An anti-aging composition and use thereof

Abstract

The application provides an anti-aging composition and application thereof, and relates to the technical field of hyaluronic acid grafts. It is found for the first time in the application that a hyaluronic acid-hexapeptide graft can effectively reduce the expression of aging-related beta-galactosidase (SA-beta-gal), so that the aging state of cells can be fundamentally reversed, a sustained anti-aging effect is brought, and a new functional ingredient is provided for developing products such as a cell aging reversal agent, a cell aging improvement agent and a product for improving the anti-aging ability of cells. Further, it is found for the first time in the application that the hyaluronic acid-hexapeptide graft and ergothioneine can be compounded to synergistically reduce the expression of SA-beta-gal, so that a more effective scheme is provided for further reversing the aging state of cells.

Description

Technical Field

[0001] This application relates to the field of hyaluronic acid grafting technology, and more particularly to an anti-aging composition and its application. Background Technology

[0002] Cellular senescence and the accumulation of senescent cells are the fundamental causes of skin aging. When senescent cells accumulate to a certain threshold in skin tissue, they further accelerate the aging process, ultimately manifesting as visible signs of aging. Therefore, the fundamental approach to skin anti-aging is to delay cellular senescence or reduce the proportion of senescent cells, thereby fundamentally promoting the rejuvenation and reversal of senescent cells.

[0003] Previous research and experience have found that existing anti-aging skin products typically do not have lasting effects. They may show effective anti-aging effects for a short period of time, but continued use does not lead to further effective anti-aging results. This may be because the products only promote collagen synthesis and secretion pathways in aging cells, without fundamentally promoting the rejuvenation and reversal of aging cells. Therefore, a "bottleneck" effect often occurs in the anti-aging effect, meaning that the anti-aging effect does not further improve with the extension of use.

[0004] Overcoming the "bottleneck" effect of anti-aging and achieving sustained improvement in aging is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This invention is the first to discover that hyaluronic acid-hexapeptide grafts can effectively reduce the expression of senescence-associated β-galactosidase (SA-β-gal), a marker of cellular senescence, thereby fundamentally reversing the senescent state of cells and bringing about a sustained improvement in aging. Further research has found that combining hyaluronic acid-hexapeptide grafts and ergothioneine in a certain ratio can synergistically reduce the expression of SA-β-gal, providing a more effective solution for further reversing the senescent state of cells.

[0006] On one hand, this application provides a composition comprising: (a) at least one hyaluronic acid-hexapeptide graft, and (b) Ergothioneine, The mass ratio of (a) and (b) is (1-30):1; The hyaluronic acid-hexapeptide graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid or its salt structure with the amino group (-NH2) in the hexapeptide structure.

[0007] Furthermore, the amino acid sequence of the hexapeptide includes His-Ala-Leu-Arg-Phe-Trp.

[0008] Furthermore, the structural formula of hyaluronic acid or its salt is shown in Formula II below: ...Form II, In the formula, n is a natural number, and R2 is H or a metal ion.

[0009] Optionally, n is selected from one or more natural numbers from 0 to 19, for example, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0010] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5.

[0011] The hyaluronic acid includes hyaluronic acid oligosaccharides, which include one or more of the following: hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, and hyaluronic acid dodecasaccharide.

[0012] Optionally, the metal ion includes Na. + K + Zn + One or more of them.

[0013] Furthermore, the hyaluronic acid-hexapeptide graft is obtained by reacting the hydroxyl group (-OH) at the non-reducing end of hyaluronic acid or its salt structure with the amino group (-NH2) in the hexapeptide structure.

[0014] Optionally, the hydroxyl group at the non-reduced end of the hyaluronic acid or its salt structure is a hydroxyl group of terminal glucuronic acid.

[0015] Furthermore, the mass ratio of (a) and (b) is (1-20):1.

[0016] The mass ratio or upper or lower limit of the mass ratio range of the hyaluronic acid-hexapeptide graft and ergothionein can be any value or range of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1.

