Preparation process of stable hydroxybutyl chitosan in full temperature range

Abstract

This application relates to the field of chitosan derivative preparation technology, specifically disclosing a preparation process for full-temperature-range stable hydroxybutyl chitosan. The preparation process for full-temperature-range stable hydroxybutyl chitosan is as follows: raw chitosan is dispersed in a solvent, Boc anhydride is added and reacted to obtain N-Boc protected chitosan, which is then added to a eutectic solvent containing basic amino acids, citric acid, and dihydroxy polymers. Butylene oxide is added dropwise to carry out the reaction. After the reaction is terminated, an intermediate product is precipitated, the Boc group is removed, and the product is purified by post-treatment to obtain the final product. The hydroxybutyl chitosan prepared by the full-temperature-range stable hydroxybutyl chitosan preparation process of this application has a narrow and stable molecular weight distribution, low impurity content, broad-spectrum antibacterial properties, and good flowability across the entire temperature range of 4-60℃. It exhibits unique application potential and advantages when used as a raw material in pharmaceutical excipients, medical devices, and cosmetic raw materials.

Description

Technical Field

[0001] This application relates to the field of chitosan derivative preparation technology, and more specifically, to a preparation process for a temperature-stable hydroxybutyl chitosan. Background Technology

[0002] With the rapid development of biomedical materials, medical dressings, as core products for wound repair, have seen their performance requirements upgraded from traditional physical barrier functions to intelligent and functional applications. Currently, natural polymer materials, represented by chitosan and its derivatives, have become important base materials in the field of medical dressings due to their excellent biocompatibility, biodegradability, and antibacterial safety. Among them, hydroxybutyl chitosan (HBC), as a chemically modified product of chitosan, significantly improves the water / alcohol solubility of chitosan by introducing hydroxybutyl groups, and has broad application prospects.

[0003] Currently, the preparation of hydroxybutyl chitosan often employs heterogeneous reaction systems. For example, the chitosan raw material is first pretreated and activated by alkali before undergoing a substitution reaction with epoxide. Regarding the aforementioned techniques, the inventors have discovered that such methods typically result in a wide distribution of hydroxybutyl substitution degrees due to factors such as heterogeneous reaction media or alkali damage to the molecular structure of the chitosan raw material. The molecular weight distribution coefficient (MDC) of the product HBC is generally higher than 1.5. Even with homogeneous catalysis, although the uniformity of etherification can be improved, local degradation of the chitosan molecular weight is prone to occur during the reaction, resulting in a still high MDC of the product HBC. This wide molecular weight distribution directly leads to a narrow and unstable gel phase transition temperature range for the HBC solution, making it difficult to control the mechanical strength, swelling degree, and degradation rate of the HBC gel dressing. It also results in insufficient antibacterial mechanisms in the HBC gel dressing. Summary of the Invention

[0004] To improve the uniformity, stability, and antibacterial activity of hydroxybutyl chitosan, this application provides a preparation process for a stable hydroxybutyl chitosan across the entire temperature range.

[0005] In a first aspect, this application provides a preparation process for a stable hydroxybutyl chitosan across the entire temperature range, employing the following technical solution: A process for preparing a temperature-stable hydroxybutyl chitosan includes the following steps: S1: Add the raw material chitosan to a mixed solvent of water and ethanol, stir evenly under an inert gas atmosphere to form a dispersion, then add anhydrous sodium carbonate, adjust the pH of the dispersion to 7.5-8, then add an ethanol solution of Boc anhydride, first react under ice-water bath conditions for 2-4 hours, then remove the ice-water bath, continue the reaction at room temperature for 10-12 hours, then neutralize, centrifuge to collect the precipitate, wash and obtain N-Boc protected chitosan. S2: The N-Boc protected chitosan is added to a eutectic solvent, heated to 55-65°C, stirred to form a blend, then cooled to 40-45°C, and epoxide is added dropwise to carry out the reaction. After the reaction is terminated, precipitation is carried out to obtain the intermediate product. S3: Add the intermediate product to water, add hydrochloric acid to adjust the pH to acidic, stir the reaction to remove the Boc group to obtain the crude extract precursor, and purify it through post-processing to obtain the final product.

[0006] By adopting the above technical solution, Boc anhydride is first used to specifically protect the N-position amino group of the raw material chitosan under weakly alkaline conditions, ensuring that the hydroxybutyl group can be substituted only at the O- site in the subsequent reaction with epoxide, avoiding interference from N-position substitution, which helps to ensure the uniformity of the degree of substitution. The prepared hydroxybutyl chitosan is mainly composed of N-free and O-substituted hydroxybutyl chitosan and has temperature stability.

