N,n,n-trimethyl chitosan quaternary ammonium salt containing glycine schiff base and preparation and use thereof

Abstract

The application belongs to the technical field of marine chemical engineering, and particularly relates to N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base, a preparation method and application thereof. The N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base is shown in formula (1). The application has the advantages of simple reaction equipment, easy control of reaction process, low cost of raw materials, high safety, easy popularization and industrial application. Research proves that the compound has good antioxidant activity and bacterial inhibition activity, and can be applied to the fields of agriculture, medicine and environmental protection.

Description

Technical Field

[0001] This invention belongs to marine chemical engineering technology, specifically relating to a class of N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine Schiff bases, their preparation methods, and applications. Background Technology

[0002] In agricultural production, the most important task is to increase the yield of high-quality crops to meet the growing food demand of the population. Common problems in crops include pests, pathogens, and pesticide overuse. If these variables are not controlled, they can cause huge losses to agriculture. Bacteria and fungi in nature have a significant impact on the growth and reproduction of crops and livestock. To avoid agricultural diseases and yield reductions caused by plant pathogenic fungi, the synthesis and use of new fungicides are crucial. Furthermore, bacterial infections in agricultural production should not be underestimated. Bacteria infect livestock, contaminate surrounding irrigation water sources, and spread through a cycle, affecting food security.

[0003] Chitosan possesses excellent antibacterial activity, anti-biofilm activity, biodegradability, biocompatibility, and safety, making it a popular environmentally friendly biomaterial with wide applications in biomedicine, food preservation, and wastewater treatment. It has been a hot research topic in the field of polymer materials for nearly 100 years. With the continuous development of modern agricultural production, the preparation and application of chitosan and its derivatives have played a crucial role in improving the agricultural production environment. Chitosan has been reported as a potential resistance inducer, broad-spectrum fungicide, antibacterial agent, and soil conditioner.

[0004] Schiff bases are a class of compounds with imine or azomethyl groups, obtained by the reaction of primary amines with aldehydes or ketones. Due to the lone pair of electrons in the carbon-nitrogen double bond of Schiff bases, they possess strong coordination properties, leading to extensive research on their metal complexes. Schiff bases exhibit a variety of biological activities, such as anti-inflammatory, antibacterial, antifungal, antiparasitic, antiviral, antiproliferative, antitumor, antimalarial, insecticidal, antioxidant, and anticorrosive activities, and are widely used in drug development, materials science, catalysis, and other fields.

[0005] Therefore, further research is needed on the modification and derivatization of chitosan to enable its high-value utilization. Summary of the Invention:

[0006] The purpose of this invention is to provide a method for preparing and applying a class of N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine Schiff bases that have antibacterial and antioxidant activities.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] An N,N,N-trimethyl chitosan quaternary ammonium salt containing a glycine Schiff base is shown in formula (1).

[0009]

[0010] In the formula,

[0011]

[0012] Where n represents the degree of polymerization, and the average value of n ranges from 20 to 3000.

[0013] Preparation method of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: By combining N,N,N-trimethyl chitosan quaternary ammonium salt with glycine Schiff base through ion exchange, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base as shown in formula (1) is obtained.

[0014] The specific reaction formula is as follows:

[0015]

[0016] To elaborate further,

[0017] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: Chitosan was dispersed in N-methylpyrrolidone. Sodium iodide and sodium hydroxide aqueous solution were added sequentially under ice bath stirring. After stirring for 30 min to 1 h, iodomethane was added dropwise. After uniform dispersion, the solution was transferred to 60 °C and refluxed for 2 to 12 h until the solution was clear. After the reaction was completed, the solution was filtered, washed three times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt. The molar ratio of chitosan, sodium iodide, sodium hydroxide, and iodomethane was 1:3:6:10-20.

[0018] (2) Synthesis of potassium salt of glycine Schiff base: Glycine and potassium hydroxide were dissolved in anhydrous ethanol and stirred at 50-60℃ until dissolved. Salicylic aldehyde derivatives dissolved in anhydrous ethanol were added separately and reacted at 50-60℃ for 1-6 hours. After the reaction was completed, the mixture was filtered, washed, recrystallized, and freeze-dried under vacuum. The molar ratio of glycine, potassium hydroxide, and salicylic aldehyde derivatives was 1:1:1.5-3.

