Polypeptide graft of hyaluronic acid as well as preparation method and application of polypeptide graft

Abstract

The invention provides a polypeptide graft of hyaluronic acid, and belongs to the technical field of hyaluronic acid modifiers. The graft is obtained by condensation reaction of carboxyl (-COOH) in a hyaluronic acid structure and amino (-NH2) in a polypeptide structure, and the number of carboxyl (-COOH) participating in the condensation reaction in the hyaluronic acid structure is at least two. Compared with unsubstituted and monosubstituted hyaluronic acid, the polypeptide graft of hyaluronic acid provided by the invention shows a remarkably improved anti-aging effect.

Description

[0001] This application claims the priority of a Chinese patent application filed with the China National Intellectual Property Administration on February 23, 2024, with the application number "202410203161.4" and the invention title "A polypeptide grafted product of hyaluronic acid, its preparation method and application", the entire content of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of hyaluronic acid modifiers, and in particular to a polypeptide grafted product of hyaluronic acid, its preparation method and application. Background Art

[0003] Hyaluronic acid (HA) is a mucopolysaccharide composed of a disaccharide repeating unit formed by one molecule of β-1,4-D-glucuronic acid and one molecule of β-1,3-D-acetylglucosamine. HA is an important structural component of the skin. Its metabolic turnover rate is very high. It is synthesized in the basolateral plasma membrane of the epithelium and then immediately excreted extracellularly, where it is degraded by hyaluronidase into fragments containing 4 to 25 disaccharides. HA can be used to smooth skin wrinkles. HA can crosslink with fragmented collagen to provide an appropriate extracellular matrix for fibroblasts and promote the production of elastin and collagen.

[0004] Since the advent of the first polypeptide-containing cosmetic in 1999, nearly a hundred polypeptide-based anti-aging raw materials have been developed so far. Compared with natural nucleic acids, proteins and other bioactive substances, small molecule active peptides are easily absorbed and utilized by the skin, soluble in water, safe and stable, and have effects such as anti-wrinkle and anti-inflammatory.

[0005] Since both hyaluronic acid and polypeptides have good effects in the anti-aging field, currently, hyaluronic acid and polypeptides are mostly used in combination, and it has also been proposed to form a grafted product of polypeptides and hyaluronic acid. For the grafted product of polypeptides and hyaluronic acid, there are many modification methods. Summary of the Invention

[0006] However, this application discovers that not any polypeptide grafted product of hyaluronic acid can obtain good anti-aging effects. Therefore, obtaining a polypeptide grafted product of hyaluronic acid with better anti-aging effects is the technical problem to be solved by the present invention.

[0007] To solve the above problems, this application provides a polypeptide grafted product of hyaluronic acid, which is obtained by a condensation reaction of the carboxyl group (-COOH) in the hyaluronic acid structure with the amino group (-NH 2 ) in the polypeptide structure, and at least two carboxyl groups (-COOH) participating in the substitution reaction in the hyaluronic acid structure.

[0008] It can be understood that the hyaluronic acid described in this application has the following structural formula:

[0009]

[0010] The carboxyl group (-COOH) that can participate in the reaction in the hyaluronic acid structure of the present application can be understood as the carboxyl group on each repeating unit in the above structural formula.

[0011] The polypeptide described in the present application is a compound formed by dehydration condensation of amino acid molecules, and it contains a reactive amino group (-NH 2 ).

[0012] Preferably, the α-amino group of the N-terminal amino acid in the polypeptide structure participates in the reaction.

[0013] In one embodiment, the condensation reaction described in the present application can be understood as a reaction in which the carboxyl group (-COOH) of hyaluronic acid and the amino group (-NH 2 ) of the polypeptide condense to form an amide bond connection and one molecule of water is removed.

[0014] Among them, the reaction of one carboxyl group (-COOH) in one molecule of hyaluronic acid with one molecule of polypeptide can be understood as monosubstitution, and the reaction of the carboxyl groups (-COOH) in multiple repeating units in one molecule of hyaluronic acid with one molecule of polypeptide respectively can be understood as polysubstitution. At least two carboxyl groups (-COOH) participating in the condensation reaction in the hyaluronic acid structure of the present application.

