL of DMSO and stir to dissolve. Then add 833.2 mg of DIEA, stir for 5 min, and then add 2441.79 mg of hyaluronic acid. Control the water bath temperature at 45 °C, and then stir and react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are as follows:

[0484] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0485] Packing: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0486] Mobile phase: A: 1% HAc; B: ACN;

[0487] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0488] Sample loading: Flow rate: 10 mL / min;

[0489] Elution: 0 - 20% B for 60 min;

[0490] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0491] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide.

[0492] Example 17:

[0493] A method for preparing a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0494] Weigh 0.5 g of H-Lys-Val-Lys-OH·2TFA, add 7 mL of DMSO and stir to dissolve. Then add 708.03 mg of DIEA, stir for 5 min, and then add 2074.97 mg of hyaluronic acid. Control the water bath temperature at 45 °C, and then stir and react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are as follows:

[0495] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0496] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0497] Mobile phase: A: 1% HAc; B: ACN;

[0498] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0499] Sample loading: Flow rate: 10 mL / min;

[0500] Elution: 0 - 20% B for 60 min;

[0501] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0502] Collect qualified products to obtain hyaluronic acid - modified beauty peptide.

[0503] Example 18:

[0504] A preparation method of hyaluronic acid - modified beauty peptide, comprising the following steps:

[0505] Weigh 0.5 g of H - Gln - Asp - Val - His - OH.TFA, add 5 mL of DMSO and stir to dissolve, add 531.55 mg of DIEA, stir for 5 min, then add 1557.76 mg of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take samples for LC - MS detection, the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples for LC - MS detection, the reaction is basically complete; then carry out reverse - phase chromatography purification, purification conditions:

[0506] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0507] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0508] Mobile phase: A: 1% HAc; B: ACN;

[0509] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0510] Sample loading: Flow rate: 10 mL / min;

[0511] Elution: 12 - 32% B for 60 min;

[0512] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0513] Collect qualified products to obtain hyaluronic acid-modified beauty peptides.

[0514] Example 19:

[0515] A preparation method of hyaluronic acid-modified beauty peptides, comprising the following steps:

[0516] Weigh 0.5 g of H-Pro-Pro-Tyr-Leu-OH.TFA, add 5 mL of DMSO and stir to dissolve. Add 541.25 mg of DIEA, stir for 5 min, then add 1586.20 mg of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, carry out a rearrangement reaction for 3 h, take a sample to detect LC-MS, and the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0517] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0518] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0519] Mobile phase: A: 1% HAc; B: ACN;

[0520] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0521] Sample loading: Flow rate: 10 mL / min;

[0522] Elution: 12 - 32% B for 60 min;

[0523] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0524] Collect qualified products to obtain hyaluronic acid-modified beauty peptides.

[0525] Example 20:

[0526] A preparation method of hyaluronic acid-modified beauty peptides, comprising the following steps:

[0527] Weigh 0.5 g of H-Pro-Lys-Glu-Lys-OH.2TFA, add 7 mL of DMSO and stir to dissolve. Then add 528.27 mg of DIEA, stir for 5 min, then add 1548.14 mg of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, carry out a rearrangement reaction for 3 h, take a sample to detect LC-MS, and the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0528] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0529] Column packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0530] Mobile phase: A: 1% HAc; B: ACN;

[0531] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0532] Sample loading: Flow rate: 10 mL / min;

[0533] Elution: 0 - 20% B for 60 min;

[0534] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0535] Collect the qualified product to obtain the hyaluronic acid - modified beauty peptide.

[0536] Example 21:

[0537] Preparation method of hyaluronic acid - modified beauty peptide, comprising the following steps:

[0538] Weigh 0.5 g of H - Val - Trp - OH·2TFA, add 7 mL of DMSO and stir to dissolve, then add 871.74 mg of DIEA, stir for 5 min, then add 2554.72 mg of hyaluronic acid, control the water bath temperature at 45 °C, and then stir and react overnight. Take a sample for LC - MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, carry out the rearrangement reaction for 3 h, take a sample for LC - MS detection, and the reaction is basically complete; then carry out reverse - phase chromatography purification, and the purification conditions are:

[0539] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0540] Column packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0541] Mobile phase: A: 1% HAc; B: ACN;

[0542] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0543] Sample loading: Flow rate: 10 mL / min;

[0544] Elution: 0 - 20% B for 60 min;

[0545] Column washing: Wash with 80% ACN until baseline balance;

[0546] Collect qualified products to obtain hyaluronic acid-modified beauty peptides.

[0547] Example 22:

[0548] A method for preparing hyaluronic acid-modified beauty peptides, comprising the following steps:

[0549] Weigh 0.5 g of H-Ser-Val-Val-Val-Arg-Thr-NH2.TFA, add 5 mL of DMSO and stir to dissolve. Add 404.41 mg of DIEA, stir for 5 min, then add 1176.38 mg of hyaluronic acid. Control the temperature in a water bath at 45 °C and stir the reaction overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the temperature in a water bath at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse-phase chromatography purification, and the purification conditions are as follows:

[0550] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0551] Packing material: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0552] Mobile phase: A: 1% HAc; B: ACN;

[0553] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0554] Sample loading: Flow rate: 10 mL / min;

[0555] Elution: 12 - 32% B for 60 min;

[0556] Column washing: Wash with 80% ACN until baseline balance;

[0557] Collect qualified products to obtain hyaluronic acid-modified beauty peptides.

