A method for preparing acetylated sodium hyaluronate

Abstract

The application belongs to the technical field of preparation of acetylated sodium hyaluronate, and discloses a preparation method of acetylated sodium hyaluronate. The method comprises the following steps: dissolving hyaluronic acid or its salt in a eutectic solvent to obtain a clear solution, then adding an acylation catalyst and acetic anhydride into the system to perform an acylation reaction, obtaining a reaction liquid, and then dialyzing the reaction liquid to obtain acetylated sodium hyaluronate. The eutectic solvent (DES) is used to replace traditional high-toxicity and high-volatility organic solvents such as DMF and DCM. The reaction composition is simple, the reaction condition is mild, the reaction medium is non-corrosive, and the material requirement of industrial equipment is greatly reduced. Meanwhile, the purification process of the application adopts membrane dialysis technology, completely abandoning the dependence on a large amount of anhydrous ethanol in the traditional precipitation method, eliminating the generation of industrial organic waste liquid from the source, and realizing the green environmental protection of the whole process.

Description

Technical Field

[0001] This invention belongs to the field of acetylated sodium hyaluronate preparation technology, and specifically relates to a method for preparing acetylated sodium hyaluronate. Background Technology

[0002] Hyaluronic acid is a natural linear high-molecular-weight mucopolysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine repeating disaccharide units linked by alternating β-1,4 and β-1,3 glycosidic bonds. This polysaccharide is widely found in the body fluids and tissues of vertebrates, with a common molecular weight range of 5 kDa to 4000 kDa. Under physiological conditions, it mainly exists stably in the form of sodium hyaluronate.

[0003] Hyaluronic acid possesses high biocompatibility, biodegradability, high hydrophilicity, and viscoelasticity, leading to its widespread application in biomedical engineering and high-end cosmetics. However, its naturally occurring structure exhibits strong hydrophilicity, resulting in insufficient lipid solubility and permeability in certain formulation systems. More critically, exogenous sodium hyaluronate is readily and rapidly hydrolyzed and degraded by endogenous hyaluronidase in vivo or on the skin surface, resulting in a short in-situ residence time and low bioavailability. To ensure a longer in-situ residence time, functionalization or cross-linking modifications of hyaluronic acid are typically required.

[0004] Acetylated sodium hyaluronate is the most common derivative of sodium hyaluronate, possessing highly effective moisturizing, skin barrier repair, and skin elasticity-enhancing properties, and is widely used in cosmetics. By acetylifying the hydroxyl groups on the hyaluronic acid backbone using acylation reagents such as acetic anhydride or acetyl chloride, the introduced acetyl groups effectively shield some strongly hydrophilic groups, thus endowing sodium hyaluronate with unique amphiphilic properties (possessing both hydrophilic and lipophilic characteristics). This structure improves the skin's bioavailability of sodium hyaluronate; the introduction of acetyl groups enhances the affinity and adsorption of sodium hyaluronate to the skin, strengthens its transdermal penetration ability, and improves its resistance to degradation by hyaluronidase in vivo.

[0005] However, existing acetylation modification processes have many problems. Traditional chemical synthesis routes mostly rely on highly polar aprotic organic solvents (such as DMF, DMSO, DCM, etc.) as reaction media, supplemented by organic bases (such as pyridine, triethylamine) or inorganic strong acids (such as concentrated sulfuric acid) as catalysts. This leads to a series of problems for the overall reaction system, including harsh reaction conditions, highly toxic and corrosive media, cumbersome post-treatment procedures, and the generation of large amounts of difficult-to-treat organic wastewater.

[0006] Chinese patent publication CN113045686A discloses a method for preparing acetylated hyaluronic acid, which involves activation with an organic base followed by acetylation with acetyl chloride. This method cannot be separated from organic solvents, and the use of dichloromethane poses a serious risk of volatile organic compound (VOC) emissions and increases environmental pressure on industry. Furthermore, due to the extremely high reactivity of acetyl chloride, its addition process must be carried out in a strictly controlled ice-water bath. This locally intense exothermic reaction places extremely stringent requirements on temperature control equipment, safe operation, and the corrosion resistance of equipment in large-scale industrial production.

