Hyaluronic acid polypeptide graft, preparation method therefor and use thereof

Abstract

The present application relates to the technical field of hyaluronic acid modifications and provides a hyaluronic acid polypeptide graft. The graft is obtained by a condensation reaction between carboxyl groups (-COOH) in a hyaluronic acid structure and an amino group (-NH2) in a polypeptide structure, and at least two carboxyl groups (-COOH) in the hyaluronic acid structure participate in the condensation reaction. Compared with unsubstituted and mono-substituted hyaluronic acids, the hyaluronic acid polypeptide graft provided by the present application exhibits a significantly improved anti-aging effect.

Description

A hyaluronic acid polypeptide graft and its preparation method and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on February 23, 2024, with application number "202410203161.4" and invention name "A hyaluronic acid polypeptide graft and its preparation method and application", the entire content of which is incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of hyaluronic acid modifications, and in particular to a hyaluronic acid polypeptide graft, a preparation method, and an application thereof. Background Art

[0003] Hyaluronic acid (HA) is a mucopolysaccharide composed of repeating disaccharide units, consisting of one molecule of β-1,4-D-glucuronic acid and one molecule of β-1,3-D-acetylglucosamine. HA is a key structural component of the skin, with a high metabolic conversion rate. It is synthesized in the basal membrane of the epithelium and then excreted outside the cell. It is then degraded into fragments containing 4 to 25 disaccharides under the catalysis of hyaluronidase. HA can be used to smooth skin wrinkles. HA can cross-link with fragmented collagen, providing an appropriate extracellular matrix for fibroblasts and promoting the production of elastin and collagen.

[0004] Since the launch of the first peptide-based cosmetic in 1999, nearly 100 peptide-based anti-aging ingredients have been developed. Compared to bioactive substances like natural nucleic acids and proteins, small-molecule active peptides are easily absorbed and utilized by the skin, are readily soluble in water, and are safe and stable, with anti-wrinkle and anti-inflammatory properties.

[0005] Because both hyaluronic acid and peptides have demonstrated promising anti-aging benefits, their combination is currently the most common approach. There are also proposals for grafting peptides onto hyaluronic acid. There are numerous methods for modifying these grafts. Summary of the Invention

[0006] However, the present application found that not all hyaluronic acid polypeptide grafts can achieve good anti-aging effects. Therefore, obtaining hyaluronic acid polypeptide grafts with better anti-aging effects is a technical problem to be solved by the present application.

[0007] In order to solve the above problems, the present application provides a hyaluronic acid polypeptide graft, which is obtained by a condensation reaction between a carboxyl group (-COOH) in the hyaluronic acid structure and an amino group (-NH2) in the polypeptide structure, and there are at least two carboxyl groups (-COOH) in the hyaluronic acid structure that participate in the substitution reaction.

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

[0009] 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.

[0010] The polypeptide described in the present application is a compound formed by dehydration condensation of amino acid molecules, which contains an active amino group (-NH2).

[0011] Optionally, the α-amino group of the N-terminal amino acid in the polypeptide structure participates in the reaction.

[0012] In one embodiment, the condensation reaction described herein can be understood as a reaction in which the carboxyl group (-COOH) of hyaluronic acid and the amino group (-NH2) of the polypeptide condense to form an amide bond and remove a molecule of water.

[0013] Among them, the reaction of one carboxyl group (-COOH) in a molecule of hyaluronic acid with a molecule of polypeptide can be understood as a single substitution, while the reaction of the carboxyl groups (-COOH) in multiple repeating units in a molecule of hyaluronic acid with a molecule of polypeptide can be understood as a multiple substitution. In the hyaluronic acid structure of the present application, there are at least two carboxyl groups (-COOH) participating in the condensation reaction.

[0014] In one embodiment, the graft has a structural formula as shown in formula (I):

[0015] In formula (I), n is a natural number selected from 2 to 2500;

[0016] 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;

[0017] The polypeptide residue is a residual group formed after an active H of an amino group (-NH2) in the polypeptide structure is replaced.