[0558] Example 23:

[0559] A method for preparing hyaluronic acid-modified beauty peptides, comprising the following steps:

[0560] Weigh 0.5 g of Pal-Lys-Met(O)2-Lys-OH.2TFA, add 7 mL of DMSO and stir to dissolve. Then add 391.22 mg of DIEA and stir for 5 min. Next, add 1146.50 mg of hyaluronic acid, control the water bath temperature at 45 °C, and stir and react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0561] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0562] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0563] Mobile phase: A: 1% HAc; B: ACN;

[0564] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0565] Sample loading: Flow rate: 10 mL / min;

[0566] Elution: 0 - 20% B for 60 min;

[0567] Column cleaning: Wash with 80% ACN until the baseline is balanced;

[0568] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide.

[0569] Example 24:

[0570] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0571] Weigh 0.5 g of H-Lys-Met(O)2-Lys-OH.2TFA, add 7 mL of DMSO and stir to dissolve. Then add 604.37 mg of DIEA and stir for 5 min. Next, add 1771.19 mg of hyaluronic acid, control the water bath temperature at 45 °C, and stir and react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification, and the purification conditions are:

[0572] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0573] Filler: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0574] Mobile phase: A: 1% HAc; B: ACN;

[0575] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0576] Sample loading: flow rate: 10 mL / min;

[0577] Elution: 0 - 20% B for 60 min;

[0578] Column cleaning: clean with 80% ACN until baseline equilibration;

[0579] Collect qualified products to obtain hyaluronic acid - modified beauty peptides.

[0580] Example 25:

[0581] A preparation method of hyaluronic acid - modified beauty peptides, comprising the following steps:

[0582] Weigh 0.5 g of H - Val - Gly - Val - Ala - Pro - Gly - OH·2TFA, add 7 mL of DMSO and stir to dissolve, then add 530.41 mg of DIEA, stir for 5 min, then add 1554.41 mg of hyaluronic acid, control the water bath temperature at 45 °C, and then stir and react overnight. Take a sample for LC - MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, carry out a rearrangement reaction for 3 h, take a sample for LC - MS detection, and the reaction is basically complete; then carry out reverse - phase chromatography purification, and the purification conditions are:

[0583] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0584] Column packing: 21.2 * 250 mm, 10 - 120, C18; flow rate: 10 mL / min; wavelength: 220 nm;

[0585] Mobile phase: A: 1% HAc; B: ACN;

[0586] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0587] Sample loading: flow rate: 10 mL / min;

[0588] Elution: 0 - 20% B for 60 min;

[0589] Column cleaning: clean with 80% ACN until baseline equilibration;

[0590] Collect qualified products to obtain hyaluronic acid - modified beauty peptides.

[0591] Example 26:

[0592] Method for preparing hyaluronic acid-modified beauty peptide, comprising the following steps:

[0593] Weigh 0.5 g of H-bataAla-His-Ser-His-OH.2TFA, add 7 mL of DMSO and stir to dissolve, then add 587.07 mg of DIEA, stir for 5 min, then add 1720.46 mg of hyaluronic acid, control the water bath temperature at 45 °C, and then stir and react overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 3 mL of acetic acid, control the water bath temperature at 35 °C, and carry out a rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse-phase chromatography purification, purification conditions:

[0594] Dissolution: Take 0.5 g of the crude product and dilute it with 100 mL of H2O;

[0595] Filler: 21.2*250 mm, 10-120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0596] Mobile phase: A: 1% HAc; B: ACN;

[0597] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0598] Sample loading: Flow rate: 10 mL / min;

[0599] Elution: 0-20% B for 60 min;

[0600] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0601] Collect the qualified product to obtain the hyaluronic acid-modified beauty peptide.

[0602] Example 27:

[0603] A synthesis method of H-Trp-Phe-Arg-D-Leu-Ala-His-NH2, comprising:

[0604] Place 5 mmol of AM resin in a 100 mL solid-phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry by suction for use.

[0605] Take 10 mmol of Fmoc-Linker and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for standby.

[0606] Take 10 mmol of Fmoc-His(Trt)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for standby.

[0607] Take 10 mmol of Fmoc-Ala-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for standby.

[0608] Take 10 mmol of Fmoc-D-Leu-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for standby.

[0609] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC. Let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time. Filter until dry for use.

[0610] Take 10 mmol of Fmoc-Phe-OH and 10 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC. Let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time. Filter until dry for use.

[0611] Take 15 mmol of Fmoc-Trp(Boc)-OH and 1 mmol of HOBt in a 100 mL beaker. Cool down to 5 °C, add 15 mL of DMF solution and 2.3 mL of DIC. Let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor. Stir and react for 1.5 h until the reaction is completed. Wash the resin with DMF solution 3 times, 30 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash with DMF solution 6 times, 40 mL each time. Filter until dry for use. Then add methanol to wash 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, and wash with methanol 2 times, 40 mL each time. Dry under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-AA5-AA6-Linker-AM resin, where AA1 is Trp(Boc); AA2 is Phe; AA3 is Arg; AA4 is D-Leu; AA5 is Ala; and AA6 is His(Trt).

