Chitosan derivative containing bifunctional groups as well as preparation and application of chitosan derivative

Abstract

The invention relates to the fields of food, cosmetics and biological medicines, in particular to a chitosan derivative containing bifunctional groups (triazole and coumarin) and a preparation method and application thereof. The structure of the derivative is shown as a formula (1). According to the method, mild reaction conditions are adopted, raw materials which are easy to obtain and low in cost are used, the prepared chitosan derivative has good water solubility and meanwhile has remarkable antioxidant activity and antifungal activity, and the application range of chitosan in all industries is effectively widened. The chitosan derivative containing bifunctional groups (triazole and coumarin) has wide application potential in the fields of cosmetics, foods, biological medicines and the like, and is convenient for industrial popularization. # imgabs0 # formula (1).

Description

Technical Field

[0001] The present invention relates to the fields of food, cosmetics and biomedicine, and in particular to a chitosan derivative containing a bifunctional group (triazole and coumarin) and a preparation method and application thereof. Background Art

[0002] Chitosan is a chitin-derived polysaccharide. It is a natural, non-toxic, abundant, renewable, biocompatible and biodegradable polysaccharide polymer biological resource. It has certain biological activities, such as antibacterial, antioxidant, immunomodulatory, food preservation, tumor growth inhibition, etc. It is widely used in agriculture, food, medicine, chemical industry, cosmetics, papermaking, textile, environmental protection and other fields. However, its solubility in neutral or alkaline pH media is low, so its application is limited. The chitosan molecular chain contains a large number of amino and hydroxyl groups, which can be modified by oxidation, esterification, etherification, alkylation, Schiff base, quaternization and other reactions to introduce different active groups, thereby obtaining chitosan derivatives with different biological activities and expanding its application range. Summary of the invention

[0003] The purpose of the present invention is to provide a chitosan derivative containing bifunctional groups (triazole and coumarin) and its preparation and application.

[0004] To achieve the above object, the technical solution adopted by the present invention is: A chitosan derivative containing a bifunctional group, wherein the chitosan derivative is represented by formula (1), Formula (1), In formula (1), R = ; R1 = H, halogen or C1-C3 alkyl; R2 = H or C1-C3 alkyl; Where n represents the degree of polymerization, and the average value range of n is 10-12000.

[0005] In the formula (1), R1 = H, Cl or CH3; R2 = H or CH3.

[0006] A method for preparing the bifunctional chitosan derivative: 1) reacting chitosan quaternary ammonium salt with N-bromosuccinimide to obtain 6-bromochitosan quaternary ammonium salt; reacting 6-bromochitosan quaternary ammonium salt with ethylenediamine to obtain 6-aminoethyl-chitosan quaternary ammonium salt; reacting 6-aminoethyl-chitosan quaternary ammonium salt with propyne bromide to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt; 2) A substituted hydroxycoumarin is subjected to a bromination reaction with tetrabutylammonium bromide to obtain a brominated coumarin product, and then the brominated coumarin product is reacted with azidotrimethylsilane to obtain an azido coumarin product; 3) The 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained above is reacted with the azidocoumarin product in the presence of a solvent to obtain a chitosan derivative containing a bifunctional group as shown in formula (1).

[0007] The 6-bromochitosan quaternary ammonium salt is prepared by using chitosan as a raw material and methyl iodide to prepare the chitosan quaternary ammonium salt, and then N -Bromosuccinimide is dissolved in 1-methyl-2-pyrrolidone, and triphenylphosphine is added, ice bathed for 30 minutes, reacted at 60-100°C for 1-4 hours, precipitated with ethanol after the reaction, and the precipitate is washed with ethanol and filtered, and dried to constant weight to obtain 6-bromochitosan quaternary ammonium salt; wherein, N - the amount of bromosuccinimide added is 2-10 times the molar amount of chitosan quaternary ammonium salt, and the amount of triphenylphosphine added is 2-10 times the molar amount of chitosan; The 6-aminoethyl-chitosan quaternary ammonium salt is prepared by dissolving 6-bromochitosan quaternary ammonium salt in ethylenediamine, reacting at 70-90° C. for 12-20 hours under nitrogen protection, and after the reaction, pouring the reaction solution into acetone for precipitation, washing twice with acetone, filtering, and drying to constant weight to obtain 6-aminoethyl-chitosan quaternary ammonium salt, wherein the amount of ethylenediamine added is 20-40 times the molar amount of 6-bromochitosan quaternary ammonium salt; The 6-aminoethyl-propynyl chitosan quaternary ammonium salt is prepared by adding sodium hydroxide and potassium iodide to the 6-aminoethyl-chitosan quaternary ammonium salt, and then adding propynyl bromide, dissolving the mixture in N,N-dimethylformamide, reacting the mixture at 70-90° C. for 18-26 hours under nitrogen protection, and after the reaction, pouring the reaction solution into excess anhydrous ethanol for precipitation, washing the mixture twice with anhydrous ethanol, filtering the mixture, and drying the mixture to a constant weight to obtain the 6-aminoethyl-propynyl chitosan quaternary ammonium salt; wherein the amount of propynyl bromide added is 2-10 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt, the amount of sodium hydroxide added is 1-5 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt, and the amount of potassium iodide added is 0.05-0.3 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt.