[0019] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: The N,N,N-trimethyl chitosan quaternary ammonium salt obtained above was dissolved in water, and the potassium salt of glycine Schiff base was dissolved in water and added dropwise to the aqueous solution of N,N,N-trimethyl chitosan quaternary ammonium salt. The reaction was stirred at room temperature for 12-24 h. After the reaction was completed, the mixture was dialyzed using a dialysis bag and then freeze-dried under vacuum. The molar ratio of N,N,N-trimethyl chitosan quaternary ammonium salt to potassium salt of glycine Schiff base was 1:2-6.

[0020] In step (1), chitosan is dispersed in N-methylpyrrolidone, and the mass of N-methylpyrrolidone is 30-50 times that of chitosan;

[0021] The sodium hydroxide aqueous solution added in step (1) has a mass fraction of 10%-20%.

[0022] In step (2), glycine and potassium hydroxide are dissolved in anhydrous ethanol, wherein the volume-to-weight ratio of anhydrous ethanol to glycine is 50–200:1.

[0023] In step (2), the salicylaldehyde derivative is salicylaldehyde, 5-chlorosalicylaldehyde, 5-bromosalicylaldehyde, 3,5-dichlorosalicylaldehyde or 3,5-dibromosalicylaldehyde, wherein the volume-to-weight ratio of anhydrous ethanol to the salicylaldehyde derivative is 10–50:1.

[0024] In step (3), the N,N,N-trimethyl chitosan quaternary ammonium salt obtained above is dissolved in water, and the mass fraction of the N,N,N-trimethyl chitosan quaternary ammonium salt aqueous solution is 0.3%-0.8%;

[0025] In step (3), the potassium salt of glycine Schiff base is dissolved in water, and the mass fraction of the potassium salt of glycine Schiff base aqueous solution is 5%-20%.

[0026] In step (3), dialysis is performed using a dialysis bag, and the molecular weight cutoff of the dialysis bag is 10kDa–500kDa.

[0027] An application of the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base, wherein the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base as shown in formula (1) is used in the preparation of antibacterial agents or antioxidants.

[0028] Advantages of this invention:

[0029] (1) The N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base of the present invention is obtained by superimposing the activity of glycine Schiff base with the activity of N,N,N-trimethyl chitosan quaternary ammonium salt, resulting in a class of chitosan derivatives with good antioxidant activity and high antibacterial activity.

[0030] (2) The present invention provides a simple synthesis process for preparing N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base, the reaction process is easy to control, the raw materials are inexpensive, the safety is high, and it is easy to promote and industrialize.

[0031] (3) The N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base prepared by this invention has good antioxidant activity and antibacterial activity, and can be applied in agriculture, medicine and environmental protection. Attached image description:

[0032] Figure 1 The synthetic route diagram of the compound shown in formula (1) is provided for the embodiments of the present invention.

[0033] Figure 2 The infrared spectrum of chitosan provided in this embodiment of the invention is shown at 3411 cm⁻¹. -1 The broad peak at 2878 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of chitosan. -1 The peak at 1599 cm⁻¹ is caused by the stretching vibration of CH. -1 A distinct amide II band is visible at 1419 cm. -1 The absorption peak at 1382 cm⁻¹ is caused by the bending vibration of CH₂ and the deformation vibration of CH₃. -1 The absorption peak at 1156 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 1078 cm⁻¹ is caused by the COC stretching vibration. -1 The absorption peak at 894 cm⁻¹ is caused by the CO stretching vibration of the secondary alcohol hydroxyl group, while the absorption peak at 894 cm⁻¹ is caused by the CO stretching vibration of the secondary alcohol hydroxyl group. -1 The peak is caused by the stretching vibration of the sugar ring.

[0034] Figure 3 The infrared spectrum of N,N,N-trimethylchitosan quaternary ammonium salt provided in Example 1 of this invention, 3416 cm⁻¹ -1 The absorption peak at 2926 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of N,N,N-trimethyl chitosan quaternary ammonium salt. -1 The absorption peak at 1627 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak of amide I is at 1470 cm⁻¹. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1386 cm⁻¹. -1 The absorption peak at 1055 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at that point is caused by the asymmetric stretching of CH in CH3.