[0612] The above peptide resin was cleaved with 110 mL of TFA / anisole / phenol / H2O / EDT (mass ratio of TFA, anisole, phenol, H2O and EDT: 87.5:5:2.5:2.5:2.5) for 2.5 h. The cleavage solution was added to 1000 mL of ether (5 °C) solution, and a white solid was precipitated. After centrifugation and vacuum drying, H-Trp-Phe-Arg-D-Leu-Ala-His-NH2 was obtained. The results of its mass spectrometry and high performance liquid chromatography characterization are as Figure 78 and Figure 79 shown.

[0613] The synthesis route of a hyaluronic acid-modified beauty peptide is as follows:

[0614] ;

[0615] wherein, -COOX is -COONa;

[0616] A preparation method of a hyaluronic acid-modified beauty peptide includes the following steps:

[0617] Weigh 1 g of H-Trp-Phe-Arg-D-Leu-Ala-His-NH2.TFA, add 10 mL of DMSO and stir to dissolve. Add 1.28 g of DIEA, stir for 5 min, then add 3.75 g of hyaluronic acid, control the water bath temperature at 45 °C, and stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 8 mL of acetic acid and carry out a rearrangement reaction at a constant temperature for 2 h. Take a sample to detect LC-MS. When the reaction is basically complete; then perform reverse chromatography purification. The purification conditions are:

[0618] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0619] Filler: 21.2*250 mm, 10-120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0620] Mobile phase: A: 1% HAc; B: ACN;

[0621] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0622] Sample loading: Flow rate: 10 mL / min;

[0623] Elution: 12-32% B for 60 min;

[0624] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0625] Collect qualified products to obtain three structures of hyaluronic acid-modified beauty peptides as follows:

[0626] E2, the characterization results of its mass spectrometry and high performance liquid chromatography are as Figure 80 and Figure 81 shown;

[0627] E4, the characterization results of its mass spectrometry and high performance liquid chromatography are as Figure 82 and Figure 83 shown;

[0628] E6, the characterization results of its mass spectrometry and high performance liquid chromatography are as Figure 84 and Figure 85 shown.

[0629] Example 28:

[0630] A synthesis method of H-Trp-Phe-Arg-Leu-Ala-His-NH2, comprising:

[0631] Placing 5 mmol of AM resin in a 100 mL solid-phase synthesis reactor, adding 30 mL of 20% Pip / DMF (v / v) solution, stirring and reacting for 30 min, filtering, removing the deprotection solution, then washing with DMF solution 6 times, 30 mL each time, and drying for later use.

[0632] Taking 10 mmol of Fmoc-Linker and 10 mmol of HOBt in a 100 mL beaker, cooling to 5 °C, adding 15 mL of DMF solution and 1.5 mL of DIC, standing and reacting for 15 min, and adding the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stirring and reacting for 1.5 h, and the reaction is completed. The resin is washed with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry for later use.

[0633] Taking 10 mmol of Fmoc-His(Trt)-OH and 10 mmol of HOBt in a 100 mL beaker, cooling to 5 °C, adding 15 mL of DMF solution and 1.5 mL of DIC, standing and reacting for 15 min, and adding the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stirring and reacting for 1.5 h, and the reaction is completed. The resin is washed with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and dry for later use.

[0634] Take 10 mmol of Fmoc-Ala-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for use.

[0635] Take 10 mmol of Fmoc -Leu-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and suction dry for use.

[0636] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 40 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and suction dry for use.

[0637] Take 10 mmol of Fmoc-Phe-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside

[0638] Take 15 mmol of Fmoc-Trp(Boc)-OH and 1 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 2.3 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 30 mL each time. After washing, proceed to the next reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain and set aside. Then add methanol to wash 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, and wash with methanol 2 times, 40 mL each time. Dry under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-AA5-AA6-Linker-AM resin, where AA1 is Trp(Boc); AA2 is Phe; AA3 is Arg; AA4 is D-Leu; AA5 is Ala; and AA6 is His(Trt).

[0639] Cut the above peptide resin with 110 mL of TFA / anisole / phenol / H2O / EDT (the mass ratio of TFA, anisole, phenol, H2O and EDT is: 87.5:5:2.5:2.5:2.5) for 2.5 h, add the cutting solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, dry under vacuum to obtain H-Trp-Phe-Arg-Leu-Ala-His-NH2, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 86 and Figure 87 shown

[0640] The synthesis route of a hyaluronic acid-modified beauty peptide is as follows: ;

[0641] wherein, -COOX is -COONa;

[0642] Preparation method of hyaluronic acid-modified beauty peptide, comprising the following steps:

[0643] Weigh 1 g of H-Trp-Phe-Arg-Leu-Ala-His-NH2.TFA, add 10 mL of DMSO and stir to dissolve, add 1.28 g of DIEA, stir for 5 min, then add 3.75 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight, take a sample to detect LC-MS, the reaction is basically complete, add 8 mL of acetic acid, carry out rearrangement reaction at constant temperature for 2 h, take a sample to detect LC-MS, the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0644] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0645] Filler: 21.2*250 mm, 10-120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0646] Mobile phase: A: 1% HAc; B: ACN;

[0647] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0648] Sample loading: Flow rate: 10 mL / min;

[0649] Elution: 12-32% B for 60 min;

[0650] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0651] Collect qualified products to obtain hyaluronic acid-modified beauty peptides with three structures, as follows:

[0652] B2, the characterization results of its mass spectrum and high performance liquid chromatography are as Figure 88 and Figure 89 shown;

[0653] B4, the characterization results of its mass spectrum and high performance liquid chromatography are as Figure 90 and Figure 91 shown;

[0654] B6, the characterization results of its mass spectrum and high performance liquid chromatography are as Figure 92 and Figure 93 shown.