[0008] The bromination reaction is as follows: substituted hydroxycoumarin and tetrabutylammonium bromide are dissolved in N, N -dimethylformamide, adding phosphorus pentoxide, reacting at 100°C for 3 hours, extracting the reaction solution repeatedly with toluene, collecting the extracts and performing reduced pressure distillation to obtain a brominated coumarin product; wherein the amount of tetrabutylammonium bromide added is 2 times the molar amount of the substituted hydroxycoumarin, and the amount of phosphorus pentoxide added is 3 times the molar amount of the substituted hydroxycoumarin.

[0009] The substituted hydroxycoumarin includes 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-7-methylcoumarin, 4-hydroxy-6-chlorocoumarin and a series of coumarins carrying a hydroxyl group.

[0010] The azidocoumarin product is: the bromocoumarin product and azidotrimethylsilane are dissolved in N,N -dimethylformamide, and then add hexamethylphosphoramide, react at 90° C. for 6.5 hours, precipitate with deionized water after the reaction, wash the precipitate with petroleum ether and filter to obtain a filter cake, dry to constant weight, and obtain an azidocoumarin product; wherein, the added amount of azidotrimethylsilane is 10 times the molar amount of the bromocoumarin product, and the added amount of hexamethylphosphoramide is 2.6 times the molar amount of the bromocoumarin product.

[0011] The chitosan derivative containing bifunctional groups is prepared by dissolving the obtained azidocoumarin product and 6-aminoethyl-propynyl chitosan quaternary ammonium salt in dimethyl sulfoxide and triethylamine, adding cuprous iodide, reacting at 50-100° C. for 20-30 hours, precipitating with acetone after the reaction, washing the precipitate with acetone, filtering to obtain a filter cake, and drying to constant weight to obtain the chitosan derivative containing bifunctional groups as shown in formula (1); wherein the amount of the azidocoumarin product added is 1.5-4 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt, the amount of the cuprous iodide added is 1-3 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt, and the amount of the triethylamine added is 5-10 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0012] An application of the chitosan derivative containing bifunctional groups, and an application of the compound represented by formula (1) in the preparation of products with antioxidant properties.

[0013] An application of the chitosan derivative containing bifunctional groups, and an application of the compound represented by formula (1) in the preparation of an antibacterial agent.

[0014] The advantages of the present invention are: (1) Chitosan has good biodegradability, biocompatibility, and non-toxicity, but its antioxidant activity is relatively weak. The present invention improves the antioxidant activity of chitosan while retaining its own advantages by chemically modifying it; the antioxidant activity of the chitosan derivative containing bifunctional groups obtained by the present invention is greatly improved compared with chitosan. The derivative can be developed into a new antioxidant to solve the problem of potential toxicity of chemical antioxidants to human health, and can also be widely used in the fields of food and medicine.

[0015] (2) The present invention adopts a sugar modification method to modify the hydroxyl groups on the polysaccharide, thereby obtaining a derivative with high antioxidant activity. The preparation process is simple and the material cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention provides a flow chart for synthesizing the derivative represented by formula (1).

[0017] Figure 2 This is an infrared spectrum of the chitosan quaternary ammonium salt provided in an embodiment of the present invention.

[0018] Figure 3 This is the infrared spectrum of 6-bromochitosan quaternary ammonium salt provided in Example 1 of the present invention.

[0019] Figure 4 This is the infrared spectrum of 6-aminoethyl-chitosan quaternary ammonium salt provided in Example 1 of the present invention.

[0020] Figure 5 This is the infrared spectrum of 6-aminoethyl-propynyl chitosan quaternary ammonium salt provided in Example 1 of the present invention.

[0021] Figure 6 This is the infrared spectrum of 4-azidocoumarin provided in Example 1 of the present invention.

[0022] Figure 7 This is the infrared spectrum of 4-azido-6-chlorocoumarin provided in Example 2 of the present invention.

[0023] Figure 8 This is the infrared spectrum of 4-azido-7-methylcoumarin provided in Example 3 of the present invention.

[0024] Fig. 9 This is the infrared spectrum of 4-azido-6-methylcoumarin provided in Example 4 of the present invention.

[0025] Fig.10 This is the infrared spectrum of the chitosan derivative containing bifunctional groups provided in Example 1 of the present invention.

[0026] Fig.11 This is the infrared spectrum of the chitosan derivative containing bifunctional groups provided in Example 2 of the present invention.

[0027] Fig.12 This is the infrared spectrum of the chitosan derivative containing bifunctional groups provided in Example 3 of the present invention.

[0028] Fig.13 This is the infrared spectrum of the chitosan derivative containing bifunctional groups provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with examples. It should be noted that the specific implementation methods described herein are only for illustrating and explaining the present invention, and are not intended to limit the present invention. Example 1

[0030] like Figure 1 The chitosan derivative containing bifunctional groups (Formula (1)) is prepared as shown: (1) Preparation of 6-bromochitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) chitosan and 2.8 g (19.7 mmol) sodium iodide, add 5.2 mL iodomethane and 4.48 mL 15% sodium hydroxide solution, add to 5.6 mL 1-methyl-2-pyrrolidone, and react at 60°C for 2 hours. After the reaction, pour the reaction solution into a dialysis membrane and dialyze for 2 days, then freeze-dry to obtain 0.64 g chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64g (2.2mmol) chitosan quaternary ammonium salt, 1.7g (9.5mmol) N -bromosuccinimide, add 7mL 1-methyl-2-pyrrolidone, mix well and slowly add 2.6g (9.9mmol) triphenylphosphine in batches. After reacting at 0℃ for 15 minutes, react at 80℃ for 2 hours. After the reaction, the reaction solution was precipitated with anhydrous ethanol, washed twice with anhydrous ethanol, and dried at 65℃ to obtain 6-bromochitosan quaternary ammonium salt (see Figure 3 ).