[0017] Optionally, the mass ratio of (a) and (b) is (1-19):1.

[0018] Furthermore, the hyaluronic acid-hexapeptide graft comprises a structure as shown in Formula I: ...Formula I, In the formula, n is a natural number, R1 is the portion of the hexapeptide structure excluding the reactive group, and R2 is H or a metal ion; Optionally, R1 is -NH-His-Ala-Leu-Arg-Phe-Trp-NH2.

[0019] Optionally, n is selected from one or more natural numbers from 0 to 19, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0020] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5.

[0021] Optionally, the metal ion includes Na. + K + Zn + One or more of them.

[0022] In one alternative embodiment, the hyaluronic acid-hexapeptide graft comprises a structure as shown in Formula III: ...Formula III, In the formula, n is a natural number, and R2 is H or a metal ion.

[0023] Optionally, n is selected from one or more natural numbers from 0 to 19, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0024] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5.

[0025] Optionally, the metal ion includes Na. + K + Zn + One or more of them.

[0026] The hyaluronic acid-hexapeptide graft used in this application can be obtained by purchase, for example, from Bloomage Biotechnology Co., Ltd.

[0027] Those skilled in the art can also prepare the hyaluronic acid-hexapeptide graft using known methods.

[0028] The preparation method of the hyaluronic acid-hexapeptide graft in this application has been described in Chinese Patent Application No. CN202311312845.X.

[0029] In one optional embodiment, the method for preparing the hyaluronic acid-hexapeptide graft includes the following steps: The hexapeptide was dissolved in an organic solvent by stirring, and then TFA (trifluoroacetic acid) was added. After stirring for 0.5-1 h, an alkaline catalyst was added, followed by the weighing of hyaluronic acid or its salt. The reaction was carried out overnight at 40℃-50℃ with stirring. After the reaction was confirmed to be complete by LC-MS, acetic acid was added, and the rearrangement reaction was carried out at 30℃-45℃ for 2-4 h. After the reaction was confirmed to be complete by LC-MS, the hyaluronic acid-hexapeptide graft was purified to obtain the hyaluronic acid-hexapeptide graft.

[0030] Optionally, the amino acid sequence of the hexapeptide includes His-Ala-Leu-Arg-Phe-Trp.

[0031] Those skilled in the art can choose conventional purification methods for purification, and optionally, reverse chromatography purification can be used.

[0032] Optionally, the organic solvents mentioned above are selected from one or more of DMF, N,N-diethylacetamide, DMSO, acetonitrile, N-methylpyrrolidone (NMP), methanol, ethanol, acetone, THF, dichloromethane, ethyl acetate, dioxane, and water, and can be single solvents or mixed solvents. Optionally, the base catalyst comprises a tertiary amine base catalyst; more preferably, the base catalyst comprises N,N-diisopropylethylamine (DIEA).

[0033] Preferably, the mass-to-volume ratio of the hexapeptide to the organic solvent is 1 g: 8-25 mL. Preferably, the mass-to-volume ratio of the hexapeptide to TFA is 1 g: 0-1.2 mL. Preferably, the mass ratio of the hexapeptide to DIEA is 1:0.5-5.5. Preferably, the molar ratio of the hexapeptide to hyaluronic acid is 1:2-8. Preferably, the mass-to-volume ratio of the hexapeptide to acetic acid is 1 g: 4-10 mL.

[0034] In one embodiment, the composition may further include a physiologically acceptable medium, such as a solvent and / or excipients, the solvent being, for example, water.

[0035] As used herein, the term "physiologically acceptable" means a molecular entity and composition that is physiologically tolerable and, when administered to humans, generally does not produce toxicity or sensitization or similar adverse reactions (such as stomach upset, dizziness, etc.). Optionally, as used herein, the term "physiologically acceptable" means approved by a government regulatory agency or approved in a pharmacopoeia or other generally recognized manner for use in animals or humans.