[0655] Example 29:

[0656] A synthesis method of H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2, comprising:

[0657] Place 5 mmol of AM resin in a 100 mL solid-phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain to dryness for standby.

[0658] Take 10 mmol of Fmoc-Linker and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain to dryness for standby.

[0659] Take 10 mmol of Fmoc-Glu(otBu)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain to dryness for standby;

[0660] Take 10 mmol of Fmoc-Glu(otBu)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain to dryness for standby;

[0661] Take 10 mmol of Fmoc-D-Met-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 40 mL each time, and drain to dry for use.

[0662] Take 10 mmol of Fmoc-Gln(Trt)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain to dry for use.

[0663] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After washing, proceed to the next reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain to dry for use.

[0664] Take 15 mmol of Fmoc-Arg(Pbf)-OH and 15 mmol of HOBt in a 100 mL beaker. Cool the temperature to 5 °C, add 20 mL of DMF solution and 2.3 mL of DIC. Let it stand for reaction for 15 min, and then add the solution in the 100 mL beaker to the above 100 mL solid-phase synthesis reactor. Stir the reaction for 1.5 h until the reaction is completed. Wash the resin 3 times with DMF solution, 20 mL each time. After the washing is completed, proceed to the next reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir the reaction for 30 min, filter by suction to remove the deprotection solution, and then wash it 6 times with DMF solution, 40 mL each time, and drain it for use. Then add methanol to wash 2 times, 40 mL each time, wash with DCM solution 2 times, 40 mL each time, and wash with methanol 2 times, 40 mL each time. Dry it under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-AA5-AA6-Linker-AM resin, where AA1 is Arg(Pbf); AA2 is Arg(Pbf); AA3 is Gln(Trt); AA4 is D-Met; AA5 is Glu(otBu); and AA6 is Glu(otBu).

[0665] Cut the above peptide resin with 110 mL of TFA / thioanisole / phenol / H2O / EDT (the mass ratio of TFA, thioanisole, phenol, H2O and EDT is: 87.5:5:2.5:2.5:2.5) for 2.5 h. Add the cutting solution to 1000 mL of ether (5 °C) solution, precipitate white solid, centrifuge, and dry it under vacuum to obtain H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2, and its mass spectrometry and high performance liquid chromatography characterization results are as Figure 94 and Figure 95 shown.

[0666] The synthesis route of a hyaluronic acid-modified beauty peptide is as follows: ;

[0667] Among them, -COOX is -COONa;

[0668] The preparation method of the hyaluronic acid-modified beauty peptide includes the following steps:

[0669] Weigh 1 g of H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2.TFA, add 10 mL of DMSO and stir to dissolve it. Add 1.25 g of DIEA, stir for 5 min, and then add 3.67 g of hyaluronic acid. Control the water bath temperature at 45 °C and stir the reaction overnight. Take samples to detect LC-MS. The reaction is basically complete. Add 8 mL of acetic acid and carry out the rearrangement reaction at a constant temperature for 2 h. Take samples to detect LC-MS. The reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0670] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0671] Column packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0672] Mobile phase: A: 1% HAc; B: ACN;

[0673] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0674] Sample loading: Flow rate: 10 mL / min;

[0675] Elution: 12 - 32% B for 60 min;

[0676] Column cleaning: Clean with 80% ACN until baseline equilibrium;

[0677] Collect qualified products to obtain beauty peptides modified with hyaluronic acid of three structures as follows:

[0678] G2, the characterization results of its mass spectrometry and high - performance liquid chromatography are as Figure 96 and Figure 97 shown;

[0679] G4, the characterization results of its mass spectrometry and high - performance liquid chromatography are as Figure 98 and Figure 99 shown;

[0680] G6, the characterization results of its mass spectrometry and high - performance liquid chromatography are as Figure 100 and Figure 101 shown.

[0681] Example 30:

[0682] A synthesis method of H - Arg - Arg - Gln - Met - Glu - Glu - NH2, comprising:

[0683] Place 5 mmol of AM resin in a 100 mL solid - phase synthesis reactor, add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction, remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and drain to dry for use.

[0684] Take 10 mmol of Fmoc-Linker and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction; add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash 6 times with 30 mL of DMF solution each time, and filter to dryness for use.

[0685] Take 10 mmol of Fmoc-Glu(otBu)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash 6 times with 40 mL of DMF solution each time, and filter to dryness for use.

[0686] Take 10 mmol of Fmoc-Glu(otBu)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash 6 times with 40 mL of DMF solution each time, and filter to dryness for use.

[0687] Take 10 mmol of Fmoc-Met-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand and react for 15 min, and then add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin 3 times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash 6 times with 40 mL of DMF solution each time, and filter to dryness for use.