[0007] Furthermore, the inventors employed a eutectic solvent system (DES) to effectively disrupt the intermolecular hydrogen bonds of the raw material chitosan through a hydrogen bond network formed by hydrogen bond acceptors and hydrogen bond donors. Compared to the heterogeneous reaction method of opening hydrogen bonds in raw material chitosan by concentrated alkali pretreatment in traditional processes, the technical solution of this application achieves homogenization of the reaction system from the traditional heterogeneous solid-liquid reaction. At the same time, it effectively avoids the random hydrolysis and destruction of the molecular structure of raw material chitosan by strong alkali pretreatment, laying the foundation for obtaining products with narrow molecular weight distribution. The reaction between raw material chitosan and epoxide butane changes from random and uncontrollable to uniform and controllable. The introduction of hydroxybutyl groups is more uniform along each polymer chain and between different polymer chains, which also means that the hydrophilicity / hydrophobicity and spatial structure of the product hydroxybutyl chitosan are more uniform, and the molecular weight distribution coefficient is lower.

[0008] In addition, the inventors discovered that the impurity content of the reactants obtained by the process of this application is significantly reduced after post-processing purification, especially the endotoxin content, which can reach medical-grade levels. This may be partly because the Boc protectant is removed in an acidic aqueous solution in step S3. This acidic environment not only restores the amino groups necessary for the antibacterial properties of chitosan, but also partially protonates the phosphate groups in the endotoxin lipopolysaccharide molecular structure, weakening the electrostatic repulsion and hydrogen bond network inside its aggregate, making its structure looser and easier to dissociate and remove in subsequent steps.

[0009] Optionally, the eutectic solvent in step S2 uses basic amino acids as hydrogen bond acceptors and citric acid and dihydroxy polymers as hydrogen bond donors.

[0010] Optionally, the basic amino acid is selected from arginine or lysine.

[0011] Optionally, the dihydroxy polymer is selected from polyethylene glycol or polypropylene glycol with a molecular weight of 400-800.

[0012] Optionally, the molar ratio of citric acid to dihydroxy polymer is 1:(0.1-0.3).

[0013] By adopting the above technical solution, when DES comes into contact with chitosan, the positively charged or strongly polar groups such as the amino and guanidine groups of basic amino acids, together with the carboxyl and hydroxyl groups of citric acid and the ether oxygen atoms and terminal hydroxyl groups of the dihydroxy polymer, jointly construct a strong exogenous hydrogen bond competition network. This network can form competitive hydrogen bonds with the hydroxyl groups on the chitosan molecular weight, directly and efficiently replacing the intermolecular hydrogen bonds between the sugar molecular chains and the molecular chains inside the raw material chitosan. This weakens the intermolecular hydrogen bond basis of Boc protecting the raw material chitosan, causing the originally tightly stacked molecular chain structure to be loosened and disassembled, forming a more reactive pre-loose state.

[0014] The linear structure of dihydroxy polymers further enhances reaction efficiency. PEG / PPG with a molecular weight of 400-800 has ether oxygen atoms that are excellent hydrogen bond acceptors and terminal hydroxyl groups that are hydrogen bond donors. The linear structure of dihydroxy polymers can quickly penetrate into the intermolecular space. It not only has sufficient density and polarity of terminal active hydroxyl groups to form a stable hydrogen bond network with citric acid (salt) and basic amino acids, establishing a eutectic solvent system, but also generates steric hindrance between chitosan chains, physically preventing the chitosan chains from re-approaching and rebuilding hydrogen bonds, thereby helping to achieve and maintain the homogenization of the reaction system.

[0015] Another reason for the significant reduction in endotoxin content may be the basic amino acids and citric acid in the DES system. Since the lipopolysaccharide structure in endotoxins relies on hydrophobic and electrostatic interactions to aggregate, and the guanidinium or amino groups in basic amino acids are positively charged, they can form electrostatic interactions with the negatively charged phosphate groups in endotoxins, while the carboxyl groups of citric acid can destroy the hydrogen bond structure between endotoxin molecules. The synergistic effect of these two factors initially loosens the aggregated structure of endotoxins, laying the foundation for subsequent adsorption and removal.

