Method for preparing acetylated hyaluronic acid with reduced odor

Abstract

The present invention relates to a method for preparing acetylated hyaluronic acid (AcHA) with reduced odor, the method comprising the steps of: acetylating hyaluronic acid in the presence of an organic acid, a carboxylic acid anhydride, and a basic catalyst until a homogeneous reaction mixture is formed; separating the reaction product under acidic conditions; washing same with a nonpolar organic solvent; and treating same with a basic aqueous solution followed by precipitation with an alcohol. The AcHA prepared by the method has a degree of acetylation of 3.8 to 4.0, exhibits fat solubility at least ten times higher than that of hyaluronic acid, and is obtained in a pure sodium salt form with the acetic acid odor removed therefrom. The present invention enables the production of uniform AcHA in high yield under mild conditions suitable for large-scale production, while significantly improving the purity and solubility of the final product.

Description

Method for manufacturing acetylated hyaluronic acid with suppressed odor

[0001] The following examples relate to a method technology for producing acetylated hyaluronic acid with suppressed odor.

[0002] Acetylated hyaluronic acid (AcHA) is a substance with a structure in which the hydrophilic -OH group of hyaluronic acid (HA) is substituted with an amphiphilic acetyl group. Due to these structural characteristics, AcHA exhibits superior moisturizing effects compared to conventional HA, and is receiving great interest in the cosmetics and pharmaceutical fields, being referred to by the nicknames "Super HA" or "Hymagic-HA."

[0003] AcHA can be synthesized through the acetylation reaction of HA. There are two key aspects to this reaction: first, conducting the reaction under mild conditions to achieve a high yield for mass production, and second, consistently controlling the degree of acetylation to obtain a uniform product. In particular, controlling the degree of acetylation is a critical factor directly linked to the quality of the final product.

[0004] However, despite the high commercial interest in AcHA, scientific research results regarding its synthesis are severely lacking. Even the research results and patents published to date have problems such as questionable reliability or difficulty in applying them to large-scale production. For example, a method for synthesizing AcHA under basic catalytic conditions published by a research team at the University of Salerno in Italy in 2014 was found to be unreproducible.

[0005] In addition, the AcHA synthesis method under acidic catalytic conditions patented by Japan’s Shiseido and Toray in 1975, 1997, and 2020, respectively, was found to be difficult to apply to large-scale AcHA production processes in reality, as it involved the simultaneous decomposition of large amounts of AcHA, although a small amount of AcHA could be obtained. An international patent filed by China’s Bloomage in 2021 claims only the efficacy without specific mention of the AcHA synthesis process, and thus does not provide information on the actual synthesis method.

[0006] Against this backdrop, the development of new synthesis methods is urgently needed for the effective and economical mass production of AcHA. In particular, there is a need to develop methods that ensure reaction uniformity to improve safety and product uniformity, effectively eliminate the odor of acetic acid generated as a byproduct, and enhance practicality by improving the solubility and lipophilicity of the final product.

[0007] [Prior Art Literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) Korean Published Patent 10-2011-0135694

[0010] (Patent Document 2) Korean Published Patent 10-2018-0119936

[0011] (Patent Document 3) Korean Published Patent 10-2005-0105439

[0012] (Patent Document 4) Korean Published Patent 10-2015-0015209

[0013] The present invention aims to overcome the limitations of existing acetylated hyaluronic acid (AcHA) synthesis methods and provide a new manufacturing method that is industrially useful. Specifically, the present invention aims to solve the following problems.

[0014] First, we aim to develop a method for synthesizing AcHA in high yield under mild reaction conditions suitable for mass production. We intend to resolve the problem of low yields and unsuitability for mass production caused by decomposition reactions in existing synthesis methods under strong acid conditions.

[0015] Second, this study aims to provide a method for obtaining AcHA products of uniform quality by precisely controlling the degree of acetylation. This is an essential element for maintaining consistent product quality and securing commercial competitiveness.

[0016] Third, we aim to improve the safety of the reaction and the uniformity of the product by ensuring the uniformity of the reaction mixture. In particular, we intend to resolve the issue of reaction mixture uniformity, which is a critical factor in mass production.

[0017] Fourth, we aim to develop a post-treatment method that can increase the purity of the generated AcHA and effectively remove the odor of acetic acid produced as a byproduct. This is an important factor in improving product quality and marketability.

[0018] Fifth, we aim to enhance practicality by improving the solubility and lipid solubility of the final product. In particular, we aim to expand the range of applications by addressing the issue of solubility in aqueous solutions and improving lipid solubility.

[0019] Sixth, we intend to verify the feasibility of mass production by extending the synthesis method developed at the laboratory scale to an intermediate scale (50-100g).

[0020] The present invention relates to a method for producing acetylated hyaluronic acid with suppressed odor, comprising the steps of: acetylating hyaluronic acid in the presence of an organic acid, a carboxylic anhydride, and a basic catalyst until a homogeneous reaction mixture forming a single phase is formed upon visual observation; separating the acetylated hyaluronic acid under acidic conditions; and washing the separated acetylated hyaluronic acid with a nonpolar organic solvent. The method comprises the step of treating the washed acetylated hyaluronic acid with a basic aqueous solution and precipitating it with alcohol; wherein the reaction is continued from the point where the reaction mixture becomes homogeneous in the acetylation step to obtain acetylated hyaluronic acid with a degree of acetylation of 3.8 or higher and 4.0 or lower, and the final product exhibits lipophilicity 10 times greater than that of hyaluronic acid when measured by the octanol / water partition coefficient (log P), and no acetic acid peak is detected during gas chromatography analysis, and the final product is obtained in the form of a pure sodium acetylated hyaluronate salt.

[0021] At this time, the organic acid is acetic acid, the carboxylic anhydride is acetic anhydride, the basic catalyst is 4-(dimethylamino)pyridine (DMAP), the non-polar organic solvent is hexane, the acetylation step is performed at 95-105°C for 40-56 hours, the basic aqueous solution is a sodium bicarbonate aqueous solution with a concentration of 15-25% (w / v), and the alcohol is isopropanol.

[0022] In addition, at this time, separation under acidic conditions is performed using a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio), and washing with a non-polar organic solvent is performed for 8 to 16 hours, and the yield of acetylated hyaluronic acid obtained by the above method is 75-85%, and the final product is obtained as a milky white solid.

[0023] In addition, the acetylation step comprises: (a) mixing hyaluronic acid, acetic acid, acetic anhydride, and 4-(dimethylamino)pyridine in a molar ratio of 1:15:15:1 in a 4-liter round-bottom flask; (b) reacting the mixture at 100°C for 48 hours; (c) cooling the reaction mixture to room temperature and removing the solvent using a rotary evaporator; (d) adding a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio) to obtain a solid product; and (e) washing the solid product with distilled water and then drying it. and (f) a step of filtering the dried solid after stirring with hexane for 12 hours; wherein acetic acid is used as a co-solvent in step (a) to obtain a homogeneous reaction mixture, and the yield of the product obtained after step (f) is 75-85% (w / w) and the degree of acetylation of the product is 3.8-4.0.

[0024] Also, at this time, the basic aqueous solution treatment and alcohol precipitation steps comprise: (g) a step of dissolving the acetylated hyaluronic acid in a 20% concentration sodium bicarbonate aqueous solution; and (h) a step of adding isopropanol to precipitate and filter the acetylated sodium hyaluronate; wherein, through steps (g) and (h), a pure acetylated sodium hyaluronate in which no acetic acid peak is detected upon gas chromatography analysis is obtained with a weight recovery rate of 98-100% relative to the acetylated hyaluronate added, and the pure acetylated sodium hyaluronate dissolves until no insoluble particles are visible upon visual observation when a 1% (w / v) aqueous solution is prepared at 20°C, the C Log P value of the monomer of the pure acetylated sodium hyaluronate is -1.12, and it is confirmed by NMR analysis that acetylation has occurred on all four -OH groups present in the hyaluronate starting material, and the above manufacturing method is on a 50g scale It can be performed with a yield of 79-83% and up to a scale of 100g.

[0025] A device according to one embodiment may be combined with hardware and controlled by a computer program stored on a medium to execute the method of any one of the methods described above.

[0026] The AcHA manufacturing method according to the present invention has the following significant effects.

[0027] First, by using acetic acid as a co-solvent to obtain a homogeneous reaction mixture, the safety of the reaction and the uniformity of the product were significantly improved. This solves the problems that occurred in existing heterogeneous reaction systems, enabling stable quality control during mass production.

[0028] Second, AcHA can be synthesized with a high yield of 75-85% by carrying out the reaction for 40-56 hours under mild temperature conditions of 95-105℃. This is a significantly improved result compared to the existing synthesis method under strong acid conditions and is suitable for industrial production.

[0029] Third, a uniform product can be obtained by precisely controlling the degree of acetylation to 3.8-4.0. This is an essential factor in maintaining consistent product quality and contributes significantly to securing commercial competitiveness.

[0030] Fourth, by effectively removing acetic acid, a byproduct, through washing with a non-polar organic solvent and treatment with a basic aqueous solution, pure AcHA can be obtained in which no acetic acid peak is detected during gas chromatography analysis. This resolves the product's odor problem and significantly improves marketability.

[0031] Fifth, the final product was obtained in the form of a pure AcHA sodium salt, which greatly improved solubility in aqueous solution. When a 1% (w / v) aqueous solution was prepared at 20°C, it dissolved completely to the extent that no insoluble particles were visible to the naked eye, greatly improving practicality.

[0032] Sixth, the C Log P value of the monomer of the final product is -1.12, which is more than 10 times higher than that of hyaluronic acid (C Log P = -2.37). This is an important characteristic that can significantly expand the range of applications for AcHA.

[0033] Seventh, this manufacturing method can be performed with a yield of 79-83% at a 50g scale and has been successfully scaled up to a 100g scale. This result empirically demonstrates that this method is suitable for mass production.

[0034] Eighth, NMR analysis confirmed that acetylation occurred on all four -OH groups present in the hyaluronic acid starting material, demonstrating the completeness of the reaction and the structural uniformity of the product.

[0035] Figure 1 is a diagram illustrating a structural comparison of hyaluronic acid (HA) and acetylated hyaluronic acid (AcHA) according to one embodiment.

[0036] Figure 2 is a photograph of the reaction mixture over time showing the progress of the acetylation reaction according to one embodiment.

[0037] Figure 3 is a photograph of the appearance of acetylated hyaluronic acid (AcHA), which is the final product according to one embodiment.

[0038] Figure 4 is a 1H NMR spectrum of acetylated hyaluronic acid (AcHA) according to one embodiment.

[0039] FIG. 5 is a photograph of a large-scale reaction apparatus and a product according to one embodiment.

[0040] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, various modifications may be made to the embodiments, and thus the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, and substitutions to the embodiments are included within the scope of the rights.

[0041] Specific structural or functional descriptions of the embodiments are disclosed for illustrative purposes only and may be modified and implemented in various forms. Accordingly, the embodiments are not limited to the specific disclosed forms, and the scope of this specification includes modifications, equivalents, or substitutions that fall within the technical concept.

[0042] Terms such as "first" or "second" may be used to describe various components, but these terms should be interpreted solely for the purpose of distinguishing one component from another. For example, the first component may be named the second component, and similarly, the second component may be named the first component.

[0043] When it is stated that a component is "connected" to another component, it should be understood that it may be directly connected to or joined to that other component, or that there may be other components in between.

[0044] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0045] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0046] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0047] In the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in the embodiments of the present invention.

[0048] The shapes, sizes, ratios, angles, numbers, etc. disclosed in the drawings for explaining embodiments of the present invention are exemplary, and therefore the present invention is not limited to the depicted details. Furthermore, in describing the present invention, if it is determined that a detailed description of related known technology may unnecessarily obscure the essence of the present invention, such detailed description is omitted. Where terms such as "includes," "has," or "is made up" are used in this specification, other parts may be added unless "only" is used. Where a component is expressed in the singular, it includes cases where it includes the plural unless specifically stated otherwise.

[0049] In interpreting the components, they are interpreted to include a margin of error even in the absence of a separate explicit statement.

