Hyaluronic acid derivatives and their preparation methods and uses

Abstract

The present application discloses hyaluronic acid derivatives, specifically hyaluronic acid esters synthesized from hyaluronic acid or its salt and salicylic acid esters. The hyaluronic acid esters disclosed in the present application are characterized by good water solubility, low irritation, and mild properties, optimizing the stratum corneum. Furthermore, the water-soluble esters inhibit the penetration of salicylic acid into the stratum corneum and extend the duration of salicylic acid's action in the stratum corneum. The unique chemical structure of the salicylic acid hyaluronic acid esters of the present application immobilizes acidic functional groups, reducing the irritation of salicylic acid. The increased hyaluronic acid polysaccharide content effectively delays the penetration of salicylic acid into the deeper layers of the skin, prolonging its action time in the epidermis, targeting the stratum corneum and contributing to the long-term regulation of keratinocytes. The improved solubility of salicylic acid itself in the aqueous phase facilitates its use in formulations.

Description

[Technical Field]

[0001] The present application relates to the field of biotechnology, in particular to hyaluronic acid derivatives, their preparation methods and their uses. [Background technology]

[0002] Hyaluronic acid (HA) is a mucopolysaccharide consisting of alternating disaccharide units of glucuronic acid and N-acetylglucosamine. Due to its unique molecular structure and physical and chemical properties, it has been effectively used in cosmetics, medical beauty, ophthalmic surgery, arthritis treatment, and more.

[0003] Hyaluronic acid is widely distributed in nature. Over 50% of hyaluronic acid in the body is found in the skin, lungs, and intestines. It is also found in interstitial tissues such as synovial fluid, cartilage, umbilical cord, and blood vessel walls. Early research identified umbilical cord as the primary source of hyaluronic acid. Currently, hyaluronic acid products can be extracted from animal tissues such as cockscomb, vitreous humor, brain cartilage, and synovial fluid, as well as by fermentation with bacteria such as Streptococcus and Pseudomonas aeruginosa. Fermentation methods for producing hyaluronic acid are gradually replacing tissue extraction methods due to their low cost, abundant raw materials, ease of large-scale production, and high molecular weight. In recent years, a variety of hyaluronic acid derivatives have been developed through various modifications of hyaluronic acid. These derivatives not only retain the functionality of hyaluronic acid itself, but also offer improved stability, biocompatibility, and water solubility.

[0004] Salicylic acid (SA), also known as o-hydroxybenzoic acid, is a white crystalline powder that exfoliates the stratum corneum, removing excessively thick layers and promoting metabolism. However, because SA is a small-molecule acid, it has high permeability, dissolving the stratum corneum and destroying the sebum film while also easily penetrating the dermis and subcutaneous tissue. As a result, it does not remain in the stratum corneum long enough to exert its characteristic exfoliating effect, and it irritates the microvessels and nerves located deep within the skin. For these reasons, its applications are limited. Summary of the Invention

[0005] In view of the above problems, the present application provides a hyaluronic acid derivative and a preparation method thereof. Specifically, the present application adopts the following technical solution:

[0006] 1. A salicylic acid hyaluronic acid ester having the structural formula (I):

[0007] [ka]

[0008] where R1 is H or

[0009] [ka]