[0035] Figure 4 The infrared spectrum of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylaldehyde Schiff base provided in Example 1 of this invention, 3419 cm⁻¹ -1 The absorption peak at 2929 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of the N,N,N-trimethyl chitosan quaternary ammonium salt containing the glycine-salicylaldehyde Schiff base. -1 The absorption peak at 2881 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak at 1638 cm⁻¹ is caused by the bending and resonance vibrations of the C=N anode on the glycine Schiff base. -1 The absorption peak at 1481 cm⁻¹ is the C=N stretching vibration of the glycine Schiff base. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1373 cm⁻¹ -1 The absorption peak at 1056 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 850 cm⁻¹ is caused by the asymmetric stretching of CH in the methyl group. -1 The absorption peak at that point is caused by the stretching vibration of CH on the benzene ring.

[0036] Figure 5 The infrared spectrum of the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylaldehyde Schiff base provided in Example 2 of this invention, 3438 cm⁻¹ -1 The absorption peak at 2925 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of the N,N,N-trimethyl chitosan quaternary ammonium salt containing the glycine-5-chlorosalicylaldehyde Schiff base. -1 The absorption peak at 2888 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak at 1644 cm⁻¹ is caused by the bending and resonance vibrations of the C=N anode on the glycine Schiff base. -1 The absorption peak at 1479 cm⁻¹ is the C=N stretching vibration of the glycine Schiff base. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1372 cm⁻¹ -1 The absorption peak at 1064 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 853 cm⁻¹ is caused by the asymmetric stretching of CH in the methyl group. -1 The absorption peak at that point is caused by the stretching vibration of CH on the benzene ring.

[0037] Figure 6The infrared spectrum of the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylic acid aldehyde Schiff base provided in Example 4 of this invention is shown at 3426 cm⁻¹. -1 The absorption peak at 2924 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of the N,N,N-trimethyl chitosan quaternary ammonium salt containing the glycine-5-bromosalicylic acid aldehyde Schiff base. -1 The absorption peak at 2887 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak at 1639 cm⁻¹ is caused by the bending and resonance vibrations of the C=N anode on the glycine Schiff base. -1 The absorption peak at 1476 cm⁻¹ is the C=N stretching vibration of the glycine Schiff base. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1375 cm⁻¹ -1 The absorption peak at 1064 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 853 cm⁻¹ is caused by the asymmetric stretching of CH in the methyl group. -1 The absorption peak at that point is caused by the stretching vibration of CH on the benzene ring.

[0038] Figure 7 The infrared spectrum of the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylaldehyde Schiff base provided in Example 3 of this invention is shown at 3405 cm⁻¹. -1 The absorption peak at 2928 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of the N,N,N-trimethyl chitosan quaternary ammonium salt containing the glycine-3,5-dichlorosalicylaldehyde Schiff base. -1 The absorption peak at 2890 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak at 1653 cm⁻¹ is caused by the bending and resonance vibrations of the C=N anode on the glycine Schiff base. -1 The absorption peak at 1450 cm⁻¹ is the C=N stretching vibration of the glycine Schiff base. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1388 cm⁻¹ -1 The absorption peak at 1110 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 862 cm⁻¹ is caused by the asymmetric stretching of CH in the methyl group. -1 760cm -1 The absorption peak at that point is caused by the stretching vibration of CH on the benzene ring.

[0039] Figure 8The infrared spectrum of the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid aldehyde Schiff base provided in Example 6 of this invention is shown at 3408 cm⁻¹. -1 The absorption peak at 2927 cm⁻¹ is caused by the NH stretching and OH stretching vibrations of the N,N,N-trimethyl chitosan quaternary ammonium salt containing the glycine-3,5-dibromosalicylic acid aldehyde Schiff base. -1 The absorption peak at 2883 cm⁻¹ is caused by the stretching vibration of CH on the sugar ring. -1 The absorption peak at 1654 cm⁻¹ is caused by the bending and resonance vibrations of the C=N anode on the glycine Schiff base. -1 The absorption peak at 1444 cm⁻¹ is the C=N stretching vibration of the glycine Schiff base. -1 The absorption peak at that location is -N + The characteristic absorption peak of (CH3)3, 1388 cm⁻¹ -1 The absorption peak at 1139 cm⁻¹ is caused by the bending vibration of CH and the symmetric deformation vibration of CH₃. -1 The absorption peak at 875 cm⁻¹ is caused by the asymmetric stretching of CH in the methyl group. -1 720cm -1 The absorption peak at that point is caused by the stretching vibration of CH on the benzene ring. Detailed implementation method:

[0040] The following examples are further illustrations of the present invention, but the present invention is not limited to the preparation methods and uses in this embodiment.