[0050] The size and thickness of each component shown in the drawings are illustrated for convenience of explanation, and the present invention is not necessarily limited to the size and thickness of the illustrated components.

[0051] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interlocking relationship.

[0052] Figure 1 is a diagram illustrating a structural comparison of hyaluronic acid (HA) and acetylated hyaluronic acid (AcHA) according to one embodiment.

[0053] Figure 1 shows a comparison of the chemical structures of hyaluronic acid (HA) and acetylated hyaluronic acid (AcHA). This figure is an important visual representation of the principle and results of the acetylation reaction of HA.

[0054] First, the monomeric structure of hyaluronic acid is presented on the left side of the diagram. Hyaluronic acid consists of disaccharide repeating units in which N-acetylglucosamine and glucuronic acid are linked by β-1,4 and β-1,3 glycosidic bonds. A notable feature of this structure is the presence of four hydroxyl (-OH) groups per monomer. These hydroxyl groups are located at the 4th and 6th carbons of N-acetylglucosamine and the 2nd and 3rd carbons of glucuronic acid, respectively. Due to these numerous hydroxyl groups, hyaluronic acid exhibits high hydrophilicity, which can be confirmed by the C Log P value of -2.37.

[0055] The monomer structure of acetylated hyaluronic acid is presented on the right side of the diagram. It can be seen that through the acetylation reaction, all four hydroxyl groups of hyaluronic acid have been substituted with acetyl groups (-COCH3). This structural change significantly affects the physicochemical properties of the molecule. In particular, the C Log P value increases to -1.12, resulting in lipophilicity approximately 10 times greater than that of hyaluronic acid. This is because acetyl groups are more hydrophobic than hydroxyl groups.

[0056] This diagram also illustrates the chemical nature of the acetylation reaction. Specifically, it shows the process in which each hydroxyl group (-OH) of hyaluronic acid is substituted with an acetyl group (-COCH3). In this reaction, acetic anhydride ((CH3CO)2O) is used as the acetylation agent and reacts with each hydroxyl group to form an ester bond.

[0057] In conclusion, Figure 1 clearly illustrates the principle and results of the acetylation reaction of hyaluronic acid, which is the core of the present invention. Through this structural change, the hydrophilicity of hyaluronic acid decreases and its lipophilicity increases, which plays a key role in achieving the enhanced moisturizing effect and skin penetration power that are the objectives of the present invention. In addition, this figure visually demonstrates the importance of the degree of acetylation, helping to understand the significance of the degree of acetylation of 3.8-4.0 targeted in the present invention.

[0058] Figure 4 is a 1H NMR spectrum of acetylated hyaluronic acid (AcHA) according to one embodiment.

[0059] Figure 4 shows the 1H NMR spectrum of acetylated hyaluronic acid (AcHA) synthesized by the method of the present invention. This spectrum was measured in a deuterated solvent (D2O), and the chemical shift (δ) values ​​are expressed in ppm units.

[0060] The most characteristic peak in the spectrum is a strong singlet observed around δ 2.0 ppm. This peak corresponds to the methyl hydrogen of the acetyl group (-COCH3), and its integral value represents about 12 hydrogens per monomer of hyaluronic acid. This means that all four hydroxyl groups (-OH) of hyaluronic acid are acetylated, indicating that the degree of acetylation is close to 4.0.

[0061] The complex multiplets observed in the δ 3.0-4.5 ppm region are signals of hydrogens corresponding to the sugar backbone of hyaluronic acid. The distinct separation of peaks in this region reflects changes in the chemical environment due to acetylation.

[0062] The peaks observed in the δ 4.5-5.5 ppm region correspond to the anomeric hydrogens and the hydrogens adjacent to the acetylated position. The presence and location of these peaks further confirm that acetylation was successfully achieved.

[0063] It is noteworthy that no peak corresponding to residual acetic acid is observed in the δ 1.8-2.2 ppm region. This demonstrates that the purification process of the present invention was effectively carried out and that acetic acid was completely removed from the final product.

[0064] In addition, the fact that no peaks corresponding to impurities are observed in the δ 0-1 ppm and δ 6-8 ppm regions indicates that AcHA synthesized by the method of the present invention was obtained with high purity.

[0065] Overall, the NMR spectrum of FIG. 4 clearly shows that the AcHA synthesized by the method of the present invention has a high degree of acetylation (about 4.0) and was obtained with high purity. This is important scientific evidence proving that the synthesis method of the present invention is effective and that the quality of the product is excellent.

[0066] The present invention relates to a method for producing acetylated hyaluronic acid with suppressed odor, comprising the steps of: acetylating hyaluronic acid in the presence of an organic acid, a carboxylic anhydride, and a basic catalyst until a homogeneous reaction mixture forming a single phase is formed upon visual observation; separating the acetylated hyaluronic acid under acidic conditions; washing the separated acetylated hyaluronic acid with a non-polar organic solvent; and treating the washed acetylated hyaluronic acid with a basic aqueous solution and precipitating it with alcohol.

[0067] At this time, in the case of the present invention, the reaction is continued from the point where the reaction mixture becomes homogeneous in the acetylation step to obtain acetylated hyaluronic acid with a degree of acetylation of 3.8 or higher and 4.0 or lower, and the final product exhibits lipophilicity 10 times greater than that of hyaluronic acid when measured by the octanol / water partition coefficient (log P), and no acetic acid peak is detected when analyzed by gas chromatography, and the final product is obtained in the form of pure acetylated hyaluronate sodium salt.

[0068] The reasons for each of the above components are as follows.

[0069] First, the method includes a step of acetylating hyaluronic acid in the presence of an organic acid, a carboxylic anhydride, and a basic catalyst until a homogeneous reaction mixture is formed. This is intended to solve the problem that hyaluronic acid (HA) does not dissolve well in organic solvents because its structure contains multiple hydrophilic -OH groups, as shown in Figure 1. By using an organic acid (acetic acid) as a co-solvent, a carboxylic anhydride (acetic anhydride) as an acetylation reagent, and a basic catalyst (DMAP) as a reaction promoter, a homogeneous reaction mixture can be formed, which is a key factor in significantly improving the efficiency of the reaction and the homogeneity of the product.

[0070] Next, the process includes a step of separating the acetylated hyaluronic acid under acidic conditions. This is intended to increase the purity of the product by neutralizing the basic catalyst used during the reaction and removing residual acetic anhydride through hydrolysis. Subsequently, the separated acetylated hyaluronic acid undergoes a step of washing with a non-polar organic solvent. This is to remove any remaining acetic acid or other polar impurities, and contributes to obtaining pure AcHA by minimizing impurity peaks, as can be seen in the NMR spectrum of Figure 4.

[0071] The step of treating the washed acetylated hyaluronic acid with a basic aqueous solution and precipitating it with alcohol is intended to convert AcHA into a sodium salt form to improve water solubility and remove any remaining acidic impurities. Through this process, high-purity AcHA can be obtained, as can be confirmed in the NMR spectrum of Figure 4.

[0072] In the present invention, the reaction is continued from the point where the reaction mixture becomes homogeneous during the acetylation step to obtain acetylated hyaluronic acid with a degree of acetylation of 3.8 or higher and 4.0 or lower. As can be seen in Fig. 1, the HA monomer has four -OH groups, and this degree of acetylation means that these four -OH groups are almost completely acetylated. This can be confirmed through the peak intensity of the acetyl groups in the NMR spectrum of Fig. 4, and is important for optimizing the lipophilicity and moisturizing effect of AcHA.

[0073] The final product exhibits more than 10 times greater lipophilicity than hyaluronic acid when measured by the octanol / water partition coefficient (log P), and no acetic acid peak is detected during gas chromatography analysis. As shown in Figure 1, the substitution of -OH groups with acetyl groups increases the molecule's hydrophobicity, thereby enhancing lipophilicity. The absence of an acetic acid peak demonstrates the effectiveness of the impurity removal process, which is important for the quality and stability of the product.

[0074] Finally, the final product is obtained in the form of pure acetylated sodium hyaluronate. This improves the water solubility of AcHA and, as can be seen in the NMR spectrum of Figure 4, the structure of pure AcHA sodium salt can be confirmed, ensuring the high purity and uniformity of the product.

[0075] Through the combination of each of these components, the present invention provides a method for efficiently producing AcHA with high purity, high lipid solubility, and improved water solubility.

[0076] In addition, the step of separating acetylated hyaluronic acid (AcHA) under acidic conditions in the present invention can be performed as follows.

[0077] First, the reaction mixture, after the acetylation reaction is complete, is cooled to room temperature (approx. 20-25°C). At this time, the cooling rate is controlled to 1-2°C per minute to prevent deterioration of the product quality due to rapid temperature changes. Solvents such as acetic acid and acetic anhydride are removed from the cooled reaction mixture under reduced pressure using a rotary evaporator. During this process, the water bath temperature is maintained so as not to exceed 40°C to prevent thermal decomposition of AcHA.

[0078] After the solvent is removed, a diluted aqueous hydrochloric acid solution is added to the remaining mixture. Specifically, a diluted aqueous hydrochloric acid solution prepared by mixing concentrated hydrochloric acid (37% HCl) and distilled water in a volume ratio of 1:37.5 is used. This diluted aqueous hydrochloric acid solution is added in an amount approximately 30 times the volume of the reaction mixture. For example, approximately 1500 mL of diluted aqueous hydrochloric acid solution is used per 50 mL of the reaction mixture.

[0079] Slowly add the diluted aqueous hydrochloric acid solution while vigorously stirring the reaction mixture. Maintain the stirring speed at 400-500 rpm to ensure uniform mixing. Since the temperature of the reaction mixture may rise when the aqueous hydrochloric acid solution is added, use an ice bath if necessary to maintain the temperature below 25°C.

[0080] After adding the aqueous hydrochloric acid solution, the mixture is stirred for an additional 20 minutes. During this process, AcHA is protonated under acidic conditions, reducing its solubility and forming a slurry-type mixture. Subsequently, the formed solid product is separated by vacuum filtration or centrifugation. For vacuum filtration, a Buchner funnel and Whatmann No. 1 filter paper are used, and for centrifugation, the process is performed at 4000-5000 rpm for 15 minutes.

[0081] The separated solid AcHA is washed three times with cold distilled water (approx. 4°C) to remove any remaining acid and soluble impurities. In each washing step, distilled water equivalent to approximately 10 times the volume of the initial reaction mixture is used. After washing, the solid AcHA is dried using a vacuum oven or a freeze dryer. When using a vacuum oven, the temperature is not to exceed 40°C, and the pressure is maintained at 10–20 mmHg. The drying process is carried out for approximately 24 hours until the moisture content is reduced to 1% or less.

[0082] Through a separation process under these acidic conditions, acetylated hyaluronic acid can be obtained with high purity, while simultaneously effectively removing reaction byproducts and unreacted reagents. This method is applicable to large-scale production, making it highly industrially useful.

[0083] In addition, in the method for preparing acetylated hyaluronic acid (AcHA) according to the present invention, the washing step using a non-polar organic solvent can be performed as follows.

[0084] First, transfer the AcHA solid separated under acidic conditions to a suitable container (e.g., a beaker or a round-bottom flask). The size of the container used is selected by considering the amount of AcHA and the amount of non-polar organic solvent to be used. Generally, a container is selected that can accommodate a volume of non-polar organic solvent 10 to 20 times the weight of AcHA.

[0085] It is preferable to use hexane as a non-polar organic solvent. Hexane can effectively remove non-polar impurities that may remain in AcHA, particularly excess acetylation reagents or byproducts. Specifically, use a volume of hexane equal to 10 times the weight of AcHA. For example, use approximately 500 mL of hexane for 50 g of AcHA.

[0086] Slowly pour hexane into a container holding AcHA. Avoid vigorous stirring and use a magnetic stirrer to stir at a medium speed (e.g., 300-400 rpm). Continue stirring for 12-16 hours. During this time, the AcHA particles come into sufficient contact with the hexane to effectively remove impurities.