[0007] Chinese patent publication CN120040619A discloses a method for preparing acetylated sodium hyaluronate, proposing a route using a recyclable catalyst to catalyze the acylation of acetyl chloride. However, this method cannot be separated from organic solvents; both the preparation of the sodium hyaluronate suspension and the dissolution of acetyl chloride require the use of DMF as a solvent, making solvent recovery difficult and requiring strict control of DMF residue in the finished product. Although the catalyst design has some innovation, the pre-synthetic process of this catalyst is extremely lengthy and cumbersome, and it uses large amounts of highly hazardous and polluting reagents such as concentrated sulfuric acid, 1-formaldehyde piperazine, 3-acetyl dimethyl ...

[0008] Chinese patent publication CN115558688A discloses a method for preparing acetylated sodium hyaluronate. This method employs a two-step process combining bio-enzymatic catalysis and chemical modification to prepare fully acetylated sodium hyaluronate with a degree of substitution of 4. Although this method uses lipase and ethyl acetate, which conform to the principles of green chemistry, it still cannot completely eliminate dependence on organic solvents during the process. The preparation of lipase magnetic microspheres uses glutaraldehyde, a highly sensitizing curing agent, and the secondary acetylation uses dichloromethane as the reaction solvent. Therefore, the regulatory risks of chemical residues in the finished product for pharmaceutical or high-end cosmetic applications have not been completely eliminated.

[0009] Chinese patent CN116444695B discloses a method for preparing acetylated sodium hyaluronate. This method rapidly prepares low molecular weight acetylated sodium hyaluronate using a highly efficient catalyst, 4-pyrrolylpyridine, in a pure aqueous alkaline environment. The process uses pure water as the reaction medium, completely avoiding the use of highly toxic and volatile organic solvents such as DMF and dichloromethane, thus eliminating the problem of organic wastewater treatment at its source. However, acylation reagents such as acetic anhydride naturally exhibit extremely high thermodynamic instability and extremely rapid hydrolysis side reactions in aqueous systems. The reaction is extremely sensitive to pH control, instantaneous feed ratio, and temperature fluctuations, resulting in an extremely narrow process window and a high dependence on specific high-valence nucleophilic catalysts, which is detrimental to the stable controlled release of quality between batches.

[0010] Eutectic solvents (DES) are low-melting-point liquid systems that form extensive hydrogen bond networks through the self-assembly of specific hydrogen bond acceptors (such as choline salts) and hydrogen bond donors (such as polyols, amides, or carboxylic acids) at specific temperatures. DES can be classified into five main categories: Category I DES consists of quaternary ammonium salts and metal halides; Category II DES consists of quaternary ammonium salts and hydrates of metal halides; Category III DES consists of quaternary ammonium salts and amines, alcohols, or carboxylic acids; Category IV DES consists of metal halides and amines, alcohols, or carboxylic acids; and Category V DES consists of nonionic compounds.

[0011] DES has advantages such as low cost, biodegradability, environmental friendliness, and simple preparation process. Currently, most research on DES in existing technologies only uses it as a mild physical extraction or recovery solvent. However, there are few reports on how to use DES directly as a reaction medium to mediate the efficient and controllable chemical functionalization modification of polymeric polysaccharides (especially sodium hyaluronate) under mild and completely VOC-free conditions. Summary of the Invention

[0012] In order to overcome a series of technical bottlenecks in the existing acetylation modification process, such as the high toxicity of the reaction medium, the strong corrosiveness of strong acids and bases, the complicated post-processing procedures, the consumption of a large amount of organic solvents (such as ethanol), and the generation of a large amount of difficult-to-treat organic waste liquid, the primary objective of this invention is to provide a green, environmentally friendly, simple process and solvent recyclable method for preparing acetylated sodium hyaluronate.

[0013] Another object of the present invention is to provide acetylated sodium hyaluronate prepared by the above method.

