Retinoic acid hyaluronate and preparation method thereof

Abstract

The invention provides retinoic acid hyaluronate and a preparation method thereof. The retinoic acid hyaluronate disclosed by the invention is high in substitution degree and high in purity, and the anti-aging and anti-inflammatory effects of the retinoic acid hyaluronate are superior to those of a product obtained in the prior art. According to the method, retinoic acid is activated through an in-situ method and then reacts with hyaluronic acid in the presence of a catalyst, and a target product is obtained. According to the method, side reactions and by-products caused by activation in the prior art can be reduced, the reaction efficiency is effectively improved, and the substitution degree of the product can be improved. Meanwhile, the reaction system is used for reacting at room temperature, low-temperature activation or high-temperature reaction is not needed, no irritant gas is released, the used reactants are also non-irritant, and energy consumption and pollutant emission can be reduced. In addition, the method is high in activation efficiency, the use amount of retinoic acid can be effectively reduced, and the material cost is reduced.

Description

Technical Field

[0001] The present application belongs to the technical field of hyaluronic acid, and specifically relates to a retinoic acid hyaluronate and a preparation method thereof. Background Art

[0002] Retinoic acid, also known as tretinoic acid or vitamin A acid, is an FDA-approved active ingredient for anti-aging. However, retinoic acid is highly irritating and cannot be added directly to cosmetics. Consequently, various vitamin A derivatives, such as retinol, retinal, and retinyl palmitate, have been developed as alternatives. While these derivatives are milder, most require one or more conversion steps before retinoic acid can bind to the relevant receptors and exert its effects, significantly reducing its efficacy.

[0003] The combination of retinoic acid and hyaluronic acid is expected to improve the irritation and solubility of retinoic acid, while maximizing its efficacy because no conversion is required. Retinoic acid hyaluronate has been shown to exhibit excellent anti-aging, anti-inflammatory, and hydrophobic penetration-promoting effects. Current preparation methods primarily include those disclosed in WO2003008457A2 and WO2020182239A1. However, the preparation method used in the former is complex and difficult to replicate. The acyl chloride used is toxic, corrosive, and unstable, and easily releases irritating gases. The activator used in the latter, benzoyl chloride, is somewhat irritating and requires activation and subsequent reactions at low temperatures. Hydrolysis of the active ingredient produces side reactions that reduce reaction efficiency. The resulting product contains benzoic acid-grafted byproducts and the maximum degree of substitution of retinoic acid is 7.2%, limiting its efficacy. CN117964796A discloses a hyaluronic acid retinoate derivative and a preparation method thereof. The method employs a two-step activation process, which is cumbersome. The first activation step requires the use of a strong acid such as sulfuric acid as an activator, which is environmentally unacceptable and carries the risk of reacting with the catalyst and introducing impurities. Both the dissolution of HA and the catalytic reaction must be performed at high temperatures, resulting in low reaction efficiency. Without subsequent meticulous purification, a large amount of retinoic acid can easily remain in the product in a free form, leading to irritation and product solubility issues. These drawbacks limit the preparation and application of retinoic acid hyaluronate. Summary of the Invention

[0004] The purpose of the present application is to provide a retinoic acid hyaluronate and a preparation method thereof.

[0005] Specifically, this application involves the following aspects:

[0006] 1. A retinoic acid hyaluronate having the structure shown in Formula I:

[0007]

[0008] Wherein, R1 is H or a metal ion, preferably, the metal ion includes sodium ion, calcium ion, zinc ion or magnesium ion;

[0009] R2 is H or a retinoic acid group represented by formula II:

[0010]

[0011] in, Represents the grafting site between retinoic acid and the hydroxyl group of hyaluronic acid or its salt;

[0012] wherein at least one R2 of the retinoic acid hyaluronate comprises a retinoic acid group;

[0013] The substitution degree of the retinoic acid hyaluronate is 8%-25%, preferably 9%-15%.

[0014] 2. The retinoic acid hyaluronate according to item 1, wherein n is an integer of 2-250, preferably an integer of 10-100.

[0015] 3. A method for preparing the retinoic acid hyaluronate according to item 1 or 2, comprising the following steps:

[0016] 1) dissolving hyaluronic acid or a salt thereof in an organic solvent to obtain a solution;

[0017] 2) dissolving retinoic acid in an organic solvent and adding an activator for activation;

[0018] 3) adding the solution obtained in step 1) to step 2), and adding a catalyst to carry out a reaction to obtain a reaction solution;

[0019] 4) Purifying the reaction solution of step 3) to obtain retinoic acid hyaluronate.