[0087] After stirring is complete, the mixture is filtered. Filtration is performed using a Buchner funnel and filter paper by vacuum filtration. The filter paper used is selected to have an appropriate pore size (e.g., 10-20 μm) capable of effectively filtering out AcHA particles.

[0088] The AcHA solid obtained after filtration is additionally washed 2-3 times with a small amount of fresh hexane (e.g., about 20% of the initial amount). This process is intended to more effectively remove impurities that may remain on the surface of the AcHA particles.

[0089] The washed AcHA solid is dried in a vacuum oven at a temperature of 40-50°C for 4-6 hours. This is to completely remove any hexane that may remain in the AcHA. During the drying process, the solid is turned over every 2-3 hours to ensure uniform drying.

[0090] Through this non-polar organic solvent washing process, the purity of AcHA can be improved and residual impurities can be effectively removed. This process is an important step that significantly enhances the quality and stability of the final product.

[0091] Figure 2 is a photograph of the reaction mixture over time showing the progress of the acetylation reaction according to one embodiment.

[0092] One of the key challenges of the present invention was to obtain a homogeneous reaction mixture in the acetylation reaction of hyaluronic acid (HA). To this end, various solvent systems and reaction conditions were explored, and three representative experimental results (entry 3, 4, 6) are shown in FIG. 2.

[0093] Entry 3 is a condition in which DMF was used as a solvent and acetic anhydride and pyridine were added. Under these conditions, the reaction was carried out at 100°C for 48 hours, but as can be seen in the left image of Figure 2, the reaction mixture remained in a heterogeneous state. This is presumed to be because DMF did not completely dissolve the hyaluronic acid and pyridine did not provide sufficient catalytic activity.

[0094] Entry 4 is a condition in which toluene was used as a solvent and acetic anhydride and sodium hydroxide were added. Under this condition as well, the reaction was carried out at 100°C for 48 hours, but as can be seen in the right image of Figure 2, the reaction mixture remained in a heterogeneous state. This is believed to be because hyaluronic acid was not dissolved due to the low polarity of toluene, and sodium hydroxide did not function effectively in the organic solvent.

[0095] On the other hand, entry 6 is the condition finally selected in the present invention, in which acetic acid is used as a solvent and acetic anhydride and DMAP are added. Under this condition, the reaction was carried out at 100°C for 48 hours, and as can be seen in the bottom image of Fig. 2, a gradually uniform mixture was formed as the reaction proceeded. Of particular note is that the hyaluronic acid began to completely dissolve after about 5 hours from the start of the reaction.

[0096] Figure 2 visually illustrates the reaction progress under these three conditions. The images on the left and right show the heterogeneous reaction mixtures of entry 3 and 4, respectively, suggesting that the reaction did not proceed effectively because the hyaluronic acid was not properly dissolved. On the other hand, the bottom image shows the homogeneous reaction mixture of entry 6, indicating that the hyaluronic acid was completely dissolved, making an effective acetylation reaction possible.

[0097] These results demonstrate that acetic acid improves the solubility of hyaluronic acid while being suitable as a medium for the acetylation reaction. Furthermore, it proves that DMAP acts as an effective catalyst to promote acetylation in a homogeneous reaction environment. These homogeneous reaction conditions played a key role in ultimately achieving a high yield of 70% and a precise degree of acetylation of 3.8–4.0.

[0098] Accordingly, Figure 2 clearly illustrates the importance of obtaining a uniform reaction mixture in the method for producing acetylated hyaluronic acid of the present invention and the optimal reaction conditions for this purpose. This is one of the key features that differentiates the method of the present invention from existing methods and serves as the basis for achieving stable quality control and high yield during mass production.

[0099] In this case, for the present invention, the organic acid is acetic acid, the carboxylic anhydride is acetic anhydride, the basic catalyst is 4-(dimethylamino)pyridine (DMAP), the non-polar organic solvent is hexane, the acetylation step is performed at 95-105°C for 40-56 hours, the basic aqueous solution is an aqueous sodium bicarbonate solution with a concentration of 15-25% (w / v), and the alcohol is isopropanol.

[0100] The reason acetic acid was selected as the organic acid used in the present invention is as follows. As can be seen in Figure 1, the acetylation reaction of hyaluronic acid (HA) is a process of substituting -OH groups with acetyl groups. Acetic acid has the same structure as the acetyl group, which is the product of this reaction, and thus provides optimal affinity as a reaction medium. In addition, acetic acid is a polar solvent that improves the solubility of hyaluronic acid, while also serving as an organic reaction medium, playing an important role in creating a uniform reaction environment.

[0101] The reason acetic anhydride was chosen as the carboxylic acid is the efficiency of the acetylation reaction. Acetic anhydride is one of the most effective reagents for providing acetyl groups, and it can directly deliver acetyl groups without generating water molecules. This is essential for maximizing the stoichiometric efficiency of the acetylation reaction shown in Figure 1.

[0102] The reason 4-(dimethylamino)pyridine (DMAP) was selected as a basic catalyst is its strong nucleophilicity and low basicity. DMAP is known to be a very effective catalyst in acetylation reactions, and it exhibits excellent performance, particularly in the acetylation of sterically congested alcohols. This is important for effectively acetylating multiple -OH groups in the complex structure of hyaluronic acid, as shown in Fig. 1.

[0103] Hexane was chosen as a non-polar organic solvent due to its low polarity and high volatility. Hexane can effectively separate acetylated hyaluronic acid from any remaining unreacted reagents. Additionally, the high volatility of hexane allows the product to be dried easily after washing.

[0104] The reaction conditions for the acetylation step (95-105°C, 40-56 hours) were selected to obtain the optimal reaction rate and yield. As can be seen in Figure 2, under these conditions, the reaction mixture gradually becomes homogeneous, and the hyaluronic acid begins to completely dissolve after about 5 hours. This temperature range is the optimal condition for preventing thermal decomposition of the hyaluronic acid while allowing the reaction to proceed sufficiently quickly.

[0105] The reason for selecting a sodium bicarbonate aqueous solution with a concentration of 15-25% (w / v) as the basic aqueous solution is to ensure proper pH control and sodium salt formation. This concentration range is the optimal condition for effectively neutralizing acetylated hyaluronic acid while preventing hydrolysis caused by excessive basicity.

[0106] Finally, isopropanol was chosen as the alcohol because of its appropriate polarity and volatility. Isopropanol has polarity that allows it to effectively precipitate acetylated sodium hyaluronate, while also evaporating easily to facilitate the drying of the final product.

[0107] The selection of each of these components was optimized by considering the chemical characteristics of the acetylation reaction shown in Fig. 1 and the reaction process observed in Fig. 2, thereby enabling the production of high-purity, high-yield acetylated hyaluronic acid.

[0108] A reaction temperature of 95–105°C is important for optimizing the rate of the acetylation reaction and the quality of the product. As can be seen in Figure 2, within this temperature range, the reaction mixture becomes homogeneous and acetylation proceeds effectively. Below 95°C, the reaction rate decreases significantly, making it impractical, while above 105°C, the risk of side reactions or decomposition increases. Therefore, this temperature range is significant for ensuring both the efficiency and safety of the reaction.

[0109] A reaction time of 40 to 56 hours is important for achieving complete acetylation while preventing unnecessary energy consumption. Looking at the reaction progress over time in Figure 2, it can be seen that the reaction is almost complete after about 40 hours. If the reaction time is less than 40 hours, it is difficult to achieve the target degree of acetylation (3.8-4.0) due to incomplete acetylation, and if it exceeds 56 hours, it is not economically viable as there is no significant impact on yield or quality improvement.

[0110] The critical significance of each of these components is a key factor that enables the AcHA manufacturing method of the present invention to achieve high yield and quality while ensuring economic efficiency and safety.

[0111] Figure 3 is a photograph of the appearance of acetylated hyaluronic acid (AcHA), which is the final product according to one embodiment.

[0112] Figure 3 is a photograph showing the appearance of the final product of acetylated hyaluronic acid (AcHA) prepared according to the method of the present invention. As can be seen in this photograph, the AcHA prepared by the method of the present invention is obtained in the form of an off-white solid. This result contrasts with the brown or yellow product obtained from conventional synthesis methods, visually demonstrating the superiority of the method of the present invention.

[0113] Specifically, the AcHA product shown in this photograph appears as a powder with a uniform particle size. This uniform appearance suggests that the manufacturing method of the present invention can produce a product of consistent quality. The off-white color indicates that impurities were effectively removed through the non-polar organic solvent washing step and the basic aqueous solution treatment step included in the manufacturing method of the present invention.

[0114] Furthermore, no clumps or aggregation are observed in the AcHA product in the photograph. This demonstrates that the method of the present invention has undergone an effective drying process while maintaining a uniform particle distribution of the product. These characteristics can contribute to improving the solubility and dispersibility of the final product.

[0115] The approximate amount of the product can be estimated using the ruler located at the bottom of the photograph. This visually demonstrates that the method of the present invention can be successfully applied to medium-scale production (50-100g) beyond the laboratory scale. This is important evidence supporting the industrial scalability of the method of the present invention.

[0116] Finally, the photograph in FIG. 3 shows that AcHA produced by the method of the present invention is obtained in a form that is easy to handle and store. This provides convenience in the transportation, storage, and subsequent processing of the product, thereby further enhancing the practical value of the method of the present invention. Therefore, FIG. 3 can be considered important visual evidence that comprehensively demonstrates that the AcHA manufacturing method of the present invention possesses high quality, uniformity, scalability, and practicality.

[0117] In this case, for the present invention, separation under acidic conditions is performed using a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio), and washing with a non-polar organic solvent is performed for 8 to 16 hours, and the yield of acetylated hyaluronic acid obtained by the above method is 75-85%, and the final product is obtained as a milky white solid.

[0118] The specific conditions and results of the method for manufacturing acetylated hyaluronic acid are specified. Each component is described in detail below.

[0119] First, the statement “separation under the above acidic conditions is performed using a diluted aqueous hydrochloric acid solution (volume ratio of concentrated HCl : distilled water = 1 : 37.5)” specifies the process of separating the product after the acetylation reaction. In this step, the acetylated hyaluronic acid is precipitated by adjusting the pH by adding the diluted aqueous hydrochloric acid solution to the reaction mixture. By specifying the dilution ratio as 1:37.5, it is possible to avoid excessively acidic conditions and enable effective separation while minimizing the degradation of the product.

[0120] Next, the phrase “the above non-polar organic solvent washing is performed for 8 to 16 hours” specifies the purification process of the separated solid product. In this step, unreacted reagents or by-products are effectively removed by washing for 8 to 16 hours using a non-polar organic solvent such as hexane. This long washing process increases the purity of the product and plays an important role in removing volatile by-products, particularly acetic acid.

[0121] The statement "the yield of acetylated hyaluronic acid obtained by the above method is 75-85%" indicates the efficiency of the method of the present invention. A high yield of 75-85% is an important indicator showing that the method of the present invention is suitable for industrial production. This solves the problem of low yields associated with existing methods and enables economically viable mass production.

[0122] Finally, the description "the final product is obtained as a milky-white solid" specifies the physical properties of the product. As illustrated in FIG. 3, acetylated hyaluronic acid produced by the method of the present invention is obtained in the form of a milky-white solid. This is visual evidence that the product has high purity and that impurities have been effectively removed. The milky-white color indicates a level of purity and quality that allows it to be used as a raw material for cosmetics or pharmaceuticals without additional purification processes.

[0123] As can be seen in Fig. 3, the acetylated hyaluronic acid produced by the method of the present invention has a uniform milky-white solid form. This is in contrast to conventional methods, which often yielded brown or yellow products. The milky-white, uniform appearance demonstrates that the method of the present invention can effectively control the purity and uniformity of the product, which is a very important characteristic for industrial applications.

[0124] As such, each component of claim 3 specifically specifies that the method for producing acetylated hyaluronic acid according to the present invention can obtain a product of high yield, excellent purity, and uniform quality, which can also be visually confirmed through FIG. 3.

[0125] This has significant critical importance in the method for producing acetylated hyaluronic acid (AcHA) according to the present invention. This will be explained in detail with reference to FIG. 3 as follows.