[0018] In one embodiment, n in formula (I) 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.

[0019] In one embodiment, when n=2, two Rs in the structure represented by formula (I) are polypeptide residues.

[0020] In one embodiment, when n=3, two Rs in the structure represented by formula (I) are polypeptide residues or three Rs are polypeptide residues.

[0021] In one embodiment, when n=4, in the structure represented by formula (I), 2 Rs are polypeptide residues, 3 Rs are polypeptide residues, or 4 Rs are polypeptide residues.

[0022] In one embodiment, when n=5, in the structure represented by formula (I), 2 Rs are polypeptide residues, 3 Rs are polypeptide residues, 4 Rs are polypeptide residues, or 5 Rs are polypeptide residues.

[0023] In one embodiment, when n=6, in the structure represented by formula (I), 2 Rs are polypeptide residues, 3 Rs are polypeptide residues, 4 Rs are polypeptide residues, 5 Rs are polypeptide residues, or 6 Rs are polypeptide residues.

[0024] In one embodiment, when n=7, in the structure represented by formula (I), 2 R are polypeptide residues, 3 R are polypeptide residues, 4 R are polypeptide residues, 5 R are polypeptide residues, 6 R are polypeptide residues, or 7 R are polypeptide residues.

[0025] In one embodiment, when n=8, in the structure represented by formula (I), 2 R are polypeptide residues, 3 R are polypeptide residues, 4 R are polypeptide residues, 5 R are polypeptide residues, 6 R are polypeptide residues, 7 R are polypeptide residues or 8 R are polypeptide residues.

[0026] In one embodiment, when n=9, in the structure represented by formula (I), 2 R are polypeptide residues, 3 R are polypeptide residues, 4 R are polypeptide residues, 5 R are polypeptide residues, 6 R are polypeptide residues, 7 R are polypeptide residues, 8 R are polypeptide residues or 9 R are polypeptide residues.

[0027] In one embodiment, when n=10, in the structure represented by formula (I), 2 R are polypeptide residues, 3 R are polypeptide residues, 4 R are polypeptide residues, 5 R are polypeptide residues, 6 R are polypeptide residues, 7 R are polypeptide residues, 8 R are polypeptide residues, 9 R are polypeptide residues or 10 R are polypeptide residues.

[0028] The same applies when n is other numbers.

[0029] In one embodiment, the polypeptide is selected from any one or more of a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide or a nonapeptide.

[0030] Optionally, the polypeptide is a hexapeptide, for example, hexapeptide-1, hexapeptide-8, hexapeptide-9, hexapeptide-11 or hexapeptide-38, optionally hexapeptide-1.

[0031] Among them, hexapeptide-1 has the following structural formula:

[0032] When hyaluronic acid is modified 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 by the circled position in formula (II).

[0033] In one embodiment, the hyaluronic acid may be one or more of hyaluronan tetrasaccharide (HA4), hyaluronan hexasaccharide (HA6), and hyaluronan octasaccharide (HA8).

[0034] In one embodiment, the hyaluronic acid polypeptide graft comprises one or more of disubstituted HA4-HP, disubstituted HA6-HP, trisubstituted HA6-HP, disubstituted HA8-HP, trisubstituted HA8-HP, and tetrasubstituted HA8-HP.

[0035] On the other hand, the present application provides a method for preparing the hyaluronic acid polypeptide graft, comprising:

[0036] The hyaluronic acid is dissolved in a solvent, and a condensing agent, a catalyst and a polypeptide are added, stirred until the reaction is completed, and purified and separated to obtain the product.

[0037] In one embodiment, the molar mass ratio of the hyaluronic acid and the polypeptide in the reaction is 1:1 or greater.

[0038] In one embodiment, the molar mass ratio of the hyaluronic acid and the polypeptide is 1:1-5.

[0039] In one embodiment, the condensing agent includes one or both of ethyl 2-oxime cyanoacetate (Oxyma) and carbodiimide hydrochloride (EDCI).

[0040] In one embodiment, the catalyst is a base catalyst.

[0041] Optionally, the base catalyst includes N,N-diisopropylethylamine (DIEA).