[0020] 4. The method according to item 3, wherein the catalyst contains at least one or two or more of 1,5-diazabicyclo[4.3.0]non-5-ene or a salt thereof, 1,8-diazabicyclo[5.4.0]undec-7-ene or a salt thereof.

[0021] 5. The preparation method according to item 3 or 4, wherein the organic solvent used to dissolve the hyaluronic acid or its salt contains at least one or two or more of N,N-dimethylformamide, N,N-dimethylacetamide, formamide, dimethyl sulfoxide, methanesulfonic acid, and trifluoromethanesulfonic acid;

[0022] The organic solvent used to dissolve retinoic acid contains at least one or two or more of methanol, ethanol, isopropanol, acetone, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, ether, methyl tert-butyl ether, formamide, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0023] 6. The method according to item 3 or 4, wherein the molar ratio of the retinoic acid to the hyaluronic acid or its salt is (0.3-5):1, preferably (0.3-2):1;

[0024] The molar ratio of the catalyst to the hyaluronic acid or its salt is (0.3-5):1, preferably (0.3-2):1;

[0025] Preferably, the reaction temperature in step 3) is 20-50°C;

[0026] Preferably, the reaction time of step 3) is 2-20 h.

[0027] 7. The preparation method according to any one of items 3 to 6, wherein the activator in step 2) is N,N'-carbonyldiimidazole; preferably, the molar ratio of the activator to the retinoic acid is (0.1-10):1;

[0028] Further preferably, the activation temperature is 0-50°C;

[0029] More preferably, the activation time is 1-6 hours.

[0030] 8. A composition comprising the retinoic acid hyaluronate described in item 1 or 2 or the retinoic acid hyaluronate prepared by the method of any one of items 3 to 7.

[0031] 9. Use of the retinoic acid hyaluronate described in item 1 or 2, or the retinoic acid hyaluronate prepared by the method of any one of items 3 to 7, or the composition described in item 8 in the preparation of cosmetics and pharmaceutical and medical products.

[0032] 10. Use of the retinoic acid hyaluronate described in item 1 or 2, or the retinoic acid hyaluronate prepared by the preparation method described in any one of items 3 to 7, or the composition described in item 8 in the preparation of anti-inflammatory and / or anti-aging products.

[0033] Effects of the Invention

[0034] The retinoic acid hyaluronate of the present application has a high degree of substitution, high purity, and better anti-aging and anti-inflammatory effects than samples obtained in the prior art. The method described in the present application first activates retinoic acid by an in situ method, and then reacts with hyaluronic acid under a catalyst to obtain the target product. The method described in the present application can reduce the side reactions and by-products caused by activation in the prior art, effectively improve the reaction efficiency, and help to increase the degree of substitution of the product. At the same time, the reaction system of the present application reacts at room temperature, does not require low-temperature activation or high-temperature reaction, no irritating gas is released, and the reactants used are also non-irritating, which can reduce energy consumption and pollutant emissions. In addition, the method has a high activation efficiency, can effectively reduce the amount of retinoic acid used, and reduce material costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the H NMR spectrum of retinoic acid hyaluronate prepared in Example 11;

[0036] Figure 2 This is the H NMR spectrum of 9000Da sodium hyaluronate;

[0037] Figure 3 This is the H NMR spectrum of retinoic acid;

[0038] Figure 4 This is a schematic diagram of the dissolution state of retinoic acid hyaluronate and retinoic acid prepared in Example 11. DETAILED DESCRIPTION

[0039] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0040] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0041] The present application provides a retinoic acid hyaluronate having the structure shown in Formula I:

[0042]

[0043] Wherein, R1 is H or a metal ion, preferably, the metal ion includes sodium ion, calcium ion, zinc ion or magnesium ion;

[0044] R2 is H or a retinoic acid group represented by formula II:

[0045]

[0046] in, Represents the grafting site between retinoic acid and the hydroxyl group of hyaluronic acid or its salt;

[0047] wherein at least one R2 of the retinoic acid hyaluronate comprises a retinoic acid group;

[0048] The degree of substitution of the retinoic acid hyaluronate is 8%-25%, for example, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, and any value therebetween.

[0049] In the present application, the degree of substitution refers to the molar ratio of the retinoic acid groups covalently linked to the disaccharide structure of hyaluronic acid or its salt to the disaccharide structure of hyaluronic acid or its salt.

[0050] In the present application, there is no limitation on the method for determining the degree of substitution, and the degree of substitution can be determined by conventional methods in the art. For example, the degree of substitution can be calculated by using H NMR spectroscopy as the ratio of half the peak area of ​​the characteristic peak of the dimethyl H on the six-membered ring of retinoic acid to the peak area of ​​the characteristic peak of the methyl H on NHCOCH3 on hyaluronic acid or its salts:

[0051]

[0052] In this application, the product sample yield can be calculated using the weight yield:

[0053]

[0054] In some embodiments, the degree of substitution of the retinoic acid hyaluronate is 9%-15%.