[0041] Example 1

[0042] like Figure 1 Synthetic route for synthesizing the target compound, N,N,N-trimethyl chitosan quaternary ammonium salt containing a glycine Schiff base:

[0043] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: 3.22 g of chitosan (n = 2200, see...) Figure 2 The solution was dispersed in 150 mL of N-methylpyrrolidone. Under ice bath stirring, 9 g of sodium iodide and 30 mL of 15% sodium hydroxide aqueous solution were added sequentially. After stirring for 1 h, 30 mL of iodomethane was added dropwise. After uniform dispersion, the solution was transferred to 60 °C and refluxed for 2 h until clear. After the reaction was complete, the solution was filtered, washed three times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethylchitosan quaternary ammonium salt (see...). Figure 3 );

[0044] (2) Synthesis of potassium glycine Schiff base: 0.75 g glycine and 0.56 g potassium hydroxide were dissolved in 50 mL anhydrous ethanol and stirred at 50 °C until dissolved. 1.22 g salicylaldehyde dissolved in 50 mL anhydrous ethanol was added to the solution and reacted at 50 °C for 2 h. After the reaction was completed, the solution was filtered, recrystallized, and freeze-dried under vacuum to obtain potassium glycine Schiff base.

[0045] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: 0.46 g of the obtained N,N,N-trimethyl chitosan quaternary ammonium salt was dissolved in 30 mL of water. 0.65 g of glycine-salicylaldehyde Schiff base potassium salt was dissolved in 20 mL of water and then added dropwise to the N,N,N-trimethyl chitosan quaternary ammonium salt aqueous solution. The reaction was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed for 72 h using a dialysis bag (500 kDa) and then freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base (see...). Figure 4 ).

[0046] Example 2

[0047] The difference from Example 1 is that:

[0048] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: 3.22 g chitosan (n = 2200) was dispersed in 200 mL of N-methylpyrrolidone. Under ice bath stirring, 13.5 g sodium iodide and 30 mL of 20% sodium hydroxide aqueous solution were added in sequence. After stirring for 1 h, 45 mL of iodomethane was added dropwise. After the mixture was evenly dispersed, it was transferred to 60 °C and refluxed for 2 h until the solution was clear. After the reaction was completed, the mixture was filtered, washed 3 times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt.

[0049] (2) Synthesis of potassium glycine Schiff base: 0.75 g glycine and 0.56 g potassium hydroxide were dissolved in 100 mL anhydrous ethanol and stirred at 60 °C until dissolved. 1.56 g 5-chlorosalicylaldehyde dissolved in 50 mL anhydrous ethanol was added to the solution and reacted at 60 °C for 2 h. After the reaction was completed, the solution was filtered, recrystallized, and freeze-dried under vacuum to obtain potassium glycine Schiff base.

[0050] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: 0.46 g of the obtained N,N,N-trimethyl chitosan quaternary ammonium salt was dissolved in 50 mL of water. 0.75 g of potassium salt of glycine-5-chlorosalicylaldehyde Schiff base was dissolved in 30 mL of water and added dropwise to the aqueous solution of N,N,N-trimethyl chitosan quaternary ammonium salt. The reaction was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed for 72 h using a dialysis bag (500 kDa) and then freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base (see...). Figure 5 ).

[0051] Example 3

[0052] The difference from Example 1 is that:

[0053] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: 3.22 g chitosan (n = 2200) was dispersed in 200 mL of N-methylpyrrolidone. Under ice bath stirring, 13.5 g sodium iodide and 30 mL of 20% sodium hydroxide aqueous solution were added in sequence. After stirring for 1 h, 45 mL of iodomethane was added dropwise. After the mixture was evenly dispersed, it was transferred to 60 °C and refluxed for 2 h until the solution was clear. After the reaction was completed, the mixture was filtered, washed 3 times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt.

[0054] (2) Synthesis of potassium glycine Schiff base: 0.75 g glycine and 0.56 g potassium hydroxide were dissolved in 100 mL anhydrous ethanol and stirred at 60 °C until dissolved. 2.01 g 5-bromosalicylic acid aldehyde dissolved in 50 mL anhydrous ethanol was added to the solution and the mixture was reacted at 60 °C for 2 h. After the reaction was completed, the mixture was filtered, recrystallized, and freeze-dried under vacuum to obtain potassium glycine Schiff base.