[0126] First, the statement "separation under the above acidic conditions is performed using a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio)" is of significant importance. This aqueous hydrochloric acid solution of a specific concentration effectively separates AcHA while preventing the decomposition of AcHA caused by excessive acidic conditions. Experimental results confirmed that if a higher concentration of hydrochloric acid is used, the decomposition of AcHA occurs, resulting in a decrease in yield, and if a lower concentration is used, the separation efficiency decreases, leading to a decrease in purity.

[0127] Next, we will examine the critical significance of the configuration "the above non-polar organic solvent washing is performed for 8 to 16 hours." This washing time is essential for effectively removing impurities that may remain in AcHA, particularly acetic acid. When washing is performed for less than 8 hours, impurities are not sufficiently removed, and an acetic acid odor is detected in the final product; when washing is performed for more than 16 hours, it is observed that some of the AcHA dissolves, resulting in a decrease in yield.

[0128] The statement "the yield of acetylated hyaluronic acid obtained by the above method is 75-85%" is an important indicator of the efficiency of the method of the present invention. This yield range is significantly higher compared to existing methods, demonstrating suitability for industrial production. A yield of less than 75% is disadvantageous in terms of economic feasibility, and a yield exceeding 85% was difficult to achieve experimentally.

[0129] Finally, the configuration that "the final product is obtained as a milky white solid" is an important feature that allows for visual verification of the purity and quality of the product. As shown in FIG. 3, AcHA produced by the method of the present invention is obtained in the form of a pure milky white solid. This implies that impurities have been effectively removed, and in particular, a brown or yellow color should be avoided as it suggests the presence of impurities or the occurrence of decomposition reactions.

[0130] By satisfying all the critical conditions of each of these components, the present invention provides a method for consistently producing high-purity, high-yield, and high-quality AcHA. As can be seen in FIG. 3, the final product obtained through these conditions is a pure, milky-white solid with high quality suitable for industrial use.

[0131] FIG. 5 is a photograph of a large-scale reaction apparatus and a product according to one embodiment.

[0132] FIG. 5 shows a large-scale reaction apparatus and a product of the method for producing acetylated hyaluronic acid (AcHA) according to the present invention. This figure is an important visual representation showing that the method of the present invention can be successfully scaled from a laboratory scale to medium-scale production.

[0133] On the left side of the drawing, a 4-liter round-bottom flask is indicated. This flask was used to perform a 50 g scale hyaluronic acid acetylation reaction. Inside the flask is a reaction mixture, which is a mixture of hyaluronic acid, acetic acid (organic acid), acetic anhydride (carboxylic anhydride), and 4-(dimethylamino)pyridine (DMAP, basic catalyst). The color of the reaction mixture is light brown, which indicates that the reaction is in progress.

[0134] A reflux condenser is installed at the top of the flask to prevent solvent evaporation at a reaction temperature of 100°C and to maintain constant reaction conditions. In addition, it can be seen that a magnetic stirrer is used to uniformly stir the reaction mixture.

[0135] A photograph of the final product, acetylated hyaluronic acid (AcHA), is presented on the right side of the drawing. The product was obtained in the form of a milky-white solid, which visually demonstrates that the method of the present invention can produce high-purity AcHA. The milky-white color of the product indicates that impurities have been effectively removed, thereby proving the effectiveness of the post-treatment process presented in the present invention (separation under acidic conditions, washing with a non-polar organic solvent, treatment with a basic aqueous solution, and alcohol precipitation).

[0136] The reaction conditions and results are briefly indicated at the bottom of the drawing. It can be seen that AcHA was obtained with a yield of 79-83% in a 50g scale reaction. This indicates that the method of the present invention can maintain high efficiency even in large-scale production.

[0137] FIG. 5 clearly shows that the method of the present invention can be successfully scaled from laboratory scale to medium-scale production, and that high-purity, high-yield AcHA can be obtained even in mass production. This is important evidence demonstrating the industrial applicability and economic feasibility of the present invention.

[0138] In this case, for the present invention, the acetylation step comprises: (a) mixing hyaluronic acid, acetic acid, acetic anhydride, and 4-(dimethylamino)pyridine in a molar ratio of 1:15:15:1 in a 4-liter round-bottom flask; (b) reacting the mixture at 100°C for 48 hours; (c) cooling the reaction mixture to room temperature and removing the solvent with a rotary evaporator; (d) adding a diluted aqueous hydrochloric acid solution (concentrated HCl: distilled water = 1:37.5 volume ratio) to obtain a solid product; (e) washing the solid product with distilled water and drying it; and (f) stirring the dried solid with hexane for 12 hours and then filtering it.

[0139] At this time, a homogeneous reaction mixture is obtained by using acetic acid as a co-solvent in step (a), and the yield of the product obtained after step (f) is 75-85% (w / w) and the degree of acetylation of the product is 3.8-4.0.

[0140] The reasons for each of the above steps are as follows.

[0141] Step (a) above involves mixing hyaluronic acid, acetic acid, acetic anhydride, and 4-(dimethylamino)pyridine in a 4-liter round-bottom flask in a molar ratio of 1:15:15:1. The large-volume flask used in this step enables uniform heat transfer and stirring while safely handling large quantities of reactants, as shown in Fig. 5. In particular, the excess amounts of acetic acid and acetic anhydride ensure the completeness of the reaction, and the acetic acid acts as a co-solvent to form a homogeneous reaction mixture.

[0142] For step (b) above, this mixture is reacted at 100°C for 48 hours. These conditions were optimized through experiments and are essential to achieve a high yield of AcHA and a precise degree of acetylation of 3.8–4.0. Figure 5 shows that these conditions can be stably maintained even in large-scale reactions.

[0143] In the case of step (c) above, the mixture is cooled to room temperature after the reaction and the solvent is removed using a rotary evaporator. As can be seen in Fig. 5, this process efficiently handles a large amount of solvent, facilitating the concentration of the product and subsequent purification.

[0144] In step (d) above, a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio) is added to obtain a solid product. This dilution ratio enables effective precipitation while minimizing the decomposition of AcHA. Figure 5 clearly shows the large amount of solid product obtained in this process.

[0145] In step (e) above, the solid product is washed with distilled water and dried to remove residual hydrochloric acid and water-soluble impurities. As can be seen in Fig. 5, this process is designed to uniformly process a large amount of product.

[0146] In step (f) above, the dried solid is stirred with hexane for 12 hours and then filtered. This step is an important process for obtaining high-purity AcHA by effectively removing residual organic impurities, particularly acetic acid. Figure 5 shows that this large-scale washing and filtration process can be effectively performed.

[0147] Through this series of processes, AcHA with a degree of acetylation of 3.8-4.0 can be obtained with a high yield of 75-85% (w / w). Figure 5 clearly demonstrates that this method can be successfully applied to actual large-scale production.

[0148] The critical significance of each of the above stages is as follows.

[0149] In step (a) above, first, hyaluronic acid, acetic acid, acetic anhydride, and 4-(dimethylamino)pyridine are mixed in a molar ratio of 1:15:15:1; this specific ratio is essential to obtain a homogeneous reaction mixture. In particular, using acetic acid as a co-solvent is key, as it improves the solubility of hyaluronic acid and provides a homogeneous reaction environment. As can be seen in Fig. 5, the reaction mixture mixed in this ratio is uniformly distributed even in a large flask.

[0150] In the case of step (b) above, the condition of reacting the mixture at 100°C for 48 hours is optimized to obtain a high yield and an appropriate degree of acetylation. If the temperature is lower than 100°C, the reaction rate is too slow, and if it is higher, side reactions increase. 48 hours is a sufficient time for the reaction to complete while preventing unnecessary energy consumption. The reaction apparatus of FIG. 5 is designed to maintain these conditions precisely.

[0151] In step (d) above, a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio) is added to obtain a solid product; this hydrochloric acid solution of a specific concentration effectively precipitates AcHA while preventing decomposition due to excessive acidic conditions. The white solid product shown in Fig. 5 is obtained through this step.

[0152] In the case of step (f) above, the step of filtering the dried solid after stirring with hexane for 12 hours is important for removing residual organic impurities, especially acetic acid. The stirring time of 12 hours is the optimized time to obtain a sufficient washing effect.

[0153] Through the conditions of each of these steps, as can be seen in Fig. 5, pure AcHA with a high yield of 79-85% and a precise degree of acetylation of 3.8-4.0 can be obtained on a 50g scale. This clearly demonstrates the potential for the method of the present invention to be extended beyond the laboratory scale to industrial production.

[0154] In addition, the specific method of implementing step (a) above is as follows.

[0155] First, prepare a 4-liter round-bottom flask. This flask is of an appropriate size to enable uniform heating and effective stirring of the reaction mixture. Clean the inside of the flask thoroughly and dry it completely so that impurities do not affect the reaction.

[0156] Next, accurately weigh 50.0 g of hyaluronic acid (0.131 mol on a monomer basis) and place it in a flask. Use the hyaluronic acid in the form of a fine powder, taking care to ensure there are no lumps. Then, slowly add 750 mL of acetic acid. At this time, use analytical grade acetic acid (purity of 99% or higher). Acetic acid acts as a co-solvent to increase the solubility of hyaluronic acid and plays a key role in forming a homogeneous reaction mixture.

[0157] Next, slowly add 750 mL of acetic anhydride. Use analytical grade acetic anhydride (purity of 99% or higher) and be careful of the exothermic reaction during addition. Finally, accurately weigh and add 16.02 g (0.131 mol) of 4-(dimethylamino)pyridine (DMAP). DMAP acts as a catalyst to increase the efficiency of the acetylation reaction.

[0158] The reaction mixture prepared in this way initially appears as a heterogeneous suspension. However, as acetic acid acts as a co-solvent, it becomes a completely homogeneous solution visible to the naked eye after about 4 to 5 hours. During this process, it is important to continuously stir the mixture using a magnetic stirrer at a speed of 200 to 250 rpm. Care must be taken, as stirring too fast can lead to excessive bubble formation, while stirring too slowly may result in uneven mixing.

[0159] The process of the reaction mixture becoming homogeneous is periodically observed and recorded over time. This ensures the reproducibility of the reaction and can be utilized as a standard for quality control during large-scale production. Once a homogeneous reaction mixture is obtained, the process proceeds to the next step, the heating reaction.

[0160] The homogeneous reaction mixture prepared by this method significantly improves the efficiency of the acetylation reaction and the uniformity of the product, and constitutes a key technical feature of the AcHA manufacturing method of the present invention.

[0161] In addition, the specific method of implementing step (b) above is as follows.

[0162] First, immerse the reaction mixture prepared in a 4-liter round-bottom flask in an oil bath. Set the temperature of the oil bath to exactly 100°C using a digital thermostat. At this time, the temperature deviation must be maintained within ±1°C. To ensure uniform heating of the reaction mixture, continuously stir it using a magnetic stirrer at a speed of 300-400 rpm.

[0163] A condenser is installed at the top of the reaction flask to maintain reflux conditions. Cooling water maintained at 10°C is circulated through the condenser to prevent steam loss. Additionally, a Dean-Stark trap is installed between the reaction flask and the condenser to remove the small amount of water generated during the reaction, thereby shifting the reaction equilibrium toward the product.

[0164] The progress of the reaction is monitored by collecting a small sample (approx. 0.5 mL) every 12 hours. The collected sample is immediately placed in an ice bath to stop the reaction, and then the degree of acetylation is confirmed through 1H NMR analysis. In this process, the reaction rate and the degree of acetylation can be precisely tracked.

[0165] After 48 hours, remove the oil bath and slowly cool the reaction mixture to room temperature (20-25°C). At this time, the cooling rate should not exceed 10°C per hour. This is because rapid temperature changes can adversely affect the quality of the product.

[0166] After the reaction is complete, the color of the mixture turns pale yellow and the viscosity increases. This is a visual indicator that the acetylation reaction has been successfully carried out.

[0167] Temperature and time are very important factors in this step; at temperatures below 100°C, the reaction rate decreases significantly, making it difficult to reach the desired degree of acetylation within 48 hours. On the other hand, at temperatures exceeding 100°C, side reactions increase, which may lower the purity of the product. Furthermore, 48 hours was identified as the optimal time, sufficient for the reaction to complete while minimizing unnecessary energy consumption and side reactions.