[0036] In one embodiment, the composition can be formulated into food, pharmaceutical, or cosmetic products, and excipients suitable for the product can be added. For example, when the composition is formulated into a cosmetic, it may also include emollients, thickeners, chelating agents, UV filters, antioxidants, emulsifiers, fillers, colorants, preservatives, fragrances, and active agents such as moisturizers, anti-aging agents, whitening agents, and / or mixtures thereof.

[0037] The compositions of this application can be prepared by common methods.

[0038] In one alternative embodiment, the composition is prepared by mixing hyaluronic acid-hexapeptide graft and ergothioneine until homogeneous.

[0039] In one alternative embodiment, the composition is prepared by mixing hyaluronic acid-hexapeptide graft and ergothionein in a physiologically acceptable medium until homogeneous.

[0040] One aspect of this application relates to compositions, which are any compositions capable of achieving the effects described in this application. The compositions include, but are not limited to, the simultaneous or sequential use of the components. "Simultaneous use" includes using them together in the same formulation or separately in different formulations. "Sequential use" includes using them sequentially in different formulations, with no restriction on the order of sequential use.

[0041] On the other hand, this application also provides the use of the described composition in the preparation of cosmetics, pharmaceuticals, health products and / or medical devices.

[0042] On the other hand, this application also provides the use of the composition in the preparation of anti-aging products.

[0043] Furthermore, the anti-aging product is a long-term anti-aging product.

[0044] Optionally, the long-term anti-aging effect refers to the effect of continuously improving aging even after use for more than one month.

[0045] Optionally, the long-term anti-aging effect refers to the continued improvement in aging even after 2 months of use.

[0046] This application is the first to discover that the composition of hyaluronic acid-hexapeptide graft and ergothionein can synergistically reduce the expression of aging-related β-galactosidase (SA-β-gal), thereby fundamentally reversing the aging state of cells, improving cell aging and / or clearing senescent cells, bringing about a sustained improvement in aging and / or anti-wrinkle effects, overcoming the "bottleneck" effect of existing anti-aging products.

[0047] Optionally, the aging includes photoaging.

[0048] On the other hand, this application also provides the use of the composition in reversing cellular senescence, improving cellular aging, and / or clearing senescent cells.

[0049] Furthermore, the composition is used to reduce β-galactosidase levels.

[0050] Optionally, reducing β-galactosidase levels includes reducing β-galactosidase expression or reducing β-galactosidase content.

[0051] The composition achieves the effects of reversing cell senescence, improving cell aging, and / or clearing senescent cells by reducing β-galactosidase levels.

[0052] The composition fundamentally reverses cell senescence, improves cell aging, and / or eliminates senescent cells, thereby achieving long-term anti-aging.

[0053] In one alternative implementation, the cells are fibroblasts.

[0054] Optionally, the aging includes photoaging.

[0055] On the other hand, this application also provides an anti-aging product comprising the aforementioned composition.

[0056] Furthermore, the content of the hyaluronic acid-hexapeptide graft in the anti-aging product can be 0.005%-10%.

[0057] The content of the hyaluronic acid-hexapeptide graft in the composition may be any value or range of 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, and 10%.

[0058] Furthermore, the content of ergothionein in the composition may be 0.005%-10%.

[0059] The content of ergothionein in the composition may be any value or range of 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0060] Optionally, the anti-aging product is a long-term anti-aging product.

[0061] Optionally, the anti-aging includes reversing cellular senescence, improving cellular aging, and / or eliminating senescent cells.

[0062] Alternatively, the anti-aging effect is achieved by reducing the level of β-galactosidase in cells.

[0063] In one alternative embodiment, the cell is a fibroblast.

[0064] Optionally, the aging includes photoaging.

[0065] On the other hand, this application also provides the application of hyaluronic acid-hexapeptide grafts in reversing cell senescence, improving cell aging and / or clearing senescent cells, wherein the hyaluronic acid-hexapeptide grafts are obtained by reacting the hydroxyl group (-OH) at the end of the structure of hyaluronic acid or its salt with the amino group (-NH2) in the structure of the hexapeptide.