[0016] Optionally, the post-processing purification process in step S3 specifically includes the following steps: The crude extract precursor was neutralized to pH 6-7, potassium chloride solution was added, followed by pre-cooled anhydrous ethanol. After stirring, the mixture was allowed to stand to allow the product to fully precipitate. The precipitate was then collected by centrifugation, washed with alcohol, and reconstituted. Finally, it was pre-filtered through a microporous membrane to obtain the crude extract. The crude extract was then dialyzed using a dialysis membrane with a molecular weight cutoff >10 kDa. An adsorbent was then added for adsorption, followed by filtration through a sterile microporous membrane and freeze-drying to obtain the final product.

[0017] Optionally, the adsorbent contains polymyxin B agarose.

[0018] By adopting the above technical solution, loose and still-unremoved endotoxins are efficiently removed through polymyxin B agarose adsorption, significantly reducing the endotoxin content level of the product.

[0019] Secondly, this application provides a temperature-stable hydroxybutyl chitosan, which is prepared by the preparation process of a temperature-stable hydroxybutyl chitosan of this application.

[0020] In summary, this application has the following beneficial effects: 1. Because this application uses Boc anhydride to protect the N-position amino group before reacting it with epoxide in the DES system, the hydrogen bond network formed by the hydrogen bond acceptor and hydrogen bond donor effectively breaks the intermolecular hydrogen bonds of the raw material chitosan, achieving homogenization of the reaction system. This effectively reduces the random degradation and damage to the chitosan molecular chain structure caused by the traditional strong alkali pretreatment. The introduction of hydroxybutyl groups can be carried out under more controllable and uniform conditions, resulting in a more uniform molecular weight distribution and a significantly reduced molecular weight distribution coefficient of the obtained hydroxybutyl chitosan.

[0021] 2. The basic amino acids and citric acid in the DES system of this application initially weaken the intermolecular hydrogen bond structure of endotoxin in the raw material chitosan, playing a pre-loosening role. The removal of Boc in the acidic environment further weakens the electrostatic repulsion and hydrogen bond network inside the endotoxin aggregate, making its structure more loose, and the endotoxin is more easily dissociated and removed through subsequent dialysis and adsorption treatment.

[0022] 3. The hydroxybutyl chitosan obtained by the preparation process of this application has a stable molecular weight distribution and can exhibit good water and alcohol solubility in the full temperature range of 4-60℃. It is liquid and easy to handle. In addition, the hydroxybutyl chitosan of this application has a broad-spectrum and strong inhibitory effect on a variety of bacteria such as Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Malassezia, and Propionibacterium acnes, which meets the requirements of medical materials and can show unique application potential and advantages in the fields of pharmaceutical excipients, medical devices and cosmetic raw materials. Detailed Implementation

[0023] The present application will be further described in detail below with reference to embodiments and comparative examples. raw material

[0024] Unless otherwise specified, all raw materials used in the embodiments and comparative examples in this application are commercially available products, specifically: Raw material chitosan, degree of deacetylation ≥80%; L-arginine was selected from Guangzhou Snoke Biotechnology Co., Ltd. Polyethylene glycol 200, 400, 600, 800, and 1000 are all sourced from Dow Chemical Company, USA. Polymyxin B agarose, from Merck Life Sciences, p. 1411; Diatomaceous earth, selected from Shijiazhuang Zhente New Material Technology Co., Ltd., 004. Example Example 1