[0031] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.17 g (0.48 mmol) of 6-bromochitosan quaternary ammonium salt and add it to 1 mL (15 mmol) of ethylenediamine. React at 80°C for 16 hours under nitrogen protection. After the reaction, pour the reaction solution into acetone for precipitation, wash twice with acetone, filter, and dry at 65°C to obtain 6-aminoethyl-chitosan quaternary ammonium salt (see Figure 4 ).

[0032] (3) Preparation of 6-aminoethyl-propynyl chitosan quaternary ammonium salt: Weigh 0.12 g (0.32 mmol) of aminoethyl-chitosan quaternary ammonium salt, 35 mg (0.88 mmol) of sodium hydroxide and 10 mg (0.06 mmol) of potassium iodide, add 150 μL (1.66 mmol) of propargyl bromide and 1 mL of N,N -dimethylformamide. Under nitrogen protection, react at 80°C for 24 hours. After the reaction, pour the reaction solution into excess anhydrous ethanol for precipitation, wash twice with anhydrous ethanol, filter, and dry at 65°C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ).

[0033] (4) Weigh 1.3 g (8 mmol) of 4-hydroxycoumarin, 5.1 g (16 mmol) of tetrabutylammonium bromide and 3.4 g (24 mmol) of phosphorus pentoxide and add them to 20 mL N,N -dimethylformamide, react at 100°C for 3 hours. After the reaction, the reaction solution was extracted three times with 20 mL of toluene, and the extracts were collected and distilled under reduced pressure to obtain 4-bromocoumarin.

[0034] (5) Weigh 1.7 g (8 mmol) of 4-bromocoumarin, 3.3 mL (21 mmol) of hexamethylphosphoramide and 10.5 mL (80 mmol) of trimethylsilyl azide and add them to 12 mL N,N -dimethylformamide, react at 90°C for 6.5 hours. After the reaction is complete, the reaction solution is poured into 65 mL of deionized water to precipitate. The precipitate is washed with petroleum ether and dried at 65°C to obtain 4-azidocoumarin (see Figure 6 ).

[0035] (6) 0.1 g (5 mmol) of 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 0.08 g (0.42 mmol) of 4-azidocoumarin obtained in step (5) and 0.05 mg (10 mmol) of cuprous iodide were added to 2.8 mL of dimethyl sulfoxide and 0.61 mL (45 mmol) of triethylamine, and the temperature was raised to 80°C for reaction for 20 hours. After the reaction was completed, the reaction solution was poured into 20 mL of acetone to precipitate. The precipitate was filtered, washed with acetone, and dried at 65°C to obtain a chitosan derivative containing a bifunctional group (see Fig.10 ), wherein R1 is hydrogen and R2 is hydrogen (see Fig.10 ).

[0036] The infrared spectrum of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt provided in Example 1 of the present invention (see Figure 5 ) and chitosan quaternary ammonium salt (see Figure 2 ) compared to 2125.02cm -1 The peak at is the stretching vibration peak of the acetylenic bond, proving that the synthesis of 6-aminoethyl-propynyl chitosan quaternary ammonium salt was successful. Figure 6 The infrared spectrum of 4-azidocoumarin provided in Example 1 of the present invention is as follows: Figure 6 It can be seen that 1724.78cm -1 The stretching vibration peak of the C=O bond on coumarin, 1610.33 cm -1 is the stretching vibration peak of the C=C bond on coumarin, 2128.02 cm -1 The peak is that of the azide group, proving that 4-azidocoumarin was successfully synthesized. Fig.10 The infrared spectrum of the chitosan derivative containing bifunctional groups provided in Example 1 of the present invention is as follows: Fig.10 It is known that with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ) compared to 1723.10cm -1 is the stretching vibration peak of C=O on coumarin, 1608.68cm -1 The stretching vibration peak of C=C on the benzene ring is 764.73 cm -1 It is the deformation vibration peak of benzene ring CH, 2125.02cm -1The peak of the alkyne bond disappeared. The above analytical data proved that the chitosan derivative containing bifunctional groups in which R1 and R2 were hydrogen was successfully synthesized. Example 2

[0037] This embodiment synthesizes a chitosan derivative containing bifunctional groups in which R1 is chlorine and R2 is hydrogen according to the above synthesis route. The difference from Example 1 is that: (1) Preparation of 6-bromochitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) chitosan and 2.8 g (19.7 mmol) sodium iodide, add 5.2 mL iodomethane and 4.48 mL 15% sodium hydroxide solution, add to 5.6 mL 1-methyl-2-pyrrolidone, and react at 60°C for 2 hours. After the reaction, pour the reaction solution into a dialysis membrane and dialyze for 2 days, then freeze-dry to obtain 0.64 g chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64g (2.2mmol) chitosan quaternary ammonium salt, 0.78g (4.4mmol) N -bromosuccinimide, add 7mL 1-methyl-2-pyrrolidone, mix well and slowly add 1.2g (4.4mmol) triphenylphosphine in batches. After reacting at 0℃ for 30 minutes, react at 90℃ for 5 hours. After the reaction, the reaction solution was precipitated with anhydrous ethanol, washed twice with anhydrous ethanol, and dried at 65℃ to obtain 6-bromochitosan quaternary ammonium salt.