[0015] In one embodiment, the graft has a structural formula shown in formula (I):

[0016]

[0017] In formula (I), n is a natural number selected from 2 to 2500;

[0018] In formula (I), R is selected from any one, two or three of polypeptide residues, -OH, -ONa, and at least two Rs are polypeptide residues;

[0019] The polypeptide residue is a residual group formed after one reactive H of the amino group (-NH 2 ) in the polypeptide structure is substituted.

[0020] In one embodiment, in formula (I), n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500.

[0021] In one embodiment, when n = 2, two Rs in the structure represented by formula (I) are polypeptide residues.

[0022] In one embodiment, when n = 3, two Rs or three Rs in the structure represented by formula (I) are polypeptide residues.

[0023] In one embodiment, when n = 4, two Rs, three Rs or four Rs in the structure represented by formula (I) are polypeptide residues.

[0024] In one embodiment, when n = 5, two Rs, three Rs, four Rs or five Rs in the structure represented by formula (I) are polypeptide residues.

[0025] In one embodiment, when n = 6, two Rs, three Rs, four Rs, five Rs or six Rs in the structure represented by formula (I) are polypeptide residues.

[0026] In one embodiment, when n = 7, two Rs, three Rs, four Rs, five Rs, six Rs or seven Rs in the structure represented by formula (I) are polypeptide residues.

[0027] In one embodiment, when n = 8, two Rs, three Rs, four Rs, five Rs, six Rs, seven Rs or eight Rs in the structure represented by formula (I) are polypeptide residues.

[0028] In one embodiment, when n = 9, two Rs, three Rs, four Rs, five Rs, six Rs, seven Rs, eight Rs or nine Rs in the structure represented by formula (I) are polypeptide residues.

[0029] In one embodiment, when n = 10, two Rs in the structure represented by formula (I) are polypeptide residues, three Rs are polypeptide residues, four Rs are polypeptide residues, five Rs are polypeptide residues, six Rs are polypeptide residues, seven Rs are polypeptide residues, eight Rs are polypeptide residues, nine Rs are polypeptide residues, or ten Rs are polypeptide residues.

[0030] And so on for other values of n.

[0031] In one embodiment, the polypeptide is selected from any one or more of dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, or nonapeptides.

[0032] Preferably, the polypeptide is a hexapeptide. For example, it can be hexapeptide-1, hexapeptide-8, hexapeptide-9, hexapeptide-11, or hexapeptide-38, and preferably hexapeptide-1.

[0033] Among them, hexapeptide-1 has the following structural formula:

[0034]

[0035] Among them, when modifying hyaluronic acid with hexapeptide-1, the α-amino group of the N-terminal histidine in its structure undergoes a condensation reaction with the carboxyl group of hyaluronic acid, as shown in the circled position in formula (II).

[0036] In one embodiment, the hyaluronic acid can be one or more of hyaluronic acid tetrasaccharide (HA4), hyaluronic acid hexasaccharide (HA6), and hyaluronic acid octasaccharide (HA8).

[0037] In one embodiment, the polypeptide grafted hyaluronic acid includes one or more of disubstituted HA4-HP, disubstituted HA6-HP, trisubstituted HA6-HP, disubstituted HA8-HP, trisubstituted HA8-HP, and tetrasubstituted HA8-HP.

[0038] On the other hand, the present application provides a method for preparing the polypeptide grafted hyaluronic acid, including:

[0039] Dissolving hyaluronic acid in a solvent, adding a condensing agent, a catalyst, and a polypeptide, stirring until the reaction is complete, and obtaining it through purification and separation.

[0040] In one embodiment, the molar mass ratio of the reaction between the hyaluronic acid and the polypeptide is 1:1 or more.

[0041] In one embodiment, the molar mass ratio of the reaction between the hyaluronic acid and the polypeptide is 1:1 to 5.

[0042] In one embodiment, the condensing agent includes one or two of ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0043] In one embodiment, the catalyst is a base catalyst.

[0044] Preferably, the base catalyst includes N,N-diisopropylethylamine (DIEA).