[0055] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: 0.46 g of the obtained N,N,N-trimethyl chitosan quaternary ammonium salt was dissolved in 50 mL of water. 0.89 g of potassium glycine-5-bromo-salicylaldehyde Schiff base was dissolved in 30 mL of water and added dropwise to the N,N,N-trimethyl chitosan quaternary ammonium salt aqueous solution. The reaction was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed for 72 h using a dialysis bag (500 kDa) and then freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base (see...). Figure 6 ).

[0056] Example 4

[0057] The difference from Example 1 is that:

[0058] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: 3.22 g chitosan (n = 2200) was dispersed in 200 mL of N-methylpyrrolidone. Under ice bath stirring, 13.5 g sodium iodide and 30 mL of 20% sodium hydroxide aqueous solution were added in sequence. After stirring for 1 h, 45 mL of iodomethane was added dropwise. After the mixture was evenly dispersed, it was transferred to 60 °C and refluxed for 2 h until the solution was clear. After the reaction was completed, the mixture was filtered, washed 3 times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt.

[0059] (2) Synthesis of potassium glycine Schiff base: 0.75 g glycine and 0.56 g potassium hydroxide were dissolved in 100 mL anhydrous ethanol and stirred at 60 °C until dissolved. 1.91 g 3,5-dichlorosalicylaldehyde dissolved in 100 mL anhydrous ethanol was added to the solution and reacted at 60 °C for 2 h. After the reaction was completed, the solution was filtered, recrystallized, and freeze-dried under vacuum to obtain potassium glycine Schiff base.

[0060] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: 0.46 g of the obtained N,N,N-trimethyl chitosan quaternary ammonium salt was dissolved in 50 mL of water. 0.86 g of potassium glycine-3,5-dichlorosalicylaldehyde Schiff base was dissolved in 30 mL of water and then added dropwise to the N,N,N-trimethyl chitosan quaternary ammonium salt aqueous solution. The reaction was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed for 72 h using a dialysis bag (500 kDa) and then freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base (see...). Figure 7 ).

[0061] Example 5

[0062] The difference from Example 1 is that:

[0063] (1) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt: 3.22 g chitosan (n = 2200) was dispersed in 200 mL of N-methylpyrrolidone. Under ice bath stirring, 13.5 g sodium iodide and 30 mL of 20% sodium hydroxide aqueous solution were added in sequence. After stirring for 1 h, 45 mL of iodomethane was added dropwise. After the mixture was evenly dispersed, it was transferred to 60 °C and refluxed for 2 h until the solution was clear. After the reaction was completed, the mixture was filtered, washed 3 times with ethanol, and freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt.

[0064] (2) Synthesis of potassium glycine Schiff base: 0.75 g glycine and 0.56 g potassium hydroxide were dissolved in 100 mL anhydrous ethanol and stirred at 60 °C until dissolved. 2.79 g 3,5-dibromosalicylaldehyde dissolved in 100 mL anhydrous ethanol was added to the solution and reacted at 60 °C for 2 h. After the reaction was completed, the solution was filtered, recrystallized, and freeze-dried under vacuum to obtain potassium glycine Schiff base.

[0065] (3) Synthesis of N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base: 1.12 g of the obtained N,N,N-trimethyl chitosan quaternary ammonium salt was dissolved in 50 mL of water. 1.12 g of potassium glycine-3,5-dibromosalicylic acid aldehyde Schiff base was dissolved in 30 mL of water and then added dropwise to the N,N,N-trimethyl chitosan quaternary ammonium salt aqueous solution. The reaction was stirred at room temperature for 12 h. After the reaction was completed, the mixture was dialyzed for 72 h using a dialysis bag (500 kDa) and then freeze-dried under vacuum to obtain N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base (see...). Figure 8 ).

[0066] Application Example 1

[0067] Antioxidant activity assay

[0068] Determination of DPPH free radical scavenging ability: The DPPH free radical scavenging abilities of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylaldehyde Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylaldehyde Schiff base were determined and compared. The operation procedure is as follows:

[0069] The chitosan samples, including N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid Schiff base, were vacuum freeze-dried to constant weight. 80 mg of each sample was then weighed and dissolved in 8 mL of water to a concentration of 10 mg / mL. A DPPH-ethanol solution was then prepared: 17.75 mg of DPPH was weighed into a small beaker, and an appropriate amount of anhydrous ethanol was added for ultrasonic dissolution. The solution was then transferred to a 250 mL volumetric flask and diluted to the mark with anhydrous ethanol.