[0042] In one embodiment, the solvent may be N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

[0043] In one embodiment, the reaction is carried out at room temperature, for example, 10-40°C.

[0044] In one embodiment, the reaction time is 10 to 20 hours, and thin layer chromatography or LC-MS can be used to detect the completion of the reaction.

[0045] In one embodiment, the steps of the preparation method include:

[0046] Hyaluronic acid is added to DMF and stirred to dissolve, and then Oxyma and DIEA are added. After stirring and activation for 10 to 30 minutes, hexapeptide-1 is added, and finally EDCI is added. The reaction is stirred for 10 to 20 hours. During the reaction, the LC-MS method is used to detect until the reaction is completed. After the reaction is completed, the pH is adjusted to weak acidity with acid and then sent for purification. Mono-substituted HA-HP, di-substituted HA-HP and / or poly-substituted HA-HP are purified and separated, and the purification method is HPLC.

[0047] Furthermore, the purity of the hyaluronic acid polypeptide graft is greater than or equal to 98%.

[0048] On the other hand, the present application provides a cosmetic composition comprising, by mass percentage, 0.01% to 10% of the hyaluronic acid polypeptide graft, or the graft prepared by the method.

[0049] In one embodiment, the composition may further include a solvent and / or an auxiliary material, such as water.

[0050] In one embodiment, the composition can be prepared into food, medicine, or cosmetics, and can be supplemented with excipients suitable for the product. For example, when the composition is prepared into cosmetics, it can further include surfactants, oils, emulsifiers, preservatives, bioactive substances, and the like.

[0051] On the other hand, the present application provides the use of the hyaluronic acid polypeptide graft, or the graft prepared by the method, or the composition in the preparation of anti-aging products.

[0052] In one embodiment, the product is used to promote the formation of type I collagen (COL1A1) in cells.

[0053] In one embodiment, the product is used to increase the relative expression level of the COL1A1 gene in cells.

[0054] In one embodiment, the dosage form of the product is not limited, for example, it can be water, lotion, cream, essence, etc., or powder, compressed tablets, pills, capsules, etc.

[0055] On the other hand, the present application provides a cosmetic with anti-aging efficacy, comprising the polypeptide graft of hyaluronic acid, or the graft prepared by the method, or the composition.

[0056] 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.

[0057] Optionally, the cells are fibroblasts, more preferably human fibroblasts.

[0058] Compared with the prior art, this application has at least the following beneficial effects:

[0059] The hyaluronic acid polypeptide grafts provided in this application exhibit significantly improved anti-aging effects compared to unsubstituted and monosubstituted hyaluronic acids;

[0060] The hyaluronic acid polypeptide graft provided in the present application has a simple preparation method, good yield and high purity, is suitable for the development of various products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0062] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. For those skilled in the art, other drawings can be obtained based on these drawings without inventive work. In the drawings:

[0063] Figure 1 is a high performance liquid chromatogram of monosubstituted HA4-HP;

[0064] FIG2 is a mass spectrum of monosubstituted HA4-HP;

[0065] FIG3 is a high performance liquid chromatogram of disubstituted HA4-HP;

[0066] FIG4 is a mass spectrum of disubstituted HA4-HP;

[0067] Figure 5 shows the NMR spectra of hexapeptide-1, monosubstituted HA4-HP, and disubstituted HA4-HP;

[0068] Figure 6 shows the NMR spectra of hexapeptide-1, monosubstituted HA4-HP, and disubstituted HA4-HP at 4.10-4.80 ppm;

[0069] FIG7 is a high performance liquid chromatogram of disubstituted HA6-HP;

[0070] FIG8 is a mass spectrum of disubstituted HA6-HP;

[0071] FIG9 is a high performance liquid chromatogram of disubstituted HA8-HP;

[0072] Figure 10 is a mass spectrum of disubstituted HA8-HP;

[0073] FIG11 is a schematic diagram of the relative expression level of the COL1A1 gene. DETAILED DESCRIPTION

[0074] In order to more clearly illustrate the overall concept of the application, the following is described in detail in the form of embodiments. In the following description, a large amount of specific details are provided so that a more thorough understanding of the application is provided. However, it will be apparent to those skilled in the art that the application can be implemented without the need for one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0075] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0076] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this application all adopt conventional analytical chemistry, cell culture, and related techniques in the art.