[0168] Through such precise control of reaction conditions, the present invention can obtain high-purity AcHA with a degree of acetylation of 3.8-4.0 in a high yield of 75-85%, which is a level suitable for industrial production.

[0169] In addition, the specific method of implementing step (c) above is as follows.

[0170] First, once the reaction proceeding at 100°C for 48 hours is complete, remove the reaction flask from the constant temperature bath and let it cool naturally at room temperature (20-25°C) for about 2 hours. At this time, cold water can be circulated outside the reaction flask to increase the cooling rate. Since the viscosity of the reaction mixture may increase during the cooling process, it is important to maintain uniformity by continuing stirring.

[0171] After cooling is complete, the reaction mixture is divided into 3 to 4 1-liter round-bottom flasks. This takes into account the capacity of the rotary evaporator and is necessary for efficient solvent removal. Each flask is mounted on the rotary evaporator in sequence, and solvent removal is initiated initially under conditions of a water bath temperature of 50°C and a reduced pressure of 150 mbar.

[0172] The solvent removal process takes a total of 3 to 4 hours, during which the bath temperature and pressure are adjusted stepwise. Specifically, after 1 hour, the bath temperature is raised to 60°C and the pressure is lowered to 100 mbar. After 2 hours, the bath temperature is adjusted to 70°C and the pressure to 50 mbar. During the final hour, the bath temperature is maintained at 80°C and the pressure is lowered to 10 mbar to remove residual solvent as much as possible.

[0173] One point to note during this process is that the reaction mixture may form foam and overflow under reduced pressure conditions. To prevent this, the degree of reduced pressure should be adjusted slowly initially, and if necessary, the pressure should be temporarily suspended to allow the foam to subside.

[0174] Once the solvent removal is complete, a viscous brown concentrate remains inside the flask. The weight of this concentrate is measured to check the extent of solvent removal. Generally, the solvent is removed until about 15-20% of the initial reaction mixture weight remains.

[0175] These cooling and solvent removal processes play a crucial role in increasing the efficiency of the subsequent acid precipitation step and improving the purity of the product. Additionally, the removed solvent can be reused if necessary, thereby enhancing economic efficiency.

[0176] In addition, the above step (d) can be specifically implemented as follows.

[0177] First, a diluted aqueous hydrochloric acid solution is prepared by mixing concentrated hydrochloric acid (35-37% HCl) and distilled water in a volume ratio of 1:37.5. This ratio is optimized to ensure effective precipitation of AcHA while simultaneously preventing decomposition caused by excessive acidic conditions. For example, to prepare 1000 mL of diluted aqueous hydrochloric acid solution, 26 mL of concentrated hydrochloric acid and 974 mL of distilled water are used.

[0178] Next, the prepared diluted aqueous hydrochloric acid solution is slowly added to the reaction mixture from which the reaction is complete and the solvent has been removed. At this time, the temperature of the reaction mixture must be maintained at room temperature (20-25°C). The addition of the diluted aqueous hydrochloric acid solution is carried out slowly over about 10-15 minutes to avoid a violent reaction or a rapid rise in temperature.

[0179] The amount of diluted aqueous hydrochloric acid solution added must be adjusted according to the volume of the reaction mixture; generally, about 2 to 3 times the volume of the reaction mixture is used. For example, for a 50g scale reaction, approximately 1500–2000 mL of diluted aqueous hydrochloric acid solution is used.

[0180] The reaction mixture is continuously stirred while adding the diluted aqueous hydrochloric acid solution to ensure uniform mixing. The stirring speed is maintained at approximately 200-300 rpm to prevent physical damage to the product caused by excessive stirring.

[0181] Once the addition of the diluted aqueous hydrochloric acid solution is complete, the mixture is stirred for an additional 30 to 60 minutes to ensure complete precipitation. During this process, the gradual formation of white solid AcHA can be observed.

[0182] After precipitation is complete, the mixture is cooled to 5-10°C to induce further precipitation. This cooling process is carried out slowly over approximately 1-2 hours to obtain a product with a uniform particle size.

[0183] Finally, the formed solid AcHA is separated by vacuum filtration. During the filtration process, the solid product is washed 2-3 times with a small amount of cold distilled water (5-10°C) to remove residual acid and impurities. The solid AcHA obtained in this way is in the form of a pure white powder and is dried in a vacuum oven at 50-60°C for 8-12 hours before undergoing a subsequent purification process.

[0184] Through such specific implementation methods, an AcHA solid product with high purity and uniform particle size can be effectively obtained.

[0185] In addition, the specific method of implementing step (e) above is as follows.

[0186] First, transfer the solid product obtained in the previous step into a Buchner funnel. Use Whatman No. 1 or a filter with a similar pore size. Connect a vacuum pump to set a reduced pressure of approximately 15-20 mmHg.

[0187] Distilled water washing is performed a total of three times. In the first wash, distilled water equivalent to about five times the weight of the product is used. For example, if the solid product is 50g, 250mL of distilled water is used. Distilled water is poured slowly to ensure that the entire surface of the solid is washed evenly. During this process, the temperature is maintained at room temperature (20-25℃).

[0188] For the second and third washes, distilled water equivalent to three times the weight of the product is used for each. Vacuum filtration is performed for about 5 minutes at each washing step to ensure that the washing solution is sufficiently removed.

[0189] After the final wash, the solid product is subjected to reduced pressure in a Buchner funnel for approximately 30 minutes to remove as much residual moisture as possible. Then, the solid product is transferred to a clean glass or ceramic drying container.

[0190] The drying process is carried out in two stages. First, the material is dried in a vacuum desiccator at room temperature (20-25°C) for 12 hours. During this time, the pressure inside the desiccator is maintained at approximately 10 mmHg. Silica gel or anhydrous calcium chloride is used as the desiccant, and it is replaced with a new desiccant every 6 hours.

[0191] In the second step, the product is dried using a vacuum oven at 40°C for an additional 12 hours. During this process, the pressure inside the oven is maintained at 5 mmHg or less. After drying is complete, the weight of the product is measured to verify that a constant weight has been reached. If necessary, drying can be extended for an additional 3 to 6 hours.

[0192] Through this washing and drying process, residual acids, salts, and organic solvents are effectively removed, and high-purity AcHA solid with a moisture content of 1% or less can be obtained. The dried product obtained in this way is placed in a sealed brown glass bottle and stored in a desiccator containing silica gel.

[0193] This washing and drying method plays a crucial role in increasing the purity and improving the stability of the product. In particular, the multi-stage washing and stepwise drying processes are essential for maintaining the uniform quality of AcHA, which directly affects the quality of subsequent processes and the final product.

[0194] In addition, the specific method of implementing step (f) above and the result thereof are as follows.

[0195] The dried solid AcHA obtained in step (e) above is placed in a 2L capacity beaker, and 1L of hexane is slowly poured in. At this time, the ratio of solid AcHA to hexane is approximately 1:10 (w / v). This mixture is continuously stirred using a magnetic stirrer at a speed of 300 rpm at room temperature (20-25℃) for 12 hours. The stirring time can be adjusted within the range of 11-13 hours, but 12 hours yielded optimal results.

[0196] After 12 hours of stirring are completed, the mixture is filtered under reduced pressure using a Buchner funnel. Whatman No. 1 filter paper is used for filtration, and the solid is washed with an additional 200 mL of hexane during the filtration process. The filtered solid is dried in a vacuum oven at 40°C for 12 hours to completely remove residual hexane.

[0197] The final product obtained through this process is a pure white solid in the form of a fine powder. The yield at this stage ranged from 75-85% (w / w) relative to the initially added hyaluronic acid, with most cases showing a yield of 79-83%. Specifically, in a 50g scale reaction, 40.5g of AcHA was obtained with an average yield of 81%.

[0198] The degree of acetylation of the product was confirmed by 1H NMR analysis. 10 mg of the product was dissolved in 0.7 mL of D2O and measured using a 400 MHz NMR instrument. The degree of acetylation was calculated by determining the integration ratio of the acetyl group peak in the δ 1.8–2.1 ppm region and the polysaccharide backbone peak in the δ 3.0–4.5 ppm region in the NMR spectrum. As a result, the degree of acetylation remained constant within the range of 3.8–4.0, and in most cases, showed a value of 3.9 ± 0.1.

[0199] These results demonstrate that the method of the present invention can stably produce AcHA with a uniform degree of acetylation at a high yield. In particular, the washing process using hexane effectively removed organic impurities such as residual acetic acid, contributing significantly to obtaining a high-purity product. Furthermore, this method was successfully applied on a 50g scale, proving its potential for expansion to an industrial scale.

[0200] In this case, for the present invention, the basic aqueous solution treatment and alcohol precipitation steps consist of (g) dissolving the acetylated hyaluronic acid in a 20% concentration sodium bicarbonate aqueous solution and (h) adding isopropanol to precipitate the sodium acetylated hyaluronate salt and filtering it.

[0201] At this time, through steps (g) and (h) above, a pure sodium acetylated hyaluronate salt in which no acetic acid peak is detected during gas chromatography analysis is obtained with a weight recovery rate of 98-100% relative to the acetylated hyaluronate added, and the pure sodium acetylated hyaluronate salt dissolves until no insoluble particles are visible when a 1% (w / v) aqueous solution is prepared at 20°C and observed visually, and the C Log P value of the monomer of the pure sodium acetylated hyaluronate salt is -1.12, and it is confirmed by NMR analysis that acetylation has occurred on all four -OH groups present in the hyaluronate starting material, and the above manufacturing method can be performed with a yield of 79-83% on a 50g scale and up to a 100g scale.

[0202] The reasons for each of the above configurations are as follows.

[0203] The above steps describe in detail the final purification and sodium salt formation process of acetylated hyaluronic acid. First, the process of dissolving acetylated hyaluronic acid in a 20% aqueous sodium bicarbonate solution in step (g) holds significant meaning as follows. This sodium bicarbonate solution completely dissolves the acetylated hyaluronic acid to form a homogeneous solution, while simultaneously converting the carboxyl groups into sodium salts with appropriate basicity. This plays a key role in improving the water solubility of the final product. Additionally, any trace acidic impurities that may remain are effectively neutralized during this process. As shown in Figure 5, the solution after this step appears uniform and transparent.

[0204] Next, the process of adding isopropanol in step (h) to precipitate sodium acetylated hyaluronate and filtering is a key step for efficiently recovering the purified final product. Isopropanol selectively precipitates polymeric electrolytes such as sodium acetylated hyaluronate while keeping water-soluble impurities dissolved, allowing only the pure target substance to be separated. Furthermore, isopropanol is suitable for industrial-scale production in terms of cost-effectiveness and ease of handling. As can be seen in Fig. 5, a pure white solid product can be obtained through this step.

[0205] The final product obtained through this purification process exhibits the following excellent characteristics. First, it can be confirmed that residual acetic acid has been completely removed, as no acetic acid peak is detected during gas chromatography analysis. Second, it demonstrates the efficiency of the purification process by showing a high weight recovery rate of 98-100%. Third, when a 1% (w / v) aqueous solution is prepared at 20°C, it dissolves completely to the extent that there are no insoluble particles observable to the naked eye, indicating very high purity and uniformity of the product.

[0206] Furthermore, the monomer of acetylated sodium hyaluronate prepared by this method exhibits a C Log P value of -1.12, showing significantly higher lipophilicity compared to hyaluronic acid (C Log P = -2.37). As can be seen from the NMR analysis results in Fig. 4, it is confirmed that the reaction proceeded completely, with all -OH groups of the hyaluronic acid starting material being acetylated. Finally, as shown in Fig. 5, this preparation method was successfully scaled from 50g to 100g, maintaining a high yield of 79–83%. This empirically demonstrates that this method is suitable for industrial-scale production.

[0207] The above characteristics demonstrate that the method for manufacturing acetylated hyaluronic acid according to the present invention can consistently produce a product with high purity, high yield, excellent solubility, and lipophilicity, and can be successfully scaled from laboratory scale to medium-scale production. Therefore, the present invention can be said to possess very high value in the industrial production and application of acetylated hyaluronic acid.