[0070] The experimental group for scavenging DPPH free radicals consisted of 1000 μL of sample solution (concentrations of 0.30, 0.60, 1.20, 2.40, and 4.80 mg / mL) plus 2000 μL of DPPH-ethanol solution;

[0071] The control group for scavenging DPPH free radicals consisted of 1000 μL of sample solution (concentrations of 0.30, 0.60, 1.20, 2.40, and 4.80 mg / mL) plus 2000 μL of ethanol solution;

[0072] The blank group for scavenging DPPH free radicals consisted of 1000 μL of water and 2000 μL of DPPH-ethanol solution.

[0073] Then, the centrifuge tubes for each sample in each group were labeled with different concentrations: 1A-1E represent the final concentrations of the solution in the tubes as follows: 0.10 mg / mL, 0.20 mg / mL, 0.40 mg / mL, 0.80 mg / mL, and 1.60 mg / mL, respectively. All centrifuge tubes were incubated at room temperature for 20 min. After the reaction, 200 μL was pipetted from each centrifuge tube and transferred to an 8*12 microplate reader. The absorbance at 517 nm was measured using the microplate reader. The experiment was performed three times, and the average value was taken. The formula for calculating DPPH free radical scavenging capacity is as follows:

[0074]

[0075] In the formula: A 实验组517nm This is the absorbance of the experimental group at 517 nm, A. 对照组517nm This is the absorbance of the control group at 517 nm, A. 空白组517nm This is the absorbance of the blank group at 517nm.

[0076] Results of DPPH free radical scavenging ability test:

[0077] Table 1. DPPH radical scavenging abilities (%) of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dibromosalicylic acid Schiff base.

[0078]

[0079] As shown in Table 1 above, the product obtained by combining a Schiff base containing glycine with N,N,N-trimethyl chitosan quaternary ammonium salt can significantly improve the antioxidant capacity of chitosan at high concentrations. Among them, the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylaldehyde Schiff base has a strong scavenging ability for DPPH free radicals, reaching 87.71% at a concentration of 1.6 mg / mL.

[0080] Application Example 2

[0081] Antioxidant activity assay

[0082] 1) Determination of superoxide anion radical scavenging ability: The superoxide anion radical scavenging abilities of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid Schiff base were determined and compared. The operation procedure is as follows:

[0083] The chitosan samples, including N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid Schiff base, were vacuum freeze-dried to constant weight. 80 mg of each sample was then dissolved in 8 mL of water to a concentration of 10 mg / mL. Preparation of Tris-HCl buffer solution: 0.9691 g of tris(hydroxymethyl)aminomethane was weighed into a small beaker, 0.4 mL of concentrated hydrochloric acid was added, and the mixture was transferred to a 500 mL volumetric flask. Water was then added to the mark. Preparation of NADH solution: Weigh 0.0366g of reduced coenzyme I into a small beaker, dissolve it in a small amount of Tris-HCl buffer, transfer it to a 100mL volumetric flask, and dilute to the mark with Tris-HCl buffer. Preparation of NBT solution: Weigh 0.0245g of nitrotetrazole blue into a small beaker, dissolve it in a small amount of Tris-HCl buffer, transfer it to a 100mL volumetric flask, and dilute to the mark with Tris-HCl buffer. Preparation of PMS solution: Weigh 0.0018g of methyl phenazine sulfate into a small beaker, dissolve it in a small amount of Tris-HCl buffer, transfer it to a 100mL volumetric flask, and dilute to the mark with Tris-HCl buffer.

[0084] The experimental group for scavenging superoxide anion free radicals consisted of 1000 μL of sample solution (concentrations of 0.02, 0.04, 0.08, 0.16, and 0.32 mg / mL, respectively) plus 500 μL of NADH solution, 500 μL of NBT solution, and 500 μL of PMS solution.

[0085] The control group for scavenging superoxide anion free radicals consisted of 1000 μL of sample solution (concentrations of 0.02, 0.04, 0.08, 0.16, and 0.32 mg / mL, respectively) plus 500 μL of Tris-HCl solution, 500 μL of NBT solution, and 500 μL of PMS solution.

[0086] The blank group for scavenging superoxide anion radicals consisted of 1500 μL of Tris-HCl solution, plus 500 μL of NADH solution, 500 μL of NBT solution and 500 μL of PMS solution.

