A chitosan derivative containing bifunctional groups, its preparation and application

Abstract

The present invention relates to the fields of food, cosmetics and biomedicine, and particularly relates to a chitosan derivative containing dual functional groups (triazole and coumarin), a preparation method thereof and an application thereof. The structure of the derivative is shown in formula (1). The present invention adopts mild reaction conditions and uses readily available and low-cost raw materials. The prepared chitosan derivative has good water solubility while having remarkable antioxidant activity and antifungal activity, effectively broadening the application scope of chitosan in various industries. The chitosan derivative containing dual functional groups (triazole and coumarin) of the present invention has wide application potential in the fields of cosmetics, food and biomedicine, and is convenient for industrial promotion. Formula (1)

Description

Technical Field

[0001] The present invention relates to the fields of food, cosmetics and biomedicine, and particularly relates to a chitosan derivative containing dual functional groups (triazole and coumarin), a preparation method thereof and an application thereof. Background Art

[0002] Chitosan is a chitin-derived polysaccharide, which is a polysaccharide polymer biological resource that is naturally non-toxic, rich in sources, renewable, has good biocompatibility and biodegradability, and has certain biological activities, such as antibacterial, antioxidant, immunomodulatory, food preservation, inhibiting tumor growth, etc., and is widely used in the fields of agriculture, food, medicine, chemical industry, cosmetics, papermaking, textile, environmental protection, etc. 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, and different active groups can be introduced through site modification by reactions such as oxidation, esterification, etherification, alkylation, Schiff base formation, quaternization, etc., so as to obtain chitosan derivatives with different biological activities and expand its application range. Summary of the Invention

[0003] The purpose of the present invention is to provide a chitosan derivative containing dual functional groups (triazole and coumarin), a preparation method thereof and an application thereof.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] A chitosan derivative containing dual functional groups, the chitosan derivative is shown in formula (1), Formula (1),

[0006] In formula (1), R = ; R1 = H, halogen or an alkyl group with 1 to 3 carbon atoms; R2 = H or an alkyl group with 1 to 3 carbon atoms;

[0007] Wherein n represents the degree of polymerization, and the average value range of n is 10 - 12000.

[0008] In the said formula (1), R1 = H, Cl or CH3; R2 = H or CH3.

[0009] A preparation method of the chitosan derivative containing dual functional groups:

[0010] 1) React chitosan quaternary ammonium salt with N-bromosuccinimide to obtain 6-bromo chitosan quaternary ammonium salt; react 6-bromo chitosan quaternary ammonium salt with ethylenediamine to obtain 6-aminoethyl-chitosan quaternary ammonium salt; react 6-aminoethyl-chitosan quaternary ammonium salt with propargyl bromide to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt;

[0011] 2) A brominated coumarin product is obtained by bromination reaction of a substituted hydroxycoumarin with tetrabutylammonium bromide, and then an azidocoumarin product is obtained by reacting the brominated coumarin product with trimethylsilyl azide;

[0012] 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 the bifunctional chitosan derivative shown in formula (1).

[0013] The 6-bromo chitosan quaternary ammonium salt is: using chitosan as a raw material to prepare chitosan quaternary ammonium salt with methyl iodide, and then dissolving the chitosan quaternary ammonium salt and N -bromosuccinimide in 1-methyl-2-pyrrolidone, adding triphenylphosphine, ice-bathing for 30 minutes, reacting at 60-100 °C for 1-4 hours, after the reaction, precipitating with ethanol, washing the precipitate with ethanol and filtering, drying to constant weight to obtain 6-bromo chitosan quaternary ammonium salt; wherein, N The addition amount of -bromosuccinimide is 2-10 times the molar amount of chitosan quaternary ammonium salt, and the addition amount of triphenylphosphine is 2-10 times the molar amount of chitosan;

[0014] The 6-aminoethyl-chitosan quaternary ammonium salt is: dissolving 6-bromo chitosan quaternary ammonium salt in ethylenediamine, reacting at 70-90 °C for 12-20 hours under nitrogen protection, after the reaction, pouring the reaction solution into acetone for precipitation, washing twice with acetone, filtering, drying to constant weight to obtain 6-aminoethyl-chitosan quaternary ammonium salt, wherein the addition amount of ethylenediamine is 20-40 times the molar amount of 6-bromo chitosan quaternary ammonium salt;

[0015] The 6-aminoethyl-propynyl chitosan quaternary ammonium salt is: adding 6-aminoethyl-chitosan quaternary ammonium salt to sodium hydroxide and potassium iodide, then adding propargyl bromide, dissolving in N,N-dimethylformamide, reacting at 70-90 °C for 18-26 hours under nitrogen protection, after the reaction, pouring the reaction solution into excessive absolute ethanol for precipitation, washing twice with absolute ethanol, filtering, drying to constant weight to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt; wherein the addition amount of propargyl bromide is 2-10 times the molar amount of 6-aminoethyl-chitosan quaternary ammonium salt, the addition amount of sodium hydroxide is 1-5 times the molar amount of 6-aminoethyl-chitosan quaternary ammonium salt, and the addition amount of potassium iodide is 0.05-0.3 times the molar amount of 6-aminoethyl-chitosan quaternary ammonium salt.

