Hydroxypropyl chitosan and its preparation process and application
By protecting and hydroxypropylating chitosan, the problems of poor water solubility and loss of amino groups in chitosan are solved, and hydroxypropyl chitosan with comprehensive advantages in multiple fields is prepared for application in cosmetics, wound healing and repair drugs and biomedical materials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO BIOTEMED BIOMATERIAL
- Filing Date
- 2026-03-07
- Publication Date
- 2026-06-09
AI Technical Summary
Chitosan's poor water solubility and the loss of active amino groups after hydroxypropylation modification limit its application in the biomedical field.
Hydroxypropyl chitosan was prepared by protecting the amino groups on chitosan, forming imine bonds with salicylaldehyde, and then hydrolyzing it in an acidic environment. The amino active sites were retained, and hydroxypropyl groups were introduced to improve water solubility.
The prepared hydroxypropyl chitosan exhibits good solubility under acidic, neutral, and alkaline conditions, retains sufficient amino active sites, expands its application range, and forms antibacterial, anti-inflammatory, and healing-promoting drug gels through cross-linking with other components.
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Abstract
Description
Technical Field
[0001] This application relates to the field of water-soluble biomaterials technology, and more specifically, to a hydroxypropyl chitosan, its preparation process, and its applications. Background Technology
[0002] Chitosan is a cationic polysaccharide that can be prepared by deacetylation of chitin, which is widely found in nature. It possesses excellent properties such as non-toxicity, good biocompatibility, biodegradability, antibacterial properties, and antioxidant effects, and has been widely used in agriculture, food, cosmetics, pharmaceuticals, and biomedical engineering. However, the extremely strong hydrogen bonds between chitosan molecular chains result in its high crystallinity, making it almost insoluble under neutral and alkaline conditions, only soluble in dilute acidic aqueous solutions. This significantly limits its widespread application.
[0003] Hydroxypropylation is a common method to improve the solubility of chitosan. Traditional methods typically use propylene oxide as a reactant to etherify chitosan. This type of reaction is highly reactive, capable of simultaneously undergoing ring-opening addition with the C6 and C3 hydroxyl groups on the chitosan molecule, introducing a hydrophilic hydroxypropyl side chain, thus achieving complete dissolution under neutral or weakly alkaline conditions. However, the epoxy reagent also reacts with the most reactive C2 primary amino group of chitosan, generating an N-hydroxypropyl-substituted byproduct, which irreversibly consumes a large amount of the primary amino group. This affects subsequent reactions of hydroxypropyl chitosan, such as grafting work molecules, cross-linking reactions, and ionic interactions. For the subsequent development of chitosan for applications in biomedical fields such as drug delivery and tissue engineering, retaining sufficient amino groups is crucial.
[0004] Regarding the aforementioned technologies, the inventors believe there is an urgent need to provide a hydroxypropyl chitosan preparation process that improves the water solubility of chitosan while preserving sufficient active sites for its amino groups in subsequent functionalization reactions. Summary of the Invention
[0005] In order to improve the water solubility of chitosan while retaining sufficient amino active sites for its subsequent biofunctionalization reaction, this application provides a hydroxypropyl chitosan, its preparation process, and its application.
[0006] In a first aspect, this application provides a preparation process for hydroxypropyl chitosan, employing the following technical solution: Chitosan was dispersed in purified water and dissolved in acetic acid under stirring to obtain a chitosan solution with a concentration of 1-3%. The chitosan solution was purified to obtain purified chitosan; The purified chitosan was added to a mixture of salicylaldehyde and 95% ethanol, stirred evenly, and reacted at 40-60℃ for 4-5 hours. After filtration and washing with 95% ethanol 2-3 times, salicylaldehyde-chitosan wet material was obtained. Salicylaldehyde chitosan wet material and isopropanol were mixed, and 10wt% sodium hydroxide solution was added to maintain the pH of the system at 10-13. Propylene oxide / 1-chloro-2-propanol was added dropwise at 40-50℃, and the reaction was stirred for 4-24 hours. After filtration and washing, amino-protected hydroxypropyl chitosan was obtained. The mass-volume ratio of chitosan, isopropanol and propylene oxide was 3-5:50-100:20-50 or the mass-volume ratio of chitosan, isopropanol and 1-chloro-2-propanol was 3-5:50-100:5-10. The amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 22-24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan.
