Water-soluble nicotinic acylated chitosan oligosaccharide quaternary ammonium salt as well as preparation method and application thereof
By precisely designing and modifying chitosan oligosaccharide molecules, water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salts were prepared, which solved the problem of insufficient biological activity of chitosan oligosaccharides, achieved efficient and safe antibacterial effects, and expanded its scope of application.
Patent Information
- Application Number
- CN202510616983.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-11-11
- Filing Date
- 2025-05-14
- Publication Date
- 2025-09-16
AI Technical Summary
The insufficient biological activity of natural chitosan oligosaccharides limits their in-depth application in high-end fields, and existing antibacterial agents have deficiencies in safety and effectiveness.
By precisely designing and modifying chitosan oligosaccharide molecules, introducing nicotinyl and quaternary ammonium groups, water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salts were prepared. N,N'-carbonyldiimidazole was used to catalyze the reaction of nicotinic acid and chitosan oligosaccharide, and then the reaction was carried out with 4-bromobutanol, 4-bromobutylamine hydrobromide and 4-bromobutyltrimethylammonium bromide to form water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salts.
It significantly improves the water solubility and biological activity of chitosan oligosaccharides, giving them broad-spectrum antibacterial properties, which can effectively inhibit Escherichia coli and Staphylococcus aureus, broaden the scope of application, partially replace commercial antibacterial agents, and improve safety and effectiveness.
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Figure CN120647804A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of daily chemicals and the pharmaceutical industry, and in particular to a water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, a preparation method thereof, and an application thereof. Background Art
[0002] Chitooligosaccharide, as a high-molecular carbohydrate derived from nature, is composed of N-acetyl-D-glucose molecules connected by glycosidic bonds. It has unique biocompatibility and degradability, and shows broad application potential in many fields such as biomedicine, food and health care, and agriculture. However, although natural chitooligosaccharides have certain biological activities, their activity intensity is often not enough to meet the needs of specific applications, which limits their in-depth application in certain high-end fields. As a major producer of chitooligosaccharides, my country has abundant output but low added value. Therefore, exploring technologies to enhance the bioactivity of chitooligosaccharides and broaden their application range is of great significance to promoting industrial upgrading and maximizing economic value.
[0003] In recent years, researchers have discovered that by precisely designing and modifying the molecular structure of chitosan oligosaccharides, their biological activity can be effectively enhanced. This discovery has opened up a new path for the high-value utilization of chitosan oligosaccharides. In particular, by using active functional groups such as amino and hydroxyl groups in chitosan oligosaccharide molecules as "anchor points" and introducing chemical groups with specific functions, such as nicotinyl and quaternary ammonium groups, not only can the water solubility of chitosan oligosaccharides be significantly improved, but also new biological functional properties can be given to them, such as enhanced antibacterial, antioxidant or immunomodulatory capabilities. This strategy not only overcomes the limitations of the mild biological activity of natural chitosan oligosaccharides, but also provides a theoretical basis and technical support for the development of efficient and multifunctional chitosan oligosaccharide-based new materials.
[0004] On this basis, the development of a highly efficient and environmentally friendly method for preparing water-soluble nicotinoyl-modified chitosan oligosaccharide quaternary ammonium salts has become a hot topic of research. This method aims to precisely control the chemical modification process, ensuring that the modified chitosan oligosaccharide derivatives retain their original biocompatibility while also achieving enhanced bioactivity and excellent water solubility. This will greatly expand their application prospects in areas such as drug delivery, biosensing, green pesticides, and functional food additives. Furthermore, the successful implementation of this technology is expected to drive the high-end and differentiated development of my country's chitosan oligosaccharide industry, bringing revolutionary technological innovation and economic benefits to related industries. Summary of the Invention
[0005] Technical problem to be solved: The present invention provides a water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt and its preparation method and application, which can be used as an antibacterial agent to partially replace commercial antibacterial agents and is safer and more effective.