[0025] A temperature-stable hydroxybutyl chitosan, the preparation process of which includes the following steps: S1: Add the raw material chitosan to a water / ethanol mixed solvent with a volume ratio of 1:10 at a material-to-liquid ratio of 1:1, stir evenly under a nitrogen-protected atmosphere to form a dispersion, then add anhydrous sodium carbonate to adjust the pH of the dispersion to 7.5-8. S2: Under the conditions of ice-water bath at 0-5℃ and continuous stirring, add a 25wt% Boc anhydride ethanol solution to the dispersion, wherein the mass ratio of Boc anhydride to raw chitosan is 2:1. First, maintain the 0-5℃ ice-water bath conditions for 3 hours, then remove the ice-water bath and continue the reaction at room temperature for 11 hours. Then, add triethanolamine dropwise to neutralize until the pH of the reaction solution is 7-8. Centrifuge to collect the precipitate, and after washing with alcohol, obtain N-Boc protected chitosan. S3: L-arginine, citric acid, and polyethylene glycol 400 were mixed in a molar ratio of 2:1:0.1 and heated in an oil bath at 80°C with magnetic stirring to form a homogeneous and transparent solution as a eutectic solvent. N-Boc protected chitosan was added to the eutectic solvent at a material-to-liquid ratio of 1:22. The mixture was heated to 57°C and stirred for 2 hours to form a blend. The temperature was then lowered to 42°C, and 1,2-epoxybutane was added dropwise. After the addition was complete, the mixture was stirred for another 22 hours. The mass ratio of 1,2-epoxybutane to the raw material chitosan was 3:1. After the reaction was terminated, the reaction solution was poured into pre-cooled anhydrous ethanol to precipitate the product. After standing for 1 hour, the precipitate was collected by centrifugation and washed with ethanol to obtain the intermediate product. S4: Add the intermediate product to deionized water at a material-to-liquid ratio of 1:10, add 1M dilute hydrochloric acid to adjust the pH to 3±0.1, stir for 2.5h to remove the Boc group and obtain the crude extract precursor; S5: Add 0.5M ammonia water dropwise to the crude extract precursor to neutralize to pH 6-7, add 1 / 5 volume of 15% potassium chloride solution to the crude extract precursor, stir well to promote subsequent precipitation, add pre-cooled anhydrous ethanol, stir and let stand at 4℃ for 10h to allow the product to fully precipitate, centrifuge to collect the precipitate and wash with alcohol, redissolve the precipitate after alcohol precipitation in deionized water at a material-to-liquid ratio of 1:10, and then pre-filter through a 0.45μm polyethersulfone (PES) membrane filter to obtain the crude extract; S6: The crude extract was placed in a dialysis bag with a molecular weight cutoff >10kDa and dialyzed for 36 hours to obtain a hydroxybutyl chitosan solution. Then, an adsorbent with a diatomaceous earth to polymyxin B agarose mass ratio of 1:0.8 was added at a mass ratio of hydroxybutyl chitosan to adsorbent of 1:5 and stirred for adsorption for 18 hours. Finally, the solution was aseptically filtered through a 0.22μm sterile PES filter membrane and freeze-dried to obtain the final product. Example 2

[0026] A temperature-stable hydroxybutyl chitosan, the preparation process of which includes the following steps: S1: Add the raw material chitosan to a water / ethanol mixed solvent with a volume ratio of 1:10 at a material-to-liquid ratio of 1:1, stir evenly under a nitrogen-protected atmosphere to form a dispersion, then add anhydrous sodium carbonate to adjust the pH of the dispersion to 7.5-8. S2: Under the conditions of ice-water bath at 0-5℃ and continuous stirring, a 25wt% Boc anhydride ethanol solution was added to the dispersion, wherein the mass ratio of Boc anhydride to raw chitosan was 1.8:1. The reaction was first maintained at 0-5℃ for 2 hours, then the ice-water bath was removed and the reaction was continued at room temperature for 10 hours. Triethanolamine was then added dropwise to neutralize the reaction solution to pH 7-8. The precipitate was collected by centrifugation and washed with alcohol to obtain N-Boc protected chitosan. S3: L-arginine, citric acid, and polyethylene glycol 600 were mixed in a molar ratio of 2:1:0.1 and heated in an oil bath at 80°C with magnetic stirring to form a homogeneous and transparent solution as a eutectic solvent. N-Boc protected chitosan was added to the eutectic solvent at a material-to-liquid ratio of 1:20. The mixture was heated to 60°C and stirred for 2 hours to form a blend. The temperature was then lowered to 42°C, and 1,2-epoxybutane was added dropwise. After the addition was complete, the reaction was stirred for another 24 hours. The mass ratio of 1,2-epoxybutane to the raw material chitosan was 3.5:1. After the reaction was terminated, the reaction solution was poured into pre-cooled anhydrous ethanol to precipitate the product. After standing for 1 hour, the precipitate was collected by centrifugation and washed with ethanol to obtain the intermediate product. S4: Add the intermediate product to deionized water at a material-to-liquid ratio of 1:10, add 1M dilute hydrochloric acid to adjust the pH to 3±0.1, stir for 2.5h to remove the Boc group and obtain the crude extract precursor; S5: Add 0.5M ammonia water dropwise to the crude extract precursor to neutralize to pH 6-7, add 1 / 5 volume of 15% potassium chloride solution to the crude extract precursor, stir well to promote subsequent precipitation, add pre-cooled anhydrous ethanol, stir and let stand at 4℃ for 10h to allow the product to fully precipitate, centrifuge to collect the precipitate and wash with alcohol, redissolve the precipitate after alcohol precipitation in deionized water at a material-to-liquid ratio of 1:10, and then pre-filter through a 0.45μm polyethersulfone (PES) membrane filter to obtain the crude extract; S6: The crude extract was placed in a dialysis bag with a molecular weight cutoff >10kDa and dialyzed for 36 hours to obtain a hydroxybutyl chitosan solution. Then, an adsorbent with a diatomaceous earth to polymyxin B agarose mass ratio of 1:0.8 was added at a mass ratio of hydroxybutyl chitosan to adsorbent of 1:5 and stirred for adsorption for 18 hours. Finally, the solution was aseptically filtered through a 0.22μm sterile PES filter membrane and freeze-dried to obtain the final product. Example 3