[0007] By adopting the above technical solution, before hydroxylation using propylene oxide / 1-chloro-2-propanol, salicylaldehyde is first used to protect the amino groups of chitosan to obtain salicylaldehyde chitosan. The salicylaldehyde chitosan is then subjected to hydroxypropylation in an alkaline environment to obtain salicylaldehyde hydroxypropyl chitosan. The salicylaldehyde is then deprotected with HCl / ethanol solution to obtain hydroxypropyl chitosan. The protection of salicylaldehyde allows the hydroxypropylation to occur on the hydroxyl groups, thus protecting the amino groups and enabling the controllable preparation of hydroxypropyl chitosan. This not only improves the water solubility of chitosan but also retains sufficient amino active sites for subsequent biofunctionalization reactions.
[0008] Preferably, the mass-to-volume ratio of chitosan to acetic acid is 1:0.5-1.
[0009] Preferably, the purification method of the chitosan solution is as follows: the chitosan solution is filtered through 1μm and 0.2μm filter membranes in sequence, then sodium hydroxide solution is added dropwise to the chitosan solution, and the mixture is stirred until a white precipitate is produced. The mixture is then filtered, washed 2-3 times with purified water, and washed 2-3 times with 95% ethanol to obtain purified chitosan.
[0010] Preferably, the volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:7-9, and the weight-volume ratio of chitosan to the mixture is 1:8-10.
[0011] Preferably, the concentration of HCl in the HCl / ethanol solution is 1-1.5M, and the concentration of ethanol is 85-90%.
[0012] By adopting the above technical solution, the acidic environment can efficiently catalyze the hydrolysis of imine bonds, while the risk of hydrolysis of the chitosan backbone is low. Ethanol can effectively dissolve the salicylaldehyde released after deprotection, making the deprotection more thorough, and at the same time maximally protecting the molecular skeleton of hydroxypropyl chitosan, so as to obtain the target product with high amino content and excellent water solubility.
[0013] Secondly, this application provides a hydroxypropyl chitosan, which adopts the following technical solution: A hydroxypropyl chitosan, prepared using the aforementioned hydroxypropyl chitosan preparation process.
[0014] Thirdly, this application provides an application of hydroxypropyl chitosan, employing the following technical solution: Application of a hydroxypropyl chitosan in cosmetics, wound healing and repair drugs, medical devices, or biomedical materials.
[0015] By adopting the above technical solution, the hydroxypropyl chitosan prepared in this application solves the problems of poor solubility of natural chitosan and loss of active amino groups after hydroxypropylation modification, and shows comprehensive advantages that traditional chitosan cannot achieve in multiple application fields.
[0016] Preferably, the wound healing and repair drug comprises the following raw materials in parts by weight: 1-2 parts hydroxypropyl chitosan, 1.1-2.2 parts nisin, 0.5-1 part transglutaminase, 3-6 parts aldehyde-modified hyaluronic acid, and 0.1-0.5 parts Sichuan pepper nano-silver.
[0017] By employing the above technical solution, amino-protected hydroxypropyl chitosan possesses excellent water solubility and high-density active amino groups. Under the catalysis of transglutaminase, hydroxypropyl chitosan is covalently fixed onto the hydroxypropyl chitosan backbone, endowing it with long-lasting and stable antibacterial activity. Then, the remaining amino groups on the hydroxypropyl chitosan undergo chemical cross-linking with the aldehyde groups of aldehyde-modified hyaluronic acid to form a three-dimensional hydrogel network, introducing the moisturizing, inflammation-regulating, and cell migration-promoting functions of hyaluronic acid. Additionally, Sichuan pepper nano-silver is introduced, which acts as a broad-spectrum antibacterial enhancer. The potent and anti-inflammatory components are physically dispersed in a hydrogel network. The nano-silver releases silver ions, generating oxidative stress and rapidly sterilizing a broad spectrum of bacteria, fungi, and even some viruses. Sichuan pepper has antibacterial and anti-inflammatory properties, which synergistically work with the nano-silver. Meanwhile, nisin can specifically target and destroy the cell membranes of Gram-positive bacteria, with a long-lasting and stable effect. Furthermore, hydroxypropyl chitosan itself is positively charged and can adsorb negatively charged bacterial cell membranes, playing a basic antibacterial role. Therefore, multiple antibacterial barriers can effectively prevent and control wound infection.