[0038] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.15 g (0.42 mmol) of 6-bromochitosan quaternary ammonium salt and add it to 0.56 mL (8.4 mmol) of ethylenediamine. React at 70°C for 12 hours under nitrogen protection. After the reaction, pour the reaction solution into acetone for precipitation, wash twice with acetone, filter, and dry at 65°C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0039] (3) Preparation of 6-aminoethyl-propynyl chitosan quaternary ammonium salt: Weigh 0.12 g (0.32 mmol) of aminoethyl-chitosan quaternary ammonium salt, 64 mg (1.6 mmol) of sodium hydroxide and 17 mg (0.10 mmol) of potassium iodide, add 287 μL (3.2 mmol) of propargyl bromide and 1 mL of N,N -dimethylformamide. Under nitrogen protection, react at 90°C for 26 hours. After the reaction is completed, pour the reaction solution into excess anhydrous ethanol for precipitation, wash twice with anhydrous ethanol, filter, and dry at 65°C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0040] (4) Weigh 1.5 g (8 mmol) 4-hydroxy-6-chlorocoumarin, 5.1 g (16 mmol) tetrabutylammonium bromide and 3.4 g (24 mmol) phosphorus pentoxide and add them to 20 mL N,N-dimethylformamide, react at 100°C for 3 hours. After the reaction, the reaction solution was extracted three times with 20 mL of toluene, and the extracts were collected and distilled under reduced pressure to obtain 4-bromo-6-chlorocoumarin.

[0041] (5) Weigh 1.9 g (8 mmol) of 4-bromo-6-chlorocoumarin, 3.3 mL (21 mmol) of hexamethylphosphoramide and 10.5 mL (80 mmol) of trimethylsilyl azide into 12 mL N,N -dimethylformamide, react at 90°C for 6.5 hours. After the reaction is complete, the reaction solution is poured into 65 mL of deionized water to precipitate. The precipitate is washed with petroleum ether and dried at 65°C to obtain 4-azido-6-chlorocoumarin (see Figure 7 ).

[0042] (6) 0.1 g (5 mmol) of 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 4 g (20 mmol) of 4-azido-6-chlorocoumarin obtained in step (5) and 0.08 mg (10 mmol) of cuprous iodide were added to 2.8 mL of dimethyl sulfoxide and 0.68 mL (50 mmol) of triethylamine, and the temperature was raised to 100° C. to react for 30 hours. After the reaction was completed, the reaction solution was poured into 20 mL of acetone to precipitate. The precipitate was filtered, washed with acetone, and dried at 65° C. to obtain a chitosan derivative containing a bifunctional group (see Fig.11 ), wherein R1 is chlorine and R2 is hydrogen.

[0043] from Fig.11 It is known that with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ) compared to 1666.14cm -1 is the stretching vibration peak of C=O on coumarin, 1620.74cm -1 The stretching vibration peak of C=C on the benzene ring is 798.86 cm -1 It is the deformation vibration peak of benzene ring CH, 2125.02cm -1 The peak of the alkyne bond disappeared. The above analytical data proved that the chitosan derivative containing bifunctional groups in which R1 was chlorine and R2 was hydrogen was successfully synthesized. Example 3

[0044] This embodiment synthesizes a chitosan derivative containing bifunctional groups in which R1 is hydrogen and R2 is methyl according to the above synthesis route. The difference from Example 1 is that: (1) Preparation of 6-bromochitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) chitosan and 2.8 g (19.7 mmol) sodium iodide, add 5.2 mL iodomethane and 4.48 mL 15% sodium hydroxide solution, add to 5.6 mL 1-methyl-2-pyrrolidone, and react at 60°C for 2 hours. After the reaction, pour the reaction solution into a dialysis membrane and dialyze for 2 days, then freeze-dry to obtain 0.64 g chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64g (2.2mmol) chitosan quaternary ammonium salt, 3.92g (22mmol) N-bromosuccinimide, add 10mL 1-methyl-2-pyrrolidone, mix well and slowly add 5.8g (22mmol) triphenylphosphine in batches. After reacting at 0℃ for 30 minutes, react at 100℃ for 4 hours. After the reaction, the reaction solution was precipitated with anhydrous ethanol, washed twice with anhydrous ethanol, and dried at 65℃ to obtain 6-bromochitosan quaternary ammonium salt.