[0066] Furthermore, the amino acid sequence of the hexapeptide includes His-Ala-Leu-Arg-Phe-Trp.

[0067] Furthermore, the structural formula of hyaluronic acid or its salt is shown in Formula II below: ...Form II, In the formula, n is a natural number, and R2 is H or a metal ion.

[0068] Optionally, n is selected from one or more natural numbers from 0 to 19, for example, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0069] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5.

[0070] The hyaluronic acid includes hyaluronic acid oligosaccharides, which include one or more of the following: hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, and hyaluronic acid dodecasaccharide.

[0071] Optionally, the metal ion includes Na. + K + Zn + One or more of them.

[0072] Furthermore, the hyaluronic acid-hexapeptide graft is obtained by reacting the hydroxyl group (-OH) at the non-reducing end of hyaluronic acid or its salt structure with the amino group (-NH2) in the hexapeptide structure.

[0073] Optionally, the hydroxyl group at the non-reduced end of the hyaluronic acid or its salt structure is a hydroxyl group of terminal glucuronic acid.

[0074] Furthermore, the application of the hyaluronic acid-hexapeptide graft in reducing β-galactosidase levels.

[0075] Furthermore, the hyaluronic acid-hexapeptide graft comprises a structure as shown in Formula I: ...Formula I, In the formula, n is a natural number, R1 is the portion of the hexapeptide structure excluding the reactive group, and R2 is H or a metal ion; Optionally, R1 is -NH-His-Ala-Leu-Arg-Phe-Trp-NH2.

[0076] Optionally, n is selected from one or more natural numbers from 0 to 19, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0077] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5.

[0078] Optionally, the metal ion includes Na. + K + Zn + One or more of them.

[0079] This application is the first to discover that hyaluronic acid-hexapeptide grafts can effectively reduce the expression of aging-related β-galactosidase (SA-β-gal), thereby fundamentally reversing the aging state of cells, improving cell aging and / or clearing senescent cells, bringing about a sustained improvement in aging, and overcoming the "bottleneck" effect of existing anti-aging products.

[0080] The present invention has the following beneficial effects: This application is the first to discover that hyaluronic acid-hexapeptide grafts can effectively reduce the expression of aging-related β-galactosidase (SA-β-gal), thereby fundamentally reversing the aging state of cells and bringing about a sustained improvement in aging. This provides a new functional ingredient for the development of cell aging reversal agents, cell aging improvement agents, and products that enhance the cells' ability to resist aging.

[0081] Furthermore, this application is the first to discover that the combination of hyaluronic acid-hexapeptide graft and ergothioneine can synergistically reduce SA-β-gal expression, providing a more effective solution for further reversing cellular senescence. Attached Figure Description

[0082] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 A bar chart showing β-Gal content; Figure 2 Bar chart showing the area of ​​skin wrinkles at different time points; Figure 3 A bar chart showing the improvement in skin wrinkle area at different time points. Detailed Implementation

[0083] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0084] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0085] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0086] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0087] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt conventional techniques in the fields of pharmacy, medicine, biochemistry, analytical chemistry, cell culture, and related fields.

[0088] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0089] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0090] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0091] Information on the reagents and equipment involved in the following examples is shown in Tables 1 and 2: Table 1

[0092] Table 2

[0093] Example 1: Cytological Testing This embodiment investigates the effects of HA-P and EGT on aging cells at the cellular level.

[0094] SA-β-gal (senescence-associated β-galactosidase) is the gold standard biomarker for identifying and quantifying senescent cells. Its activity is specifically increased in senescent cells, and its expression level directly reflects the degree of cellular aging and is positively correlated with the proportion of senescent cells, making it an effective indicator for evaluating anti-aging efficacy.