[0018] Hyaluronic acid can lock in moisture, creating a moist healing environment for the wound, while absorbing excess exudate to keep the wound moist without soaking. It also regulates immunity, reduces excessive inflammatory response, and, in combination with Sichuan pepper components, eliminates free radicals, reduces oxidative stress damage at the wound site, and promotes cell proliferation. Moreover, both hyaluronic acid and hydroxypropyl chitosan are biodegradable materials that can be broken down by enzymes during wound healing. Furthermore, the drug is formulated as a gel that can be injected into irregular wounds, perfectly conforming to any wound shape. It has good adhesion and a certain degree of self-repairing properties, thus creating a drug gel that integrates antibacterial, anti-inflammatory, moisturizing, hemostatic, and healing-promoting functions.
[0019] Preferably, the Sichuan pepper nano-silver undergoes the following pretreatment: asiaticoside was added to anhydrous ethanol and ultrasonically dispersed to obtain asiaticoside solution. The nano-silver of Sichuan pepper was dispersed in deionized water to prepare a dispersion; zinc chloride hexahydrate aqueous solution was added to the dispersion and stirred evenly; under light-protected conditions, asiaticoside solution was added, stirred evenly, centrifuged, washed, and dried.
[0020] By employing the above-mentioned technical solutions, zinc ions can disrupt cell membranes, generate reactive oxygen species, and inhibit key bacterial enzymes, thereby producing multiple antibacterial mechanisms. This results in a more direct synergistic antibacterial effect with nano-silver. Zinc ions act as cofactors for over 300 enzymes, particularly those related to cell proliferation, differentiation, and antioxidant activity. They promote epithelial cell migration and regeneration, possess strong anti-inflammatory properties, scavenge free radicals, and participate in the balance between collagen synthesis and degradation. Aspartame is a hydrophobic molecule; direct addition to gel-like repair drugs may lead to uneven distribution or easy loss. By loading it onto the surface of nano-silver, its dispersibility and stability in the aqueous system are improved, achieving sustained release. The healing-promoting zinc ions and asiaticoside, combined with the antibacterial nano-silver and zinc ions, achieve synergistic effects, regulate inflammatory responses, promote rapid angiogenesis in the early stages of wound healing, and further accelerate wound healing.
[0021] Preferably, the method for preparing the wound healing and repair drug includes the following steps: Hydroxypropyl chitosan and lactic acid nisin were dissolved in phosphate buffer, stirred evenly, and then purified transglutaminase was added. The mixture was stirred at 50°C for 4 hours, heated to 100°C and kept at that temperature for 10 minutes, cooled, filtered, dialyzed in deionized water for 3 days, and freeze-dried to obtain the complex. The complex was dissolved in distilled water to prepare a 10 wt% complex solution. Aldehyaluronic acid was dissolved in phosphate buffer, and then the complex solution and Sichuan pepper nano-silver were added and mixed evenly to prepare a wound healing and repair drug.
[0022] By adopting the above technical solution, using hydroxypropyl chitosan as the backbone, and through enzyme-catalyzed grafting and Schiff base cross-linking, and loading a gel made of Sichuan pepper nano-silver, it can fill irregular wounds, provide multiple antibacterial barriers, improve the healing microenvironment, accelerate healing, and improve healing quality.
[0023] In summary, this application has the following beneficial effects: 1. Because this application uses salicylaldehyde to protect chitosan with amino groups, an imine bond is formed between the aldehyde group of salicylaldehyde and the amino group in chitosan. Then, the imine bond is hydrolyzed and broken in an acidic environment, thereby removing the salicylaldehyde protecting group and obtaining hydroxypropyl chitosan. The retained amino groups provide sufficient active sites for covalent grafting growth factors, antimicrobial peptides, and targeting molecules. Moreover, the introduced hydroxypropyl group is a strong hydrophilic group, which can effectively destroy the crystalline region of chitosan, making the product soluble in water and greatly expanding its application range. Thus, a hydroxypropyl chitosan with abundant amino groups and good water solubility is obtained.