[0014] The objective of this invention is achieved through the following solution:

[0015] A method for preparing acetylated sodium hyaluronate includes the following steps:

[0016] S1: Hyaluronic acid or its salt is dissolved in a eutectic solvent (DES) to form a homogeneous system, and then an acylation reaction catalyst is added to carry out an acylation reaction with acetic anhydride to obtain a reaction solution;

[0017] S2: Place the reaction solution in a dialysis bag and use pure water as the dialysis medium for dialysis separation. The component retained inside the dialysis bag is phase A, which is rich in acetylated sodium hyaluronate, and the dialysate that seeps out of the dialysis bag is phase B, which is rich in DES.

[0018] S3: After collecting phase A and filtering to remove trace amounts of insoluble matter, freeze-dry the product to obtain acetylated sodium hyaluronate.

[0019] S4: Collect the B phase and desalinate it to recover DES. The recovered DES can be directly recycled in step S1.

[0020] In step S1, the molecular weight of hyaluronic acid is 1 kDa to 100 kDa.

[0021] In step S1, the hyaluronic acid salt is at least one of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, and zinc hyaluronate, preferably sodium hyaluronate.

[0022] In step S1, the DES used is either type III DES or type V DES.

[0023] When the DES in step S1 is a type III DES, the type III DES uses a quaternary ammonium salt as the hydrogen bond acceptor and an organic acid or imidazole compound as the hydrogen bond donor. The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:0.5 to 1:3. The quaternary ammonium salt is selected from any one of tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, and tetrabutylammonium chloride. The organic acid is selected from any one or more combinations of acetic acid, propionic acid, oxalic acid, or malonic acid. The imidazole compound is selected from any one of imidazole, 2-methylimidazolium, 4-methylimidazolium, and 5-methylimidazolium.

[0024] When the DES in step S1 is a type 5 DES, the type 5 DES uses an imidazole compound as a hydrogen bond acceptor and an organic acid as a hydrogen bond donor. The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:0.5 to 1:3. The imidazole compound is selected from any one of imidazole, 1-methylimidazolium, 2-methylimidazolium, 4-methylimidazolium, and 5-methylimidazolium. The organic acid is selected from any one or a combination of acetic acid, propionic acid, oxalic acid, or malonic acid.

[0025] In step S1, the mass concentration of hyaluronic acid or its salt in the DES is 1% to 15%, and the amount of acetic anhydride used is 2 to 12 times the mass of hyaluronic acid or its salt.

[0026] In step S1, the acylation reaction catalyst is one of pyridine, 4-dimethylaminopyridine, and 4-pyrrolidinylpyridine. The amount of the acylation reaction catalyst used is 0% to 2% of the mass of acetic anhydride.

[0027] In step S1, the acylation reaction temperature is 25~80℃ and the acylation reaction time is 4~20 hours.

[0028] In step S2, when the raw material in step S1 is hyaluronic acid or other non-sodium hyaluronate, it is preferable to add 0.5 mol / L sodium chloride solution to the reaction solution before dialysis in order to form the final sodium salt, namely acetylated sodium hyaluronate.

[0029] In step S2, the molecular weight cutoff of the dialysis bag is 1 kDa to 10 kDa. Dialysis is stopped when the conductivity of the external fluid of the dialysis bag is <5 μS / cm.

[0030] An acetylated sodium hyaluronate prepared by the above method, wherein the degree of substitution of the acetylated sodium hyaluronate is 2.33-3.63.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] (1) This invention uses a eutectic solvent (DES) to replace traditional highly toxic and volatile organic solvents such as DMF and DCM. The reaction composition is simple, the reaction conditions are mild, and the reaction medium is non-corrosive, which greatly reduces the material requirements for industrial equipment. At the same time, the purification process of this invention adopts membrane dialysis technology, which completely eliminates the dependence on large amounts of anhydrous ethanol in the traditional precipitation method, eliminates the generation of industrial organic waste liquid from the source, and realizes green and environmentally friendly operation throughout the entire process.