[0045] In one embodiment, the solvent can be N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

[0046] In one embodiment, the reaction is carried out at room temperature, for example, it can be 10 - 40 °C.

[0047] In one embodiment, the reaction time is 10 - 20 h, and thin layer chromatography or LC-MS method can be used to detect until the reaction is completed.

[0048] In one embodiment, the specific steps of the preparation method include:

[0049] Add hyaluronic acid to DMF and stir to dissolve it, then add Oxyma and DIEA, stir and activate for 10 - 30 min, then add hexapeptide-1, and finally add EDCI, stir and react for 10 - 20 h. During the reaction process, LC-MS method is used to detect until the reaction is completed. After the reaction is completed, adjust the pH to weakly acidic with acid and send it for purification. The purified and separated products are mono-substituted HA-HP, di-substituted HA-HP and / or multi-substituted HA-HP, and the purification method is HPLC.

[0050] Furthermore, the purity of the polypeptide grafted product of hyaluronic acid is greater than or equal to 98%.

[0051] On the other hand, the present application provides a cosmetic composition, by mass percentage, which includes 0.01% - 10% of the polypeptide grafted product of hyaluronic acid, or the grafted product obtained by the method.

[0052] In one embodiment, the composition may further include a solvent and / or excipients, such as water.

[0053] In one embodiment, the composition can be made into food, medicine, cosmetics, and excipients suitable for the product can be added. For example, when the composition is made into cosmetics, it may further include surfactants, oils, emulsifiers, preservatives, bioactive substances, etc.

[0054] On the other hand, the present application provides the use of the polypeptide grafted product of hyaluronic acid, or the grafted product obtained by the method, or the composition in the preparation of anti-aging products.

[0055] In one embodiment, the product is used to promote the formation of type I collagen (COL1A1) in cells.

[0056] In one embodiment, the product is used to increase the relative expression level of the COL1A1 gene in cells.

[0057] In one embodiment, the dosage form of the product is not limited. For example, it can be water, emulsion, cream, essence, etc., or powder, tablet, pill, capsule, etc.

[0058] On the other hand, the present application provides a cosmetic having anti-aging efficacy, containing the polypeptide graft of hyaluronic acid, or the graft obtained by the method, or the composition.

[0059] Optionally, the cosmetic can be used to promote the formation of type I collagen in cells, or to increase the relative expression level of the COL1A1 gene in cells.

[0060] Optionally, the cells are fibroblasts, more preferably human fibroblasts.

[0061] Compared with the prior art, the present invention has at least the following beneficial effects:

[0062] The polypeptide graft of hyaluronic acid provided by the present application shows a significantly improved anti-aging effect compared with unsubstituted and monosubstituted hyaluronic acid;

[0063] The polypeptide graft of hyaluronic acid provided by the present application has a simple preparation method, good yield, high purity, is suitable for the development of various products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The drawings herein are incorporated into the specification and form a part of the specification, showing embodiments in accordance with the present invention, and are used together with the specification to explain the principles of the present invention.

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts. In the drawings:

[0066] Figure 1 is the high performance liquid chromatography (HPLC) chromatogram of monosubstituted HA4-HP;

[0067] Figure 2 is the mass spectrum of monosubstituted HA4-HP;

[0068] Figure 3 is the high performance liquid chromatography (HPLC) chromatogram of disubstituted HA4-HP;

[0069] Figure 4Mass spectrum of double-substituted HA4-HP;

[0070] Figure 5 NMR spectra of hexa-peptide-1, mono-substituted HA4-HP, and double-substituted HA4-HP;

[0071] Figure 6 NMR spectra of hexa-peptide-1, mono-substituted HA4-HP, and double-substituted HA4-HP at 4.10 - 4.80 ppm;

[0072] Figure 7 High-performance liquid chromatography (HPLC) chart of double-substituted HA6-HP;

[0073] Figure 8 Mass spectrum of double-substituted HA6-HP;

[0074] Figure 9 High-performance liquid chromatography (HPLC) chart of double-substituted HA8-HP;

[0075] Figure 10 Mass spectrum of double-substituted HA8-HP;

[0076] Figure 11 Schematic diagram of relative expression level of COL1A1 gene. Specific implementation manners

[0077] In order to more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples. In the following description, a large number of specific details are given to provide a more thorough understanding of this application. However, it is obvious to those skilled in the art that this application can be implemented without one or more of these details. In other examples, in order to avoid confusion with this application, some well-known technical features in the art are not described.