[0024] 2. In this application, transglutaminase is preferably used as a catalyst to graft lactic acid nisin onto hydroxypropyl chitosan, then crosslink it with aldehyde-modified hyaluronic acid, and add Sichuan pepper nano-silver as an additive, thereby obtaining a drug gel with antibacterial, anti-inflammatory, and antioxidant properties that effectively promotes wound healing.
[0025] 3. In this application, asiaticoside and zinc ions are preferred to pretreat the nano-silver of Sichuan pepper. Zinc ions and nano-silver produce an antibacterial synergistic effect, and zinc ions and asiaticoside can synergistically accelerate wound closure and provide stronger antioxidant defense for the wound, protecting newly formed tissue, thereby having a stronger anti-inflammatory and epithelialization-promoting effect. Detailed Implementation
[0026] The present application will be further described in detail below with reference to the embodiments.
[0027] Example 1: Dissolve 1g of hyaluronic acid in 100mL of deionized water, add 0.536g of sodium periodate, place in a light-protected environment, stir and react at room temperature for 2h, add 0.5mL of ethylene glycol and continue stirring for 1h, dialyze in deionized water for 3 days using a dialysis bag (molecular weight cutoff 8000Da), and freeze-dry to obtain aldehyde-modified hyaluronic acid.
[0028] Example 2 of preparation of nano-silver of Sichuan pepper: Sichuan pepper leaves were washed and dried at 50℃ for 12h, pulverized into powder, 2.5g of Sichuan pepper leaf powder was added to 100mL of deionized water, heated in a water bath at 80℃ for 20min, cooled to room temperature, centrifuged at 6000rpm for 10min, and the supernatant was taken as Sichuan pepper leaf extract. 10 mL of Sichuan pepper leaf extract was added to 20 mL of 5 mmol / L silver nitrate solution, stirred evenly, and stirred at 60 °C for 1 h. The mixture was then centrifuged at 10,000 rpm for 10 min. The precipitate was washed three times with anhydrous ethanol and purified water, respectively. The mixture was then frozen at -80 °C for 12 h and freeze-dried for 12 h to obtain Sichuan pepper nano-silver. Example
[0029] Example 1: A preparation process for hydroxypropyl chitosan, comprising the following steps: S1. Disperse 10g of chitosan in purified water, add 5mL of acetic acid under stirring to dissolve, and obtain a chitosan solution with a concentration of 2wt% and a degree of deacetylation of chitosan ≥90.0%; S2. Filter the chitosan solution through 1μm and 0.2μm filter membranes in sequence. Then add 2.5M sodium hydroxide solution dropwise to the chitosan solution and stir until a white precipitate is formed. Filter, wash twice with purified water, and then wash twice with 95% ethanol to obtain purified chitosan. S3. Add the purified chitosan to 80 mL of a mixture of salicylaldehyde and 95% ethanol, stir well, react at 40°C for 5 h, filter, wash three times with 95% ethanol to obtain salicylaldehyde-chitosan wet material. The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:7, and the weight-volume ratio of chitosan to the mixture is 1:8. S4. Mix the salicylaldehyde chitosan wet material with 100 mL of isopropanol, add 10 wt% sodium hydroxide solution, maintain the pH value of the system at 10, add 40 mL of propylene oxide dropwise at 40 °C, stir and react for 24 h, filter, and wash 3 times with 95% ethanol to obtain amino-protected hydroxypropyl chitosan. S6. Amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan. The concentration of HCl in the HCl / ethanol solution was 1M and the concentration of ethanol was 85%.
[0030] Example 2: A preparation process for hydroxypropyl chitosan, comprising the following steps: S1. Disperse 10g of chitosan in purified water, add 10mL of acetic acid under stirring to dissolve, and obtain a chitosan solution with a concentration of 3wt% and a degree of deacetylation of chitosan ≥90.0%; S2. Filter the chitosan solution through 1μm and 0.2μm filter membranes in sequence. Then add 2.5M sodium hydroxide solution dropwise to the chitosan solution and stir until a white precipitate is formed. Filter, wash three times with purified water, and then wash three times with 95% ethanol to obtain purified chitosan. S3. Add the purified chitosan to 100 mL of a mixture of salicylaldehyde and 95% ethanol, stir well, react at 50°C for 5 h, filter, wash three times with 95% ethanol to obtain salicylaldehyde-chitosan wet material. The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:9, and the weight-volume ratio of chitosan to the mixture is 1:10. S4. Mix the salicylaldehyde chitosan wet material with 200 mL of isopropanol, add 10 wt% sodium hydroxide solution, maintain the pH value of the system at 13, add 100 mL of propylene oxide dropwise at 50 °C, stir and react for 4 h, filter, and wash 3 times with 95% ethanol to obtain amino-protected hydroxypropyl chitosan. S6. Amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan. The concentration of HCl in the HCl / ethanol solution was 1.5M and the concentration of ethanol was 90%.