[0033] (2) In the process design of this invention, the excess acetic anhydride not consumed during the acylation reaction will naturally hydrolyze to generate acetic acid during dialysis. Ingeniously, the generated acetic acid happens to be the hydrogen bond donor component of the DES selected in this invention. This means that the byproduct not only does not become a difficult-to-separate waste liquid impurity, but is instead transformed in situ into a component of the solvent itself. This makes it easier to separate water and acid during subsequent dehydration and recovery of the dialysate (phase B). After recovery, only the corresponding molar amount of hydrogen bond acceptor needs to be added to the system to maintain a constant DES component ratio, thereby achieving low-cost, high-efficiency closed-loop recycling of the reaction solvent. Attached Figure Description

[0034] Figure 1 A standard curve plotted according to the O-acetyl determination method in General Chapter 3117 of the Chinese Pharmacopoeia;

[0035] Figure 2 This is an image of the freeze-dried sample of acetylated sodium hyaluronate prepared according to the present invention.

[0036] Figure 3 The 1H NMR spectrum of sodium hyaluronate used in this invention;

[0037] Figure 4 The image shows the 1H NMR spectrum of the acetylated sodium hyaluronate prepared according to this invention. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0039] Example 1

[0040] A method for preparing acetylated sodium hyaluronate, comprising the following steps:

[0041] (1) Weigh 5.85 g of tetrapropylammonium chloride and 3.15 g of acetic acid into a reaction flask, stir at 75 °C for 30 min to obtain a clear eutectic solvent (DES), then add 1 g of sodium hyaluronate with an average molecular weight of 50 kDa to the DES, continue stirring for 30 min to dissolve until it is completely dissolved and forms a clear solution, cool the system to 40 °C, add 0.015 g of 4-dimethylaminopyridine to the solution, then slowly add 3.23 g of acetic anhydride to the solution, and react at 40 °C for 10 hours to obtain the reaction solution;

[0042] (2) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 for dialysis purification. When the conductivity of the liquid outside the dialysis bag was less than 5 μS / cm, the liquid inside the dialysis bag was collected and freeze-dried to obtain a white powdery sodium acetylated hyaluronic acid product.

[0043] Example 2

[0044] A method for preparing acetylated sodium hyaluronate, with the same process steps as in Example 1, except that: in step (1), the raw material sodium hyaluronate is replaced with 0.3 g of hyaluronic acid with an average molecular weight of 3 kDa; the DES preparation components are replaced with 4.03 g of tetraethylammonium chloride and 4.97 g of imidazole. 10 mL of 0.5 mol / L sodium chloride aqueous solution is added to the reaction solution before dialysis to form the corresponding sodium salt.

[0045] Example 3

[0046] A method for preparing acetylated sodium hyaluronate, the specific process steps are the same as in Example 1, the difference being that: in step (1), the DES preparation components are replaced with 3.97 g of 4-methylimidazole and 5.03 g of malonic acid; the acylation reaction catalyst is replaced with an equal mass of pyridine.

[0047] Example 4

[0048] A method for preparing acetylated sodium hyaluronate, the specific process steps are the same as in Example 1, the difference being that: in step (1), the DES preparation components are replaced with 3.74 g of tetrapropylammonium chloride, 1.01 g of acetic acid and 1.24 g of propionic acid.

[0049] Example 5

[0050] A method for preparing acetylated sodium hyaluronate, the specific process steps are the same as in Example 1, the difference being that: in step (1), the DES preparation components are replaced with 4.60 g of 2-methylimidazole, 3.36 g of acetic acid and 5.04 g of oxalic acid; the amount of 4-dimethylaminopyridine is 0 g.

[0051] Example 6

[0052] A method for preparing acetylated sodium hyaluronate, with the same process steps as in Example 1, except that: in step (1), the raw material sodium hyaluronate is replaced with 1 gram of hyaluronic acid with an average molecular weight of 90 kDa; the isothermal reaction time after adding acetic anhydride is shortened to 4 hours, and the isothermal temperature is 25°C; the amount of acetic anhydride is replaced with 10.76 grams; and the amount of 4-dimethylaminopyridine is replaced with 0.13 grams. Before dialysis, 10 mL of 0.5 mol / L sodium chloride aqueous solution is added to the reaction solution to form the corresponding sodium salt.

[0053] Example 7

[0054] A method for preparing acetylated sodium hyaluronate, the specific process steps are the same as in Example 1, the difference being that: in step (1), the amount of acetic anhydride used is replaced with 5.38g; and the acylation reaction catalyst is replaced with 0.081g of pyridine.