[0010] wherein R2 is H or a metal ion, x1≧0, x2≧0, and y≧1. 2. The hyaluronic acid ester according to Item 1, wherein the substitution degree of salicylic acid in the hyaluronic acid or a salt thereof is 15% to 60%, preferably 25% to 50%. 3. The hyaluronic acid ester according to any one of items 1 to 3, wherein the molecular weight of the hyaluronic acid ester is 500 KDa or less, preferably 5 KDa to 300 KDa, and more preferably 5 KDa to 100 KDa. 4. A method for preparing a salicylic acid hyaluronic acid ester, wherein the hyaluronic acid ester is an ester synthesized from hyaluronic acid or its salt and a salicylic acid ester. Item 5. A method for preparing a hyaluronic acid ester according to Item 4, wherein the hyaluronic acid ester is obtained by reacting a quaternary ammonium salt of hyaluronic acid with a salicylic acid ester in the presence of an inorganic base. 6. The method for preparing a hyaluronic acid ester according to item 4 or 5, wherein the molar ratio of the quaternary ammonium salt of hyaluronic acid to the salicylic acid ester is 1:(1 to 10). 7. The preparation method according to any one of items 4 to 6, wherein the molar ratio of the inorganic base to the quaternary ammonium salt of hyaluronic acid is 1:(2 to 10). 8. The preparation method according to any one of items 4 to 7, wherein the inorganic base comprises one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate. 9. The preparation method according to any one of items 4 to 8, wherein the reaction temperature is 50 to 100°C, preferably 60 to 80°C. 10. The inorganic base is treated, and the treatment is Item 10. The preparation method according to any one of Items 4 to 9, comprising mixing and dissolving an inorganic base and a tetrabutylammonium salt, followed by freeze-drying. 11. The preparation method according to any one of items 4 to 10, wherein in the treatment with the inorganic base, the molar ratio of the inorganic base to the tetrabutylammonium salt is 1:(0.1 to 1). 12. Use of the hyaluronic acid ester according to any one of items 1 to 3 or the hyaluronic acid ester prepared by the preparation method according to any one of items 4 to 11 in a skin care product. 13. Use of the hyaluronic acid ester according to any one of items 1 to 6 or the hyaluronic acid ester prepared by the preparation method according to any one of items 7 to 15 in the preparation of an exfoliating and / or mild topical preparation. 14. A composition comprising the hyaluronic acid ester according to any one of items 1 to 3, or the hyaluronic acid ester prepared by the preparation method according to any one of items 4 to 11. [Effects of the Invention]

[0011] 1. The salicylic acid hyaluronic acid ester of the present application has good water solubility, low irritation, mild properties, and optimizes the stratum corneum. Furthermore, the water-soluble ester inhibits the penetration of salicylic acid into the stratum corneum, thereby prolonging the duration of the action of salicylic acid in the stratum corneum. 2. The unique chemical structure of the salicylic acid hyaluronic acid ester of the present application can fix the acidic functional group and reduce the irritation of salicylic acid. The increase in hyaluronic acid polysaccharides effectively delays the penetration of salicylic acid into the deeper layers of the skin, prolonging its action time in the epidermis, targeting the stratum corneum and helping to regulate keratinocytes for a long time. The solubility of salicylic acid itself in the aqueous phase is improved, making it easier to use in formulations. 3. In the preparation method of salicylic acid hyaluronic acid ester of the present application, the inorganic base is mixed and freeze-dried to improve the catalytic effect of the inorganic base in the reaction solvent, reduce the reaction temperature, and improve the reaction efficiency. In comparison, its catalytic effect is much higher than that of untreated inorganic base and even higher than that of organic base. Furthermore, the preparation process of the present application is simple to operate, suitable for industrial production, and has broad prospects in the cosmetic raw materials market. [Brief explanation of the drawings]

[0012] The accompanying drawings are used for a better understanding of the present application and shall not constitute undue limitations to the present application. [Figure 1] 1H NMR spectrum of salicylic acid hyaluronic acid ester prepared in Example 18. [Figure 2] 1 is a full wavelength ultraviolet scanning spectrum of salicylic acid hyaluronic acid ester prepared in Example 18. Details of the invention

[0013]

[0033] Exemplary embodiments of the present application will be described below. These embodiments include various details of the embodiments of the present application for ease of understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will understand that various changes and modifications to the embodiments described herein can be made without departing from the scope and spirit of the present application. In addition, in the following description, for the sake of clarity and conciseness, descriptions of well-known functions and structures will be omitted.

[0014] The present application provides hyaluronic acid derivatives, and more particularly, provides hyaluronic acid esters, which are esters synthesized from hyaluronic acid or its salt and salicylic acid ester.