[0078] Unless otherwise specified, in the following implementation manners, for reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase. For those not indicating specific conditions in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0079] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt the conventional techniques in the fields of conventional analytical chemistry, cell culture, and related fields in this technical field.

[0080] The information of the reagents and equipment involved in the following examples is shown in Table 1 and Table 2:

[0081] Table 1

[0082] Materials Source Information HA Tetrasaccharide (HA4) Bloomage Biotechnology Corporation Limited HA Hexasaccharide (HA6) Bloomage Biotechnology Corporation Limited HA Octasaccharide (HA8) Bloomage Biotechnology Corporation Limited Hexapeptide-1 Zhejiang Peptide Biotechnology Co., Ltd. Fibroblasts Lot No.: Fb22113007, Guangdong Boxi Biotechnology Co., Ltd. DMEM Basic Culture Medium Gibco, Thermo Fisher D'Hanks Buffer Gibco, Thermo Fisher Reverse Transcription Kit RNA-Quick Purification Kit, Yishan Biotech

[0083] Table 2

[0084] Main Equipment Manufacturer & Model Mass Spectrometer AXIMA-PerformanceMA High Performance Liquid Chromatograph Hitachi Laminar Flow Hood Suzhou Sujing Purification Technology Co., Ltd., SW-CJ-2D Inverted Microscope OLYMPUS, CKX53 Precision Electronic Balance OHAUS, CP214 <![CDATA[CO 2 Incubator]]> Thermo, 3111 Ultraviolet Light Therapy Instrument Keno Medical Instrument Equipment Co., Ltd., KN-4006BL1 Microplate Reader Tecan, SPARK Real-Time Fluorescent Quantitative PCR Instrument BioRad, CFX Connect

[0085] Example 1

[0086] This example provides a method for preparing a polypeptide graft of hyaluronic acid. Among them, the structural general formula of the graft is shown in formula (I):

[0087]

[0088] In formula (I), n is selected from natural numbers from 2 to 2500, and R is selected from any two or three of polypeptide residues, -OH, and -ONa, and at least two Rs are polypeptide residues. Among them, the polypeptide residue is a residual group formed after one active H of the amino group (-NH 2 ) in the polypeptide structure is substituted.

[0089] In this example, the values of n are 2, 3, and 4 respectively, that is, hyaluronic acid tetrasaccharide (HA4), hyaluronic acid hexasaccharide (HA6), and hyaluronic acid octasaccharide (HA8) are used.

[0090] In this example, the polypeptide is hexapeptide-1. In other embodiments, the polypeptide can also be any one or several of hexapeptide-8, hexapeptide-9, hexapeptide-11, hexapeptide-38, or dipeptide, tripeptide, tetrapeptide, pentapeptide, heptapeptide, octapeptide, nonapeptide.

[0091] Among them, hexapeptide-1 has the following structural formula:

[0092]

[0093] In this example, hexapeptide-1 is used to modify hyaluronic acid, and the α-amino group of the N-terminal histidine in its structure undergoes a condensation reaction with the carboxyl group of hyaluronic acid, as shown in the circled position in formula (II).

[0094] To prepare a single hexapeptide-1 substituted and double hexapeptide-1 substituted tetrasaccharide hyaluronic acid graft (HA4-HP), the specific preparation method is as follows:

[0095] Dissolve tetrasaccharide hyaluronic acid (HA4) in DMF with stirring, then add Oxyma and DIEA, stir and activate for 20 min, add hexapeptide-1, and finally add EDCI. Stir and react for 16 h. During the reaction process, use LC-MS method to detect until the reaction is completed. After the reaction is completed, adjust the pH to weakly acidic with acid and send it for purification. The purified product is separated to obtain mono-substituted HA4-HP and di-substituted HA4-HP. The purification method is HPLC. Among them, the feeding amount of HA4 is 9 g, the amount of DMF solvent is 200 ml, Oxyma is 3.3 g, DIEA is 3.83 ml, hexapeptide-1 is 13.5 g, and EDCI is 2.23 g. Finally, 2.075 g of mono-substituted HA4-HP and 1.594 g of di-substituted HA4-HP are separated.