[0055] Application Experiment Example 1

[0056] Sodium hyaluronate contains four free hydroxyl groups that can be acetylated in one repeating disaccharide unit. Therefore, the theoretical degree of substitution (DS) of the resulting acetylated sodium hyaluronate product ranges from 0 to 4 (i.e., covering the unsubstituted to fully substituted state). The degree of substitution of the acetylated sodium hyaluronate products in the above seven examples was determined and calculated according to the O-acetyl determination method in General Chapter 3117 of the Chinese Pharmacopoeia, including the following steps:

[0057] (1) Draw the standard curve

[0058] Accurately weigh 22.7 mg of acetylcholine chloride, which has been dried to constant weight, and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with 0.001 mol / L sodium acetate solution, and shake well to obtain the acetylcholine chloride standard solution (hereinafter referred to as the standard solution).

[0059] Prepare 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of precise standard solutions in separate test tubes. Add water to a final volume of 1 ml, then add 2 ml of freshly prepared alkaline hydroxylamine solution. Shake well and allow to stand at room temperature for 4 minutes. Add 1 ml of 4 mol / L acid to adjust the pH to 1.2 ± 0.2, shake well, and then add 1 ml of 0.37 mol / L ferric chloride-hydrochloric acid solution. Shake well and measure the absorbance at a wavelength of 540 nm.

[0060] In addition, accurately measure the corresponding series of standard solutions mentioned above, and from "add water to 1 ml", operate in the same manner except that the order of adding acid and basic hydroxylamine is reversed, and use them as blank controls for the standard solutions.

[0061] Subtract the absorbance of the corresponding blank control tube from each absorbance of the standard tube, and plot a standard curve by performing a linear regression on the corresponding absorbance with respect to the volume of the standard solution contained in the standard tube. Figure 1 ).

[0062] (2) Determination of O-acetyl content in acetylated sodium hyaluronate samples

[0063] Take 0.3 g (dry basis) of acetylated sodium hyaluronate and place it in a 100 ml volumetric flask. Dissolve and dilute with water to the mark, shake well, and obtain the acetylated sodium hyaluronate test solution (hereinafter referred to as the test solution).

[0064] Accurately measure 1.0 ml of the test solution and place it in a test tube. Starting from the step described above, "add 2 ml of freshly prepared alkaline hydroxylamine solution", proceed in the same manner. Take another 1 ml of the test solution and proceed in the same manner as the blank control of the standard solution. Use this as a blank control for the test solution.

[0065] Subtract the absorbance of the corresponding blank control tube from the absorbance of the test solution, and then input the result into the standard curve to calculate the volume (V, ml) of the standard solution equivalent to 1 ml of the test solution. Calculate the O-acetyl content in the test solution using Formula 1.

[0066] The O-acetyl content (c, mmol / L) in the test solution = V × 2.5 (Formula 1)

[0067] In the formula 2.5: the content of acetylcholine chloride in the standard solution (mmol / L).

[0068] (3) Calculate the degree of substitution of acetylated sodium hyaluronate samples

[0069] The mass fraction of acetylated sodium hyaluronate can be calculated using Formula 2; the degree of substitution of acetylated sodium hyaluronate can be calculated using Formula 3.

[0070] Acetyl mass fraction (A, %) = 43 × c × V × 1000 / M × 100% (Formula 2)

[0071] Degree of substitution (DS) = 401.31 × A / [4300 - (43 - 1) × A] (Formula 3)

[0072] In the formula, 43 represents the relative molecular mass of the acetyl group;

[0073] c: O-acetyl content in the test solution (mmol / L);

[0074] V: Volume of the solution to be tested (L);

[0075] M: Mass (g, dry basis) of acetylated sodium hyaluronate weighed in preparation of the test solution;

[0076] 1000: Conversion factor between mmol and mol;

[0077] 100%: Conversion factor for mass fraction;

[0078] 401.31: The relative molecular mass of one unsubstituted repeating disaccharide unit of sodium hyaluronate;

[0079] 1: The relative molecular mass of a hydrogen atom.