[0045] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.2 g (0.56 mmol) of 6-bromochitosan quaternary ammonium salt and add it to 1.1 mL (22.4 mmol) of ethylenediamine. React at 90°C for 20 hours under nitrogen protection. After the reaction, pour the reaction solution into acetone for precipitation, wash twice with acetone, filter, and dry at 65°C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0046] (3) Preparation of 6-aminoethyl-propynyl chitosan quaternary ammonium salt: weigh 0.12 g (0.32 mmol) of aminoethyl-chitosan quaternary ammonium salt, 13 mg (0.32 mmol) of sodium hydroxide and 3 mg (0.02 mmol) of potassium iodide, add 58 μL (1.28 mmol) of propargyl bromide and 1 mL of N,N -dimethylformamide. Under nitrogen protection, react at 70°C for 18 hours. After the reaction is completed, pour the reaction solution into excess anhydrous ethanol for precipitation, wash twice with anhydrous ethanol, filter, and dry at 65°C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0047] (4) Weigh 1.4 g (8 mmol) of 4-hydroxy-7-methylcoumarin, 5.1 g (16 mmol) of tetrabutylammonium bromide and 3.4 g (24 mmol) of phosphorus pentoxide and add them to 20 mL N,N -dimethylformamide, react at 100°C for 3 hours. After the reaction, the reaction solution was extracted three times with 20 mL of toluene, and the extracts were collected and distilled under reduced pressure to obtain 4-bromo-7-methylcoumarin.

[0048] (5) Weigh 1.5 g (8 mmol) of 4-bromo-7-methylcoumarin, 3.3 mL (21 mmol) of hexamethylphosphoramide and 10.5 mL (80 mmol) of trimethylsilyl azide into 12 mL N,N-dimethylformamide, react at 90°C for 6.5 hours. After the reaction is complete, the reaction solution is poured into 65 mL of deionized water to precipitate. The precipitate is washed with petroleum ether and dried at 65°C to obtain 4-azido-7-methylcoumarin (see Figure 8 ).

[0049] (6) Add 0.1 g (5 mmol) of 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 1.5 g (7.5 mmol) of 4-azido-7-methylcoumarin obtained in step (5) and 0.3 mg (5 mmol) of cuprous iodide to 2.8 mL of dimethyl sulfoxide and 0.34 mL (25 mmol) of triethylamine, and heat to 50° C. to react for 20 hours. After the reaction is completed, pour the reaction solution into 20 mL of acetone to precipitate. The precipitate is filtered, washed with acetone, and dried at 65° C. to obtain a chitosan derivative containing a bifunctional group (see Fig.12 ), wherein R1 is hydrogen and R2 is methyl.

[0050] from Fig.12 It is known that with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ) compared to 1720.25cm -1 is the stretching vibration peak of C=O on coumarin, 1625.43cm -1 The stretching vibration peak of C=C on the benzene ring is 815.90 cm -1 It is the deformation vibration peak of benzene ring CH, 2125.02cm -1 The peak of the acetylene bond disappeared. The above analytical data proved that the chitosan derivative containing bifunctional groups in which R1 was hydrogen and R2 was methyl was successfully synthesized. Example 4

[0051] This example synthesizes a chitosan derivative containing bifunctional groups in which R1 is methyl and R2 is hydrogen according to the above synthesis route. The difference from Example 1 is that: (1) Preparation of 6-bromochitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) chitosan and 2.8 g (19.7 mmol) sodium iodide, add 5.2 mL iodomethane and 4.48 mL 15% sodium hydroxide solution, add to 5.6 mL 1-methyl-2-pyrrolidone, and react at 60°C for 2 hours. After the reaction, pour the reaction solution into a dialysis membrane and dialyze for 2 days, then freeze-dry to obtain 0.64 g chitosan quaternary ammonium salt (see Figure 2). Weigh 0.64g (2.2mmol) chitosan quaternary ammonium salt, 2.0g (11mmol) N-bromosuccinimide, add 7mL 1-methyl-2-pyrrolidone, mix well and slowly add 2.9g (11mmol) triphenylphosphine in batches. After reacting at 0℃ for 20 minutes, react at 85℃ for 3 hours. After the reaction, the reaction solution was precipitated with anhydrous ethanol, washed twice with anhydrous ethanol, and dried at 65℃ to obtain 6-bromochitosan quaternary ammonium salt.

[0052] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: 0.17 g (0.48 mmol) of 6-bromochitosan quaternary ammonium salt was weighed and added to 0.96 mL (14.4 mmol) of ethylenediamine, and reacted at 85°C for 18 hours under nitrogen protection. After the reaction, the reaction solution was poured into acetone for precipitation, washed twice with acetone, filtered, and dried at 65°C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0053] (3) Preparation of 6-aminoethyl-propynyl chitosan quaternary ammonium salt: Weigh 0.12 g (0.32 mmol) of aminoethyl-chitosan quaternary ammonium salt, 38 mg (0.96 mmol) of sodium hydroxide and 13 mg (0.08 mmol) of potassium iodide, add 143 μL (1.6 mmol) of propargyl bromide and 1 mL of N,N -dimethylformamide. Under nitrogen protection, react at 75°C for 20 hours. After the reaction is completed, pour the reaction solution into excess anhydrous ethanol for precipitation, wash twice with anhydrous ethanol, filter, and dry at 65°C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0054] (4) Weigh 1.4 g (8 mmol) of 4-hydroxy-6-methylcoumarin, 5.1 g (16 mmol) of tetrabutylammonium bromide and 3.4 g (24 mmol) of phosphorus pentoxide and add them to 20 mL N,N -dimethylformamide, react at 100°C for 3 hours. After the reaction, the reaction solution was extracted three times with 20 mL of toluene, and the extracts were collected and distilled under reduced pressure to obtain 4-bromo-6-methylcoumarin.