[0688] Take 10 mmol of Fmoc-Gln(Trt)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 1.5 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After the washing is completed, proceed to the next reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and filter to dryness for standby.

[0689] Take 10 mmol of Fmoc-Arg(Pbf)-OH and 10 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 15 mL of DMF solution and 10 mmol of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After the washing is completed, proceed to the next reaction. Add 30 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with DMF solution 6 times, 30 mL each time, and filter to dryness for standby.

[0690] Take 15 mmol of Fmoc-Arg(Pbf)-OH and 15 mmol of HOBt in a 100 mL beaker, cool down to 5 °C, add 20 mL of DMF solution and 2.3 mL of DIC, let it stand for reaction for 15 min, and add the solution in the 100 mL beaker to the above-mentioned 100 mL solid-phase synthesis reactor, stir and react for 1.5 h, and the reaction is completed. Wash the resin with DMF solution 3 times, 20 mL each time. After the washing is completed, proceed to the next reaction. Add 40 mL of 20% Pip / DMF (v / v) solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with 40 mL of DMF solution 6 times, and filter to dryness for standby. Then add 40 mL of methanol to wash 2 times, 40 mL of DCM solution to wash 2 times, and 40 mL of methanol to wash 2 times. Dry under vacuum to obtain the peptide resin of H-AA1-AA2-AA3-AA4-AA5-AA6-Linker-AM resin, where AA1 is Arg(Pbf); AA2 is Arg(Pbf); AA3 is Gln(Trt); AA4 is Met; AA5 is Glu(otBu); and AA6 is Glu(otBu).

[0691] The above peptide resin was cleaved with 110 mL of TFA / anisole / phenol / H2O / EDT (mass ratio of TFA, anisole, phenol, H2O and EDT: 87.5:5:2.5:2.5:2.5) for 2.5 h. The cleavage solution was added to 1000 mL of ether (5 °C) solution, and a white solid was precipitated. After centrifugation and vacuum drying, H-Arg-Arg-Gln-Met-Glu-Glu-NH2 was obtained. The results of its mass spectrometry and high performance liquid chromatography characterization are as Figure 102 and Figure 103 shown.

[0692] The synthesis route of a hyaluronic acid-modified beauty peptide is as follows: ;

[0693] Among them, -COOX is -COONa;

[0694] The preparation method of the hyaluronic acid-modified beauty peptide includes the following steps:

[0695] Weigh 1 g of H-Arg-Arg-Gln-Met-Glu-Glu-NH2.TFA, add 10 mL of DMSO and stir to dissolve. Add 1.25 g of DIEA, stir for 5 min, then add 3.67 g of hyaluronic acid, control the water bath temperature at 45 °C, stir and react overnight. Take a sample to detect LC-MS. When the reaction is basically complete, add 8 mL of acetic acid and carry out a rearrangement reaction at a constant temperature for 2 h. Take a sample to detect LC-MS. When the reaction is basically complete; then carry out reverse chromatography purification, purification conditions:

[0696] Dissolution: Take 0.2 g of the crude product and dilute it with 100 mL of H2O;

[0697] Packing: 21.2*250 mm, 10 - 120, C18; Flow rate: 10 mL / min; Wavelength: 220 nm;

[0698] Mobile phase: A: 1% HAc; B: ACN;

[0699] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 10 mL / min;

[0700] Sample loading: Flow rate: 10 mL / min;

[0701] Elution: 12 - 32% B for 60 min;

[0702] Column cleaning: Clean with 80% ACN until the baseline is balanced;

[0703] Collect the qualified products to obtain three structures of hyaluronic acid-modified beauty peptides, as shown below:

[0704] F2, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 104 and Figure 105 shown;

[0705] F4, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 106 and Figure 107 shown;

[0706] F6, whose mass spectrometry and high performance liquid chromatography characterization results are as Figure 108 and Figure 109 shown.

[0707] Example 31:

[0708] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0709] Weigh 0.56 g of H-Phe-Val-Ala-Pro-Phe-Pro-OH.TFA (prepared in Example 8), add 6 mL of DMSO and stir to dissolve, add 0.75 g of DIEA, then add 2.2 g of hyaluronic acid (y = 1), control the water bath temperature at 45 °C, stir and react overnight, take samples for LC-MS detection, the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples for LC-MS detection, the reaction is basically complete; then carry out reverse chromatography purification to obtain the hyaluronic acid-modified beauty peptide.

[0710] Example 32:

[0711] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0712] Weigh 0.56 g of H-Phe-Val-Ala-Pro-Phe-Pro-OH.TFA (prepared in Example 8), add 6 mL of DMSO and stir to dissolve, add 0.75 g of DIEA, then add 2.2 g of hyaluronic acid (y = 2), control the water bath temperature at 45 °C, stir and react overnight, take samples for LC-MS detection, the reaction is basically complete, add 4 mL of acetic acid, control the water bath temperature at 35 °C, carry out rearrangement reaction for 3 h, take samples for LC-MS detection, the reaction is basically complete; then carry out reverse chromatography purification to obtain the hyaluronic acid-modified beauty peptide.