[0006] Technical solution: water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, the structural formula of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt is shown in (1):
[0007]
[0008] Among them, n ranges from 6 to 20, R= R1= Any one of them.
[0009] The preparation method of the water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salt comprises the following steps: 1) firstly using N,N'-carbonyldiimidazole to catalyze nicotinic acid and then reacting it with chitosan oligosaccharide to obtain a chitosan oligosaccharide nicotinic acid graft copolymer, wherein the molar ratio of the N,N'-carbonyldiimidazole to chitosan oligosaccharide is 1.5-2.5; and the molar ratio of nicotinic acid to chitosan is 1.5-2.5; 2) reacting the chitosan oligosaccharide nicotinic acid graft copolymer with 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide to obtain the water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salt represented by formula (1); the molar amounts of the 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide are 2.0-4.0 times the molar amount of the chitosan oligosaccharide nicotinic acid graft copolymer.
[0010] Preferably, N,N'-carbonyldiimidazole is added to a dimethyl sulfoxide solution of nicotinic acid and mixed, and the mixture is reacted at 55-65°C in a nitrogen atmosphere for 8.0-16.0 hours, wherein the molar ratio of nicotinic acid to N,N'-carbonyldiimidazole is 5.0:3.0-3.0:5.0; chitosan oligosaccharide is dissolved in dimethyl sulfoxide, and then the solution obtained in the previous step is added dropwise, and the reaction is continued at 55-65°C for 8.0-16.0 hours. After the reaction is completed and cooled to room temperature, the mixture is precipitated with ethanol, washed, cooled and dried to obtain a chitosan oligosaccharide nicotinic acid graft copolymer for later use.
[0011] Preferably, the chitosan oligosaccharide nicotinic acid graft copolymer is reacted with 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide under nitrogen protection at 55-65° C. for 18-30 hours, precipitated with ethanol, washed and freeze-dried to obtain three water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salts.
[0012] Application of the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt in the preparation of antibacterial agents.
[0013] An antibacterial agent, the active ingredient of which contains the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt.
[0014] Beneficial effects: The present invention provides a water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salt and its efficient preparation method and broad application prospects. This innovative product not only shows great potential as a new antibacterial agent and can partially replace the commercial antibacterial agents currently on the market, but also achieves significant improvements in safety and effectiveness. Its unique molecular structure gives it excellent biocompatibility and broad-spectrum antibacterial properties, making it more targeted and durable when combating various microbial contaminations. In addition, its water-soluble characteristics greatly broaden the scope of application, making it easy to disperse evenly on different materials and environments, thereby effectively inhibiting bacterial growth, protecting human health and extending the service life of the product. In summary, the water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salt of the present invention not only opens up a new path in the antibacterial field, but also makes an important contribution to promoting the development of green and safe antibacterial technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the infrared spectrum of chitosan oligosaccharide.
[0016] Figure 2 The infrared spectrum of chitosan oligosaccharide nicotinic acid graft copolymer is provided for the embodiment of the present invention. -1 The new peaks appearing at 400 nm are the stretching vibration peaks of amide bond (C=O), ester bond (C=O) and ester bond (CO), respectively. This proves that the chitosan oligosaccharide nicotinic acid grafted derivatives were successfully synthesized.
[0017] Figure 3 The present invention provides an infrared spectrum of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt A. At 1477 cm -1 The peak at 40° is the absorption peak of pyridinium cation, which proves that the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt A was successfully synthesized.
[0018] Figure 4 The infrared spectrum of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt B is provided for the embodiment of the present invention. -1 The peak at 40° is the absorption peak of pyridinium cation, which proves that the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt A was successfully synthesized.
[0019] Figure 5 The present invention provides an infrared spectrum of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt C. At 1478 cm -1 The peak at 40° is the absorption peak of pyridinium cation, which proves that the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt A was successfully synthesized. DETAILED DESCRIPTION
[0020] The present invention will be further explained below with reference to the accompanying drawings and examples.