[0015] The hyaluronic acid referred to in this application refers to a biopolymer material composed of linearly linked repeating units of N-acetyl-D-glucosamine and D-glucuronic acid. The hyaluronic acid or its salts include hyaluronic acid itself, its salts, or combinations thereof. Examples of hyaluronate salts include, but are not limited to, inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, gold hyaluronate, and cobalt hyaluronate, as well as organic salts such as quaternary ammonium hyaluronate. In this application, hyaluronic acid itself or its salts may be used alone, or two or more types of hyaluronic acid or their salts may be used in combination.

[0016] The hyaluronic acid or its salt described in this application is not limited. In a preferred embodiment, the hyaluronic acid salt is a water-soluble salt of hyaluronic acid, more preferably a quaternary ammonium salt of hyaluronic acid.

[0017] The salicylate esters described in this application are preferably alkyl or alkenyl esters, where the alkyl or alkenyl group is C1-6, and include, but are not limited to, pentyl salicylate, ethyl salicylate, cis-3-hexenyl salicylate, hexyl salicylate, isobutyl salicylate, and methyl salicylate.

[0018] In a preferred embodiment, the alkyl salicylate is methyl salicylate, i.e., methyl o-hydroxybenzoate, also known as methyl salicylate, an organic compound having the chemical formula CHO, a clear, colorless to pale yellow liquid with a strong wintergreen oil odor.

[0019] The present application also provides a salicylic acid hyaluronate having the structural formula shown in formula (I):

[0020] [ka]

[0021] where R1 is H or

[0022] [ka]

[0023] wherein R2 is H or a metal ion, x1≧0, x2≧0, y≧1, and x1, x2, and y are all independent repeating units.

[0024] The term "grafting" refers to a reaction in which appropriate branch chains or functional side groups are attached to a polymer chain via chemical bonds, and the resulting product is called a graft copolymer. The grafting rate generally refers to the grafting efficiency, which is calculated as follows: grafting rate = [amount of graft monomer or copolymer branch chain grafted onto the graft copolymer / (total amount of grafted monomer or grafted polymer initially added)] × 100%. In this application, the concept of substitution degree is used to express the grafting efficiency. The term "substitution degree" refers to the amount of substance replacing the active hydroxyl group of each D-glucose unit of cellulose. In this application, the substitution degree specifically refers to [amount of graft monomer or copolymer branch chain grafted onto the graft copolymer / (total amount of grafted monomer or grafted polymer initially added)] × 100%.

[0025] The degree of substitution of the salicylic acid hyaluronic acid ester in the present application is 15% to 60%, and may be, for example, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59%. Preferably, the degree of substitution is 25% to 50%. The molecular weight of the salicylic acid hyaluronic acid ester of the present application is 500 KDa or less, for example, 1 KDa, 2 KDa, 3 KDa, 4 KDa, 5 KDa, 6 KDa, 7 KDa, 8 KDa, 9 KDa, 10 KDa, 20 KDa, 30 KDa, 40 KDa, 50 KDa, 60 KDa, 70 KDa, 80 KDa, 90 KDa, 100 KDa, 200 KDa, 300 KDa, 400 KDa, or 500 KDa, preferably 5 KDa to 300 KDa, more preferably 5 KDa to 100 KDa.

[0026] The present application further provides a method for preparing the hyaluronic acid ester described in the present application, the method comprising: using an inorganic base as a catalyst to carry out a grafting reaction of a salicylic acid ester onto a quaternary ammonium salt of hyaluronic acid, i.e., grafting methyl salicylate onto a quaternary ammonium salt of hyaluronic acid by a covalent bond to prepare the hyaluronic acid ester.

[0027] The hyaluronic acid quaternary ammonium salt refers to a hyaluronic acid graft polymer formed by linking a positively charged quaternary ammonium group to the active group of hyaluronic acid. By combining hyaluronic acid with a quaternary ammonium base, it can enhance its excellent moisturizing properties, improve the adhesion of hyaluronic acid to the surface of skin and hair, and enhance the moisturizing, lubricating, nourishing, and repairing effects of hyaluronic acid. Patent documents such as CN101316864 (Shiseido, Japan), CN101715457A (Kewpie, Japan), CN107739417A (Yangzhou Zhongfu Biotechnology Co., Ltd.), and CN107556402A (Chongqing Technological and Commercial University) disclose methods for preparing hyaluronic acid quaternary ammonium salt. The quaternary ammonium salt of hyaluronic acid used in the preparation method of the present application can be the quaternary ammonium salt of hyaluronic acid prepared by any method in the prior art, including the above-mentioned patent documents, and can also be any of the quaternary ammonium salt of hyaluronic acid that is commonly available commercially.In a preferred embodiment, the present application uses the quaternary ammonium salt of hyaluronic acid prepared by the method disclosed in patent document CN107459590B.