[0031] Example 3: A preparation process for hydroxypropyl chitosan, comprising the following steps: S1. Disperse 10g of chitosan in purified water, add 8mL of acetic acid under stirring to dissolve, and obtain a chitosan solution with a concentration of 1wt% and a degree of deacetylation of chitosan ≥90.0%; S2. Filter the chitosan solution through 1μm and 0.2μm filter membranes in sequence. Then add 2.5M sodium hydroxide solution dropwise to the chitosan solution and stir until a white precipitate is formed. Filter, wash three times with purified water, and then wash three times with 95% ethanol to obtain purified chitosan. S3. Add the purified chitosan to 90 mL of a mixture of salicylaldehyde and 95% ethanol, stir well, react at 60°C for 4 h, filter, wash three times with 95% ethanol to obtain salicylaldehyde-chitosan wet material. The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:8, and the weight-volume ratio of chitosan to the mixture is 1:9. S4. Mix the salicylaldehyde chitosan wet material with 160 mL of isopropanol, add 10 wt% sodium hydroxide solution, maintain the pH value of the system at 12, add 80 mL of propylene oxide dropwise at 45 °C, stir and react for 10 h, filter, and wash three times with 95% ethanol to obtain amino-protected hydroxypropyl chitosan. S6. Amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan. The concentration of HCl in the HCl / ethanol solution was 1.5M and the concentration of ethanol was 90%.
[0032] Example 4: A preparation process for hydroxypropyl chitosan, comprising the following steps: S1. Disperse 10g of chitosan in purified water, add 10mL of acetic acid under stirring to dissolve, and obtain a chitosan solution with a concentration of 1wt% and a degree of deacetylation of chitosan ≥90.0%; S2. Filter the chitosan solution through 1μm and 0.2μm filter membranes in sequence. Then add 2.5M sodium hydroxide solution dropwise to the chitosan solution and stir until a white precipitate is formed. Filter, wash three times with purified water, and then wash three times with 95% ethanol to obtain purified chitosan. S3. Add the purified chitosan to 100 mL of a mixture of salicylaldehyde and 95% ethanol, stir well, react at 60°C for 4 h, filter, wash 3 times with 95% ethanol to obtain salicylaldehyde chitosan wet material. The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:7, and the weight-volume ratio of chitosan to the mixture is 1:10. S4. Mix the salicylaldehyde chitosan wet material with 120 mL of isopropanol, add 10 wt% sodium hydroxide solution, maintain the pH value of the system at 13, add 50 mL of propylene oxide dropwise at 45 °C, stir and react for 20 h, filter, and wash three times with 95% ethanol to obtain amino-protected hydroxypropyl chitosan. S6. Amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan. The concentration of HCl in the HCl / ethanol solution was 1.3M and the concentration of ethanol was 90%.
[0033] Example 5: A preparation process for hydroxypropyl chitosan, comprising the following steps: S1. Disperse 10g of chitosan in purified water, add 10mL of acetic acid under stirring to dissolve, and obtain a chitosan solution with a concentration of 3wt% and a degree of deacetylation of chitosan ≥90.0%; S2. Filter the chitosan solution through 1μm and 0.2μm filter membranes in sequence. Then add 2.5M sodium hydroxide solution dropwise to the chitosan solution and stir until a white precipitate is formed. Filter, wash three times with purified water, and then wash three times with 95% ethanol to obtain purified chitosan. S3. Add the purified chitosan to 80 mL of a mixture of salicylaldehyde and 95% ethanol, stir well, react at 60°C for 4 h, filter, wash three times with 95% ethanol to obtain salicylaldehyde-chitosan wet material. The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:9, and the weight-volume ratio of chitosan to the mixture is 1:8. S4. Mix the salicylaldehyde chitosan wet material with 140 mL of isopropanol, add 10 wt% sodium hydroxide solution, maintain the pH value of the system at 13, add 60 mL of propylene oxide dropwise at 40 °C, stir and react for 24 h, filter, wash 3 times with 95% ethanol to obtain amino-protected hydroxypropyl chitosan. S6. Amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan. The concentration of HCl in the HCl / ethanol solution was 1.5M and the concentration of ethanol was 90%.