[0021] First, N,N'-carbonyldiimidazole is used to catalyze the carboxyl groups on the nicotinic acid molecules, and then directly reacts with chitosan oligosaccharide molecules to obtain chitosan oligosaccharide nicotinic acid graft copolymers. The chitosan oligosaccharide nicotinic acid graft copolymers are then reacted with 4-bromobutanol, 4-bromobutylamine hydrobromide and 4-bromobutyltrimethylammonium bromide to obtain water-soluble nicotinated chitosan oligosaccharide quaternary ammonium salts.
[0022] The water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt obtained above has the ability to inhibit Escherichia coli and Staphylococcus aureus.
[0023] The synthesis route of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt is as follows:
[0024]
[0025] Wherein, R is nicotinyl, R1 is hydroxyl, amino and quaternary ammonium salt at the end of the alkyl group, R2 is N-n-butylnicotinyl, and the average degree of polymerization n ranges from 6 to 20.
[0026] Example 1
[0027] In this example, the target compound, water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, was synthesized according to the above synthesis route.
[0028] 1) Preparation of chitosan oligosaccharide-nicotinic acid graft copolymer: 4.92 g of nicotinic acid was dispersed in 10 mL of dimethyl sulfoxide, 6.48 g of N,N'-carbonyldiimidazole was added, and a clear solution was obtained by stirring with a heat-collecting constant temperature heating magnetic stirrer. The solution was reacted at 60°C for 12 h under a nitrogen atmosphere. 3.22 g of chitosan oligosaccharide (see Figure 1 ) was dissolved in 20 mL of dimethyl sulfoxide, and the solution obtained in the previous step was added dropwise to the starch dimethyl sulfoxide solution, and the reaction was continued at 60 ° C for 12 h under a nitrogen atmosphere. Then, it was precipitated with anhydrous ethanol, washed, and freeze-dried to obtain 3.14 g of chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ), for future use. Wherein, R1 is nicotinoyl.
[0029] 2) Preparation of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt: 1.06 g chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ) was added to 6 mL of dimethyl sulfoxide, and then 1.71 mL of 4-bromo-n-butanol, 2.80 g of 4-bromo-n-butylamine hydrobromide and 4.41 g of 4-bromobutyltrimethylammonium bromide were added respectively, and the mixture was reacted at 60 ° C for 24 hours under nitrogen protection. After the reaction, the mixture was precipitated with anhydrous ethanol, filtered, washed, and vacuum freeze-dried to obtain a water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt (see Figure 3-5), 0.96, 1.04, and 1.15 g, for later use. Wherein, R is a nicotinyl group, R1 is an alkyl terminal hydroxyl group (water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salt A), an amino group (water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salt B), or a quaternary ammonium salt (water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salt C), and R2 is an N-butylnicotinyl group.
[0030] Example 2
[0031] In this example, the target compound, water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, was synthesized according to the above synthesis route.
[0032] 1) Preparation of chitosan oligosaccharide-nicotinic acid graft copolymer: 3.28 g of nicotinic acid was dispersed in 8 mL of dimethyl sulfoxide, 4.32 g of N,N'-carbonyldiimidazole was added, and a clear solution was obtained by stirring with a heat-collecting constant temperature heating magnetic stirrer. The solution was reacted at 65°C for 16 h under a nitrogen atmosphere; 3.22 g of chitosan oligosaccharide (see Figure 1 ) was dissolved in 20 mL of dimethyl sulfoxide, and the solution obtained in the previous step was added dropwise to the starch dimethyl sulfoxide solution, and the reaction was continued at 65 ° C for 16 hours under a nitrogen atmosphere. Then, it was precipitated with anhydrous ethanol, washed, and freeze-dried to obtain 2.79 g of chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ), for future use. Wherein, R1 is nicotinoyl.