[0713] Example 33:

[0714] A preparation method of a hyaluronic acid-modified beauty peptide, comprising the following steps:

[0715] Weigh 0.56 g of H-Phe-Val-Ala-Pro-Phe-Pro-OH.TFA (prepared in Example 8), add 6 mL of DMSO and stir to dissolve. Add 0.75 g of DIEA, then add 2.2 g of hyaluronic acid (y = 3). Control the temperature of the water bath at 45 °C and stir the reaction overnight. Take a sample for LC-MS detection. When the reaction is basically complete, add 4 mL of acetic acid, control the temperature of the water bath at 35 °C, and carry out the rearrangement reaction for 3 h. Take a sample for LC-MS detection. When the reaction is basically complete; then carry out reverse chromatography purification to obtain the hyaluronic acid-modified beauty peptide.

[0716] Test Example 1:

[0717] 1. Moisture retention performance test

[0718] The AQP3 content test is as follows:

[0719] (1) Cell seeding: Seed the cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2);

[0720] (2) Solution preparation: Prepare the test substance working solution according to the experimental design (as shown in Table 1);

[0721] Table 1 AQP3 experimental design table

[0722]

[0723] (3) Add the test substance: After culturing in an incubator (37 °C, 5% CO2) for 24 h, add the test substance according to the table and continue culturing for 24 h;

[0724] (4) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS;

[0725] (5) Immunofluorescence staining:

[0726] a. Add methanol to fix the cells, rinse 3 times with PBS, and add 1 mL of BSA to each well for 1 hour of blocking;

[0727] b. Discard the blocking solution, add the primary antibody to each well, and place it in a 4 °C refrigerator overnight. Discard the primary antibody and rinse 3 times with PBS;

[0728] c. Add the secondary antibody to each well and let it act for 2 hours; discard the secondary antibody and rinse 3 times with PBS;

[0729] d. Add DAPI to each well for nuclear staining, let it act for 10 min, discard DAPI, rinse 3 times with PBS, and then take pictures using a fluorescence microscope;

[0730] (6) Result analysis: Use Image Pro Plus software to quantitatively analyze the AQP3 fluorescence intensity.

[0731] The HA content was tested as follows:

[0732] (1) Cell seeding: Seed the cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2);

[0733] (2) Solution preparation: Prepare the test article working solution according to the experimental design (Table 2);

[0734] Table 2 HA experimental design table

[0735]

[0736] (3) Addition of the test article: After culturing in an incubator (37 °C, 5% CO2) for 24 h, add the test article according to the table and continue culturing for 24 h;

[0737] (4) Sample collection: Collect the supernatant and determine the HA content using an ELISA kit.

[0738] Result analysis:

[0739] Table 3-1 AQP3 test results

[0740]

[0741] From the data analysis in Table 3-1, it can be seen that the hyaluronic acid-modified beauty peptides prepared in the embodiments of the present invention have excellent moisturizing properties and can effectively increase the content of AQP3. Specifically, the hyaluronic acid-modified beauty peptide D4 and the hyaluronic acid-modified beauty peptide D6 prepared in Example 4 of the present invention have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, among the hyaluronic acid-modified beauty peptides with three structures (D2, D4, D6), after being mixed in a certain mass ratio, they also have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (E2, E4, E6) of hyaluronic acid-modified beauty peptides prepared in Example 27 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the three structures (B2, B4, B6) of hyaluronic acid-modified beauty peptides prepared in Example 28 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (G2, G4, G6) of hyaluronic acid-modified beauty peptides prepared in Example 29 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (F2, F4, F6) of hyaluronic acid-modified beauty peptides prepared in Example 30 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The hyaluronic acid-modified beauty peptide A4 and the hyaluronic acid-modified beauty peptide A6 prepared in Example 14 of the present invention have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, among the hyaluronic acid-modified beauty peptides with three structures (A2, A4, A6), after being mixed in a certain mass ratio, they also have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0742] Table 3-2 AQP3 Test Results

[0743]

[0744] From the data analysis in Table 3-2, it can be seen that the hyaluronic acid-modified beauty peptides prepared in the embodiments of the present invention show excellent promoting effects on AQP3, and have a better effect on increasing its content. Specifically, the hyaluronic acid-modified beauty peptides prepared in Examples 1-2, Examples 5-7, and Examples 10-13 of the present invention have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, the three structures (D2, D4, D6) of the hyaluronic acid-modified beauty peptides prepared in Example 4 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the three structures (H2, H4, H6) of the hyaluronic acid-modified beauty peptides prepared in Example 8 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the three structures (L2, L4, L6) of the hyaluronic acid-modified beauty peptides prepared in Example 9 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0745] Table 4 HA test results

[0746]

[0747] From the data analysis in Table 4, it can be seen that the hyaluronic acid-modified beauty peptides prepared in Examples 2 and 8 of the present invention show excellent promoting effects on HA, and have a better effect on increasing its content. Specifically, the hyaluronic acid-modified beauty peptide prepared in Example 2 of the present invention has excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, the three structures (H2, H4, H6) of the hyaluronic acid-modified beauty peptides prepared in Example 8 of the present invention, after being mixed in a certain mass ratio, have excellent moisturizing effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0748] 2. Determination of anti-wrinkle and firming performance

[0749] The content tests of type I collagen and MMP-1 are as follows:

[0750] (1) Cell seeding: Seed the cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2);

[0751] (2) Solution preparation: Prepare the test substance working solution according to the experimental design (Table 5);

[0752] Table 5 Experimental design table

[0753]