[0055] In some specific embodiments, n is an integer from 2 to 250, for example, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, and any integer therebetween.

[0056] In some embodiments, n is an integer from 10 to 100.

[0057] The present application also provides a method for preparing the above-mentioned retinoic acid hyaluronate, comprising the following steps:

[0058] 1) dissolving hyaluronic acid or a salt thereof in an organic solvent to obtain a solution;

[0059] 2) dissolving retinoic acid in an organic solvent and adding an activator for activation;

[0060] 3) adding the solution obtained in step 1) to step 2), and adding a catalyst to carry out a reaction to obtain a reaction solution;

[0061] 4) Purifying the reaction solution of step 3) to obtain retinoic acid hyaluronate.

[0062] In step 1), the organic solvent contains at least one or two or more of N,N-dimethylformamide, N,N-dimethylacetamide, formamide, dimethyl sulfoxide, methanesulfonic acid, and trifluoromethanesulfonic acid.

[0063] In step 2), the organic solvent contains at least one or more of methanol, ethanol, isopropanol, acetone, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, ether, methyl tert-butyl ether, formamide, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0064] In some specific embodiments, the activating agent used for activation is N,N'-carbonyldiimidazole.

[0065] In some embodiments, the molar ratio of the activator to the retinoic acid is (0.1-10):1, for example, 0.1:1, 0.5:1, 1:1, 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, 9.5:1, 10:1, and any values ​​therebetween.

[0066] In some embodiments, the activation temperature is 0-50°C, for example, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and any value therebetween.

[0067] In some specific embodiments, the activation time is 1-6 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, and any value therebetween.

[0068] In step 3), the catalyst contains at least one or two or more of 1,5-diazabicyclo[4.3.0]non-5-ene or a salt thereof, 1,8-diazabicyclo[5.4.0]undec-7-ene or a salt thereof.

[0069] In some specific embodiments, the molar ratio of the retinoic acid to the hyaluronic acid or its salt is (0.3-5):1, for example, it can be 0.3:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, and any value in between these values.

[0070] In some specific embodiments, the molar ratio of the retinoic acid to the hyaluronic acid or a salt thereof is (0.3-2):1.

[0071] In some specific embodiments, the molar ratio of the catalyst to the hyaluronic acid or its salt is (0.3-5):1, for example, it can be 0.3:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, and any value in between these values.

[0072] In some specific embodiments, the molar ratio of the catalyst to the hyaluronic acid or its salt is (0.3-2):1.

[0073] In some specific embodiments, the concentration of the hyaluronic acid or its salt in the initial reaction solution of step 3) is 1-10 wt%, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, and any value therebetween.

[0074] In some specific embodiments, the reaction temperature of step 3) is 20-50°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and any value therebetween.

[0075] In some specific embodiments, the reaction time of step 3) is 2-20 h, for example, it can be 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, and any value in between.

[0076] In step 4), purification methods include precipitation by adding a precipitant, dialysis, nanofiltration, ultrafiltration, and the like.

[0077] Furthermore, the precipitant can be ethanol, methanol, isopropanol, acetone, ethyl acetate, ethyl ether, or methyl tert-butyl ether, preferably ethanol.

[0078] In some specific embodiments, the method for preparing retinoic acid hyaluronate of the present application comprises the following steps:

[0079] 1) dissolving hyaluronic acid or a salt thereof in an organic solvent to obtain a solution;

[0080] 2) dissolving retinoic acid in an organic solvent and adding an activator for activation; wherein the activator used for activation is N,N'-carbonyldiimidazole, the molar ratio of the activator to the retinoic acid is (0.1-10):1, the activation temperature is 0-50°C, and the activation time is 1-6 hours;

[0081] 3) adding the solution obtained in step 1) to step 2), and adding a catalyst to react to obtain a reaction solution; wherein the catalyst contains at least one or more of 1,5-diazabicyclo[4.3.0]non-5-ene or a salt thereof, 1,8-diazabicyclo[5.4.0]undec-7-ene or a salt thereof, the molar ratio of the retinoic acid to the hyaluronic acid or a salt thereof is (0.3-5):1, the molar ratio of the catalyst to the hyaluronic acid or a salt thereof is (0.3-5):1, the reaction temperature is 20-50° C., and the reaction time is 2-20 h;

[0082] 4) Purifying the reaction solution of step 3) to obtain retinoic acid hyaluronate.