[0028] The type of inorganic base is not limited, and any inorganic base known in the art that can promote the reaction in the preparation method of the present application can be used. In the present application, the inorganic base is a catalyst in the reaction process, but the term catalyst does not limit the inorganic base. Those skilled in the art will understand that catalysis refers to the process of changing the rate of a chemical reaction. A substance that can play such a role in the reaction process can be called a catalyst and can be used in the reaction process of the present application. In some preferred embodiments, the inorganic base can include one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate. For example, the inorganic base can be sodium hydroxide. The inorganic base can be potassium hydroxide. The inorganic base can be ammonium hydroxide. The inorganic base can be barium hydroxide. The inorganic base can be sodium carbonate. The inorganic base can be potassium carbonate. The inorganic base can be potassium bicarbonate.

[0029] In the preparation method of the present application, the specific implementation method is as follows: firstly, dissolve described quaternary ammonium salt of hyaluronic acid in organic solvent, then add methyl salicylate to the organic solvent that dissolves quaternary ammonium salt of hyaluronic acid, then add inorganic base to form reaction solution, stir under heating, react for a certain time, then add salt solution, continue stirring, then carry out alcohol precipitation, alcohol washing, dehydration, suction filtration and drying to obtain described hyaluronic acid ester.

[0030] In the above-mentioned preparation method, the organic solvent used to dissolve the quaternary ammonium salt of hyaluronic acid is not limited.In a preferred embodiment, the organic solvent is DMSO (dimethyl sulfoxide) or DMF (dimethylformamide).

[0031] The heating can be performed using any method known in the art, such as water bath heating, oil bath heating, air bath heating, sand bath heating, or electrical heating. The heating temperature, i.e., the reaction temperature between the quaternary ammonium salt of hyaluronic acid and methyl salicylate in the preparation method of the present application, is not limited as long as the two can undergo a covalent bond reaction. In a preferred embodiment, the reaction temperature is 50 to 100°C, and can be, for example, 55°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 85°C, 90°C, or 95°C. In a preferred embodiment, the reaction temperature is 60 to 80°C.

[0032] The stirring can be carried out by any method known in the art, so long as the reactants are uniformly mixed and heated.

[0033] The term "reacted for a certain period of time" refers to the time required for the quaternary ammonium salt of hyaluronic acid and methyl salicylate to react completely, and there is no limitation as long as they can react completely.

[0034] The salt solution is not limited, and may be, for example, a sodium chloride solution, a potassium chloride solution, a calcium chloride solution, etc. In a preferred embodiment of the reaction, the salt solution is an NaCl solution, the concentration of the salt solution is 2-5%, and the volume is at least half the volume of the reaction solution.

[0035] In the above preparation method, a salt solution is added and stirring is continued for a certain period of time, for example, 10 to 50 minutes, to promote precipitation. After completing the ion exchange, an alcohol solution can be used for precipitation and washing. The concentration of the alcohol solution is not limited, but is preferably 60 to 90%, and the number of washings is not limited, but is preferably 1 to 10. In a preferred preparation method, after washing with alcohol, the product is dehydrated once with ethanol, then suction filtered and vacuum dried to obtain the final product.

[0036] In the above-mentioned preparation method, the molar ratio of the quaternary ammonium salt of hyaluronic acid to methyl salicylate is not limited. In a preferred embodiment, the molar ratio of the quaternary ammonium salt of hyaluronic acid to methyl salicylate is 1: (1-10), for example, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5.