[0027] A temperature-stable hydroxybutyl chitosan, the preparation process of which includes the following steps: S1: Add the raw material chitosan to a water / ethanol mixed solvent with a volume ratio of 1:10 at a material-to-liquid ratio of 1:1, stir evenly under a nitrogen-protected atmosphere to form a dispersion, then add anhydrous sodium carbonate to adjust the pH of the dispersion to 7.5-8. S2: Under the conditions of ice-water bath at 0-5℃ and continuous stirring, a 25wt% Boc anhydride ethanol solution was added to the dispersion, wherein the mass ratio of Boc anhydride to raw chitosan was 2.2:1. The reaction was first maintained at 0-5℃ for 4 hours, then the ice-water bath was removed and the reaction was continued at room temperature for 12 hours. Triethanolamine was then added dropwise to neutralize the reaction solution to pH 7-8. The precipitate was collected by centrifugation and washed with alcohol to obtain N-Boc protected chitosan. S3: L-arginine, citric acid, and polyethylene glycol 800 were mixed in a molar ratio of 2:1:0.1 and heated in an oil bath at 80°C with magnetic stirring to form a homogeneous and transparent solution as a eutectic solvent. N-Boc protected chitosan was added to the eutectic solvent at a material-to-liquid ratio of 1:22. The mixture was heated to 55°C and stirred for 2 hours to form a blend. The temperature was then lowered to 40°C, and 1,2-epoxybutane was added dropwise. After the addition was complete, the mixture was stirred for another 20 hours. The mass ratio of 1,2-epoxybutane to the raw material chitosan was 3:1. After the reaction was terminated, the reaction solution was poured into pre-cooled anhydrous ethanol to precipitate the product. After standing for 1 hour, the precipitate was collected by centrifugation and washed with ethanol to obtain the intermediate product. S4: Add the intermediate product to deionized water at a material-to-liquid ratio of 1:10, add 1M dilute hydrochloric acid to adjust the pH to 3±0.1, stir for 2.5h to remove the Boc group and obtain the crude extract precursor; S5: Add 0.5M ammonia water dropwise to the crude extract precursor to neutralize to pH 6-7, add 1 / 5 volume of 15% potassium chloride solution to the crude extract precursor, stir well to promote subsequent precipitation, add pre-cooled anhydrous ethanol, stir and let stand at 4℃ for 10h to allow the product to fully precipitate, centrifuge to collect the precipitate and wash with alcohol, redissolve the precipitate after alcohol precipitation in deionized water at a material-to-liquid ratio of 1:10, and then pre-filter through a 0.45μm polyethersulfone (PES) membrane filter to obtain the crude extract; S6: The crude extract was placed in a dialysis bag with a molecular weight cutoff >10kDa and dialyzed for 36 hours to obtain a hydroxybutyl chitosan solution. Then, an adsorbent with a diatomaceous earth to polymyxin B agarose mass ratio of 1:0.8 was added at a mass ratio of hydroxybutyl chitosan to adsorbent of 1:5 and stirred for adsorption for 18 hours. Finally, the solution was aseptically filtered through a 0.22μm sterile PES filter membrane and freeze-dried to obtain the final product. Example 4