[0754] (3) UVA radiation: After culturing for 24 hours, the negative control group, positive control group, and sample group were irradiated with UVA at a total dose of 9 J / cm 2 . Meanwhile, the blank control group was placed in the same environment (UVA radiation dose: 0 J / cm 2 );

[0755] (4) Adding test substances: According to the experimental design, after irradiation, the test substances were added to each group. The blank control group and negative control group were each added with 1 mL of cell culture medium per well; the positive control group was added with 1 mL of cell culture medium containing vitamin C and vitamin E per well; the sample group was added with 1 mL of cell culture medium containing the test substance at the corresponding concentration per well. After adding the test substances, the 24-well plates were placed in an incubator (37°C, 5% CO2) and cultured for 24 h;

[0756] (5) Collecting the supernatant for determination of the contents of type I collagen and MMP-1;

[0757] (6) Result analysis: The t-test statistical analysis was used for comparison among groups, and all statistical analyses were two-tailed.

[0758] Table 6 MMP-1 test results

[0759]

[0760] Table 7-1 Type I collagen test results

[0761]

[0762] From the data analysis in Table 6 and Table 7-1, it can be seen that the hyaluronic acid-modified beauty peptides prepared in the embodiments of the present invention show excellent inhibitory effects on MMP-1 and excellent promoting effects on Collagen I. Specifically, the hyaluronic acid-modified beauty peptides D4 and D6 prepared in Example 4 of the present invention have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, for the hyaluronic acid-modified beauty peptides with three structures (D2, D4, D6), after being mixed in a certain mass ratio, they also have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (E2, E4, E6) of hyaluronic acid-modified beauty peptides prepared in Example 27 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the three structures (B2, B4, B6) of hyaluronic acid-modified beauty peptides prepared in Example 28 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (G2, G4, G6) of hyaluronic acid-modified beauty peptides prepared in Example 29 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The three structures (F2, F4, F6) of hyaluronic acid-modified beauty peptides prepared in Example 30 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The hyaluronic acid-modified beauty peptides A4 and A6 prepared in Example 14 of the present invention have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, for the hyaluronic acid-modified beauty peptides with three structures (A2, A4, A6), after being mixed in a certain mass ratio, they also have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0763] Table 7-2 Test Results of Type I Collagen

[0764]

[0765] From the data analysis in Table 7-2, it can be seen that the hyaluronic acid-modified beauty peptides prepared in Examples 1-2, Examples 5-7, and Examples 10-13 of the present invention have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. Moreover, the hyaluronic acid-modified beauty peptides with three structures (D2, D4, D6) prepared in Example 4 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the hyaluronic acid-modified beauty peptides with three structures (H2, H4, H6) prepared in Example 8 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the hyaluronic acid-modified beauty peptides with three structures (L2, L4, L6) prepared in Example 9 of the present invention, after being mixed in a certain mass ratio, have excellent anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0766] 3. Soothing performance test

[0767] IL-6 content test

[0768] (1) Cell seeding: Seed cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2);

[0769] (2) Solution preparation: Prepare the test substance working solution according to the experimental design (as shown in Table 8);

[0770] Table 8 Experimental design table for IL-6 synthesis

[0771]

[0772] (3) Add the test substance: According to the experimental grouping, when the cell confluence rate in the 24-well plate reaches 40% - 60%, add the test substance in groups, with 3 replicates in each group, and place the 24-well plate in an incubator (37 °C, 5% CO2) and incubate for 24 h;

[0773] (4) Detection: After culturing for 24 hours, collect the supernatant and measure the IL-6 content using an ELISA kit.

[0774] Table 9 Soothing performance test results

[0775]

[0776] From the data analysis in Table 9, it can be seen that the hyaluronic acid-modified beauty peptides prepared in Example 2 of the present invention have a soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 5 of the present invention have a soothing effect at a concentration of 0.063 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 6 of the present invention have a soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 7 of the present invention have a soothing effect at a concentration of 0.125 mg / mL; the three-structured hyaluronic acid-modified beauty peptides prepared in Example 9 of the present invention, when mixed in a certain mass ratio, have a soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 10 of the present invention have a soothing effect at a concentration of 0.063 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 13 of the present invention have a soothing effect at a concentration of 0.125 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 11 of the present invention have a soothing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL; the hyaluronic acid-modified beauty peptides prepared in Example 12 of the present invention have a soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL.

[0777] 4. Whitening performance test

[0778] The melanin content was tested as follows:

[0779] Table 10 Experimental design table for melanin content

[0780]

[0781] Collect cells in the logarithmic growth phase and inoculate them into a 24-well plate. After culturing in an incubator (37°C, 5% CO2) for 24 h, according to the cytotoxicity results, add the test substances as shown in the table, using untreated cells as the blank control, and set 3 parallels for each group.

[0782] After adding the drugs, continue to culture in an incubator (37°C, 5% CO2) for 24 h. Discard the supernatant, add 0.5 mL of 1 M NaOH containing 10% DMSO, incubate at 80°C for 1 h, using 1 M NaOH containing 10% DMSO as the solvent control, and read the absorbance value under an enzyme-linked immunosorbent assay (ELISA) reader and calculate the relative inhibition rate of cellular melanin.