[0083] In some specific embodiments, the method for preparing retinoic acid hyaluronate of the present application comprises the following steps:

[0084] 1) dissolving hyaluronic acid or a salt thereof in an organic solvent to obtain a solution;

[0085] 2) dissolving retinoic acid in an organic solvent and adding an activator for activation; wherein the activator used for activation is N,N'-carbonyldiimidazole, the molar ratio of the activator to the retinoic acid is (0.1-10):1, the activation temperature is 0-50°C, and the activation time is 1-6 hours;

[0086] 3) adding the solution obtained in step 1) to step 2), and adding a catalyst to react to obtain a reaction solution; wherein the catalyst contains at least one or more of 1,5-diazabicyclo[4.3.0]non-5-ene or a salt thereof, 1,8-diazabicyclo[5.4.0]undec-7-ene or a salt thereof, the molar ratio of the retinoic acid to the hyaluronic acid or a salt thereof is (0.3-5):1, the molar ratio of the catalyst to the hyaluronic acid or a salt thereof is (0.3-5):1, the reaction temperature is 20-50° C., and the reaction time is 2-20 h;

[0087] 4) The reaction solution of step 3) is purified, washed, filtered, and dried to obtain retinoic acid hyaluronate.

[0088] The present application also provides a composition comprising the above-mentioned retinoic acid hyaluronate or the retinoic acid hyaluronate prepared by the above-mentioned method.

[0089] The present application also provides the use of the above-mentioned retinoic acid hyaluronate, or the retinoic acid hyaluronate prepared by the above-mentioned method, or the above-mentioned composition in the preparation of cosmetics and pharmaceutical and medical products.

[0090] The present application also provides the use of the above-mentioned retinoic acid hyaluronate, or the retinoic acid hyaluronate prepared by the above-mentioned method, or the above-mentioned composition in the preparation of anti-inflammatory and / or anti-aging products.

[0091] Example

[0092] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0093] Example 1

[0094] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0095] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 100 kDa in 80 mL of formamide to form a homogeneous solution.

[0096] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a uniform solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0097] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0098] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, and the solution was washed with 150 mL of ethanol and filtered to obtain a bright yellow wet product, which was then vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 91.52% and the degree of substitution was 9.13%.

[0099]

[0100] The degree of substitution was calculated using H NMR spectroscopy by the ratio of half the peak area of ​​the characteristic peak of the dimethyl H on the six-membered ring of retinoic acid to the peak area of ​​the characteristic peak of the methyl H on NHCOCH3 on hyaluronic acid or its salts:

[0101]

[0102] The yield and degree of substitution were calculated using the same method in the following examples and comparative examples.

[0103] Example 2

[0104] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0105] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0106] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0107] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0108] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 86.68% and the degree of substitution was 23.01%.

[0109] Example 3

[0110] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0111] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0112] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 1 h to obtain a retinoic acid solution.

[0113] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0114] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain a retinoic acid hyaluronate sample with a yield of 85.25% and a degree of substitution of 15.63%.

[0115] Example 4

[0116] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0117] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0118] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 6 h to obtain a retinoic acid solution.

[0119] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0120] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 85.01% and the degree of substitution was 23.82%.

[0121] Example 5

[0122] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0123] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0124] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 0°C in the dark for 2 h to obtain a retinoic acid solution.

[0125] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0126] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 83.19% and the degree of substitution was 17.77%.

[0127] Example 6

[0128] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0129] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0130] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 50°C in the dark for 2 h to obtain a retinoic acid solution.

[0131] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 3 g (0.02 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0132] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 82.99% and the degree of substitution was 20.01%.

[0133] Example 7

[0134] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0135] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0136] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0137] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 0.45 g (0.003 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0138] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 84.64% and the degree of substitution was 8.98%.

[0139] Example 8

[0140] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0141] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0142] 2) 6 g (0.02 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 3.2 g (0.02 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0143] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0144] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 86.07% and the degree of substitution was 22.82%.

[0145] Example 9

[0146] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0147] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0148] 2) Dissolve 0.9 g (0.003 mol) of retinoic acid in 20 mL of N,N-dimethylformamide in the dark to form a homogeneous solution, add 0.48 g (0.003 mol) of N,N'-carbonyldiimidazole, and activate at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0149] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0150] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, and the solution was washed with 150 mL of ethanol and filtered to obtain a bright yellow wet product, which was then vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 85.04% and the degree of substitution was 9.18%.

[0151] Example 10

[0152] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0153] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0154] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0155] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0156] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 85.76% and the degree of substitution was 12.25%.

[0157] Example 11

[0158] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0159] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0160] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0161] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 20° C. in the dark for 4 h.

[0162] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 82.84% and the degree of substitution was 9.50%.