[0037] In the above-mentioned preparation method, the molar ratio of the inorganic base to the quaternary ammonium salt of hyaluronic acid is not limited.In a preferred embodiment, the molar ratio of the inorganic base to the quaternary ammonium salt of hyaluronic acid is 1:(2-10), and can be, for example, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5.

[0038] In a preferred embodiment, the inorganic base is a treated inorganic base.

[0039] The treatment method includes a method in which an inorganic base and a tetrabutylammonium salt are mixed and dissolved, and then the mixture is freeze-dried.

[0040] The mixing can be carried out by any conventional method in the art, and the dissolution involves mixing and dissolving the inorganic base and the tetrabutylammonium salt in water.

[0041] The type of the tetrabutylammonium salt is not limited, and may be, for example, tetrabutylammonium fluoride, tetrabutylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium iodide, or the like.

[0042] Lyophilization is a drying method in which a substance containing water is frozen to form a solid, and then dehydrated and dried at a low temperature by utilizing the sublimation property of water under low temperature and low pressure conditions. The freeze-drying process of the present application can be carried out by any method known in the art, for example, by freezing the substance in advance in a refrigerator, a freeze-dryer, or liquid nitrogen, and then freeze-drying the substance.

[0043] In the above treatment process, the molar ratio of the inorganic base to the tetrabutylammonium salt is not limited. In a preferred embodiment, the molar ratio of the inorganic base to the tetrabutylammonium salt is 1:0.1 to 1:1, for example, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, or 1:0.95.

[0044] The present application further provides for the use of any of the hyaluronic acid esters described above, or hyaluronic acid esters prepared using the preparation methods provided herein, in skin care or skin care products.

[0045] The skin care products include, but are not limited to, water, lotion, ointment, cream, essence, sunscreen, oil, makeup remover, solution, gel, shower gel, shampoo, facial cleanser, shampoo cream, shaving cream, shaving foam, shaving water, moisturizing lotion, moisturizing spray, moisturizing mask, toner, lotion, softener, exfoliating gel, acne gel, facial cream, body cream, hand cream, foot cream, repair essence, moisturizing lotion, moisturizing cream, repair lotion, moisturizing lipstick, lip gloss, lip glaze, hair removal cream, and the like.

[0046] In a preferred embodiment, the skin care product is an exfoliating skin care product.

[0047] The hyaluronic acid ester provided in this application is a novel hyaluronic acid derivative that can be completely dissolved in water and has good water solubility. It can be prepared as a cosmetic raw material, and can also be blended with various cosmetic raw materials to prepare cosmetics. Furthermore, the molecular weight of the hyaluronic acid ester can be selected according to various needs, providing further possibilities for the cosmetic raw material market.

[0048] The hyaluronic acid ester of the present application has the properties of both hyaluronic acid and salicylic acid, and its unique chemical structure allows the immobilization of acidic functional groups, reducing the irritation of salicylic acid.The increase in hyaluronic acid polysaccharides effectively delays the penetration of salicylic acid into the deeper layers of the skin, prolonging its action time in the epidermis, targeting the stratum corneum, and exhibiting low irritation and mild properties, optimizing the stratum corneum.Human skin tests have shown that the hyaluronic acid ester of the present application has lower irritation and better exfoliation rate than a composition of salicylic acid and hyaluronic acid ester.

[0049] This application provides a method for preparing said salicylic acid hyaluronic acid ester, and optimizes the type and dosage of raw materials in the preparation method.By mixing and lyophilizing inorganic base, the catalytic effect of inorganic base in the reaction solvent is improved, the reaction temperature is reduced, and the reaction efficiency is improved.By comparison, its catalytic effect is much higher than that of untreated inorganic base and higher than that of organic base.At the same time, by optimizing the preparation conditions, it is also possible to prepare hyaluronic acid ester with a higher degree of substitution. [Example]