[0028] A temperature-stable hydroxybutyl chitosan, differing from Example 1 in that its preparation step S3 is specifically as follows: L-arginine, citric acid, and polyethylene glycol 800 were mixed in a molar ratio of 2:1:0.1 and heated in an oil bath at 80°C with magnetic stirring to form a homogeneous and transparent solution as a eutectic solvent. N-Boc protected chitosan was added to the eutectic solvent at a feed-to-liquid ratio of 1:18. The mixture was heated to 63°C and stirred for 2 hours to form a blend. The temperature was then lowered to 45°C, and 1,2-epoxybutane was added dropwise. After the addition was complete, the reaction was stirred for another 26 hours. The mass ratio of 1,2-epoxybutane to the raw chitosan was 4:1. After the reaction was terminated, the reaction solution was poured into pre-cooled anhydrous ethanol to precipitate the product. After standing for 1 hour, the precipitate was collected by centrifugation and washed with ethanol to obtain an intermediate product. All other steps were the same as in Example 1. Example 5

[0029] A temperature-stable hydroxybutyl chitosan, differing from Example 1 in that its preparation step S3 is specifically as follows: L-arginine, citric acid, and polyethylene glycol 800 were mixed in a molar ratio of 2:1:0.1 and heated in an oil bath at 80°C with magnetic stirring to form a homogeneous and transparent solution as a eutectic solvent. N-Boc protected chitosan was added to the eutectic solvent at a feed-to-liquid ratio of 1:18. The mixture was heated to 65°C and stirred for 2 hours to form a blend. The temperature was then lowered to 45°C, and 1,2-epoxybutane was added dropwise. After the addition was complete, the reaction was stirred for another 28 hours. The mass ratio of 1,2-epoxybutane to the raw chitosan was 5:1. After the reaction was terminated, the reaction solution was poured into pre-cooled anhydrous ethanol to precipitate the product. After standing for 1 hour, the precipitate was collected by centrifugation and washed with ethanol to obtain the intermediate product. All other steps were the same as in Example 1. Example 6

[0030] A temperature-stable hydroxybutyl chitosan, differing from Example 1 only in that the molar ratio of citric acid and polyethylene glycol 400 in the eutectic solvent of step S3 is 1:0.2. Example 7

[0031] A temperature-stable hydroxybutyl chitosan, differing from Example 1 only in that the molar ratio of citric acid and polyethylene glycol 400 in the eutectic solvent of step S3 is 1:0.3. Example 8

[0032] A temperature-stable hydroxybutyl chitosan, differing from Example 1 only in that the molecular weight of polyethylene glycol in the eutectic solvent of step S3 is 200. Example 9

[0033] A temperature-stable hydroxybutyl chitosan, differing from Example 3 only in that the molecular weight of polyethylene glycol in the eutectic solvent of step S3 is 1000. Example 10

[0034] A temperature-stable hydroxybutyl chitosan, differing from Example 1 only in that polyethylene glycol was not added to the eutectic solvent in step S3. Example 11

[0035] A temperature-stable hydroxybutyl chitosan, differing from Example 1 only in that polymyxin B agarose in step S6 is replaced with an equal mass of diatomaceous earth. Comparative Example

[0036] Comparative Example 1 A hydroxybutyl chitosan, the preparation process of which includes the following steps: S1: Add raw chitosan to 45wt% sodium hydroxide solution at a material-to-liquid ratio of 1:10. Under nitrogen protection and stirring, microwave at 600W power for 24 hours. Filter and squeeze out excess alkali solution to obtain pretreated chitosan. S2: Add pretreated chitosan to a water / isopropanol solution with a volume ratio of 1 / 19 at a material-to-liquid ratio of 1:10, add 1,2-epoxybutane dropwise, the mass ratio of 1,2-epoxybutane to pretreated chitosan is 5:1, react for 1 hour, then heat to 50°C under microwave power of 1200W and react for 24 hours. After cooling to room temperature, add 1M dilute hydrochloric acid dropwise to the reaction solution to adjust the pH to 6-7, and obtain a mixed solution. S3: The mixture was placed in a dialysis bag with a molecular weight cutoff >10kDa and dialyzed for 36 hours to obtain a hydroxybutyl chitosan solution. Then, an adsorbent with a diatomaceous earth ratio of 1:0.8 and a polymyxin B agarose ratio of 1:5 was added and stirred for 18 hours. Finally, the solution was aseptically filtered through a 0.22μm sterile PES filter membrane and freeze-dried to obtain the final product.

[0037] Comparative Example 2 A hydroxybutyl chitosan differs from Comparative Example 1 only in that polymyxin B agarose in the adsorbent in step S3 is replaced with an equal mass of diatomaceous earth. Performance testing Test Example 1

[0038] The hydroxybutyl chitosan prepared by the preparation processes of Examples 1-9 and Comparative Examples 1-2 was subjected to the following related performance tests. Each test was performed 3 times, and the average value of the 3 test results was taken as the final result and recorded in Table 1.