[0096] Prepare mono-hexapeptide-1-substituted, di-hexapeptide-1-substituted, and tri-hexapeptide-1-substituted hexasaccharide hyaluronic acid grafts (HA6-HP). The specific preparation method is as follows:

[0097] Dissolve hexasaccharide hyaluronic acid (HA6) in DMF with stirring, then add Oxyma and DIEA, stir and activate for 20 min, add hexapeptide-1, and finally add EDCI. Stir and react for 16 h. During the reaction process, use LC-MS method to detect until the reaction is completed. After the reaction is completed, adjust the pH to weakly acidic with acid and send it for purification. The purified product is separated to obtain mono-substituted HA6-HP, di-substituted HA6-HP, and tri-substituted HA6-HP. The purification method is HPLC. Among them, the feeding amount of HA6 is 15 g, the amount of DMF solvent is 290 ml, Oxyma is 3.95 g, DIEA is 4.83 ml, hexapeptide-1 is 24.5 g, and EDCI is 3.83 g. Finally, 1.892 g of mono-substituted HA6-HP, 1.223 g of di-substituted HA6-HP, and 0.835 g of tri-substituted HA6-HP are separated.

[0098] Prepare mono-hexapeptide-1-substituted, di-hexapeptide-1-substituted, tri-hexapeptide-1-substituted, and tetra-hexapeptide-1-substituted octasaccharide hyaluronic acid grafts (HA8-HP). The specific preparation method is as follows:

[0099] Dissolve octasaccharide hyaluronic acid (HA8) in DMF with stirring, then add Oxyma and DIEA, stir and activate for 20 min, add hexapeptide-1, and finally add EDCI. Stir and react for 16 h. During the reaction process, use LC-MS method to detect until the reaction is completed. After the reaction is completed, adjust the pH to weakly acidic with acid and send it for purification. The purified and separated products are monosubstituted HA8-HP, disubstituted HA8-HP, trisubstituted HA8-HP, and tetrasubstituted HA8-HP. The purification method is HPLC. Among them, the feeding amount of HA8 is 28 g, the solvent amount of DMF is 390 ml, Oxyma is 5.25 g, DIEA is 5.53 ml, hexapeptide-1 is 35.6 g, and EDCI is 5.61 g. Finally, 1.592 g of monosubstituted HA8-HP, 1.135 g of disubstituted HA8-HP, 0.825 g of trisubstituted HA8-HP, and 0.426 g of tetrasubstituted HA8-HP are separated.

[0100] Perform mass spectrometry analysis and high performance liquid chromatography analysis on different HA-HP prepared by the above method respectively. The methods are as follows:

[0101] Detection method of high performance liquid chromatography:

[0102] Accurately weigh the above-mentioned HA-HP respectively, make up the volume to 1 mg / ml with purified water, and detect by high performance liquid chromatography. The chromatographic conditions are as follows: the chromatographic column is a C18 3.5 μm chromatographic column, 4.6 mm * 100 mm; mobile phase A is 0.1% TFA + H 2 2O, mobile phase B is 0.1% TFA + acetonitrile, the detection wavelength is 220 nm, and the column temperature is 25 °C.

[0103] Mass spectrometry detection method:

[0104] Take 1 mg of the sample and dissolve it in a centrifuge tube. Take 1 μL with a syringe and inject it for mass spectrometry measurement.

[0105] The obtained results are shown in Table 3, Figures 1 to 10 specifically:

[0106] As Figure 1 shown, the high performance liquid chromatography result of the prepared monosubstituted HA4-HP shows that a chromatographic peak appears at 10.780 min, and the purity is 98.79%.

[0107] Perform mass spectrometry detection on this substance. As Figure 2 shown, the mass spectrometry result shows: MS: [M + H] + , m / z: 1586.84, confirmed as monosubstituted HA4-HP.