[0077] The information of the reagents and equipment involved in the following examples is shown in Table 1 and Table 2:

[0078] Table 1

[0079] Table 2

[0080] Example 1

[0081] This embodiment provides a method for preparing a polypeptide graft of hyaluronic acid, wherein the general structural formula of the graft is shown in formula (I):

[0082] In formula (I), n is selected from a natural number of 2 to 2500, and R is selected from any two or three of a polypeptide residue, -OH, and -ONa, with at least two Rs being polypeptide residues. A polypeptide residue is a residual group formed by replacing an active hydrogen of an amino group (-NH2) in a polypeptide structure.

[0083] In this embodiment, 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.

[0084] In this embodiment, the polypeptide is hexapeptide-1. In other embodiments, the polypeptide may also be hexapeptide-8, hexapeptide-9, hexapeptide-11, hexapeptide-38, or any one or more of a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a heptapeptide, an octapeptide, or a nonapeptide.

[0085] Among them, hexapeptide-1 has the following structural formula:

[0086] In this example, hexapeptide-1 was used to modify hyaluronic acid. The α-amino group of the N-terminal histidine in the structure underwent a condensation reaction with the carboxyl group of hyaluronic acid, as shown by the circled position in formula (II).

[0087] The preparation method of the tetrasaccharide hyaluronic acid grafted with single hexapeptide-1 substitution and double hexapeptide-1 substitution (HA4-HP) is as follows:

[0088] Hyaluronic acid tetrasaccharide (HA4) was added to DMF and stirred to dissolve, and then Oxyma and DIEA were added. After stirring and activating for 20 minutes, hexapeptide-1 was added, and finally EDCI was added. The reaction was stirred for 16 hours. During the reaction, LC-MS was used to detect the completion of the reaction. After the reaction was completed, the pH was adjusted to weak acidity with acid and then sent for purification. Monosubstituted HA4-HP and disubstituted HA4-HP were purified and separated by HPLC. Among them, the HA4 feed amount was 9g, the DMF solvent amount was 200ml, Oxyma 3.3g, DIEA 3.83ml, 13.5g hexapeptide-1, and EDCI was 2.23g. Finally, 2.075g monosubstituted HA4-HP and 1.594g disubstituted HA4-HP were separated.

[0089] The preparation methods of single hexapeptide-1 substitution, double hexapeptide-1 substitution and triple hexapeptide-1 substitution hexasaccharide hyaluronic acid grafts (HA6-HP) are as follows:

[0090] Hyaluronic acid hexasaccharide (HA6) was added to DMF and stirred to dissolve, and then Oxyma and DIEA were added. After stirring and activating for 20 minutes, hexapeptide-1 was added, and finally EDCI was added. The reaction was stirred for 16 hours. During the reaction, LC-MS was used to detect until the reaction was complete. After the reaction was completed, the pH was adjusted to weak acidity with acid and then sent for purification. Monosubstituted HA6-HP, disubstituted HA6-HP and trisubstituted HA6-HP were purified and separated. The purification method was HPLC. Among them, the HA6 feed amount was 15g, the DMF solvent amount was 290ml, Oxyma 3.95g, DIEA 4.83ml, 24.5g hexapeptide-1, and EDCI was 3.83g. Finally, 1.892g monosubstituted HA6-HP, 1.223g disubstituted HA6-HP, and 0.835g trisubstituted HA6-HP were separated.