[0095] 1. Solution preparation Case 1 Weigh 0.05 g of palmitoyl tripeptide-5 (CAS: 623172-56-5) powder and prepare it into a working solution for case 1 using culture medium solution, wherein the content of palmitoyl tripeptide-5 is 100 ppm.

[0096] Case 2 Weigh 0.05 g of EGCG (epigalocatechin gallate, CAS: 989-51-5) powder and prepare the case 2 working solution with culture medium solution, wherein the content of EGCG is 100 ppm.

[0097] Case 3 Weigh 0.05 g of HA-P powder and prepare the case 3 working solution with culture medium solution, wherein the HA-P content is 100 ppm.

[0098] Case 4 Weigh 0.05 g of EGT powder and prepare the working solution of case 4 with culture medium solution, wherein the content of EGT is 100 ppm.

[0099] Case 5 The Case 5 working solutions of HA-P and EGT were prepared using culture medium solution, with HA-P content of 95 ppm and EGT content of 5 ppm.

[0100] Case 6 The Case 6 working solutions of HA-P and EGT were prepared using culture medium solution, with HA-P content of 90 ppm and EGT content of 10 ppm.

[0101] Case 7 Prepare Case 7 working solutions of HA-P and EGT using culture medium solution, wherein the content of HA-P is 80 ppm and the content of EGT is 20 ppm.

[0102] Case 8 Prepare Case 8 working solutions of HA-P and EGT using culture medium solution, wherein the content of HA-P is 50 ppm and the content of EGT is 50 ppm.

[0103] Case 9 Prepare Case 9 working solutions of HA-P and EGT using culture medium solution, wherein the content of HA-P is 10 ppm and the content of EGT is 90 ppm.

[0104] Case 10 The Case 10 working solution of hexapeptide, hyaluronic acid and EGT was prepared using culture medium solution, wherein the content of hyaluronic acid was 43.2 ppm, the content of hexapeptide was 46.8 ppm and the content of EGT was 10 ppm.

[0105] Case 11 The Case 11 working solution of hexapeptide and hyaluronic acid was prepared using a culture medium solution, with the hyaluronic acid content at 48 ppm and the hexapeptide content at 52 ppm.

[0106] Case 12 Case 12 working solutions of HA-P, EGT, and palmitoyl tripeptide-5 were prepared using culture medium solutions, with HA-P at 90 ppm, EGT at 10 ppm, and palmitoyl tripeptide-5 at 10 ppm.

[0107] 2. Cytological testing 1) Cell seeding: Fibroblasts were seeded into 6-well plates containing DMEM-H complete medium (3 × 10⁻⁶ cells / well). 5 (each well) was incubated at 37°C with 5% CO2 for 24 h. 2) Modeling: Remove the culture medium from each well, wash away any residual culture medium with PBS, and irradiate the cells with UVB (except for the normal control group (BC)) at a dose of 30 mJ / cm². 2 (Equipment power: 1.5 mW / cm) 2 Irradiation time: 20 s.

[0108] 3) Drug administration: PBS was removed. The normal control group (BC) and model group (NC) were supplemented with DMEM-H complete medium containing 2% serum. The sample groups (cases 1-12) were supplemented with working solutions of different concentrations prepared from DMEM-H complete medium containing 2% serum. After drug administration, the 6-well plates were placed in an incubator (37℃, 5% CO2) and incubated for 48 h. 4) Viable cell count: After culture, aspirate the cell culture supernatant. Wash twice with PBS, add 300 μL of trypsin-EDTA to each well, and digest at 37°C for 2 min. After digestion, add 600 μL of DMEM-H complete medium to each well to terminate digestion. Transfer the digested cell suspension to centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 1 mL of PBS, and count using a hemocytometer.

[0109] β-Gal content assay: After viable cell counting, the β-Gal content was measured using an ELISA kit (Huamei Biotechnology, Human β-galactosidase (β-Gal) ELISA Kit) according to the kit instructions. The measured content was then averaged to the number of viable cells per well to obtain the average β-Gal content. The results were recorded as shown in Table 3. A graph was plotted from Table 3 to obtain the β-Gal content. Figure 1 .