[0108] As Figure 3As shown, the high performance liquid chromatography result of the prepared double-substituted HA4-HP shows that a chromatographic peak appears at 12.653 min, and the purity is 99.05%.

[0109] The mass spectrometry detection was carried out on this substance, as Figure 4 shown. The mass spectrometry result shows: MS: [M+H] + , m / z: 2397.10, which was confirmed to be double-substituted HA4-HP.

[0110] As Figure 7 shown, the high performance liquid chromatography result of the prepared double-substituted HA6-HP shows that a chromatographic peak appears at 8.555 min, and the purity is 99.10%.

[0111] The mass spectrometry detection was carried out on this substance, as Figure 8 shown. The mass spectrometry result shows: MS: [M+2H] 2+ , m / z: 1388.3, which was confirmed to be double-substituted HA6-HP, and the molecular weight M = m*n - n (m is the m / z value of the peak, n is the charge number).

[0112] As Figure 9 shown, the high performance liquid chromatography result of the prepared double-substituted HA8-HP shows that a chromatographic peak appears at 7.575 min, and the purity is 98.20%.

[0113] The mass spectrometry detection was carried out on this substance, as Figure 10 shown. The mass spectrometry result shows: MS: [M+2H] 2+ , m / z: 1578.8, which was confirmed to be double-substituted HA8-HP, and the molecular weight M = m*n - n (m is the m / z value of the peak, n is the charge number).

[0114] The purity and molecular weight of the HA-HP prepared by the above method are shown in Table 3:

[0115] Table 3

[0116] No Component Purity (%) Molecular Weight 1 Monosubstituted HA4-HP 98.79 1585.84 2 Disubstituted HA4-HP 99.05 2396.10 3 Monosubstituted HA6-HP 98.89 1964.9 4 Disubstituted HA6-HP 99.10 2774.6 5 Trisubstituted HA6-HP 99.20 3585.7 6 Monosubstituted HA8-HP 99.13 2344.4 7 Disubstituted HA8-HP 98.20 3155.6 8 Trisubstituted HA8-HP 98.97 3965.0 9 Tetrasubstituted HA8-HP 99.02 4774.9

[0117] Furthermore, nuclear magnetic resonance analysis was carried out on the single-substituted HA4-HP and double-substituted HA4-HP to confirm the connection position of hyaluronic acid and hexapeptide-1. The nuclear magnetic resonance method is as follows:

[0118] Take 5 mg of the sample and dissolve it in 300 μL of D 2 O in a vial. After complete dissolution, transfer it to a nuclear magnetic resonance tube with a pipette and inject it for nuclear magnetic resonance measurement.

[0119] The results are as Figure 5 、 Figure 6 shown. For Figure 5 and Figure 6Analysis shows that the α-amino group (-CH-NH 2 ) of the N-terminal histidine of hexa-peptide-1 participated in the condensation reaction with hyaluronic acid, and -CH-NH 2 changed to -CH-NH-CO-, as shown in the dashed boxes of Figure 5 and 6 . The chemical shift of -CH- changed from 4.40 ppm to 4.72 ppm and 4.65 ppm respectively. The above results indicate that through the analysis and confirmation of the molecular weight and chemical structure, the target product was obtained by using the preparation method provided in this example.

[0120] Example 2

[0121] In this example, the relative expression level of intracellular COL1A1 gene of the target product prepared in Example 1 was tested by using a cytological test method. The specific method is as follows:

[0122] a) Cell seeding: Fibroblasts were seeded into a 6-well plate at an inoculation density of 3×10 5 cells / well and incubated in an incubator (37 °C, 5% CO 2 ) for 24 h;

[0123] b) Preparation of solutions: HA4 (tetrasaccharide hyaluronic acid), HA6 (hexasaccharide hyaluronic acid), HA8 (octasaccharide hyaluronic acid), hexa-peptide-1, mono-substituted HA4-HP, di-substituted HA4-HP, mono-substituted HA6-HP, di-substituted HA6-HP, tri-substituted HA6-HP, mono-substituted HA8-HP, di-substituted HA8-HP, tri-substituted HA8-HP, and tetra-substituted HA8-HP powders were weighed separately and dissolved in a complete DMEM medium solution to form a test mixture with a mass percentage concentration of 0.01%;