[0208] The critical significance for each component is as follows.

[0209] The critical significance of using a 20% concentration aqueous sodium bicarbonate solution in the above composition is as follows. The inventors conducted experiments with aqueous sodium bicarbonate solutions of various concentrations and discovered that optimal results could be obtained at a 20% concentration. At lower concentrations, complete dissolution of acetylated hyaluronic acid was difficult, and at higher concentrations, there was a risk of partial hydrolysis of the acetyl group due to excessive basicity. A 20% concentration aqueous sodium bicarbonate solution provides optimal conditions for completely dissolving acetylated hyaluronic acid while preserving the acetyl group. This can be confirmed by the fact that the acetyl group peak remains intact in the 1H NMR spectrum of Figure 4.

[0210] The critical significance of the process of precipitating acetylated sodium hyaluronate using isopropanol is as follows. Experiments with various alcohols revealed that isopropanol most effectively precipitates acetylated sodium hyaluronate while also effectively removing impurities. Methanol and ethanol showed low precipitation efficiency, while higher alcohols such as butanol presented a problem in that they were difficult to remove during the post-treatment process. Isopropanol possesses appropriate polarity, allowing it to effectively precipitate acetylated sodium hyaluronate while keeping water-soluble impurities in a dissolved state for removal. The appearance of the pure acetylated sodium hyaluronate obtained through this process can be seen in Fig. 5.

[0211] The critical significance of obtaining pure acetylated sodium hyaluronate with a weight recovery rate of 98-100% is as follows. This high recovery rate demonstrates that the method of the present invention is highly efficient and economical. A recovery rate of less than 98% is uneconomical for industrial production, while a rate exceeding 100% implies that impurities have not been completely removed. Therefore, a recovery rate of 98-100% is the optimal range that simultaneously satisfies purity and economic feasibility. This can be confirmed through the quantity and quality of the large-scale production results shown in Fig. 5.

[0212] The critical significance of the characteristic of dissolving until no insoluble particles are visible to the naked eye when a 1% (w / v) aqueous solution is prepared at 20°C is as follows. This demonstrates that the sodium acetylated hyaluronate prepared by the method of the present invention possesses excellent water solubility. Practicality is reduced at concentrations below 1%, and complete dissolution at concentrations above 1% is very important for industrial applications. 20°C is a temperature representative of room temperature, meaning that it dissolves easily without special heating. This characteristic can be indirectly confirmed by the fact that the NMR spectrum in Fig. 4 was measured clearly in the D2O solvent.

[0213] The critical significance of the monomer having a C Log P value of -1.12 is as follows. This value is significantly higher than the C Log P value of hyaluronic acid, which is -2.37, indicating that the acetylated hyaluronic acid of the present invention has improved lipophilicity by more than 10 times compared to the original hyaluronic acid. If the C Log P value is lower than -1.12, the effect of improving lipophilicity is negligible, and if it is higher, water solubility is excessively reduced, resulting in reduced practicality. Therefore, a C Log P value of -1.12 represents the optimal balance between water solubility and lipophilicity. This characteristic can be indirectly confirmed through the peak intensity of the acetyl group in the NMR spectrum of Figure 4.

[0214] The critical significance of confirming through NMR analysis that acetylation has occurred on all four -OH groups present in the hyaluronic acid starting material is as follows. This demonstrates that the method of the present invention has achieved complete acetylation. Partial acetylation has a limited effect on improving physical properties, and excessive acetylation can lead to molecular degradation. Therefore, a state in which all four -OH groups are acetylated provides optimal physical properties. This can be clearly confirmed through the peak integral value of the acetyl group in the NMR spectrum of Figure 4.

[0215] The critical significance of being able to achieve a yield of 79-83% at a 50g scale and up to a 100g scale is as follows. This demonstrates that the method of the present invention can be successfully scaled beyond the laboratory scale to medium-scale production. A yield of 79-83% is sufficiently high for industrial production, and the 100g scale is a scale that can serve as the basis for pilot production. Industrial applications are limited at smaller scales, and additional process optimization may be required at larger scales. Therefore, the 50-100g scale is an important indicator proving industrialization potential. This can be clearly confirmed through the large-scale reaction apparatus shown in Fig. 5 and the amount of acetylated hyaluronic acid produced.

[0216] In addition, the specific method of implementing step (g) above is as follows.

[0217] First, prepare a 20% aqueous solution of sodium bicarbonate. To do this, slowly add 200 g of sodium bicarbonate to 800 mL of purified water and dissolve it completely. Maintain the temperature of the solution at room temperature (20-25°C). Use a pH meter to check that the pH of the prepared solution is within the range of 8.0-8.5.

[0218] Next, take 50 g of the previously prepared acetylated hyaluronic acid and place it in a 1 L capacity beaker. Then, begin slowly adding the previously prepared 20% sodium bicarbonate aqueous solution. Initially, add 100 mL first and stir slowly at 200-300 rpm using a stirrer. During this process, stir carefully to prevent the acetylated hyaluronic acid from clumping on the surface of the solution.

[0219] As dissolution proceeds, slowly add an additional 50 mL of sodium bicarbonate aqueous solution. After each addition, stir for 5–10 minutes to observe the state of dissolution. The total amount added is approximately 400–450 mL of 20% sodium bicarbonate aqueous solution per 50 g of acetylated hyaluronic acid. The exact amount is adjusted while observing the state of dissolution.

[0220] During the dissolution process, the temperature of the solution should be maintained at 25-30°C. If necessary, dissolution can be promoted by slight heating (below 35°C), but care should be taken as excessive heating may cause hydrolysis of the acetyl group.

[0221] Complete dissolution is generally achieved within 1 to 2 hours. Once dissolution is complete, the solution is stirred for an additional 30 minutes to ensure uniformity. Finally, the solution is visually inspected to check for the absence of insoluble particles. If necessary, it can be filtered using a 0.45 μm filter.

[0222] Once dissolution is complete, the solution can be immediately processed to the next step, alcohol precipitation, or stored at 4°C for up to 24 hours if necessary. For long-term storage, freezing (-20°C) is recommended to ensure the stability of the acetyl group.

[0223] Through this process, acetylated hyaluronic acid is completely dissolved and converted into a sodium salt form, which plays an important role in increasing the efficiency of the purification and separation process in the next step.

[0224] In addition, the specific implementation method for the above step (h) is as follows.

[0225] First, prepare an acetylated hyaluronic acid solution dissolved in a 20% aqueous sodium bicarbonate solution. Slowly add isopropanol to this solution, adjusting the addition rate to 10-20 mL per minute. The amount of isopropanol added should be 2-3 times the volume of the acetylated hyaluronic acid solution. For example, if the acetylated hyaluronic acid solution is 100 mL, add 200-300 mL of isopropanol.

[0226] When adding isopropanol, the solution is stirred at 400-600 rpm to ensure uniform mixing. After the addition of isopropanol is complete, the mixture is cooled to 0-5°C and maintained for 2-3 hours to complete precipitation. During this process, sodium acetylated hyaluronate is formed as a fine white precipitate.

[0227] After precipitation is complete, the mixture is filtered under reduced pressure using a Buchner funnel equipped with filter paper with a pore size of 5-10 μm. During filtration, the pressure is maintained at 400-600 mmHg. The solid obtained after filtration is washed 2-3 times with 50-100 mL of cold isopropanol (0-5°C) to remove any remaining water-soluble impurities.

[0228] The solid, after filtration and washing, is dried in a vacuum oven at 40-50°C for 12-24 hours. During this time, the vacuum level is maintained at 10-20 mmHg. After drying is complete, the product is cooled to room temperature in a desiccator.

[0229] The finally obtained sodium acetylated hyaluronate is in the form of a fine white powder, with a moisture content of 1% or less and a particle size ranging from 50 to 200 μm. Through this process, pure sodium acetylated hyaluronate can be obtained with a weight recovery rate of 98-100% relative to the initially added acetylated hyaluronate.

[0230] The conditions presented in this embodiment were experimentally derived to obtain optimal results and can be adjusted within a range of ±10% of each condition. The acetylated sodium hyaluronate obtained through this method has high purity and uniform physical properties and can be ideally used for subsequent processes or product manufacturing.

[0231] In addition, the process of obtaining pure acetylated sodium hyaluronate through the above steps (g) and (h) is carried out as follows.

[0232] First, in step (g), the previously obtained acetylated hyaluronic acid is dissolved in a 20% (w / v) aqueous sodium bicarbonate solution. In this process, the volume of the sodium bicarbonate solution used is five times the weight of the acetylated hyaluronic acid. For example, 50 g of acetylated hyaluronic acid is added to 250 mL of a 20% aqueous sodium bicarbonate solution. The dissolution process is carried out at room temperature (20-25°C), and the stirring speed is maintained at 200-300 rpm. Stirring is continued for 2-3 hours to ensure complete dissolution.

[0233] Next, in step (h), isopropanol is added to the solution to precipitate the sodium acetylated hyaluronate salt. The amount of isopropanol added is four times the volume of the sodium bicarbonate aqueous solution. Following the previous example, 1000 mL of isopropanol is slowly added dropwise to 250 mL of aqueous solution. The dropwise addition rate is maintained at 20-30 mL per minute, and the stirring speed during addition is increased to 400-500 rpm to induce uniform precipitation.

[0234] After the precipitation is complete, the mixture is left at 4°C for 2 hours to complete the precipitation. Subsequently, the precipitate is separated by filtration using a Buchner funnel and a vacuum filtration apparatus. The solid obtained after filtration is washed twice with cold isopropanol (0-5°C) to remove any remaining water-soluble impurities.

[0235] The washed solid is dried in a vacuum oven at 40°C for 12 hours to completely remove residual solvent. The final product obtained in this way is in the form of a fine white powder and is obtained with a weight recovery rate of 98-100% relative to the input acetylated hyaluronic acid.

[0236] Gas chromatography analysis for purity verification is performed as follows. Accurately weigh 0.1 g of the sample, dissolve it in 1 mL of methanol, and filter it through a 0.45 μm filter. Inject this solution into a gas chromatograph (e.g., Agilent 7890B) for analysis. A DB-WAX column (30 m × 0.25 mm, 0.25 μm) is used, and the oven temperature is maintained at 40°C for 2 minutes, then increased to 220°C at a rate of 10°C / min and maintained for 5 minutes. The injection port and detector (FID) temperatures are each set to 250°C. When analyzed under these conditions, it can be confirmed that no peak corresponding to acetic acid (approx. 2.5 min) is detected in the final product.

[0237] Through such specific implementation methods, high-purity acetylated sodium hyaluronate salt can be obtained with a high recovery rate, and a pure product with residual acetic acid completely removed can be manufactured.

[0238] In addition, a specific method for confirming the solubility characteristics of the pure acetylated sodium hyaluronate salt is as follows.

[0239] First, accurately weigh 1.00 g of pure acetylated sodium hyaluronate using a precise electronic balance. Add 99 mL of distilled water to a clean 100 mL beaker and place the beaker in a constant temperature water bath maintained at 20 ± 0.5 °C. Use a thermometer to check if the temperature of the distilled water has reached 20 °C.

[0240] Next, the measured 1.00g of sodium acetylated hyaluronate is added little by little to distilled water while stirring slowly with a glass rod. Care must be taken to prevent lumps from forming, and if necessary, ultrasonic treatment can be applied for 10 seconds to disperse the lumps.

[0241] During the dissolution process, observe the solution at 5-minute intervals to check for the presence of insoluble particles. This observation is performed by carefully examining the solution through the bottom and sides of the beaker under bright lighting. If insoluble particles are observed, continue stirring with a glass rod while proceeding with the dissolution process.

[0242] Once it is determined that complete dissolution has occurred, let the solution stand for 15 minutes to stabilize it, and then observe it carefully once again. At this time, no insoluble particles should be observed at all.

[0243] After the dissolution process is complete, the final volume of the solution is accurately adjusted using a 100 mL volumetric flask. The solution prepared in this way becomes a 1% (w / v) aqueous solution of acetylated sodium hyaluronate.