[0055] (5) Weigh 1.5 g (8 mmol) of 4-bromo-6-methylcoumarin, 3.3 mL (21 mmol) of hexamethylphosphoramide, and 10.5 mL (80 mmol) of trimethylsilyl azide into 1 mL N,N -dimethylformamide, react at 90°C for 6.5 hours. After the reaction is complete, the reaction solution is poured into 65 mL of deionized water to precipitate. The precipitate is washed with petroleum ether and dried at 65°C to obtain 4-azido-6-methylcoumarin (see Fig. 9 ).

[0056] (6) 0.1 g (5 mmol) of 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 0.08 g (0.38 mmol) of 4-azido-6-methylcoumarin obtained in step (5) and 0.05 mg (10 mmol) of cuprous iodide were added to 2.8 mL of dimethyl sulfoxide and 0.61 mL of triethylamine, and the temperature was raised to 80° C. to react for 20 hours. After the reaction was completed, the reaction solution was poured into 20 mL of acetone to precipitate. The precipitate was filtered, washed with acetone, and dried at 65° C. to obtain a chitosan derivative containing a bifunctional group (see Fig.13 ), wherein R1 is hydrogen and R2 is methyl.

[0057] from Fig.13 It is known that with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ) compared to 1720.48cm -1 is the stretching vibration peak of C=O on coumarin, 1607.21cm -1 The stretching vibration peak of C=C on the benzene ring is 820.08 cm -1 It is the deformation vibration peak of benzene ring CH, 2125.02cm -1 The peak of the alkyne bond disappeared. The above analytical data proved that the chitosan derivative containing bifunctional groups in which R1 was methyl and R2 was hydrogen was successfully synthesized. Application Examples

[0058] Antioxidant activity assay (1) Determination of superoxide anion scavenging ability: The superoxide anion scavenging ability of chitosan and the chitosan derivative containing bifunctional groups obtained in the above examples were respectively determined and compared (Table 1): The chitosan and the chitosan derivative containing bifunctional groups in the examples were prepared into a 1 mg / mL solution. Tris-HCl buffer solution, 0.969 g Tris and 0.4 mL concentrated HCl were added with water to make the volume to 500 mL. Different volumes of sample solution were taken according to the preset proportion, 0.5 mL NBT solution (300 μM), 0.5 ml NADH (550 μM), and then 0.5 mL PMS (60 μM) was added to the reaction solution. After mixing in a test tube, the final concentrations of the samples were 0.001, 0.005, 0.01, 0.05, and 0.1 mg / mL. The samples were allowed to stand at room temperature for 5 minutes, and the absorbance was measured at 560 nm. In the blank group, 0.5 mL Tris-HCl buffer solution was used instead of NADH (Note: all samples were tested three times and the average value was taken).

[0059] Superoxide anion scavenging capacity (%) = [(A 空白 -A 样品 ) / A 空白 ]×100 The results of the determination of the antioxidant capacity of scavenging superoxide anions are shown in Table 1.

[0060] Table 1 Superoxide anion scavenging ability of chitosan and chitosan derivatives containing bifunctional groups (%)

[0061] (2) Determination of the ability to scavenge hydroxyl radicals: The scavenging abilities of chitosan and chitosan derivatives containing bifunctional groups were determined and compared (Table 2): The chitosan and chitosan derivatives containing bifunctional groups used in the experiment in the example were prepared into a 5 mg / mL sample solution. Phosphate buffer solution (pH 7.4) was prepared by diluting 20.79 g of disodium hydrogen phosphate dodecahydrate and 2.644 g of sodium dihydrogen phosphate dihydrate with water to 500 ml. Different volumes of sample solution were taken according to the preset ratio, 0.5 mL of phosphate buffer solution, 0.25 ml of EDTA-Fe 2+ solution (2mmol / L, prepared with phosphate buffer solution), 0.5mlH2O2 solution (3%, prepared with phosphate buffer solution), and then 0.5mL of saffron T solution (360μg / ml, prepared with phosphate buffer solution) was added to the reaction solution. After mixing in a test tube, the final concentration of the sample was 0.1, 0.2, 0.4, 0.6, and 0.8mg / mL. It was allowed to stand at 40℃ for 30 minutes, and the absorbance was measured at 520nm. The blank group was replaced with 0.5mL of phosphate buffer solution instead of the sample solution, and the control group was a mixture of 1mL of phosphate buffer solution, 0.5mL of saffron T solution and 0.5mL of H2O2 solution (Note: the tested samples were tested three times and the average value was taken).

[0062] Hydroxyl free radical scavenging ability (%) = [(A 样品 -A 空白 ) / (A 对照 -A 空白 )]×100 The results of the hydroxyl radical scavenging ability are shown in Table 2.