[0783]

[0784] Table 11 Whitening performance test results

[0785]

[0786] From the data analysis in Table 11, it can be seen that the hyaluronic acid-modified beauty peptide prepared in Example 7 of the present invention has excellent whitening effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; and the whitening effects at concentrations of 0.125 mg / mL and 0.25 mg / mL are equivalent to or higher than those of the positive control group.

[0787] 5. Anti-glycation performance test

[0788] Prepare a mixed solution containing bovine serum albumin and glucose using PBS, filter it through a 0.22 μm filter membrane, and use it as a 2× glycation reaction solution. Prepare the reaction systems for each group according to the table.

[0789] Table 12 Reaction system table for AGEs scavenging test

[0790]

[0791] After mixing evenly, incubate at 55 °C for 4 days. Use PBS instead of the sample as the negative control, use aminoguanidine hydrochloride (100 mg / mL) as the positive control, and use PBS instead of the glycation reaction solution as the control system. After the reaction is completed, cool the incubated solution to room temperature, centrifuge at 2000 r / min for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane, then take 200 μL of the reaction solution and add it to a 96-well plate in sequence. Use a fluorescence microplate reader to detect at an excitation wavelength of 320 nm and an emission wavelength of 460 nm, and calculate the AGEs inhibition rate according to the following formula.

[0792]

[0793] In the formula:

[0794] A - Fluorescence intensity of the glycation system with the test substance added;

[0795] B - Fluorescence intensity of the PBS solution with the test substance added;

[0796] C - Fluorescence intensity of the glycation system without the test substance added;

[0797] D - Fluorescence intensity of the PBS solution without the test substance added.

[0798] Table 13 Results of anti-glycation performance test

[0799]

[0800] From the data analysis in Table 13, it can be seen that the hyaluronic acid-modified beauty peptide prepared in Example 11 of the present invention has a scavenging effect on advanced glycation end products at concentrations of 0.0625 mg / mL and 0.25 mg / mL.

[0801] 6. Antioxidant performance test

[0802] The test of ROS content is as follows:

[0803] (1) Cell seeding: Seed the cells into a 24-well plate and incubate overnight in an incubator (37°C, 5% CO2);

[0804] (2) Solution preparation: Prepare the test substance working solution according to the experimental design (Table 13).

[0805] Table 14 ROS experimental design table

[0806]

[0807] (3) Adding the test substance: After culturing for 24 h in an incubator (37°C, 5% CO2), add the test substance according to the table and continue culturing for 24 h;

[0808] (4) Model establishment: Wash twice with PBS. Use the untreated cells as the blank control, and the remaining groups are stimulated with UVB according to the conditions in the table, with the VC+VE group as the positive control;

[0809] (5) Detection of ROS content: Dilute the DCFH-DA probe stock solution with serum-free culture medium. Add 500 μL of the diluted DCFH-DA probe to each well and incubate in a 37°C cell incubator; After 30 min, wash the cells 3 times with serum-free DMEM culture medium to fully remove the probe that has not entered the cells; Use a fluorescence microscope to observe and take pictures under the condition of an excitation wavelength of 488 nm.

[0810] (6) Result analysis: Use Image Pro Plus software to quantitatively analyze the ROS fluorescence intensity.

[0811] Table 15 Antioxidant performance test results

[0812]

[0813] It can be seen from the data analysis in Table 15 that the hyaluronic acid-modified beauty peptide prepared in Example 11 of the present invention has better antioxidant ability at concentrations of 0.125 mg / mL and 0.25 mg / mL, and its antioxidant ability level at a concentration of 0.25 mg / mL is comparable to that of the positive control.

[0814] The conventional techniques in the above embodiments are the existing techniques well known to those skilled in the art, so they will not be elaborated in detail here.

[0815] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A hyaluronic acid-modified beauty peptide, the structural formula of the hyaluronic acid-modified beauty peptide is shown as formula (I): M-C (I); The compound shown in formula (I) includes the structure shown in the following formula (III): (III); Wherein, The n is a natural number; The R is the remaining part of the beauty peptide structure excluding the reactive group amino group; The beauty peptide includes a polypeptide having beauty and / or skin care effects, the polypeptide includes a hexapeptide; the hexapeptide includes any one of H-Glu-Glu-Met-Gln-Arg-Arg-NH2, H-Gly-Pro-Gln-Gly-Pro-Gln-OH, H-Phe-Val-Ala-Pro-Phe-Pro-OH, H-His-Ala-Leu-Arg-Phe-Trp-NH2, H-Trp-Phe-Arg-D-Leu-Ala-His-NH2, H-Trp-Phe-Arg-Leu-Ala-His-NH2, H-Arg-Arg-Gln-D-Met-Glu-Glu-NH2 and hexapeptide H-Arg-Arg-Gln-Met-Glu-Glu-NH2.

2. Use of the hyaluronic acid-modified beauty peptide according to claim 1 in the preparation of cosmetics and / or skin care products.

3. Use of the hyaluronic acid-modified beauty peptide according to claim 1 in enhancing the moisturizing performance or firming and anti-wrinkle performance or anti-aging performance of cosmetics and / or skin care products.

4. Use of the hyaluronic acid-modified beauty peptide according to the claim in enhancing the soothing performance or antioxidant performance or whitening performance of cosmetics and / or skin care products.

5. A cosmetic comprising the hyaluronic acid-modified beauty peptide according to claim 1.

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