[0033] 2) Preparation of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt: 1.06 g chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ) was added to 6 mL of dimethyl sulfoxide, and then 1.14 mL of 4-bromo-n-butanol, 1.87 g of 4-bromo-n-butylamine hydrobromide and 2.20 g of 4-bromobutyltrimethylammonium bromide were added respectively, and the mixture was reacted at 65 ° C for 30 hours under nitrogen protection. After the reaction, the mixture was precipitated with anhydrous ethanol, filtered, washed, and vacuum freeze-dried to obtain a water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt (see Figure 3-5 ), 0.91, 0.94, and 1.07 g, for later use. Wherein, R is a nicotinyl group, R1 is a hydroxyl group, an amino group, or a quaternary ammonium salt at the end of an alkyl group, and R2 is an N-n-butylnicotinyl group.
[0034] Example 3
[0035] In this example, the target compound, water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, was synthesized according to the above synthesis route.
[0036] 1) Preparation of chitosan oligosaccharide-nicotinic acid graft copolymer: 6.15 g of nicotinic acid was dispersed in 12 mL of dimethyl sulfoxide, 7.20 g of N,N'-carbonyldiimidazole was added, and a clear solution was obtained by stirring with a heat-collecting constant temperature heating magnetic stirrer. The solution was reacted at 55°C for 8 h under a nitrogen atmosphere. 3.22 g of chitosan oligosaccharide (see Figure 1) was dissolved in 20 mL of dimethyl sulfoxide, and the solution obtained in the previous step was added dropwise to the starch dimethyl sulfoxide solution, and the reaction was continued at 55 ° C for 8 h under a nitrogen atmosphere. Then, it was precipitated with anhydrous ethanol, washed, and freeze-dried to obtain 3.34 g of chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ), for future use. Wherein, R1 is nicotinoyl.
[0037] 2) Preparation of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt: 1.06 g chitosan oligosaccharide nicotinic acid graft copolymer (see Figure 2 ) was added to 6 mL of dimethyl sulfoxide, and then 2.27 mL of 4-bromo-n-butanol, 3.73 g of 4-bromo-n-butylamine hydrobromide and 4.41 g of 4-bromobutyltrimethylammonium bromide were added respectively, and the mixture was reacted at 55 ° C for 18 hours under nitrogen protection. After the reaction, the mixture was precipitated with anhydrous ethanol, filtered, washed, and vacuum freeze-dried to obtain a water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt (see Figure 3-5 ), 1.02, 1.09, and 1.24 g, for future use. Wherein, R is a nicotinyl group, R1 is a hydroxyl group, an amino group, or a quaternary ammonium salt at the end of an alkyl group, and R2 is an N-n-butylnicotinyl group.
[0038] Application Case 1
[0039] Determination of antibacterial ability against Escherichia coli:
[0040] Chitosan oligosaccharide, the chitosan oligosaccharide nicotinic acid graft copolymer prepared in Example 1, and three water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salts were prepared into corresponding sample solutions at 32 mg / mL. Serial dilutions were performed in a suitable liquid culture medium in a 96-well plate. A certain amount of Escherichia coli culture was added to each tube, and the final sample concentrations were controlled at 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.078125 mg / mL. After 18 hours of incubation, bacterial growth was observed and compared with the blank control. The lowest concentration at which the culture medium was clear and no bacterial growth was observed was determined as the minimum inhibitory concentration (MIC) of the drug. The drug and bacterial strains without bacterial growth were streaked onto new agar plates for incubation to further determine the minimum bactericidal concentration (MBC). The experiment was typically repeated three times for each compound.
[0041] Table 1. Minimum inhibitory concentration and minimum bactericidal concentration of chitosan oligosaccharide derivatives and chitosan oligosaccharides against Escherichia coli
[0042]
[0043] Application Case 2
[0044] Determination of antibacterial ability against Staphylococcus aureus:
[0045] Chitosan oligosaccharide, the chitosan oligosaccharide nicotinic acid graft copolymer prepared in Example 1, and three water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salts were prepared into corresponding sample solutions at 32 mg / mL. Serial dilutions were performed in a suitable liquid culture medium in a 96-well plate. A certain amount of Escherichia coli culture was added to each tube, and the final sample concentrations were controlled at 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.078125 mg / mL. After 18 hours of incubation, bacterial growth was observed and compared with the blank control. The lowest concentration at which the culture medium was clear and no bacterial growth was observed was determined as the minimum inhibitory concentration (MIC) of the drug. The drug and bacterial strains without bacterial growth were streaked onto new agar plates for incubation to further determine the minimum bactericidal concentration (MBC). The experiment was typically repeated three times for each compound.