[0063] Table 2 Hydroxyl radical scavenging ability of chitosan and chitosan derivatives containing bifunctional groups (%)

[0064] (3) Determination of the ability to scavenge DPPH free radicals: The scavenging DPPH free radical abilities of chitosan and chitosan derivatives containing bifunctional groups were determined and compared (Table 3): The chitosan and chitosan derivatives containing bifunctional groups used in the experiment in the example were prepared into a sample solution of 1 mg / mL. Then, the DPPH ethanol solution, the sample solution and distilled water were mixed in proportion and allowed to stand at 25°C for 30 minutes. The final concentrations of the samples were 0.01, 0.05, 0.1, 0.2 and 0.3 mg / mL, respectively. The sample solution was replaced with an equal volume of distilled water as a blank group. In addition, an equal volume of ethanol was used to replace the DPPH ethanol solution as a control group. The absorbance of each sample at different concentrations was measured at a wavelength of 517 nm. (Note: All samples were measured three times and the average value was taken).

[0065] DPPH free radical scavenging ability (%) = [(A 样品 -A 空白 ) / (A 对照 -A 空白 )]×100 The results of the DPPH free radical scavenging ability are shown in Table 3.

[0066] Table 3 DPPH free radical scavenging ability of chitosan and chitosan derivatives containing bifunctional groups (%)

[0067] The results show that, since triazole and coumarin have strong antioxidant capacity, the antioxidant capacity of the chitosan derivative containing bifunctional groups synthesized by the present invention is significantly improved compared with the chitosan raw material.

[0068] Antifungal activity assay (1) Anti-Fusarium oxysporum activity: The anti-Fusarium oxysporum ability of chitosan and chitosan derivatives containing bifunctional groups was determined and compared (Table 4): 200 μL of Fusarium oxysporum bacterial solution was transferred to the solidified solid culture medium with a pipette, spread evenly, and cultured in a mold incubator at 28°C and 60% humidity for 48 h. The chitosan and chitosan derivatives containing bifunctional groups used in the experiment in the example were then prepared into a 4 mg / mL sample solution. Then, the sample solution and solid culture medium (28.5 g fungal culture medium plus 18 g agar plus 1000 ml water) were mixed according to the preset concentration and poured into the labeled sample culture dish to be cooled and solidified. A 5 mm diameter bacterial cake was punched out from the periphery of the fresh colony with a sterile puncher, and the bacterial cake was carefully inoculated into the sample culture medium with sterile tweezers, and cultured at 28°C and 60% humidity. The sample solution was replaced with an equal volume of solid culture medium as a blank group. After 48 hours of cultivation, the diameter of the hyphae growth area was measured by the cross method (Note: all samples were measured three times and the average value was taken).

[0069] Table 4 Anti-Fusarium oxysporum ability of chitosan and chitosan derivatives containing bifunctional groups (%)

[0070] (2) Anti-Asparagus stem blight activity: The anti-Asparagus stem blight ability of chitosan and chitosan derivatives containing bifunctional groups was measured and compared (Table 5): 1000 mL of solid culture medium was prepared: 28.5 g of fungal culture medium plus 18 g of agar plus 1000 ml of water, and poured into a culture dish while hot after sterilization for use. Use a pipette to transfer 200 μL of the bacterial solution of Asparagus stem blight obtained in a conventional manner to the solidified solid culture medium, spread it evenly, and culture it in a mold incubator at 28°C and 60% humidity for 48 hours. Then, the experimental chitosan and chitosan derivatives containing bifunctional groups in the example were prepared into a 4 mg / mL sample solution. Then, the sample solution and solid culture medium were mixed at a preset concentration and poured into a labeled sample culture dish and allowed to cool and solidify. Use a sterile puncher to punch a 5mm diameter bacterial cake on the periphery of the fresh colony, and use sterile tweezers to carefully inoculate the bacterial cake into the sample culture medium, and culture it at 28°C and 60% humidity. The sample solution is replaced with an equal volume of solid culture medium as a blank group. After 48 hours of culture, the diameter of the hyphae growth area is measured by the cross method (Note: the samples tested are measured three times and the average value is taken).

[0071] Table 5 Anti-asparagus stem blight activity of chitosan and chitosan derivatives containing bifunctional groups (%)

[0072] The results show that, due to the strong antioxidant capacity of triazole and coumarin, the antifungal capacity of the chitosan derivative containing bifunctional groups synthesized by the present invention is significantly improved compared with the chitosan raw material.

[0073] The above embodiments are preferred implementation modes of the present invention, but the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions and combinations made in accordance with the essence and principles of the present invention are equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A chitosan derivative containing bifunctional groups, characterized in that: The chitosan derivative is represented by formula (1), Formula (1), In formula (1), R = ; R1 = H, halogen or C1-C3 alkyl; R2 = H or C1-C3 alkyl; Where n represents the degree of polymerization, and the average value range of n is 10-12000.

2. The chitosan derivative containing bifunctional groups according to claim 1, characterized in that: In formula (1), R1 = H, Cl or CH3; R2 = H or CH3.

3. A method for preparing a chitosan derivative containing bifunctional groups according to claim 1, characterized in that: 1) reacting chitosan quaternary ammonium salt with N-bromosuccinimide to obtain 6-bromochitosan quaternary ammonium salt; reacting 6-bromochitosan quaternary ammonium salt with ethylenediamine to obtain 6-aminoethyl-chitosan quaternary ammonium salt; reacting 6-aminoethyl-chitosan quaternary ammonium salt with propyne bromide to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt; 2) A substituted hydroxycoumarin is subjected to a bromination reaction with tetrabutylammonium bromide to obtain a brominated coumarin product, and then the brominated coumarin product is reacted with azidotrimethylsilane to obtain an azido coumarin product; 3) The 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained above is reacted with the azidocoumarin product in the presence of a solvent to obtain a chitosan derivative containing a bifunctional group as shown in formula (1).