[0244] The entire dissolution process must not exceed one hour, and the dissolution time must be recorded. To minimize temperature fluctuations during the dissolution process, the temperature of the constant temperature water bath must be continuously monitored and maintained.

[0245] Finally, transfer the prepared 1% solution into a clear glass vial, place it against a bright background, and visually inspect it to confirm that it is a completely clear solution free of insoluble particles or turbidity. Record the results of this observation, and if necessary, use a digital camera to photograph the transparency of the solution.

[0246] Through this method, it can be confirmed that the sodium acetylated hyaluronate of the present invention is completely dissolved at a concentration of 1% (w / v) at 20°C to form a transparent aqueous solution free of insoluble particles. This is an important characteristic that demonstrates the excellent water solubility and uniformity of the product of the present invention.

[0247] In addition, a specific method for obtaining the characteristic of the monomer of the pure acetylated sodium hyaluronate salt having a C Log P value of -1.12 is as follows.

[0248] In addition, a specific method for confirming the structure of pure acetylated sodium hyaluronate salt according to the present invention is as follows.

[0249] First, 10 mg of the prepared sodium hyaluronate acetylated sample is taken and completely dissolved in 0.7 mL of water deuteride (D2O). This solution is transferred to a 5 mm NMR tube, and the spectrum is measured at room temperature using a 400 MHz 1H-NMR spectrometer. For the measurement, 3-(trimethylsilyl)propionicate-d4 sodium salt (TSP-d4) is used as the internal standard, and the chemical shift value (δ) is expressed in ppm units.

[0250] Figure 4 shows a representative 1H-NMR spectrum of acetylated sodium hyaluronate measured by this method. A strong single peak observed in the δ 1.8-2.1 ppm region of the spectrum corresponds to the methyl hydrogen of the acetyl group. The degree of acetylation can be quantitatively evaluated by comparing the integral value of this peak with the integral values ​​of other characteristic peaks present in the monosaccharide units of hyaluronic acid.

[0251] Specifically, the complex multiplets observed in the δ 3.2-4.5 ppm region correspond to hydrogens on the sugar ring of the hyaluronic acid backbone. Among these, the peak near δ 4.3-4.5 ppm corresponds to anomer hydrogen, and when the integral value of this peak is set to 1, it can be confirmed that the integral value of the methyl hydrogen peak of the acetyl group (δ 1.8-2.1 ppm) approaches 12. This means that four acetyl groups have been introduced per hyaluronic acid monomer unit.

[0252] In addition, it can be confirmed that the hydroxyl group (-OH) peak, which should be observed in the δ 4.5-5.5 ppm region, has completely disappeared. This indicates that all hydroxyl groups originally present in hyaluronic acid have been acetylated.

[0253] When these NMR analysis results are interpreted comprehensively, it can be clearly confirmed that the acetylated sodium hyaluronate prepared according to the present invention has undergone acetylation on all four -OH groups present in the hyaluronic acid starting material. This is evidenced in [Fig. 4] by the fact that the integral value of the methyl hydrogen peak of the acetyl group (δ 1.8-2.1 ppm) is close to 12 times the integral value of the anomeric hydrogen peak (δ 4.3-4.5 ppm), and the -OH peak (δ 4.5-5.5 ppm) has completely disappeared.

[0254] This NMR analysis method is an essential process for accurately confirming the structure of the prepared acetylated sodium hyaluronate and verifying the completeness of the acetylation reaction. The results obtained through this method serve as important scientific evidence proving that the manufacturing method of the present invention has achieved complete acetylation of hyaluronic acid.

[0255] In addition, the methods for manufacturing acetylated hyaluronic acid on 50g and 100g scales are specifically described as follows.

[0256] In the 50g scale example, a 4L round-bottom flask is used as shown in FIG. 5. First, 50.0g of hyaluronic acid (0.131 mol based on monomer) is placed in the flask, and 750mL of acetic acid, 750mL of acetic anhydride, and 16.02g (0.131 mol) of 4-(dimethylamino)pyridine (DMAP) are added in sequence. When this mixture is heated to 100°C, a homogeneous solution is formed after about 4.5 hours. The reaction is continued for 48 hours in this state.

[0257] After the reaction is complete, the mixture is cooled to room temperature and acetic acid and acetic anhydride are removed using a rotary evaporator. A diluted aqueous hydrochloric acid solution (40 mL of concentrated HCl diluted in 1500 mL of distilled water) is added to the remaining mixture and stirred for 20 minutes to obtain a solid product from the reaction mixture in the form of a slurry. This solid is separated by filtration, washed with 500 mL of distilled water, and then dried.

[0258] 500 mL of hexane is added to the dried solid and stirred for 12 hours, then filtered to obtain the final acetylated hyaluronic acid product. As shown on the right side of [Fig. 5], the product obtained through this process is in the form of a milky white solid, and the yield reaches 57 g (79%).

[0259] In the example of scaling up to a 100g scale, a reactor with a larger capacity than that shown in FIG. 5 is used. Specifically, a 10L round-bottom flask or a jacketed reactor of equivalent capacity is used. The amount of hyaluronic acid added is increased to 100.0g (0.262 mol based on monomer), and the amounts of other reagents are increased proportionally. That is, 1500mL of acetic acid, 1500mL of acetic anhydride, and 32.04g (0.262 mol) of DMAP are used.

[0260] The reaction conditions and post-treatment process are maintained identically to the 50g scale example, but the amounts of solvent and reagent used in each step are increased proportionally. For example, 1000 mL of distilled water and 1000 mL of hexane are used in the washing step.

[0261] In a 100g scale reaction, the reaction time and temperature are maintained the same as in a 50g scale, and the time to form a uniform solution is not significantly different. However, considering the heat transfer efficiency, it is necessary to slightly extend the heating and cooling times.

[0262] Through this 100g scale operation, an amount of acetylated hyaluronic acid equivalent to about twice the 50g scale result shown in Fig. 5 can be obtained. Specifically, the final yield is about 115-120g (79-83%).

[0263] As such, the successful scaling up from 50g to 100g demonstrates that the manufacturing method of the present invention has a high potential for transition to mass production. In particular, the fact that the yield tends to be maintained or even slightly increased with scale expansion is an important indicator of the excellent scalability of the present method.

[0264] Hereinafter, the structure of the present invention and the resulting effects will be explained in more detail through specific embodiments and comparative examples. However, these embodiments are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these embodiments.

[0265] [Example 1] Method for manufacturing AcHA according to the present invention

[0266] 50.0 g (0.131 mol, monomer basis) of hyaluronic acid was placed in a 4-liter round-bottom flask, followed by the addition of 750 mL of acetic acid, 750 mL of acetic anhydride, and 16.02 g (0.131 mol) of 4-(dimethylamino)pyridine (DMAP). The reaction mixture was heated to 100°C and stirred for 48 hours. A homogeneous solution was formed approximately 4.5 hours after the start of the reaction. After the reaction was complete, the mixture was cooled to room temperature, and acetic acid and acetic anhydride were removed using a rotary evaporator. A diluted aqueous hydrochloric acid solution (40 mL of concentrated HCl diluted in 1500 mL of distilled water) was added to the residue and stirred for 20 minutes to obtain a solid product. This solid was filtered, washed with 500 mL of distilled water, and dried. 500 mL of hexane was added to the dried solid and stirred for 12 hours, after which it was filtered. The obtained solid was dissolved in 500 mL of a 20% aqueous sodium bicarbonate solution, and then 1500 mL of isopropanol was slowly added to precipitate acetylated sodium hyaluronate. The precipitate was filtered and dried under vacuum to obtain a milky white solid of 39.5 g (yield 81%) as the final product.

[0267] [Comparative Example 1] Acetylation under strong acid conditions

[0268] 50.0 g of hyaluronic acid was placed in a 1-liter round-bottom flask, and 50 mL of sulfuric acid and 750 mL of acetic anhydride were slowly added. The reaction mixture was heated to 50°C and stirred for 24 hours. After the reaction was complete, the mixture was poured into 2 L of ice water to form a precipitate. The precipitate was filtered, washed with 500 mL of cold water, and dried under vacuum. Finally, 18.5 g of a brown solid product (yield 38%) was obtained.

[0269] [Comparative Example 2] Acetylation under basic conditions

[0270] 50.0 g of hyaluronic acid was placed in a 2-liter round-bottom flask, and 1,000 mL of pyridine and 750 mL of acetic anhydride were added. The reaction mixture was heated to 100°C and stirred for 48 hours. After the reaction was complete, the mixture was poured into 2 L of ethanol to form a precipitate. The precipitate was filtered, washed with 500 mL of ethanol, and dried under vacuum. Finally, 47.5 g of a light brown solid product (yield 97%) was obtained.

[0271] [Comparative Example 3] Reaction without acetic acid

[0272] 50.0 g of hyaluronic acid, 1500 mL of acetic anhydride, and 16.02 g of DMAP were added to a 4-liter round-bottom flask. The reaction mixture was heated to 100°C and stirred for 48 hours. The reaction mixture remained heterogeneous throughout the entire process. After the reaction was complete, unreacted hyaluronic acid was removed by filtration, and the filtrate was poured into 2 L of ice water to form a precipitate. The precipitate was filtered, washed with 500 mL of water, and dried under vacuum. Finally, 22.0 g of a light brown solid product (yield 45%) was obtained.

[0273] [Comparative Example 4] Prepared without basic post-treatment

[0274] The reaction was carried out in the same manner as in Example 1, but the step of treating with a 20% sodium bicarbonate aqueous solution was omitted. Instead, the solid obtained after hexane washing was directly dried under vacuum to obtain a light brown solid of 36.5 g (yield 75%) as the final product.

[0275] [Comparative Example 5] Reaction at low temperature

[0276] The reaction was carried out with the same composition as in Example 1, but the reaction temperature was lowered to 50°C and stirred for 48 hours. The reaction mixture maintained a heterogeneous state throughout the entire process. After the reaction was completed, a light brown solid of 24.5 g (yield 50%) was obtained as a final product through the same post-treatment process as in Example 1.

[0277] [Yield and Acetylation Measurement]

[0278] The yield was calculated by weighing the final product obtained in each example and comparative example using a precision electronic balance. To measure the degree of acetylation, 10 mg of each sample was dissolved in 0.7 mL of deuterated dimethyl sulfoxide (DMSO-d6) and 1H NMR analysis was performed. Spectra were obtained using a Bruker Avance III 400 MHz NMR spectrometer, and chemical shift values ​​were corrected using tetramethylsilane (TMS) as an internal standard. The degree of acetylation was calculated using the integration ratio of the hyaluronic acid N-acetyl peak (δ 2.0 ppm) and the newly introduced O-acetyl peak (δ 2.1-2.3 ppm).

[0279]

[0280] The acetylated hyaluronic acid (AcHA) obtained in Example 1 showed an excellent yield of 81% and a high degree of acetylation of 3.92. This demonstrates that the method of the present invention enables an efficient acetylation reaction and simultaneously allows for obtaining the target product with a high yield. In the case of Comparative Example 1 (strong acid conditions), a low yield of 38% and an incomplete degree of acetylation of 2.15 were observed. This suggests that under strong acid conditions, the degradation of hyaluronic acid occurs, significantly reducing the yield, and simultaneously, the acetylation reaction proceeds incompletely.

[0281] Comparative Example 2 (basic conditions) showed a very high yield of 97%, but the degree of acetylation was very low at 1.78. This means that although pyridine acted as an effective solvent enabling a high yield, the efficiency of the acetylation reaction itself was low.

[0282] In the case of Comparative Example 3 (reaction without acetic acid), a low yield of 45% and an incomplete degree of acetylation of 3.45 were observed. This demonstrates that acetic acid plays an important role in ensuring the uniformity of the reaction mixture and in efficient acetylation.

[0283] Comparative Example 4 (prepared without basic post-treatment) showed a relatively high yield of 75% and a high degree of acetylation of 3.88. However, the yield was somewhat lower compared to Example 1, which suggests that basic post-treatment contributes to both the improvement of product purity and the improvement of yield.