[0163] Example 12

[0164] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0165] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0166] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0167] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 50° C. in the dark for 4 h.

[0168] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 83.51% and the degree of substitution was 10.13%.

[0169] Example 13

[0170] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0171] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0172] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0173] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 2 h.

[0174] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 83.39% and the degree of substitution was 9.01%.

[0175] Example 14

[0176] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0177] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0178] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0179] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 20 h.

[0180] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 84.69% and the degree of substitution was 12.99%.

[0181] Example 15

[0182] A hyaluronic acid retinoic acid ester, the preparation method of which is as follows:

[0183] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0184] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0185] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.2 g (0.01 mol) of 1,5-diazabicyclo[4.3.0]non-5-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0186] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, and the solution was washed with 150 mL of ethanol and filtered to obtain a bright yellow wet product, which was then vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 83.96% and the degree of substitution was 10.72%.

[0187] Comparative Example 1

[0188] 1) 4 g of sodium hyaluronate with a molecular weight of 10 kDa was dissolved in 400 mL of dimethyl sulfoxide in a brown reaction flask under argon protection and light protection. 0.1 g of sulfuric acid was added and stirred for 2 hours.

[0189] 2) Dissolve 3 g of retinoic acid in 15 mL of dimethyl sulfoxide in a brown reaction flask, add 3.79 g of benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate, and stir for 1 hour.

[0190] 3) The retinoic acid solution from step 2) was added to the sodium hyaluronate from step 1), and 1.26 g of diisopropylcarbodiimide and 2.9 g of 10X molecular sieves were added. The mixture was stirred and reacted at 80° C. in the dark for 4 hours. The reaction was stopped, and the insoluble matter was filtered out after cooling.

[0191] 4) The filtrate was protected from light and precipitated with methyl tert-butyl ether, filtered, and dried under vacuum to obtain retinoic acid hyaluronate. The sample yield was 43.90% and the degree of substitution was 13.41%.

[0192] Comparative Example 2

[0193] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0194] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0195] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 0.15 g (0.001 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0196] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 84.15% and the degree of substitution was 1.21%.

[0197] Comparative Example 3

[0198] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0199] 2) Dissolve 0.3 g (0.001 mol) of retinoic acid in 20 mL of N,N-dimethylformamide in the dark to form a uniform solution. Add 0.16 g (0.001 mol) of N,N'-carbonyldiimidazole and activate at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0200] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0201] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 85.90% and the degree of substitution was 4.13%.

[0202] Comparative Example 4

[0203] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0204] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0205] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 0° C. in the dark for 4 h.

[0206] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 82.04% and the degree of substitution was 5.80%.

[0207] Comparative Example 5

[0208] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0209] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0210] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 1.5 g (0.01 mol) of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the mixture was reacted at 30° C. in the dark for 1 h.

[0211] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 81.77% and the degree of substitution was 5.32%.

[0212] Comparative Example 6

[0213] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0214] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0215] 3) The activated retinoic acid solution of step 2) was mixed with the sodium hyaluronate solution of step 1) and 2.4 g (0.02 mol) of 4-dimethylaminopyridine was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0216] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 80.03% and the degree of substitution was 5.01%.

[0217] Comparative Example 7

[0218] 1) Dissolve 4 g (0.01 mol) of sodium hyaluronate with a molecular weight of 9 kDa in 80 mL of formamide to form a homogeneous solution.

[0219] 2) 1.5 g (0.005 mol) of retinoic acid was dissolved in 60 mL of N,N-dimethylformamide in the dark to form a homogeneous solution. 0.81 g (0.005 mol) of N,N'-carbonyldiimidazole was added and activated at 30°C in the dark for 2 h to obtain a retinoic acid solution.

[0220] 3) The activated retinoic acid solution from step 2) was mixed with the sodium hyaluronate solution from step 1) and 2.7 g (0.02 mol) of 1-hydroxybenzotriazole was added, and the mixture was reacted at 30° C. in the dark for 4 h.

[0221] 4) After the reaction, 150 mL of sodium chloride solution and 600 mL of ethanol were added to the solution for precipitation, washed with 150 mL of ethanol, filtered to obtain a bright yellow wet product, and vacuum dried to obtain retinoic acid hyaluronate. The sample yield was 72.18% and the degree of substitution was 2.57%.

[0222] Specifically, the reaction conditions of the above examples are shown in Table 1, and the yields and substitution degrees of the examples and comparative examples are shown in Table 2.