[0050] Example 1 1) Treatment of inorganic base: 5 mmol of potassium bicarbonate and 2.5 mmol of tetrabutylammonium chloride were mixed and dissolved in 10 ml of purified water. After complete dissolution, the mixture was pre-frozen in liquid nitrogen and freeze-dried to obtain the treated inorganic base. 2) Preparation of hyaluronic acid quaternary ammonium salt: Hyaluronic acid with a molecular weight of 100 KDa was selected, and hyaluronic acid quaternary ammonium salt was prepared according to the method disclosed in Chinese patent CN107459590B. 3) Preparation of water-soluble hyaluronic acid ester: 10 mmol of the quaternary ammonium salt of hyaluronic acid prepared in step 2) was dissolved in DMSO (dimethyl sulfoxide). After complete dissolution, 50 mmol of methyl salicylate was added. All of the inorganic base prepared in step 1) was added, and the mixture was stirred at 80°C and reacted for 4 hours. Half the total volume of 2% NaCl solution was added, and the mixture was stirred for 20 minutes. After that, ethanol was added to cause precipitation, the mixture was allowed to stand, the supernatant was removed, and the mixture was washed with 80% ethanol. This process was repeated five times, and then the mixture was dehydrated once with ethanol, suction filtered, and vacuum dried to obtain a water-soluble hyaluronic acid ester powder.

[0051] The degree of substitution of the product was detected by quantitative NMR analysis, which compares the intensities of different absorption peaks. When performing quantitative analysis, the integral of the absorption peak intensity of the H atom is proportional to its molar concentration, which is then converted into the degree of substitution. The degree of substitution was 15%.

[0052] Examples 2 to 27 A water-soluble hyaluronic acid powder was prepared according to the method of Example 1, except that the types and amounts of raw materials shown in Table 2 were used, and the rest was the same as in Example 1 unless otherwise specified.

[0053] The structural formula of the water-soluble hyaluronic acid ester prepared in Example 18 is as follows:

[0054] [ka]

[0055] 1 The HNMR spectrum is shown in Figure 1.

[0056] [Table 1]

[0057] 1 The H spectrum shows 16 hydrogen groups, with the integral ratios from low-field to high-field hydrogens being 1:1:1:1:2:2:4:2:2:2:2:2:2:2:2:2:2:6, which is consistent with the structure of this product. Among these, the hydrogen at δ 8.015 is a doublet peak group with one proton and assigned to the 7' hydrogen. The hydrogen at δ 7.600 is a triplet peak group with one proton and assigned to the 5' hydrogen. The hydrogen at δ 7.053 is a multiplet peak group with one proton and assigned to the 6' hydrogen. The hydrogen at δ 7.032 is a multiplet peak group with one proton and assigned to the 4' hydrogen. Chemical shifts in the δ 7 to δ 8.5 range are generally caused by the benzene ring structure. The above results indicate that this product contains a benzene ring structure and has a 1:1 relationship with hyaluronic acid molecules.

[0058] The ultraviolet absorption spectrum of the benzene ring: 203 nm (ε = 8700) is called the E2 band. There is a series of relatively weak absorption bands called fine structures between 230 and 270 nm, centered at 254 nm (ε = 204) and called the B band. When groups such as -OCH3, -CHO, -COOH, and -NO2 are introduced, both the E2 band and the B band generally undergo a red shift. The full-wavelength ultraviolet scan spectrum of this product is shown in Figure 2. The peak in the range of 230 to 240 nm is the E2 band of the benzene ring, and the peak in the range of 300 to 310 nm is the B band of the benzene ring. The above results indicate that this product contains carbonyl and substituted benzene ring structures.

[0059] [Table 2-1]

[0060] [Table 2-2]

[0061] [Table 2-3]

[0062] The substitution degree of the prepared hyaluronic acid ester was detected by nuclear magnetic resonance spectroscopy and is shown in Table 3.