4. The method for preparing a chitosan derivative containing bifunctional groups according to claim 3, characterized in that: The 6-bromochitosan quaternary ammonium salt is prepared by using chitosan as a raw material and methyl iodide to prepare the chitosan quaternary ammonium salt, and then N -Bromosuccinimide is dissolved in 1-methyl-2-pyrrolidone, and triphenylphosphine is added, ice bathed for 30 minutes, reacted at 60-100°C for 1-4 hours, precipitated with ethanol after the reaction, and the precipitate is washed with ethanol and filtered, and dried to constant weight to obtain 6-bromochitosan quaternary ammonium salt; wherein, N - the amount of bromosuccinimide added is 2-10 times the molar amount of chitosan quaternary ammonium salt, and the amount of triphenylphosphine added is 2-10 times the molar amount of chitosan; The 6-aminoethyl-chitosan quaternary ammonium salt is prepared by dissolving 6-bromochitosan quaternary ammonium salt in ethylenediamine, reacting at 70-90° C. for 12-20 hours under nitrogen protection, and after the reaction, pouring the reaction solution into acetone for precipitation, washing twice with acetone, filtering, and drying to constant weight to obtain 6-aminoethyl-chitosan quaternary ammonium salt, wherein the amount of ethylenediamine added is 20-40 times the molar amount of 6-bromochitosan quaternary ammonium salt; The 6-aminoethyl-propynyl chitosan quaternary ammonium salt is prepared by adding sodium hydroxide and potassium iodide to the 6-aminoethyl-chitosan quaternary ammonium salt, and then adding propynyl bromide, dissolving the mixture in N,N-dimethylformamide, reacting the mixture at 70-90° C. for 18-26 hours under nitrogen protection, and after the reaction, pouring the reaction solution into excess anhydrous ethanol for precipitation, washing the mixture twice with anhydrous ethanol, filtering the mixture, and drying the mixture to a constant weight to obtain the 6-aminoethyl-propynyl chitosan quaternary ammonium salt; wherein the amount of propynyl bromide added is 2-10 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt, the amount of sodium hydroxide added is 1-5 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt, and the amount of potassium iodide added is 0.05-0.3 times the molar amount of the 6-aminoethyl-chitosan quaternary ammonium salt.

5. The method for preparing a chitosan derivative containing bifunctional groups according to claim 3, characterized in that: The bromination reaction is as follows: substituted hydroxycoumarin and tetrabutylammonium bromide are dissolved in N,N -dimethylformamide, adding phosphorus pentoxide, reacting at 100°C for 3 hours, extracting the reaction solution repeatedly with toluene, collecting the extracts and performing reduced pressure distillation to obtain a brominated coumarin product; wherein the amount of tetrabutylammonium bromide added is 2 times the molar amount of the substituted hydroxycoumarin, and the amount of phosphorus pentoxide added is 3 times the molar amount of the substituted hydroxycoumarin.

6. The method for preparing a chitosan derivative containing bifunctional groups according to claim 5, characterized in that: The substituted hydroxycoumarin includes 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-7-methylcoumarin, 4-hydroxy-6-chlorocoumarin and a series of coumarins carrying a hydroxyl group.

7. The method for preparing a chitosan derivative containing bifunctional groups according to claim 3, characterized in that: The azidocoumarin product is: the bromocoumarin product and azidotrimethylsilane are dissolved in N,N -dimethylformamide, and then add hexamethylphosphoramide, react at 90° C. for 6.5 hours, precipitate with deionized water after the reaction, wash the precipitate with petroleum ether and filter to obtain a filter cake, dry to constant weight, and obtain an azidocoumarin product; wherein, the added amount of azidotrimethylsilane is 10 times the molar amount of the bromocoumarin product, and the added amount of hexamethylphosphoramide is 2.6 times the molar amount of the bromocoumarin product.

8. The method for preparing a chitosan derivative containing bifunctional groups according to claim 3, characterized in that: The obtained azidocoumarin and 6-aminoethyl-propynyl chitosan quaternary ammonium salt product are dissolved in dimethyl sulfoxide and triethylamine, and then cuprous iodide is added, and the reaction is carried out at 50-100° C. for 20-30 hours. After the reaction, the precipitate is precipitated with acetone, and the precipitate is washed with acetone and filtered to obtain a filter cake, which is dried to constant weight to obtain a chitosan derivative containing a bifunctional group as shown in formula (1); wherein the amount of the azidocoumarin product added is 1.5-4 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt, the amount of cuprous iodide added is 1-3 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt, and the amount of triethylamine added is 5-10 times the molar amount of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

9. An application of the chitosan derivative containing bifunctional groups according to claim 1, characterized in that: Application of the compound represented by formula (1) in the preparation of products with antioxidant properties.

10. An application of the chitosan derivative containing bifunctional groups according to claim 1, characterized in that: Use of the compound represented by formula (1) in the preparation of antibacterial agents.

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