[0223] Table 1 Example reaction conditions

[0224]

[0225]

[0226] Table 2 Yield and degree of substitution of various examples and comparative examples

[0227] Degree of substitution / % Yield / % Example 1 9.13 91.52 Example 2 23.01 86.68 Example 3 15.63 85.25 Example 4 23.82 85.01 Example 5 17.77 83.19 Example 6 20.01 82.99 Example 7 8.98 84.64 Example 8 22.82 86.07 Example 9 9.18 85.04 Example 10 12.25 85.76 Example 11 9.50 82.84 Example 12 10.13 83.51 Example 13 9.01 83.39 Example 14 12.99 84.69 Example 15 10.72 83.96 Comparative Example 1 13.41 43.90 Comparative Example 2 1.21 84.15 Comparative Example 3 4.13 85.90 Comparative Example 4 5.80 82.04 Comparative Example 5 5.32 81.77 Comparative Example 6 5.01 80.03 Comparative Example 7 2.57 72.18

[0228] Experimental Example 1 Nuclear Magnetic Field Measurement

[0229] NMR (liquid) spectra were performed using a Bruker Avance III HD 500 MHz instrument in Germany, using deuterated water or deuterated DMSO as the solvent. Weigh 5-10 mg of the sample to be tested and dissolve it in 0.6 mL of the deuterated solvent. Once dissolved, transfer the sample to a 5 mm NMR tube. Set the scan count to 16, select the desired solvent, and begin the test.

[0230] The H NMR spectra of retinoic acid hyaluronate, 9000 Da molecular weight sodium hyaluronate and retinoic acid obtained in Example 11 are as follows: Figure 1 、 Figure 2 、 Figure 3 shown.

[0231] from Figure 1It can be seen that 0.97ppm corresponds to the absorption peak of the dimethyl group on the six-membered ring of retinoic acid, 1.42ppm and 1.54ppm correspond to the characteristic absorption peaks of the methylene group on the six-membered ring of retinoic acid, 1.67ppm corresponds to the absorption peak of the methyl group connected to the double bond of the six-membered ring of retinoic acid, the sharp peak of 1.9-2.0ppm is attributed to the absorption peak corresponding to the methyl group on NHCOCH3 on sodium hyaluronate, and 3.0-5.0ppm is attributed to the characteristic peak of the sodium hyaluronate skeleton. The peak at 5.5-7.5ppm corresponds to the characteristic peak of the double bond of retinoic acid. In summary, the synthesized sample has multiple characteristic absorption peaks of retinoic acid in the H NMR spectrum and retains the absorption of the sodium hyaluronate skeleton, indicating that the obtained sample is retinoic acid hyaluronate.

[0232] Experimental Example 2 Determination of Retinoic Acid Residues

[0233] Chromatographic column: Agilent Eclipse Plus C18 (4.6×250 mm, 5 μm); mobile phase: methanol: 2% acetic acid solution (V:V) = 90:10; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 355 nm; injection volume: 10 μL.

[0234] Accurately weigh an appropriate amount of retinoic acid reference substance and prepare a solution containing 0.1 mg per mL in methanol. Accurately pipette 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 mL of this standard solution into 10 mL brown volumetric flasks. Dilute to the mark with methanol, shake well, and inject 20 μL of each sample into the chromatographic analysis. Construct a standard curve using sample concentration and corresponding peak area as coordinates.

[0235] Accurately transfer 0.2 g of the samples from Examples 2, 5, 6, 11, and Comparative Example 1 to a 10 mL brown volumetric flask. Add methanol to the mark and shake well. Pass the supernatant through a 0.45 μm filter to serve as the test solution. Inject into a high-performance liquid chromatograph, and calculate the content using the external standard method. The results are shown in Table 3.

[0236] Table 3 Example sample retinoic acid residue results

[0237] sample Retinoic acid content / ppm Example 2 23.2 Example 5 40.9 Example 6 33.4 Example 11 21.6 Comparative Example 1 25227.3

[0238] The presence of residual retinoic acid not only affects sample solubility but also the results of subsequent cytotoxicity, efficacy, and safety tests. The retinoic acid content of the example samples was determined to be less than 50 ppm, indicating that the free form of retinoic acid was substantially removed by purification and washing. Combined with the NMR spectrum, this indicates that retinoic acid is primarily present in a grafted form. The comparative example 1 sample had a high residual retinoic acid content, indicating that a portion of the retinoic acid content is present in the product in a free form.

[0239] Experimental Example 3 Solubility Determination

[0240] The sample of Example 11 and the retinoic acid sample were added to an appropriate amount of purified water, stirred at room temperature, and heated appropriately to prepare a retinoic acid sample solution of Example 11 and a retinoic acid sample solution having a retinoic acid content equivalent to that of Example 11. Figure 4 (0.1%), as shown in Table 4.