[0080] The experimental results of the above seven embodiments are shown in Table 1 below:

[0081] Table 1: Experimental Results

[0082]

[0083] Application Experiment Example 2

[0084] The acetylated sodium hyaluronate obtained in Example 5 above is a white solid, such as... Figure 2 As shown. The raw material sodium hyaluronate and the acetylated sodium hyaluronate obtained in Example 5 above were dissolved in heavy water and subjected to 1H NMR spectroscopy to obtain the corresponding spectra, as shown. Figure 3 and Figure 4 As shown.

[0085] from Figure 3 The 1H NMR spectrum of sodium hyaluronate shows that the peaks with chemical shifts of 3.2-4.1 ppm correspond to hydrogens on the hyaluronic acid glycan ring, and the peaks with chemical shifts of 1.8-2.1 ppm correspond to hydrogens on the acetyl groups of sodium hyaluronate itself. The integral of the glycan ring region should roughly match the integral of the acetyl group, that is, theoretically, 10 hydrogens on the glycan ring of each repeating unit correspond to 3 hydrogens on the acetyl groups of sodium hyaluronate itself.

[0086] Figure 4 and Figure 3In comparison, due to the modification of the acetyl group, the hydrogen chemical shifts on the sugar ring all increase, corresponding to 3.6-4.7 ppm, while the hydrogen chemical shifts on the acetyl group remain basically unchanged, corresponding to 1.8-2.3 ppm. The increased integral area is contributed by the modified acetyl group. Based on the integral ratio, the degree of substitution can be directly calculated from the graph: DS = 13.75 / 3 - 1 = 3.58. This result matches the result calculated according to the O-acetyl determination method in General Chapter 3117 of the Chinese Pharmacopoeia. In the above formula for calculating the degree of substitution, 13.75 refers to the integral of acetylated sodium hyaluronate at the 1.8-2.3 ppm shift in the 1H NMR spectrum, representing the number of hydrogen atoms attributable to the acetyl group; 3 refers to the conversion factor between the acetyl group and hydrogen atoms, i.e., one acetyl group contains 3 hydrogen atoms; 1 refers to the one acetyl group inherent in the sodium hyaluronate raw material. Therefore, the overall formula represents the net increase in the number of acetyl groups in the acetylated sodium hyaluronate product compared to the sodium hyaluronate raw material, i.e., the degree of substitution.

[0087] Comparative Example 1

[0088] The difference between Comparative Document 1 and Example 1 lies in the different eutectic solvents (DES) used in step (1). The specific steps of step (1) in Comparative Example 1 are as follows: 2.60 g of choline chloride and 1.12 g of acetic acid are weighed into a reaction flask and stirred at 75°C for 30 min to obtain clear DES. Then, 0.5 g of sodium hyaluronate with an average molecular weight of 50 kDa is added to the DES and stirred for another 30 min until it is completely dissolved and forms a clear solution. The system is cooled to 60°C and 0.018 g of 4-pyrrolidinylpyridine is added to the solution. Then, 1.39 g of acetic anhydride is slowly added dropwise to the solution and the reaction is carried out at 60°C for 10 hours. After the reaction is completed, the reaction solution is obtained.

[0089] (2) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 for dialysis purification. When the conductivity of the liquid outside the dialysis bag was less than 5 μS / cm, the liquid inside the dialysis bag was collected and freeze-dried to obtain a white powdery sodium acetylated hyaluronic acid product.

[0090] The degree of substitution of the resulting acetylated sodium hyaluronate product is 0.58.

[0091] Comparative Example 2

[0092] The difference between Comparative Document 2 and Example 1 is that the eutectic solvent (DES) used in step (1) is different. The specific steps of step (1) in Comparative Example 2 are as follows: 2.18 g of betaine and 1.94 g of malonic acid were weighed into a reaction flask and stirred at 75°C for 30 min to obtain clear DES. Then, 0.5 g of sodium hyaluronate with an average molecular weight of 50 kDa was added to the DES. After stirring for another 30 min, there was still undissolved sodium hyaluronate. The system was cooled to 60°C and 0.018 g of 4-pyrrolidinylpyridine was added to the solution. Then, 1.58 g of acetic anhydride was slowly added dropwise to the solution. The reaction was carried out at 60°C for 10 hours. After the reaction was completed, the reaction solution was obtained.