[0034] The solubility of the prepared hydroxypropyl chitosan was tested to determine its water solubility. 0.01 g of both the chitosan raw material and the prepared hydroxypropyl chitosan sample were accurately weighed using a balance and added to a 25 mL beaker. 9 mL of distilled water and 1 mL of 0.3 M HCl solution were added. The pH was measured using a pH meter, with an initial value of 2. The transmittance was measured at 660 nm using a UV-2600 microscope with a cuvette, and the pH value and transmittance at 660 nm were recorded. After recording, the solution in the cuvette was poured back into the beaker, and 0.5 M sodium hydroxide solution was added dropwise. After thorough stirring, the pH and transmittance were measured again. The amount of sodium hydroxide added should not be excessive when the pH value is close to the abrupt change point. The pH range was tested between 4 and 10. The transmittance of the dissolved hydroxypropyl chitosan at different pH values is recorded in Table 1.
[0035] Table 1
[0036] As shown in Table 1, chitosan is soluble in acidic conditions. With the addition of sodium hydroxide solution, the transmittance of chitosan decreases rapidly at pH 6. However, after hydroxypropylation, the transmittance of chitosan is still above 98% at pH 7-10. This indicates that the prepared hydroxypropyl chitosan has good solubility in acidic, neutral and alkaline environments.
[0037] Application Example 1: A wound healing and repair drug comprising the following raw materials by weight: 1g hydroxypropyl chitosan, 1.1g nisin, 0.5g transglutaminase, 3g aldehyde-modified hyaluronic acid prepared in Example 1, and 0.1g Sichuan pepper nano-silver prepared in Example 2. The transglutaminase was selected from Guangzhou Longde Biotechnology, catalog number 5688, the nisin was selected from Anhui Zhanhao Bioengineering, catalog number 669, and the hydroxypropyl chitosan was prepared in Example 1.
[0038] The preparation method of the above-mentioned wound healing and repair drugs is as follows: Purification of transglutaminase: Dissolve transglutaminase in phosphate buffer (0.2 mol / L, pH=6), and after complete dissolution, centrifuge at 4000 rpm for 10 min. Transfer the supernatant into a dialysis bag and dialyze in deionized water for 3 days, changing the water every 8 hours. Then freeze-dry. Hydroxypropyl chitosan and lactic acid nisin were dissolved in 500 mL of phosphate buffer (0.2 mol / L, pH=6), stirred evenly, and then purified transglutaminase was added. The mixture was stirred at 50 °C for 4 h, heated to 100 °C and kept at that temperature for 10 min, cooled, filtered, dialyzed in deionized water for 3 days, and then freeze-dried to obtain the complex. The complex was dissolved in distilled water to prepare a 10 wt% complex solution. Aldehyaluronic acid was dissolved in 100 mL of phosphate buffer (0.2 mol / L, pH=6), and the complex solution and Sichuan pepper nano-silver were added and mixed evenly to prepare a wound healing and repair drug.
[0039] Application Examples 2-5: A wound healing and repair drug, which differs from Application Example 1 in that it uses hydroxypropyl chitosan prepared in Examples 2-5.
[0040] Application Example 6: A wound healing and repair drug, which differs from Application Example 1 in that it does not contain Sichuan pepper nano-silver.
[0041] Application Example 7: A wound healing and repair drug, which differs from Application Example 1 in that an equal amount of nano-silver is used instead of pepper nano-silver.