[0241] The results showed that retinoic acid hyaluronate (left image) was soluble in water, forming a clear solution. However, retinoic acid (right image) failed to dissolve effectively at room temperature or under heated conditions, remaining dispersed in the solution. This indicates that the water solubility of retinoic acid grafted onto hyaluronic acid was significantly improved.

[0242] Table 4 Example 11 Sample and retinoic acid dissolution state

[0243]

[0244] Experimental Example 4 Cytotoxicity Test

[0245] The experimental method was based on the "People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SNT 2328-2009" Cosmetics Acute Toxicity Test for Keratinocytes. Samples from Examples 5, 6, and 11 were prepared in serum-free culture medium to concentrations of 0.001%, 0.005%, 0.01%, and 0.05%. The solutions were sterilized by filtration through a 0.22 μm filter. After 24 hours of regular culture, the old culture medium was discarded and replaced with 100 μL of the experimental group sample. The normal control group (control) was treated with an equal amount of serum-free culture medium. Six parallel wells were plated on each level. After a further 24 hours of culture, the relative cell proliferation rate was measured using CCK-8. The culture medium was discarded, and the cells were washed twice with PBS. 100 μL of CCK-8 diluted 10-fold with serum-free culture medium was added to each well. The cells were placed in a cell culture incubator and incubated for 2 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The relative proliferation rate (RGR) was the ratio of the absorbance of the experimental group to that of the normal control group. The results are shown in Table 5.

[0246] Table 5 Relative cell proliferation rate (%)

[0247]

[0248] According to GB / T 16886.5-2017, a sample is considered cytotoxic when its RGR is below 70%. Results showed that cytotoxicity increased with increasing sample concentration and degree of substitution. The Example group showed no potential cytotoxicity within the 0.001%-0.01% concentration range, but cytotoxicity increased to varying degrees as the concentration increased to 0.05%.

[0249] Experimental Example 5 Anti-aging Effect Test

[0250] Refer to T / SHRH031-2020 Cosmetic firming and anti-wrinkle efficacy test-in vitro fibroblast type I collagen content determination.

[0251] The determination method of MMP-1 refers to the "Study on the Inhibitory Effect of Ferulic Acid on UVA-Induced Photoaging of Human Fibroblasts", as follows:

[0252] Plating: Take HSF in logarithmic growth period and use 1×10 5 Cells were seeded at a density of 100 μL / well in a 96-well plate using DMEM supplemented with 10% fetal bovine serum. The cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 24 hours. Irradiation: The culture medium in the 96-well plate was aspirated and PBS was added. An irradiation control group, an experimental group, and a negative control group were set up. Both the irradiation control group and the experimental group were illuminated with UVA light at an intensity of 2000 μW / cm 2 , the illumination time was 60 min, and the negative control group was not illuminated.

[0253] Dosing: After 24 hours of standard culture, discard the old culture medium and replace with 100 μL of the sample solution in the experimental group. Add an equal volume of serum-free culture medium to the irradiated control and negative control groups and continue culturing for another 24 hours. Prepare a 0.1% stock solution in serum-free DMEM medium, sterilize by filtration through a 0.22 μm filter, and dilute to the desired final concentration of 0.005% and 0.01% immediately before use.

[0254] Detection: Collect cell culture supernatant and determine the MMP-1 content according to the human MMP-1 kit.

[0255] The samples of Example 11, Comparative Example 1, and the control group were taken to measure the content of type I collagen and MMP-1. The results are shown in Tables 6 and 7.

[0256] Table 6 Effects of samples on type I collagen production

[0257]

[0258] Note: * indicates p < 0.05 compared with Control; ** indicates p < 0.01 compared with Control; *** indicates p < 0.001 compared with Control. # Indicates p < 0.05 compared with Example 11; ## Indicates p < 0.01 compared with Example 11; ### Compared with Example 11, p<0.001.

[0259] Table 7 Effects of samples on MMP-1

[0260]

[0261] Note: ** indicates p < 0.01 compared with the Control group; # indicates p < 0.05 compared with the irradiation group; ## indicates p < 0.01 compared with the irradiation group.

[0262] As shown in Tables 6 and 7, the retinoic acid hyaluronate sample significantly promoted type I collagen production while inhibiting the production of MMP-1, demonstrating potential anti-aging properties. Furthermore, the sample prepared in this application exhibited superior collagen-stimulating effects compared to Comparative Example 1.

[0263] Experimental Example 6 Anti-inflammatory Effect Test

[0264] Refer to the T / SHRH 034-2021 Cosmetic Soothing Efficacy Test - In Vitro TNF-α Inflammatory Factor Content Determination in Lipopolysaccharide (LPS)-Induced Macrophage RAW264.7 Test Method. LPS is used to stimulate macrophages to secrete inflammatory factors. The samples are then treated and the anti-inflammatory effects of the samples are evaluated by quantitatively detecting the expression of the inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), and TNF-α.