[0039] 1. Degree of hydroxybutyl substitution: Based on the carbon-nitrogen ratio determined by an elemental analyzer, i.e., X=C / N, the formula for calculating the degree of substitution is DS=(X-6) / 4; 2. Molecular weight distribution coefficient: The molecular weight of hydroxybutyl chitosan was determined by light scattering, and the molecular weight distribution coefficient of hydroxybutyl chitosan was determined by laser scattering-gel permeation chromatography. 3. Endotoxins: Endotoxins were detected using a bacterial endotoxin detector according to the turbidimetric method specified in Section 1143 of the General Chapter of Part IV of the Pharmacopoeia of the People's Republic of China (2015 edition).

[0040] 4. Antibacterial activity: Antibacterial activity was determined according to the quantitative suspension antibacterial test in section 5.1.1 of WST 650-2019, Evaluation Methods for Antibacterial and Antimicrobial Efficacy. A positive control group and an experimental group were set up. The target bacteria in the experimental group were: Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Malassezia, and Propionibacterium acnes. The antibacterial rate (X) was calculated using the formula: X = [A0 - A1 / A0] × 100%, where A0 is the amount of bacteria recovered in the positive control group and A1 is the amount of bacteria recovered in the experimental group, both in CFU / mL.

[0041] Table 1

[0042] As can be seen from the performance test results in Table 1, compared with the traditional method of preparing hydroxybutyl chitosan by reacting it with epoxide after alkali pretreatment, this application adopts the method of protecting the N-position amino group and reacting it with epoxide in the DES system. This method opens the intermolecular hydrogen bonds of the raw material chitosan in a more gentle and orderly manner, avoiding the random degradation and destruction of the chitosan molecular chain structure by the strong alkaline environment. The introduction of hydroxybutyl groups can be carried out under more controllable and uniform conditions. The resulting hydroxybutyl chitosan has a more uniform molecular weight distribution and a significantly reduced molecular weight distribution coefficient, which ensures the stable application performance of the product hydroxybutyl chitosan.

[0043] Furthermore, compared to traditional hydroxybutyl chitosan, it is evident that the hydroxybutyl chitosan of this application exhibits an antibacterial rate of over 90% against Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Malassezia, and Propionibacterium acnes. The antibacterial effect of hydroxybutyl chitosan produced by traditional processes is extremely low. This highlights the excellent broad-spectrum antibacterial properties of the hydroxybutyl chitosan of this application, making it safer for use in pharmaceutical excipients, medical devices, and cosmetic raw materials.

[0044] According to the performance test results of Examples 1, 11, and Comparative Examples 1-2, the chitosan raw material of this application undergoes a homogeneous reaction in a eutectic solvent, which improves the removal effect of endotoxin. This is because the basic amino acids and citric acid initially weaken the intermolecular hydrogen bond structure of endotoxin in the raw material chitosan, playing a pre-loosening role. Subsequently, the removal of Boc in an acidic environment further weakens the electrostatic repulsion and hydrogen bond network inside the endotoxin aggregate, making its structure more loose. After subsequent dialysis and adsorption with an adsorbent containing polymyxin B agarose, the endotoxin is more easily dissociated and removed through specific adsorption treatment. The endotoxin content in the product hydroxybutyl chitosan is <0.05 EU / mg, which meets the requirements for use in medical materials.

[0045] Comparative Example 1 also used an adsorbent containing polymyxin B agarose, but its specific removal effect on endotoxins was significantly insufficient. This may be because although alkalization treatment may destroy the structure of endotoxins, strong alkali may cause partial degradation or conformational changes in endotoxins. At the same time, the reaction always takes place in a solid-liquid heterogeneous phase, and the harsh conditions may also cause endotoxins to form more complex and compact complexes or aggregates with partially degraded chitosan fragments and impurities through hydrophobic interactions and ionic interactions. Endotoxins are likely to be physically trapped or encapsulated inside or on the surface of incompletely dissolved chitosan particles, reducing their possibility of contact with subsequent adsorbents.