[0284] Comparative Example 5 (reaction at low temperature) showed a low yield of 50% and an incomplete degree of acetylation of 2.67. This demonstrates that the reaction temperature has a significant effect on the efficiency and yield of the acetylation reaction.

[0285] Therefore, the method of the present invention (Example 1) showed the best results in terms of yield and degree of acetylation compared to other comparative examples. In particular, the degree of acetylation was 3.92, a value close to 4, demonstrating that all hydroxyl groups of hyaluronic acid were effectively acetylated. These results clearly show that the method of the present invention is highly suitable for the efficient and high-quality production of AcHA.

[0286] [Uniformity Assessment]

[0287] For each example and comparative example, the state of the reaction mixture was observed immediately after the start of the reaction, and at 2, 4, 6, 24, and 48 hours. At each time point, a portion of the reaction mixture (about 1 mL) was taken, placed in a glass vial, and photographed. The uniformity of the mixture, changes in color, and changes in viscosity were recorded through visual observation.

[0288]

[0289] As a result of the uniformity evaluation, Example 1, which is the method of the present invention, exhibited the best uniformity. In the case of Example 1, most of the hyaluronic acid began to dissolve after 4 hours from the start of the reaction, and a completely uniform transparent solution was formed after 6 hours. This is attributed to the effect of using acetic acid as a co-solvent. In the case of Comparative Example 1 (strong acid conditions) and Comparative Example 3 (reaction without acetic acid), a non-uniform state was maintained throughout the entire reaction process. This suggests that the acetylation reaction did not proceed efficiently due to the incomplete dissolution of hyaluronic acid.

[0290] Comparative Example 2 (basic conditions) showed a homogenization process similar to Example 1, but turned a darker brown in the latter part of the reaction. This suggests the possibility that side reactions or decomposition reactions occurred under basic conditions.

[0291] Comparative Example 4 (no basic post-treatment) showed a homogenization process similar to Example 1, but this is judged to be because the reaction conditions were identical. However, it is expected that there will be differences in the characteristics of the final product due to differences in the post-treatment process.

[0292] In the case of Comparative Example 5 (low-temperature reaction), the homogenization rate was significantly slow, and it took 24 hours to reach a completely homogenized state. This indicates that the dissolution and acetylation reaction rates of hyaluronic acid were significantly reduced due to the low reaction temperature.

[0293] These results demonstrate that the method of the present invention can carry out the acetylation reaction of hyaluronic acid more efficiently and uniformly by effectively ensuring the uniformity of the reaction mixture. In particular, it can be seen that using acetic acid as a co-solvent and maintaining an appropriate reaction temperature plays an important role in ensuring the uniformity of the reaction. It is believed that these uniform reaction conditions will serve as a key factor in achieving uniform quality of the final product and high yield.

[0294] [Purity Analysis]

[0295] Purity analysis was performed using gas chromatography (GC) and high-performance liquid chromatography (HPLC). For GC analysis, an Agilent 7890B gas chromatograph was used, equipped with an HP-5 column (30 m x 0.32 mm, 0.25 μm) and detected using a flame ionization detector (FID). The oven temperature was maintained at 50°C for 2 minutes and then increased to 250°C at a rate of 10°C / min. For HPLC analysis, a Waters Alliance e2695 system was used, equipped with a C18 reverse-phase column (150 mm x 4.6 mm, 5 μm) and detected using a UV detector (λ = 210 nm). An acetonitrile / water (40:60, v / v) mixed solvent was used as the mobile phase, and the flow rate was set to 1.0 mL / min.

[0296]

[0297] As a result of gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis, acetylated hyaluronic acid prepared by the method of the present invention (Example 1) exhibited the highest purity. GC analysis showed that in Example 1, residual acetic acid was below the detection limit (0.01%). This demonstrates that the method of the present invention can effectively remove acetic acid. In contrast, residual acetic acid of 0.32-0.85% was detected in Comparative Examples 1-5. In particular, the highest residual acetic acid content (0.85%) was observed in Comparative Example 1, in which the reaction was carried out under strong acid conditions. This suggests that the reaction under strong acid conditions makes it difficult to completely remove acetic acid.

[0298] HPLC analysis results showed that the purity of Example 1 was the highest at 99.5%. This demonstrates that the method of the present invention can produce high-purity acetylated hyaluronic acid. The purity of Comparative Examples 1-5 ranged from 95.2% to 98.1%, showing lower purity than Example 1. In particular, the lowest purity (95.2%) was observed in Comparative Example 1, which was reacted under strong acid conditions. This suggests that reaction under strong acid conditions can induce side reactions that reduce purity.

[0299] The high purity and low residual acetic acid content observed in the method of the present invention (Example 1) are determined to be the result of the formation of a uniform reaction mixture, appropriate reaction temperature and time, and an effective post-treatment process. In particular, it is presumed that the main factors are the formation of a uniform reaction mixture using acetic acid as a co-solvent and the effective removal of residual acetic acid through treatment with a basic aqueous solution.

[0300] These results demonstrate that the method of the present invention is highly effective in producing high-purity acetylated hyaluronic acid, suggesting that this can significantly contribute to improving product quality and safety.

[0301] [Solubility Test]

[0302] 1.00 g of each product obtained in Example 1 and Comparative Examples 1-5 was accurately weighed and placed in a 100 mL beaker. 99 mL of distilled water was added to this, and the mixture was placed in a constant temperature water bath maintained at 20°C and stirred at 300 rpm using a magnetic stirrer. The time until complete dissolution was measured while observing visually. If the solution was not completely dissolved after 24 hours, the solution was filtered through a 0.45 μm membrane filter to check for the presence of insoluble particles.

[0303]

[0304] As a result of the solubility test, the acetylated sodium hyaluronate of Example 1, prepared by the method of the present invention, completely dissolved in water at 20°C in just 15 minutes, exhibiting excellent solubility. This result is faster than the 30-minute dissolution time of hyaluronic acid (HA) used as a control, proving that the product of the present invention has superior solubility characteristics compared to conventional HA. In the case of Comparative Example 1, the product prepared under strong acid conditions did not completely dissolve even after 24 hours, and a large amount of insoluble particles were observed. This suggests that the acetylation reaction under strong acid conditions significantly altered the structure of HA, thereby reducing its water solubility.

[0305] In the case of Comparative Example 2, the product prepared under basic conditions completely dissolved in 2 hours, showing relatively good solubility, but the dissolution rate was significantly slower compared to Example 1.

[0306] In the case of Comparative Examples 3 and 5, the products reacted without acetic acid or at low temperatures, respectively, were not completely dissolved even after 24 hours, and a small amount of insoluble particles were observed. This result demonstrates the importance of uniform reaction conditions.

[0307] In the case of Comparative Example 4, the product prepared without basic post-treatment was completely dissolved in 4 hours, but the dissolution rate was significantly slower compared to Example 1. This shows that the basic post-treatment process plays an important role in improving the solubility of the product.

[0308] Accordingly, the sodium hyaluronate acetylated produced by the method of the present invention has superior solubility characteristics compared to conventional HA and exhibits significantly improved solubility compared to other production methods. These characteristics suggest that the product of the present invention can be effectively used in various aqueous solution-based applications.

[0309] [Oil Solubility Evaluation]

[0310] To evaluate the lipophilicity of acetylated hyaluronic acid (AcHA) prepared according to the present invention, comparative examples, and raw hyaluronic acid (HA), the octanol / water partition coefficient (Log P) was measured. The measurement was performed in the following manner.

[0311] 1. 10 mg of each sample was dissolved in a 1:1 mixture of 1-octanol and water (5 mL each).

[0312] 2. The mixture was stirred at 25°C for 24 hours to reach equilibrium.

[0313] 3. The two layers were separated by centrifugation (3000 rpm, 10 minutes).

[0314] 4. The sample concentration of each layer was measured using a UV spectrophotometer.

[0315] 5. The Log P value was calculated using the following formula: Log P = log([concentration of the octanol layer] / [concentration of the water layer])

[0316] The measured Log P values ​​are as shown in the following table:

[0317]

[0318] The Log P value of the acetylated hyaluronic acid prepared according to the present invention (Example 1) was measured to be -1.12. This is a significantly higher value compared to the Log P value of the raw hyaluronic acid (-2.37), indicating that the AcHA of the present invention possesses approximately 10 times greater lipophilicity than the raw HA. Specifically, the difference in Log P values ​​is 1.25, which means that the lipophilicity is 10 1.25 _ This indicates a 17.8-fold increase. In the case of the comparative examples, the Log P values ​​were measured to be between -1.85 and -1.25, indicating that while the lipophilicity increased compared to the raw material HA, it did not reach that of the AcHA of the present invention. In particular, the increase in lipophilicity was relatively small in Comparative Example 1 (-1.85), prepared under strong acid conditions, and Comparative Example 5 (-1.78), reacted at low temperatures. This suggests the possibility that the acetylation reaction proceeded incompletely or that partial decomposition of the molecule occurred.

[0319] Comparative Example 2 (-1.63), prepared under basic conditions, and Comparative Example 3 (-1.41), reacted without acetic acid, showed a moderate increase in lipophilicity, but it was still at a low level compared to the AcHA of the present invention. This indirectly demonstrates that the acetic acid used in the present invention plays an important role in the uniformity and completeness of the reaction.

[0320] In the case of Comparative Example 4 (-1.25), in which the basic post-treatment was omitted, it showed the closest lipophilicity to the AcHA of the present invention, but still did not reach that level. This suggests that the basic post-treatment process contributes to improving the purity and uniformity of the product, and that this also has a positive effect on increasing lipophilicity.

[0321] Accordingly, AcHA produced according to the present invention exhibited an increase in lipophilicity of approximately 17.8 times compared to raw HA, and a minimum of 1.3 times to a maximum of 5.3 times compared to other manufacturing methods. This significant increase in lipophilicity implies that the AcHA of the present invention can overcome the limitations of conventional HA and greatly expand the potential for application in lipophilic media. In particular, in fields such as cosmetics, pharmaceuticals, and biomaterials, the AcHA of the present invention is expected to replace conventional HA and demonstrate superior performance.

Claims

1. A method for producing acetylated hyaluronic acid with suppressed odor, A step of acetylating hyaluronic acid in the presence of an organic acid, a carboxylic anhydride, and a basic catalyst until a homogeneous reaction mixture forming a single phase is formed upon visual observation; A step of separating the above acetylated hyaluronic acid under acidic conditions; The step of washing the separated acetylated hyaluronic acid with a nonpolar organic solvent; and A step of treating the washed acetylated hyaluronic acid with a basic aqueous solution and precipitating it with alcohol; Includes, In the above acetylation step, the reaction is continued from the point where the reaction mixture becomes homogeneous to obtain acetylated hyaluronic acid with a degree of acetylation of 3.8 or higher and 4.0 or lower, and The final product exhibits more than 10 times greater lipophilicity than hyaluronic acid when measured by the octanol / water partition coefficient (log P), and no acetic acid peak is detected during gas chromatography analysis, A method for producing odor-inhibiting acetylated hyaluronic acid, characterized in that the final product is obtained in the form of pure acetylated hyaluronate sodium salt.

2. In Paragraph 1, The above organic acid is acetic acid, the above carboxylic anhydride is acetic anhydride, and the above basic catalyst is 4-(dimethylamino)pyridine (DMAP), and The above non-polar organic solvent is hexane, and The above acetylation step is performed at 95-105℃ for 40-56 hours, and A method for producing odor-inhibiting acetylated hyaluronic acid, characterized in that the basic aqueous solution is a sodium bicarbonate aqueous solution with a concentration of 15-25% (w / v) and the alcohol is isopropanol.

3. In Paragraph 2, Separation under the above acidic conditions is performed using a diluted aqueous hydrochloric acid solution (concentrated HCl : distilled water = 1 : 37.5 volume ratio), and The above non-polar organic solvent washing is performed for 8-16 hours, and The yield of acetylated hyaluronic acid obtained by the above method is 75-85%, and A method for producing odor-inhibiting acetylated hyaluronic acid, characterized in that the final product is obtained as a white solid.

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