[0042] Application Example 8: A wound healing and repair drug, differing from Application Example 1 in that the Sichuan pepper nano-silver undergoes the following pretreatment: Add asiaticoside to anhydrous ethanol and sonicate for 30 min to prepare a asiaticoside solution with a concentration of 10 mg / ml. The nano-silver of Sichuan pepper was dispersed in deionized water to obtain a dispersion with a concentration of 2wt%. A zinc chloride hexahydrate aqueous solution with a concentration of 0.01mol / L was added to the dispersion until the zinc ion concentration was 60mM. The mixture was stirred evenly to obtain a composite solution. Add asiaticoside solution under light-protected conditions, with a volume ratio of 1:1 between the compound solution and the asiaticoside solution. Stir well, centrifuge, wash, and dry.
[0043] Application Example 9: A wound healing and repair drug, differing from Application Example 1 in that the Sichuan pepper nano-silver undergoes the following pretreatment: Add asiaticoside to anhydrous ethanol and ultrasonically disperse for 30 min to obtain a asiaticoside solution with a concentration of 10 mg / ml. The nano-silver of Sichuan pepper was dispersed in deionized water to prepare a dispersion with a concentration of 2wt%. Centella asiatica glycoside solution was added under light-protected conditions, with a volume ratio of 1:1 between the dispersion and the centella asiatica glycoside solution. After stirring evenly, the mixture was centrifuged, washed, and dried.
[0044] Application Example 10: A wound healing and repair drug, differing from Application Example 1 in that the Sichuan pepper nano-silver undergoes the following pretreatment: The nano-silver of Sichuan pepper was dispersed in deionized water to obtain a dispersion with a concentration of 2wt%. A zinc chloride hexahydrate aqueous solution with a concentration of 0.01mol / L was added to the dispersion until the zinc ion concentration reached 60mM. The mixture was stirred evenly, centrifuged, washed, and dried.
[0045] Performance testing
[0046] Prepare the wound healing and repair drug according to the method in the application example, and conduct a wound healing speed test according to the following method. Record the test results in Table 2.
[0047] (1) Establishment of wound model: SD rats were randomly numbered after being fed for one week and divided into groups of 4. Group 1 was the saline group, Group 2 was the hydroxypropyl chitosan group, and Groups 3-12 were respectively treated with the repair drugs prepared in Examples 1-10. 0.9 mL of 3% sodium pentobarbital solution was injected into the peritoneum of each SD rat. After complete anesthesia, the back was shaved and the skin was prepared. Finally, a constant temperature electric iron was set to 75°C and applied vertically to the skin of the rats for 15 seconds to establish a symmetrical deep second-degree burn model on the back of each rat.
[0048] (2) Administration regimen: The wounds on the backs of the rats were disinfected, and then different rats were given corresponding medications. Physiological saline was applied to the wounds of the rats in group 1, hydroxypropyl chitosan solution was applied to the wounds of the rats in group 2, and drug gel was applied to the rats in groups 3-12. The amount of medication applied was the same. Each wound was bandaged with sterile gauze and fixed with elastic bandages. Each rat was housed in a separate cage and given normal water and feed. The wounds were cleaned and dressed every 2 days.
[0049] (3) Wound healing rate: On days 0, 7, 14 and 21, standard transparent grid paper was used to cover each wound and trace the shape of each wound. The wound healing rate was calculated by calculating the area of the figure on the transparent grid paper according to the following formula: Wound healing rate = (A0-At) / A0×100%, where A0 is the area of the wound on day 0 and At are the areas of the wound on days 7, 14 and 21 respectively.
[0050] (4) Wound healing time: Wound healing time is determined by visual observation and the time required for complete wound healing is recorded.
[0051] Table 2
[0052] As can be seen from the data in Table 2, the healing and repair drugs prepared using Examples 1-5 have anti-inflammatory effects and inhibit excessive proliferation of fibroblasts in rats, which can reduce the inflammatory response of the wound and accelerate the healing of the wound.
[0053] In Application Example 6, no Sichuan pepper nano-silver was added. The data in Table 2 shows that the healing rate of the prepared repair drug gel on the burn wound was slightly lower than that in Application Example 1, indicating that the anti-inflammatory and antibacterial effects of Sichuan pepper nano-silver can improve the wound healing rate.
[0054] Compared with Application Example 1, Application Example 7 used nano-silver instead of Sichuan pepper nano-silver. The data in Table 2 shows that the healing effect of the repair drug gel prepared in Application Example 7 on burn wounds decreased, but was higher than that in Application Example 6, indicating that Sichuan pepper and nano-silver have a synergistic effect.