[0046] According to the performance test results of Examples 1-10, when the raw material chitosan reacts in a eutectic solvent containing polyethylene glycol or polypropylene glycol with a molecular weight of 400-800, hydroxybutyl chitosan with a more uniform and stable molecular weight distribution can be obtained. This is because polyethylene glycol or polypropylene glycol has a unique linear straight-chain structure that can quickly penetrate between molecules. It not only has sufficient end-active hydroxyl density and polarity to form a stable hydrogen bond network with citric acid (salt) and basic amino acids to establish a eutectic solvent system, but also generates steric hindrance between chitosan chains, physically preventing the chitosan chains from re-approaching and rebuilding hydrogen bonds, thereby helping to achieve and maintain the homogenization of the reaction system.

[0047] According to the performance test results of Examples 8-9, it can be seen that both excessively high and excessively low molecular weight of the dihydroxy polymer will lead to an increase in the molecular weight distribution coefficient. This may be because when the chain length is too short, the steric hindrance is insufficient, and the chitosan chain cannot be effectively maintained in an extended state. The decrease in hydrogen bond network strength makes the chitosan chain easy to re-aggregate. On the other hand, excessively long chains will lead to an increase in system viscosity, and long chains are prone to entanglement, affecting mass transfer. Test Example 2

[0048] Take 1.25g of hydroxybutyl chitosan prepared by the preparation processes of Examples 1-11 and Comparative Examples 1-2 respectively, add it to different test tubes, add 50mL of deionized water to each and stir to dissolve, and keep warm at 4℃, 15℃, 20℃, 30℃, 40℃, 50℃ and 60℃ for 2h respectively, observe the state of the solution, and determine whether the solution is a flowing solution or a gel based on whether the solution is in a flowing state.

[0049] Table 2

[0050] As can be seen from the performance test results in Table 2, the hydroxybutyl chitosan of this application exhibits good fluidity across the entire temperature range of 4-60℃ as the dissolution temperature increases, and is liquid and easy to handle under a wider temperature range. This also indicates that the hydroxybutyl chitosan of this application can demonstrate unique application potential and advantages when used as a raw material in pharmaceutical excipients, medical devices and cosmetic raw materials.

[0051] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A preparation process for a temperature-stable hydroxybutyl chitosan, characterized in that, Includes the following steps: S1: Add the raw material chitosan to a mixed solvent of water and ethanol, stir evenly under an inert gas atmosphere to form a dispersion, then add anhydrous sodium carbonate to adjust the pH of the dispersion to 7.5-8, then add an ethanol solution of Boc anhydride, first react under ice-water bath conditions for 2-4 hours, then remove the ice-water bath and continue the reaction at room temperature for 10-12 hours, then neutralize, centrifuge to collect the precipitate, wash and obtain N-Boc protected chitosan. S2: The N-Boc protected chitosan is added to a eutectic solvent, heated to 55-65°C, stirred to form a blend, then cooled to 40-45°C, and epoxide is added dropwise to carry out the reaction. After the reaction is terminated, precipitation is carried out to obtain the intermediate product. S3: Add the intermediate product to water, add hydrochloric acid to adjust the pH to acidic, stir the reaction to remove the Boc group to obtain the crude extract precursor, and purify it through post-processing to obtain the final product.

2. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 1, characterized in that, In step S2, the eutectic solvent uses basic amino acids as hydrogen bond acceptors and citric acid and dihydroxy polymers as hydrogen bond donors.

3. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 2, characterized in that, The basic amino acid is selected from arginine or lysine.

4. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 2, characterized in that, The dihydroxy polymer is selected from polyethylene glycol or polypropylene glycol with a molecular weight of 400-800.

5. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 2, characterized in that, The molar ratio of citric acid to dihydroxy polymer is 1:(0.1-0.3).

6. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 1, characterized in that, The post-processing purification process in step S3 specifically includes the following steps: The crude extract precursor was neutralized to pH 6-7, potassium chloride solution was added, followed by pre-cooled anhydrous ethanol. After stirring, the mixture was allowed to stand to allow the product to fully precipitate. The precipitate was then collected by centrifugation, washed with alcohol, and reconstituted. Finally, it was pre-filtered through a microporous membrane to obtain the crude extract. The crude extract was then dialyzed using a dialysis membrane with a molecular weight cutoff >10 kDa. An adsorbent was then added for adsorption, followed by filtration through a sterile microporous membrane and freeze-drying to obtain the final product.

7. The preparation process of the full-temperature-range stable hydroxybutyl chitosan according to claim 6, characterized in that, The adsorbent contains polymyxin B agarose.

8. A temperature-range stable hydroxybutyl chitosan, characterized in that, It is prepared by the preparation process of the full-temperature-range stable hydroxybutyl chitosan described in any one of claims 1-7.

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