[0265] Relative expression level = content / LPS content*100%

[0266] Relative expression level (normalized) = relative expression level / cell proliferation rate

[0267] Inhibition rate (normalized) = 100% - relative expression level

[0268] The results are shown in Tables 8, 9 and 10.

[0269] Table 8 Effects of samples on IL-6 secretion

[0270]

[0271]

[0272] Note: * indicates p < 0.05 compared with LPS control; ** indicates p < 0.01 compared with LPS control; *** indicates p < 0.001 compared with LPS control.

[0273] Table 9 Effects of samples on IL-1β secretion

[0274]

[0275] Note: * indicates p < 0.05 compared with LPS control; ** indicates p < 0.01 compared with LPS control; *** indicates p < 0.001 compared with LPS control.

[0276] Table 10 Effects of samples on TNF-α secretion

[0277]

[0278] Note: * indicates p < 0.05 compared with LPS control; ** indicates p < 0.01 compared with LPS control at the same concentration; *** indicates p < 0.001 compared with LPS control at the same concentration.

[0279] The results showed that retinoic acid hyaluronate had a good effect on inflammatory factors at three concentrations, indicating that the studied samples had excellent anti-inflammatory effects.

[0280] In summary, the retinoic acid hyaluronate prepared in the present application has advantages in anti-aging and anti-inflammatory effects.

Claims

1. A retinoic acid hyaluronate having the structure shown in Formula I: in, R1 is H or a metal ion, preferably, the metal ion includes sodium ion, calcium ion, zinc ion or magnesium ion; R2 is H or a retinoic acid group represented by formula II: in, Represents the grafting site between retinoic acid and the hydroxyl group of hyaluronic acid or its salt; wherein at least one R2 of the retinoic acid hyaluronate comprises a retinoic acid group; The substitution degree of the retinoic acid hyaluronate is 8%-25%, preferably 9%-15%.

2. The retinoic acid hyaluronate according to claim 1, wherein n is an integer of 2-250, preferably an integer of 10-100.

3. A method for preparing the retinoic acid hyaluronate according to claim 1 or 2, comprising the following steps: 1) dissolving hyaluronic acid or a salt thereof in an organic solvent to obtain a solution; 2) dissolving retinoic acid in an organic solvent and adding an activator for activation; 3) adding the solution obtained in step 1) to step 2), and adding a catalyst to carry out a reaction to obtain a reaction solution; 4) Purifying the reaction solution of step 3) to obtain retinoic acid hyaluronate.

4. The method according to claim 3, wherein the catalyst contains at least one or more of 1,5-diazabicyclo[4.3.0]non-5-ene or a salt thereof, 1,8-diazabicyclo[5.4.0]undec-7-ene or a salt thereof.

5. The preparation method according to claim 3 or 4, wherein the organic solvent used to dissolve the hyaluronic acid or its salt contains at least one or more of N,N-dimethylformamide, N,N-dimethylacetamide, formamide, dimethyl sulfoxide, methanesulfonic acid, and trifluoromethanesulfonic acid; in, The organic solvent used to dissolve retinoic acid contains at least one or two or more of methanol, ethanol, isopropanol, acetone, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, ether, methyl tert-butyl ether, formamide, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

6. The method according to claim 3 or 4, wherein the molar ratio of the retinoic acid to the hyaluronic acid or its salt is (0.3-5):1, preferably (0.3-2):1; The molar ratio of the catalyst to the hyaluronic acid or its salt is (0.3-5):1, preferably (0.3-2):1; Preferably, the reaction temperature of step 3) is 20-50°C; and the reaction time of step 3) is 2-20h.

7. The preparation method according to any one of claims 3 to 6, wherein the activator in step 2) is N,N'-carbonyldiimidazole; preferably, the molar ratio of the activator to the retinoic acid is (0.1-10):1; More preferably, the activation temperature is 0-50° C.; and the activation time is 1-6 h.

8. A composition comprising the retinoic acid hyaluronate according to claim 1 or 2 or the retinoic acid hyaluronate prepared by the method according to any one of claims 3 to 7.

9. Use of the retinoic acid hyaluronate according to claim 1 or 2, or the retinoic acid hyaluronate prepared by the method according to any one of claims 3 to 7, or the composition according to claim 8 in the preparation of cosmetics, pharmaceuticals and medical devices.

10. Use of the retinoic acid hyaluronate according to claim 1 or 2, or the retinoic acid hyaluronate prepared by the preparation method according to any one of claims 3 to 7, or the composition according to claim 8 in the preparation of anti-inflammatory and / or anti-aging products.

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