[0091] Octasaccharide hyaluronic acid grafts (HA8-HP) with single hexapeptide-1 substitution, double hexapeptide-1 substitution, triple hexapeptide-1 substitution, and quadruple hexapeptide-1 substitution were prepared. The preparation method is as follows:

[0092] Hyaluronic acid octasaccharide (HA8) was added to DMF and stirred to dissolve, and then Oxyma and DIEA were added. After stirring and activating for 20 minutes, hexapeptide-1 was added, and finally EDCI was added. The reaction was stirred for 16 hours. During the reaction, LC-MS was used to detect the completion of the reaction. After the reaction was completed, the pH was adjusted to weak acidity with acid and then sent for purification. Monosubstituted HA8-HP, disubstituted HA8-HP, trisubstituted HA8-HP and tetrasubstituted HA8-HP were purified and separated. The purification method was HPLC. Among them, the HA8 feed amount was 28g, the DMF solvent amount was 390ml, Oxyma 5.25g, DIEA 5.53ml, 35.6g hexapeptide-1, and EDCI was 5.61g. Finally, 1.592g monosubstituted HA8-HP, 1.135g disubstituted HA8-HP, 0.825g trisubstituted HA8-HP, and 0.426g tetrasubstituted HA8-HP were separated.

[0093] The different HA-HP prepared by the above method were subjected to mass spectrometry analysis and high performance liquid chromatography analysis, respectively, as follows:

[0094] HPLC detection method:

[0095] The above HA-HP was accurately weighed and diluted to 1 mg / ml with purified water. The HPLC was used for detection. The chromatographic conditions were as follows: C18 3.5 μm column, 4.6 mm*100 mm; mobile phase A was 0.1% TFA+H2O, mobile phase B was 0.1% TFA+acetonitrile, detection wavelength was 220 nm, and column temperature was 25°C.

[0096] Mass spectrometry detection method:

[0097] Take 1 mg of sample and dissolve it in a centrifuge tube, take 1 μL of it with a syringe and inject it, and measure the mass spectrum.

[0098] The results are shown in Table 3 and Figures 1 to 10.

[0099] As shown in FIG1 , the HPLC results of the prepared monosubstituted HA4-HP showed that a chromatographic peak appeared at 10.780 min and the purity was 98.79%.

[0100] The substance was subjected to mass spectrometry detection, as shown in Figure 2. The mass spectrometry results showed: MS: [M+H] + , m / z: 1586.84, confirmed to be monosubstituted HA4-HP.

[0101] As shown in FIG3 , the HPLC results of the prepared disubstituted HA4-HP showed that a chromatographic peak appeared at 12.653 min and the purity was 99.05%.

[0102] The substance was subjected to mass spectrometry detection, as shown in Figure 4. The mass spectrometry results showed: MS: [M+H] + , m / z: 2397.10, confirmed to be disubstituted HA4-HP.

[0103] As shown in FIG7 , the HPLC results of the prepared disubstituted HA6-HP showed that a chromatographic peak appeared at 8.555 min and the purity was 99.10%.

[0104] The substance was subjected to mass spectrometry detection, as shown in Figure 8. The mass spectrometry results showed: MS: [M+2H] 2+ , m / z: 1388.3, ​​confirmed to be disubstituted HA6-HP, molecular weight M=m*nn (m is the m / z value of the peak, n is the charge number).

[0105] As shown in FIG9 , the HPLC results of the prepared disubstituted HA8-HP showed that a chromatographic peak appeared at 7.575 min and the purity was 98.20%.

[0106] The substance was subjected to mass spectrometry detection, as shown in Figure 10. The mass spectrometry results showed: MS: [M+2H] 2+ , m / z: 1578.8, confirmed to be disubstituted HA8-HP, molecular weight M=m*nn (m is the m / z value of the peak, n is the charge number).

[0107] The purity and molecular weight of HA-HP prepared by the above method are shown in Table 3:

[0108] Table 3

[0109] Further NMR analysis was performed on the monosubstituted HA4-HP and disubstituted HA4-HP to confirm the connection position of hyaluronic acid and hexapeptide-1. The NMR method is as follows:

[0110] Take 5 mg of sample in a vial and dissolve it in 300 μL D2O. After it is fully dissolved, transfer it to the NMR tube with a pipette and inject it for NMR measurement.