[0046] Table 2. Minimum inhibitory concentration and minimum bactericidal concentration of chitosan oligosaccharide derivatives and chitosan oligosaccharides against Escherichia coli
[0047]
[0048] Experimental results: The antibacterial activities of the chitosan oligosaccharide derivatives and chitosan oligosaccharides synthesized by the present invention against Escherichia coli and Staphylococcus aureus are shown in Tables 1 and 2. The antibacterial activities of the three water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salts synthesized by the present invention are stronger than those of the chitosan oligosaccharide raw material and the chitosan oligosaccharide nicotinic acid graft copolymer, proving that the presence of quaternary ammonium salt can improve the antibacterial activity of chitosan oligosaccharides; and the antibacterial activity of the water-soluble nicotinoylated chitosan oligosaccharide quaternary ammonium salt C is significantly better than that of the first two chitosan oligosaccharide derivatives, with MIC and MBC against Staphylococcus aureus of 0.5 and 1 mg / mL, respectively, indicating that the introduction of a higher concentration of positive charge density can significantly improve the antibacterial ability.
Claims
1. A water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt, characterized in that: The structural formula of water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt is shown in (1): Among them, n ranges from 6 to 20, R= R1= Any one of them.
2. The method for preparing the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt according to claim 1, characterized in that: The method comprises the following steps: 1) firstly using N,N'-carbonyldiimidazole to catalyze nicotinic acid and then reacting it with chitosan oligosaccharide to obtain a chitosan oligosaccharide nicotinic acid graft copolymer, wherein the molar ratio of the N,N'-carbonyldiimidazole to chitosan oligosaccharide is 1.5-2.5; and the molar ratio of nicotinic acid to chitosan is 1.5-2.5; 2) reacting the chitosan oligosaccharide nicotinic acid graft copolymer with 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide to obtain a water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salt represented by formula (1); wherein the molar amounts of the 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide are 2.0-4.0 times the molar amount of the chitosan oligosaccharide nicotinic acid graft copolymer.
3. The method for preparing the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt according to claim 2, characterized in that: N,N'-carbonyldiimidazole is added to a dimethyl sulfoxide solution of nicotinic acid and mixed evenly, and the mixture is reacted at 55-65°C in a nitrogen atmosphere for 8.0-16.0 hours, wherein the molar ratio of nicotinic acid to N,N'-carbonyldiimidazole is 5.0:3.0-3.0:5.0; chitosan oligosaccharide is dissolved in dimethyl sulfoxide, and then the solution obtained in the previous step is added dropwise, and the reaction is continued at 55-65°C for 8.0-16.0 hours. After the reaction is completed and cooled to room temperature, the mixture is precipitated with ethanol, washed, cooled and dried to obtain a chitosan oligosaccharide nicotinic acid graft copolymer for later use.
4. The method for preparing the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt according to claim 2, characterized in that: The chitosan oligosaccharide nicotinic acid graft copolymer is reacted with 4-bromo-n-butanol, 4-bromo-n-butylamine hydrobromide and 4-bromobutyltrimethylammonium bromide at 55-65° C. for 18-30 hours under nitrogen protection, and then precipitated with ethanol, washed and freeze-dried to obtain three water-soluble nicotinyl chitosan oligosaccharide quaternary ammonium salts.
5. Use of the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt according to claim 1 in the preparation of an antibacterial agent.
6. An antibacterial agent, characterized in that The active ingredient contains the water-soluble nicotinoyl chitosan oligosaccharide quaternary ammonium salt according to claim 1.