[0055] Compared with Application Example 1, Application Example 8 uses asiaticoside and zinc ions to pretreat the nano-silver of Sichuan pepper. As shown in Table 2, the wound healing speed is accelerated, and healing can be completed in 17 days, indicating that asiaticoside and zinc ions can accelerate wound healing.
[0056] Compared with Application Example 1, Application Examples 9 and 10 used only asiaticoside and zinc ions to pretreat the nano-silver of Sichuan pepper. It can be seen that the wound healing rate increased and the healing time was shortened. However, compared with Application Example 8, the healing speed was slower, indicating that the combination of asiaticoside and zinc ions can accelerate the wound healing speed.
[0057] 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 hydroxypropyl chitosan, characterized in that, Includes the following steps: Chitosan was dispersed in purified water and dissolved in acetic acid under stirring to obtain a chitosan solution with a concentration of 1-3%. The chitosan solution was purified to obtain purified chitosan; The purified chitosan was added to a mixture of salicylaldehyde and 95% ethanol, stirred evenly, and reacted at 40-60℃ for 4-5 hours. After filtration and washing with 95% ethanol 2-3 times, salicylaldehyde-chitosan wet material was obtained. Salicylaldehyde chitosan wet material and isopropanol were mixed, and 10wt% sodium hydroxide solution was added to maintain the pH of the system at 10-13. Propylene oxide / 1-chloro-2-propanol was added dropwise at 40-50℃, and the reaction was stirred for 4-24 hours. After filtration and washing, amino-protected hydroxypropyl chitosan was obtained. The mass-volume ratio of chitosan, isopropanol and propylene oxide was 3-5:50-100:20-50 or the mass-volume ratio of chitosan, isopropanol and 1-chloro-2-propanol was 3-5:50-100:5-10. The amino-protected hydroxypropyl chitosan was soaked in an HCl / ethanol solution for 22-24 hours, then dialyzed and freeze-dried to obtain hydroxypropyl chitosan.
2. The preparation process of hydroxypropyl chitosan according to claim 1, characterized in that: The mass-to-volume ratio of chitosan to acetic acid is 1:0.5-1.
3. The preparation process of hydroxypropyl chitosan according to claim 1, characterized in that: The purification method of the chitosan solution is as follows: the chitosan solution is filtered through 1μm and 0.2μm filter membranes in sequence, then sodium hydroxide solution is added dropwise to the chitosan solution, and the mixture is stirred until a white precipitate is produced. The mixture is then filtered, washed 2-3 times with purified water, and washed 2-3 times with 95% ethanol to obtain purified chitosan.
4. The preparation process of hydroxypropyl chitosan according to claim 1, characterized in that: The volume ratio of salicylaldehyde to 95% ethanol in the mixture is 1:7-9, and the weight-volume ratio of chitosan to the mixture is 1:8-10.
5. The preparation process of hydroxypropyl chitosan according to claim 1, characterized in that: The concentration of HCl in the HCl / ethanol solution is 1-1.5M, and the concentration of ethanol is 85-90%.
6. A hydroxypropyl chitosan, characterized in that, It is prepared using the preparation process of hydroxypropyl chitosan according to any one of claims 1-5.
7. The use of hydroxypropyl chitosan prepared by the preparation process according to any one of claims 1-5 in cosmetics, wound healing and repair drugs, medical devices, or biomedical materials.
8. The application of the hydroxypropyl chitosan according to claim 7, characterized in that, The wound healing and repair drug contains the following raw materials in parts by weight: 1-2 parts hydroxypropyl chitosan, 1.1-2.2 parts nisin, 0.5-1 part transglutaminase, 3-6 parts aldehyde-modified hyaluronic acid, and 0.1-0.5 parts Sichuan pepper nano-silver.
9. The application of hydroxypropyl chitosan according to claim 8, characterized in that, The Sichuan pepper nano-silver underwent the following pretreatment: asiaticoside was added to anhydrous ethanol and ultrasonically dispersed to obtain asiaticoside solution. The nano-silver of Sichuan pepper was dispersed in deionized water to prepare a dispersion; zinc chloride hexahydrate aqueous solution was added to the dispersion and stirred evenly; under light-protected conditions, asiaticoside solution was added, stirred evenly, centrifuged, washed, and dried.