[0093] (2) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 for dialysis purification. When the conductivity of the liquid outside the dialysis bag was less than 5 μS / cm, the liquid inside the dialysis bag was collected and freeze-dried to obtain a white powdery sodium acetylated hyaluronic acid product.

[0094] The degree of substitution of the resulting acetylated sodium hyaluronate product was 0.07.

[0095] Comparative Examples 1 and 2 show that not all DES solvents can yield acetylated sodium hyaluronate with a high degree of substitution.

[0096] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing acetylated sodium hyaluronate, characterized in that... Includes the following steps: S1: Hyaluronic acid or its salt is dissolved in the eutectic solvent DES to form a homogeneous system, and then an acylation reaction catalyst is added to carry out an acylation reaction with acetic anhydride to obtain a reaction solution; S2: Place the reaction solution in a dialysis bag and perform dialysis separation using water as the dialysis medium. The component retained in the dialysis bag is phase A, which is rich in acetylated sodium hyaluronate. S3: After collecting phase A and filtering to remove insoluble matter, freeze-dry the product to obtain acetylated sodium hyaluronate.

2. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S1, the DES used is either type III DES or type V DES.

3. The method for preparing acetylated sodium hyaluronate according to claim 2, characterized in that: When the DES in step S1 is a type III DES, the type III DES uses a quaternary ammonium salt as a hydrogen bond acceptor and an organic acid or imidazole compound as a hydrogen bond donor; the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:0.5~1:

3. When the DES in step S1 is a type 5 DES, the type 5 DES uses imidazole compounds as hydrogen bond acceptors and organic acids as hydrogen bond donors, and the molar ratio of hydrogen bond acceptors to hydrogen bond donors is 1:0.5 to 1:

3.

4. The method for preparing acetylated sodium hyaluronate according to claim 3, characterized in that: When the DES in step S1 is a type III DES, the quaternary ammonium salt is selected from any one of tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, and tetrabutylammonium chloride; the organic acid is selected from any one or a combination of acetic acid, propionic acid, oxalic acid, or malonic acid; and the imidazole compound is selected from any one of imidazole, 2-methylimidazolium, 4-methylimidazolium, and 5-methylimidazolium. When the DES in step S1 is a Class 5 DES, the imidazole compound is selected from any one of imidazole, 1-methylimidazolium, 2-methylimidazolium, 4-methylimidazolium, and 5-methylimidazolium; the organic acid is selected from any one or a combination of acetic acid, propionic acid, oxalic acid, or malonic acid.

5. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S1, the molecular weight of hyaluronic acid is 1 kDa to 100 kDa; In step S1, the hyaluronic acid salt is at least one of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, and zinc hyaluronate, preferably sodium hyaluronate; The catalyst for the acylation reaction is one of pyridine, 4-dimethylaminopyridine, or 4-pyrrolidinylpyridine.

6. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S1, the mass concentration of hyaluronic acid or its salt in the DES is 1% to 15%, and the amount of acetic anhydride is 2 to 12 times the mass of hyaluronic acid or its salt; the amount of the acylation reaction catalyst is 0% to 2% of the mass of acetic anhydride.

7. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S1, the acylation reaction temperature is 25~80℃ and the acylation reaction time is 4~20 hours.

8. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S2, the molecular weight cutoff of the dialysis bag is 1kDa~10kDa; dialysis is stopped when the conductivity of the external fluid of the dialysis bag is <5μS / cm.

9. The method for preparing acetylated sodium hyaluronate according to claim 1, characterized in that: In step S2, when the raw material in step S1 is hyaluronic acid or other non-sodium salt hyaluronic acid salts, 0.5 mol / L sodium chloride solution is added to the reaction solution before dialysis in order to form the final sodium salt, namely acetylated sodium hyaluronate.

10. An acetylated sodium hyaluronate prepared according to any one of claims 1-9, characterized in that... The degree of substitution of acetylated sodium hyaluronate is 2.33-3.63.

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