[0063] [Table 3-1]

[0064] [Table 3-2]

[0065] Test Example Test Example 1 Cytotoxicity test 1.1 Sample: Salicylic acid hyaluronic acid ester obtained in Example 18. 1.2 Experimental methods and procedures: 1.2.1 Plating HaCaT cells in logarithmic growth phase were harvested and plated in 96-well plates at 1 x 10 5 The cells were seeded at a density of 100 μL per well. The culture system was DMEM basal medium supplemented with 10% fetal bovine serum. The seeded cells were placed in a carbon dioxide incubator at 37°C and 5% CO2 and typically cultured for 24 hours. 1.2.2 Preparation of sample solution The samples were prepared in serum-free medium at a concentration of 2.0% and sterilized by filtration through a 0.22 μm filter membrane. The final concentrations were 0.05%, 0.1%, 0.2%, and 0.5%, and the samples were prepared immediately before use. 1.2.3 Administration After 24 hours of normal culture, the old culture medium was discarded, and 100 μL of sample was added to the experimental group, and an equal volume of serum-free medium was added to the normal control group (control), with six parallel wells at each level. 1.3 Detection After 24 hours of incubation, relative cell proliferation was measured using CCK-8. The culture medium was discarded, and 100 μL of CCK-8 diluted 1:10 with serum-free medium was added to each well. The wells were then placed in a cell culture incubator and incubated for 2 hours. Absorbance was measured at 450 nm using a plate reader. The relative proliferation rate (RGR) is the ratio of the absorbance of the experimental group to that of the normal control group. According to GB / T 16886.5-2017, a sample is considered cytotoxic if its RGR is less than 70%. 1.4 Results and Analysis: A relative proliferation rate of 70% or higher is considered to be non-cytotoxic. Salicylic acid hyaluronate is not cytotoxic at concentrations of 0.5% or less.

[0066] [Table 4]

[0067] Test Example 2: Irritation test 1.1 Sample: Sample 1: The salicylic acid hyaluronic acid ester obtained in Example 18 was dissolved in water to a mass concentration of 5%. Sample 2: A composition of hyaluronic acid and salicylic acid was dissolved in water, where the molecular weight of the hyaluronic acid was 100 KDa, the mass concentration of the hyaluronic acid was 4.35%, and the mass concentration of the salicylic acid was 0.7%. 1.2 Experimental methods and procedures: Healthy volunteers were recruited to voluntarily participate in this project. Exclusion criteria: (1) Persons with severe systemic illness, immunodeficiency or autoimmune disease, or skin disease or history of such illness (severe freckles, atopic skin disease, psoriasis, eczema, severe acne, etc.). (2) Those who have undergone dermatological treatment or cosmetic surgery, or who have used hormones or immunosuppressants within the past month. (3) People with allergies, allergic dermatitis, or active allergic diseases. (4) Those who are pregnant, breastfeeding, or in menopause. (5) Any person who has other skin conditions that may affect the study and is deemed inappropriate for participation in this project through clinical evaluation. Number of valid subjects: 30. Subjects washed their faces with cleanser, wiped them with tissue, and waited 15 minutes. 20 μL of each of the two samples was dispensed onto 7 mm diameter circular filter paper and randomly attached to the subject's nasolabial folds on both sides. Subjects rated the discomfort levels of itching, tingling, and burning sensations at the test site at 0, 1, 5, and 10 minutes. The sum of the sensory scores at each time point was the total sensory score. Scoring scale: 0 points: No tingling, itching, or burning sensation. 1 point: Mild tingling, itching, or burning sensation (slightly felt). 2 points: Moderate tingling, itching, or burning sensation (strong sensation, but tolerable). 3 points: Definite tingling, itching, or burning sensation (relatively strong sensation, unbearable). 1.3 Statistical methods: Paired t-tests were used for statistical analysis, with a test level of α = 0.05. If the test mean value is in the beneficial direction and the test result is p<0.05, it means that there is an effect. 1.4 Results and Analysis: Within 10 minutes of application, the sample containing 5% salicylic acid hyaluronate had a lower overall skin irritation score, demonstrating that salicylic acid hyaluronate is less irritating.