[0111] The results are shown in Figures 5 and 6. Analysis of Figures 5 and 6 shows that the α-amino group (-CH-NH2) of the N-terminal histidine of hexapeptide-1 participates in the condensation reaction with hyaluronic acid, converting -CH-NH2 to -CH-NH-CO-. As shown in the dotted boxes in Figures 5 and 6, the chemical shift of -CH- changes from 4.40 ppm to 4.72 ppm and 4.65 ppm, respectively. These results demonstrate that the target product was prepared using the preparation method provided in this example, as confirmed by analysis of the molecular weight and chemical structure.

[0112] Example 2

[0113] In this example, the target product prepared in Example 1 was tested for relative expression of the COL1A1 gene in cells using a cytological test method as follows:

[0114] a) Cell seeding: 3×10 5 Fibroblasts were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO2) for 24 h;

[0115] b) Liquid preparation: Weigh HA4 (hyaluronic acid tetrasaccharide), HA6 (hyaluronic acid hexasaccharide), HA8 (hyaluronic acid octasaccharide), hexapeptide-1, monosubstituted HA4-HP, disubstituted HA4-HP, monosubstituted HA6-HP, disubstituted HA6-HP, trisubstituted HA6-HP, monosubstituted HA8-HP, disubstituted HA8-HP, trisubstituted HA8-HP, and tetrasubstituted HA8-HP powders respectively, and dissolve them in complete DMEM medium to form a test mixture at a mass percentage concentration of 0.01%;

[0116] c) Modeling: According to the experimental groups, the sample group and the model (negative) control group were exposed to ultraviolet light therapy device (wavelength 320-400nm, 5J / cm 2 ) were irradiated with UVA; the blank control group was not irradiated;

[0117] d) Administration: Dosing was performed in groups according to the test protocol in Table 4. Complete medium containing the test substance working solution was added to the sample group wells, with 1 mL of sample added to each well. Three replicates were set up for each group. After administration, the 6-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 h.

[0118] e) Cell collection: After 24 h of culture, collect the cell supernatant, wash twice with 1 mL / well D'Hanks buffer, lyse the cells by pipetting, and collect the sample;

[0119] f) Gene expression detection: After the cells were treated with RNA-Quick Purification Kit, samples were collected and RNA extraction, reverse transcription and fluorescence quantitative PCR were performed according to the kit instructions. -△ △Ct Methods are used to calculate the results and obtain the test results;

[0120] g) Statistical analysis of results: t-test statistical analysis was used for comparison between groups with a confidence level of 95%;

[0121] Use letters to indicate the difference between the results. There is no significant difference between two groups of results with the same letters, such as a vs a, or a vs ab; there is a significant difference between two groups of results with completely different letters, such as a vs b.

[0122] The results are shown in Table 4 and Figure 11.

[0123] Table 4

[0124] The results in Table 4 and Figure 11 show that the hyaluronic acid or polypeptide in sample groups 1 to 4 had similar effects on promoting COL1A1 gene expression; the hyaluronic acid-peptide grafts in sample groups 5, 7, and 10 did not achieve a better effect on promoting COL1A1 gene expression than the corresponding ungrafted hyaluronic acid or polypeptide; the hyaluronic acid-peptide grafts obtained in sample groups 6, 8, 9, 11 to 13 exceeded the effect of the corresponding ungrafted hyaluronic acid or polypeptide in promoting COL1A1 gene expression.

[0125] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A hyaluronic acid polypeptide graft, characterized in that: The graft 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 compound has a structural formula as shown in formula (I): In formula (I), n is a natural number selected from 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 an 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, and can be a hexapeptide.

4. The method for preparing a hyaluronic acid polypeptide graft according to any one of claims 1 to 3, characterized in that: include: The hyaluronic acid is dissolved in a solvent, and a condensing 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 and the polypeptide is greater than 1:1, and can be selected from 1:1 to 5.

6. A cosmetic composition, characterized in that Calculated by mass percentage, it comprises 0.01% to 10% of the hyaluronic acid polypeptide graft according to any one of claims 1 to 3, or the graft prepared by the method according to 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 Type 1 collagen production.

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