[0110] 5) Statistical analysis of results: t-tests were used for comparisons between groups. A p-value < 0.05 was considered statistically significant. Groups with the same letter were considered not significantly different; for example, there was no significant difference between groups a and ab.

[0111] Table 3 Cell Experiment Design

[0112] From Table 3 and Figure 1 As shown, after photoaging induction, the SA-β-Gal single cell content in the NC group was significantly higher than that in the BC group, indicating that the cells in the NC group were in a state of aging or even death.

[0113] Compared to the NC group, the anti-aging ingredient palmitoyl tripeptide-5 had no effect on the single-cell SA-β-Gal content, while the single-cell SA-β-Gal content of cells treated with the anti-aging ingredient EGCG was significantly increased. This indicates that anti-aging ingredients do not necessarily reduce SA-β-Gal levels or reverse cellular senescence. Cases 3-12 showed a significant reduction in SA-β-Gal content compared to the NC group, implying a decrease in the average degree of cellular senescence (clearing senescent cells, reversing cellular senescence, and increasing cell number). Furthermore, cases 5-8 showed significant differences compared to cases 3 and 4, indicating that a synergistic effect can occur when HA-P and EGT are mixed in a certain proportion. Example 2 Clinical Testing This embodiment explores the effects of HA-P and EGT on photoaging in clinical trials.

[0114] 1. Solution preparation Matrix formulation: According to the formulation in Table 4, weigh out 25 g butanediol, 5 g glycerol, 0.25 g sodium EDTA-2, 0.25 g sodium sulfite, 2.5 g polyacrylate crosspolymer-6, 2.5 g p-hydroxyacetophenone, 2.5 g hexanediol, and 462 g water, and stir to dissolve evenly.

[0115] TP group: According to the formula in Table 4, weigh out 25 g butanediol, 5 g glycerol, 0.25 g sodium EDTA-2, 0.25 g sodium sulfite, 2.5 g polyacrylate crosspolymer-6, 2.5 g p-hydroxyacetophenone, 2.5 g hexanediol, 0.5 g palmitoyl tripeptide-5, and 461.5 g water, and stir to dissolve evenly.

[0116] EGT group: According to the formula in Table 4, weigh out 25 g butanediol, 5 g glycerol, 0.25 g sodium EDTA-2, 0.25 g sodium sulfite, 2.5 g polyacrylate crosspolymer-6, 2.5 g p-hydroxyacetophenone, 2.5 g hexanediol, 0.5 g EGT, and 461.5 g water, and stir to dissolve evenly.

[0117] HA-P group: According to the formula in Table 4, weigh out 25 g butanediol, 5 g glycerol, 0.25 g sodium EDTA-2, 0.25 g sodium sulfite, 2.5 g polyacrylate crosspolymer-6, 2.5 g p-hydroxyacetophenone, 2.5 g hexanediol, 0.5 g HA-P, and 461.5 g water, and stir to dissolve evenly.

[0118] Combined formulation group (EGT+HA-P): According to the formulation in Table 4, weigh out 25 g butanediol, 5 g glycerol, 0.25 g sodium EDTA-2, 0.25 g sodium sulfite, 2.5 g polyacrylate crosspolymer-6, 2.5 g p-hydroxyacetophenone, 2.5 g hexanediol, 0.45 g HA-P, 0.05 g EGT, and 461.5 g water, and stir to dissolve evenly.

[0119] Table 4 Clinical Trial Formulation Design

[0120] 2. Human testing 1) Volunteer selection: 24 volunteers were selected, aged 22-40, with no obvious facial scars. All volunteers were randomly assigned to 4 groups.