[0068] [Table 5]

[0069] Test Example 3: Exfoliation Ability on Keratinocytes - Stratum Corneum Desquamation Assay 1.1 Sample: Sample 1: The salicylic acid hyaluronic acid ester obtained in Example 18 was dissolved in water to a mass concentration of 5%. Sample 2: A composition of hyaluronic acid and salicylic acid was dissolved in water, where the molecular weight of the hyaluronic acid was 100 KDa, the mass concentration of the hyaluronic acid was 4.35%, and the mass concentration of the salicylic acid was 0.7%. 1.2 Experimental methods and procedures: Twenty-five healthy subjects were recruited. After wiping the flexor sides of their forearms with a dry tissue, a 3 cm × 3 cm test area was marked at the same position on the left and right flexor sides of their forearms. Sample application: 20 μL of each of the two samples was applied to the test areas of the left and right arms, massaged for 30 seconds, waited for 1 minute, then rinsed with warm water for 10 seconds and absorbed with a dry tissue. 1.3 Desquamation Measurement Method: After 15 minutes of absorbing moisture, the peel film was gently pressed against the test area to collect the desquamation, and the percentage of keratinocyte peeling was measured using a Visioscope PC 35. 1.4 Results and Analysis: The 5% concentration of salicylic acid hyaluronic acid ester has powerful exfoliating properties.

[0070] [Table 6]

[0071] Although the embodiments of the present application have been described above, the present application is not limited to the specific embodiments or application fields described above. The specific embodiments described above are merely examples and guidelines, and are not limiting. Those skilled in the art can create many forms according to the guidance of this specification without departing from the scope of protection of the claims of the present application, and all of them are protected by the present application.

Claims

1. A salicylic acid hyaluronic acid ester having the structural formula (I): 【Chemistry 1】 In the formula, R １ is H or 【Chemistry 2】 and R ２ is H or a metal ion, and x １ ≧0, x ２ ≧0, y≧1.

2. 2. The hyaluronic acid ester according to claim 1, wherein the degree of substitution of salicylic acid in the hyaluronic acid or its salt is 15% to 60%.

3. 3. The hyaluronic acid ester according to claim 2, wherein the degree of substitution is 25% to 50%.

4. The hyaluronic acid ester according to any one of claims 1 to 3, characterized in that the molecular weight of the hyaluronic acid ester is 500 KDa or less.

5. The hyaluronic acid ester according to claim 4, characterized in that the molecular weight of the hyaluronic acid ester is 5 KDa to 300 KDa.

6. The hyaluronic acid ester according to claim 4, characterized in that the molecular weight of the hyaluronic acid ester is 5 KDa to 100 KDa.

7. A method for preparing a salicylic acid hyaluronic acid ester, characterized in that the hyaluronic acid ester is an ester synthesized from hyaluronic acid or its salt and a salicylic acid ester.

8. 8. The method for preparing a hyaluronic acid ester according to claim 7, characterized in that the hyaluronic acid ester is obtained by reacting a quaternary ammonium salt of hyaluronic acid with a salicylic acid ester in the presence of an inorganic base.

9. The preparation method according to claim 7 or 8, characterized in that the molar ratio of the quaternary ammonium salt of hyaluronic acid to the salicylic acid ester is 1:(1-10).

10. The preparation method according to claim 9, characterized in that the molar ratio of the inorganic base to the quaternary ammonium salt of hyaluronic acid is 1:(2-10).

11. 8. The process of claim 7, wherein the inorganic base comprises one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate.

12. The preparation method according to claim 7, characterized in that the reaction temperature is 50-100°C.

13. The preparation method according to claim 12, characterized in that the reaction temperature is 60-80°C.

14. The inorganic base is treated, and the treatment comprises:

8. The method according to claim 7, comprising mixing and dissolving an inorganic base and a tetrabutylammonium salt, followed by freeze-drying.

15. 15. The method according to claim 14, wherein in the treatment with the inorganic base, the molar ratio of the inorganic base to the tetrabutylammonium salt is 1:(0.1-1).

16. Use of a hyaluronic acid ester according to any one of claims 1 to 6 or a hyaluronic acid ester prepared by the preparation method according to any one of claims 7 to 15 in a skin care product.

17. Use of a hyaluronic acid ester according to any one of claims 1 to 6 or a hyaluronic acid ester prepared by the preparation method according to any one of claims 7 to 15 in the preparation of an exfoliating and / or mild topical preparation.

18. A composition comprising a hyaluronic acid ester according to any one of claims 1 to 6, or a hyaluronic acid ester prepared by the preparation method according to any one of claims 7 to 15.

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