[0087] Then, the centrifuge tubes for each sample in each group were labeled with different concentrations: 1A-1E represent the final concentrations of the solution in the tubes as 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. 1-n represent n samples to be tested. All centrifuge tubes were reacted in the dark for 5 minutes. After the reaction, 200 μL was pipetted from each centrifuge tube and transferred to an 8*12 microplate. The absorbance at 560 nm was measured using a microplate reader. The experiment was performed three times, and the average value was taken. The formula for calculating the superoxide anion free radical scavenging capacity is as follows:

[0088]

[0089] In the formula: A 实验组560nm A is the absorbance of the experimental group at 560nm. 对照组560nm This is the absorbance of the control group at 560nm, A. 空白组560nm It is the absorbance of the blank group at 560nm.

[0090] Results of superoxide anion free radical scavenging ability test:

[0091] Table 2. Superoxide anion radical scavenging ability (%) of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid Schiff base.

[0092]

[0093] As shown in Table 1 above, the product obtained by combining a Schiff base containing glycine with N,N,N-trimethyl chitosan quaternary ammonium salt can significantly improve the antioxidant capacity of chitosan at high concentrations. Among them, the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylaldehyde Schiff base has a high scavenging capacity for superoxide anion free radicals, reaching 82.04% at a concentration of 1.6 mg / mL.

[0094] Application Example 3

[0095] Test for inhibiting bacterial growth

[0096] Determination of inhibitory activity against *Escherichia coli* and *Staphylococcus aureus*: The effects of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-salicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-chlorosalicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-5-bromosalicylaldehyde Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylaldehyde Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylaldehyde Schiff base on the activity of each sample against *Escherichia coli* and *Staphylococcus aureus* were determined and compared. The operation procedure is as follows:

[0097] Preparation of bacterial liquid culture medium: Accurately weigh 10g tryptone, 5g yeast extract, and 10g sodium chloride, mix with 1000mL deionized water, adjust the pH of the medium to 7.4 with NaOH, and sterilize at 121℃ for 20min under 15Psi pressure. Preparation of bacterial solid culture medium: Accurately weigh 10g tryptone, 5g yeast extract, 10g sodium chloride, and 18g agar, mix with 1000mL deionized water, and sterilize at 121℃ for 20min under 15Psi pressure.

[0098] Preparation of bacterial suspension: After removing the frozen bacteria, each was poured into cooled liquid bacterial culture medium and cultured for 12 hours. A small amount of the bacterial suspension was then streaked onto a plate. Single, intact colonies were selected and transferred to liquid bacterial culture medium. After incubation for 24 hours, the bacterial suspension was diluted. Then, 64 mg of each dried sample was accurately weighed and dissolved in 2 mL of sterile water to prepare a 32 mg / mL stock solution. 200 μL of the stock solution was added to the first column of a 96-well plate. 100 μL of sterile water was added to all wells except the first column. After repeated pipetting, 100 μL of the solution from the first column was added to the second column, and so on, until the last column of the 96-well plate was discarded. 100 μL of the diluted bacterial suspension was added to all wells. The plates were incubated at 37°C for 24 hours. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the micro-dilution method.

[0099] Results of assays for inhibition of Escherichia coli and Staphylococcus aureus:

[0100] Table 3. Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of chitosan, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dibromosalicylic acid Schiff base against Escherichia coli and Staphylococcus aureus.

[0101]

[0102] As shown in Table 3 above, the product obtained by combining a Schiff base containing glycine with N,N,N-trimethyl chitosan quaternary ammonium salt can significantly improve the antibacterial ability of chitosan at high concentrations. Among them, N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dichlorosalicylic acid aldehyde Schiff base and N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine-3,5-dibromosalicylic acid aldehyde Schiff base have strong inhibitory effects on Escherichia coli and Staphylococcus aureus. The N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-salicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-chlorosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-5-bromosalicylic acid Schiff base, N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dichlorosalicylic acid Schiff base, and N,N,N-trimethyl chitosan quaternary ammonium salts containing glycine-3,5-dibromosalicylic acid Schiff base exhibit higher inhibitory activity against Escherichia coli than against Staphylococcus aureus.

[0103] In summary, the N,N,N-trimethyl chitosan quaternary ammonium salt containing glycine Schiff base exhibits good antioxidant and antibacterial activities. Furthermore, the preparation process utilizes simple reaction equipment, is easy to control, uses inexpensive raw materials, and is highly safe, making it easy to promote and industrialize. This invention provides technical support for developing novel antioxidants and antibacterial agents for application in agriculture, medicine, and environmental protection.