[0121] 2) Drug Administration: Following the test protocol in Table 4, a left-right control drug administration was conducted. Group 1 volunteers: left cheek used the formulation matrix, right cheek used the formulation case (combination formulation). Group 2 volunteers: left cheek used the formulation matrix, right cheek used the TP group. Group 3 volunteers: left cheek used the formulation matrix, right cheek used the HA-P group. Group 4 volunteers: left cheek used the formulation matrix, right cheek used the EGT group. Two pumps of working solution were used on each side of the face, with six parallel trials per group. Application was continuous for 56 days, and volunteers were photographed on days 0, 14, 28, 42, and 56.

[0122] 3) Data collection: Volunteer testing was conducted using VISIA 7, and wrinkle area data were recorded, as shown in Table 5. Graphs were then created from Table 5. Figure 2 By comparing the reduction in wrinkle area after 28 days and 56 days of use compared to before use, where the reduction in wrinkle area after 28 days of use (D28 vs D0 wrinkle reduction) = D0 - D28, and the reduction in wrinkle area after 56 days of use (D56 vs D0 wrinkle reduction) = D0 - D56, the differences between different drug administration groups were compared horizontally. The results are shown in Table 6, and a graph was plotted from Table 6. Figure 3 .

[0123] 4) Data Processing and Statistical Analysis: t-tests were used for comparisons between groups. For comparisons at different time points, p < 0.05 was indicated by **. For comparisons with individual groups (EGT, HA-P), p < 0.05 was indicated by ##.

[0124] Table 5

[0125] Table 6

[0126] From Tables 5 and 6 Figure 2 , 3 It was found that the average SA-β-Gal content of palmitoyl tripeptide-5 in the cell assay was not significantly different from that in the NC group, and it did not reverse the aging state of cells. Human assay results also showed that the anti-wrinkle effect only lasted for one month, and the effect became slower or ineffective over time. The combination of HA-P and EGT significantly reduced the average SA-β-Gal content and maintained its anti-wrinkle effect in humans for a longer period. Furthermore, the improvement effect of the HA-P and EGT combination was significantly better than that of the monomers, indicating that HA-P and EGT also have a synergistic effect in human assays.

[0127] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A composition, characterized in that, The composition comprises: (a) at least one hyaluronic acid-hexapeptide graft, and (b) Ergothioneine, The mass ratio of (a) and (b) is (1-30):1; The hyaluronic acid-hexapeptide graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid or its salt structure with the amino group (-NH2) in the hexapeptide structure.

2. The composition according to claim 1, characterized in that, The mass ratio of (a) to (b) is (1-20):

1.

3. The composition according to claim 1, characterized in that, The hyaluronic acid-hexapeptide graft comprises a structure as shown in Formula I: ...Equation I, In the formula, n is a natural number, R1 is the portion of the hexapeptide structure excluding the reactive group, and R2 is H or a metal ion; Optionally, R1 is -NH-His-Ala-Leu-Arg-Phe-Trp-NH2.

4. The use of the composition according to any one of claims 1-3 in the preparation of anti-aging products.

5. The use of the composition according to any one of claims 1-3 in reversing cellular senescence, improving cellular aging, and / or clearing senescent cells.

6. The application according to claim 5, characterized in that, The application of the composition in reducing β-galactosidase levels.

7. An anti-aging product, characterized in that, Includes the composition according to any one of claims 1-3.

8. The application of hyaluronic acid-hexapeptide grafts in reversing cell senescence, improving cell aging, and / or clearing senescent cells, characterized in that, The hyaluronic acid-hexapeptide graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid or its salt structure with the amino group (-NH2) in the hexapeptide structure.

9. The application according to claim 8, characterized in that, The application of the hyaluronic acid-hexapeptide graft in reducing β-galactosidase levels.

10. The application according to claim 8, characterized in that, The hyaluronic acid-hexapeptide graft comprises a structure as shown in Formula I: ...Equation I, In the formula, n is a natural number, R1 is the portion of the hexapeptide structure excluding the reactive group, and R2 is H or a metal ion; Optionally, R1 is -NH-His-Ala-Leu-Arg-Phe-Trp-NH2.

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