[0124] c) Modeling: According to the experimental grouping, the sample group and the model (negative) control group were exposed to a UV light therapy instrument (wavelength 320 - 400 nm, 5 J / cm 2 ) for UVA irradiation; the blank control group was not irradiated;

[0125] d) Administration: According to the test scheme in Table 4, grouped administration was carried out. A complete medium containing the test substance working solution was added to the wells of the sample group, 1 mL of sample was added to each well, and 3 replicate wells were set for each group; after administration, the 6-well plate was placed in an incubator (37 °C, 5% CO 2 ) and cultured for 24 h;

[0126] e) Cell collection: After culturing for 24 h, the cell supernatant was collected, and then the cells were washed twice with 1 mL / well of D'Hanks buffer. After pipetting and lysing the cells, the samples were collected;

[0127] f) Gene expression detection: After treating the cells with the RNA-Quick Purification Kit, samples were collected. According to the kit instructions, RNA extraction, reverse transcription, and fluorescence quantitative PCR operations were carried out. The 2 -△△Ct method was used for result calculation to obtain the detection results;

[0128] g) Result statistical analysis: The t-test statistical analysis was used for comparison between groups, and the confidence level was 95%;

[0129] Letters were used to represent the differences between the results. There was no significant difference between two groups with the same letter, such as a vs a, or a vs ab; there was a significant difference between two groups with completely different letters, such as a vs b.

[0130] The results are shown in Table 4, Figure 11 as follows.

[0131] Table 4

[0132]

[0133]

[0134] As shown in Table 4 and Figure 11 the results show that the hyaluronic acid or polypeptide in sample groups 1 to 4 had similar effects on promoting COL1A1 gene expression; the hyaluronic acid-polypeptide grafts in sample groups 5, 7, and 10 did not show better effects on promoting COL1A1 gene expression than the corresponding ungrafted hyaluronic acid and polypeptide; the hyaluronic acid-polypeptide grafts obtained in sample groups 6, 8, 9, and 11 to 13 exceeded the effects of the corresponding ungrafted hyaluronic acid and polypeptide on promoting COL1A1 gene expression.

[0135] The above are only examples of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A hyaluronic acid polypeptide graft, characterized in that: The grafted product is obtained by condensation reaction between the carboxyl group (-COOH) in the hyaluronic acid structure and the amino group (-NH2) in the polypeptide structure, and there are at least two carboxyl groups (-COOH) in the hyaluronic acid structure participating in the condensation reaction.

2. The graft according to claim 1, characterized in that The grafted material has a structural formula as shown in formula (I): In formula (I), n is selected from a natural number of 2 to 2500; In formula (I), R is selected from any one, two or three of a polypeptide residue, -OH, and -ONa, and at least two Rs are polypeptide residues; The polypeptide residue is a residual group formed after an active H of the amino group (-NH2) in the polypeptide structure is replaced.

3. The graft according to claim 1 or 2, characterized in that The polypeptide is selected from any one or more of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide or nonapeptide, preferably hexapeptide.

4. The method for preparing a polypeptide graft of hyaluronic acid according to any one of claims 1 to 3, characterized in that: include: The hyaluronic acid is dissolved in a solvent, a condensation agent, a catalyst and a polypeptide are added, stirred until the reaction is completed, and purified and separated to obtain the product.

5. The method according to claim 4, characterized in that The reaction molar mass ratio of the hyaluronic acid to the polypeptide is greater than 1:1, preferably 1:1-5.

6. A cosmetic composition, characterized in that By mass percentage, it comprises 0.01% to 10% of the polypeptide graft of hyaluronic acid as claimed in any one of claims 1 to 3, or the graft prepared by the method as claimed in claim 4 or 5.

7. Use of the hyaluronic acid polypeptide graft according to any one of claims 1 to 3, or the graft obtained by the method according to claim 4 or 5, or the composition according to claim 6 in the preparation of anti-aging products.

8. The use according to claim 7, characterized in that: The product is used to promote the production of type I collagen.

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