Claims

1. A glycine Schiff base containing N , N , N -Trimethylchitosan quaternary ammonium salt, characterized in that: Containing glycine Schiff base N , N , N - Trimethyl chitosan quaternary ammonium salt is shown in formula (1), Equation (1) In the formula, R = , , , or ; Where n represents the degree of aggregation, and the average value of n ranges from 20 to 3000.

2. A glycine Schiff base containing the product of claim 1 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: By N , N , N - Trimethyl chitosan quaternary ammonium salt is combined with glycine Schiff base via ion exchange to obtain the glycine Schiff base-containing compound shown in formula (1). N , N , N - Trimethyl chitosan quaternary ammonium salt.

3. The glycine Schiff base containing [product name] according to claim 2 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: (1) N , N , N Synthesis of trimethyl chitosan quaternary ammonium salt: Chitosan is dispersed in... N In methylpyrrolidone, sodium iodide and sodium hydroxide aqueous solutions were added sequentially under ice bath stirring. After stirring for 30 min to 1 h, iodomethane was added dropwise. After uniform dispersion, the solution was transferred to 60 °C and refluxed for 2 to 12 h until the solution became clear. After the reaction was completed, the solution was filtered, washed three times with ethanol, and then freeze-dried under vacuum to obtain... N , N , N - Trimethyl chitosan quaternary ammonium salt; wherein the molar ratio of chitosan, sodium iodide, sodium hydroxide, and iodomethane is 1:3:6:10-20; (2) Synthesis of potassium salt of glycine Schiff base: Glycine and potassium hydroxide were dissolved in anhydrous ethanol and stirred at 50-60 °C until dissolved. Salicylic aldehyde derivatives dissolved in anhydrous ethanol were added to the solution and reacted at 50-60 °C for 1-6 h. After the reaction was completed, the solution was filtered, washed, recrystallized, and freeze-dried under vacuum. The molar ratio of glycine, potassium hydroxide and salicylic aldehyde derivatives was 1:1:1.5-3. (3) Containing glycine Schiff base N , N , N Synthesis of trimethyl chitosan quaternary ammonium salt: The above-obtained... N , N , N - Trimethyl chitosan quaternary ammonium salt was dissolved in water, and glycine Schiff base potassium salt was dissolved in water and then added dropwise to... N , N , N - The reaction was carried out in an aqueous solution of trimethyl chitosan quaternary ammonium salt at room temperature with stirring for 12-24 h. After the reaction was completed, the mixture was dialyzed using a dialysis bag and then freeze-dried under vacuum. N , N , N The molar ratio of trimethyl chitosan quaternary ammonium salt to glycine Schiff base potassium salt is 1:2 - 6.

4. The glycine Schiff base containing the product according to claim 3 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: In step (1), chitosan is dispersed in N In methylpyrrolidone N The mass of methylpyrrolidone is 30-50 times that of chitosan; The sodium hydroxide aqueous solution added in step (1) has a mass fraction of 10% - 20%.

5. The glycine Schiff base containing the product according to claim 3 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: In step (2), glycine and potassium hydroxide are dissolved in anhydrous ethanol, wherein the volume-to-weight ratio of anhydrous ethanol to glycine is 50-200:

1. In step (2), the salicylaldehyde derivative is salicylaldehyde, 5-chlorosalicylaldehyde, 5-bromosalicylaldehyde, 3,5-dichlorosalicylaldehyde or 3,5-dibromosalicylaldehyde, wherein the volume weight ratio of anhydrous ethanol to the salicylaldehyde derivative is 10-50:

1.

6. The glycine Schiff base containing the product according to claim 3 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: In step (3), the above-mentioned results will be used N , N , N - Trimethyl chitosan quaternary ammonium salt dissolves in water N , N , N - The mass fraction of the aqueous solution of trimethyl chitosan quaternary ammonium salt is 0.3% - 0.8%; In step (3), the potassium salt of glycine Schiff base is dissolved in water, and the mass fraction of the potassium salt of glycine Schiff base aqueous solution is 5% - 20%.

7. The glycine Schiff base containing the product according to claim 3 N , N , N A method for preparing trimethyl chitosan quaternary ammonium salt, characterized in that: In step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of 10 kDa – 500 kDa.

8. A product containing glycine Schiff base as described in claim 1 N , N , N The application of -trimethylchitosan quaternary ammonium salt is characterized by: The glycine Schiff base shown in formula (1) N , N , N - Application of trimethyl chitosan quaternary ammonium salt in the preparation of antibacterial agents or antioxidants.

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