[0016] The bromination reaction is: a substituted hydroxycoumarin and tetrabutylammonium bromide are dissolved in N, NIn dimethylformamide, phosphorus pentoxide was added, and the reaction was carried out at 100 °C for 3 hours. After the reaction, the reaction solution was repeatedly extracted with toluene, and the collected extract was subjected to vacuum distillation to obtain the bromocoumarin product; among them, the addition amount of tetrabutylammonium bromide was 2 times the molar amount of the hydroxycoumarin with substituents, and the addition amount of phosphorus pentoxide was 3 times the molar amount of the hydroxycoumarin with substituents.

[0017] The hydroxycoumarin with substituents is 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-7-methylcoumarin, 4-hydroxy-6-chlorocoumarin, and a series of coumarins carrying hydroxy groups.

[0018] The azidocoumarin product is: the bromocoumarin product and trimethylsilyl azide are dissolved in N, N -dimethylformamide, and then hexamethylphosphoramide is added, and the reaction is carried out at 90 °C for 6.5 hours. After the reaction, it is precipitated with deionized water, and the precipitate is washed with petroleum ether and filtered to obtain a filter cake, which is dried to constant weight to obtain the azidocoumarin product; among them, the addition amount of trimethylsilyl azide is 10 times the molar amount of the bromocoumarin product, and the addition amount of hexamethylphosphoramide is 2.6 times the molar amount of the bromocoumarin product.

[0019] The chitosan derivative containing bifunctional groups is obtained by dissolving the obtained azidocoumarin product and 6-aminoethyl-propynyl chitosan quaternary ammonium salt in dimethyl sulfoxide and triethylamine, and then adding copper iodide, and reacting at 50-100 °C for 20-30 hours. After the reaction, it 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 the chitosan derivative containing bifunctional groups shown in formula (1); among them, the addition amount of the azidocoumarin product is 1.5-4 times the molar amount of 6-aminoethyl-propynyl chitosan quaternary ammonium salt, the addition amount of copper iodide is 1-3 times the molar amount of 6-aminoethyl-propynyl chitosan quaternary ammonium salt, and the addition amount of triethylamine is 5-10 times the molar amount of 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0020] An application of the chitosan derivative containing bifunctional groups, the application of the compound shown in formula (1) in the preparation of products with antioxidant properties.

[0021] An application of the chitosan derivative containing bifunctional groups, the application of the compound shown in formula (1) in the preparation of antibacterial agents.

[0022] The advantages of the present invention:

[0023] (1) Chitosan has good biodegradability, biocompatibility, and non-toxicity, but its antioxidant activity is weak. Through chemical modification of chitosan in the present invention, while retaining its own advantages, its antioxidant activity is improved; the antioxidant activity of the obtained chitosan derivative containing bifunctional groups is greatly improved compared with that of chitosan. The said derivative can be developed into a new type of antioxidant to solve the problems such as potential toxicity of chemical antioxidants to human health, and can also be widely applied in the fields of food and medicine, etc.

[0024] (2) The present invention uses a sugar modification method to modify the hydroxyl groups on polysaccharides, and then obtains derivatives with high antioxidant activity. The preparation process is simple and the cost of the materials used is low. Description of the Drawings

[0025] Figure 1 It is a flow chart for synthesizing the derivative shown in formula (1) provided by the embodiment of the present invention.

[0026] Figure 2 It is an infrared spectrum diagram of chitosan quaternary ammonium salt provided by the embodiment of the present invention.

[0027] Figure 3 It is an infrared spectrum diagram of 6-bromo chitosan quaternary ammonium salt provided by Example 1 of the present invention.

[0028] Figure 4 It is an infrared spectrum diagram of 6-aminoethyl-chitosan quaternary ammonium salt provided by Example 1 of the present invention.

[0029] Figure 5 It is an infrared spectrum diagram of 6-aminoethyl-propynyl chitosan quaternary ammonium salt provided by Example 1 of the present invention.

[0030] Figure 6 It is an infrared spectrum diagram of 4-azidocoumarin provided by Example 1 of the present invention.

[0031] Figure 7 It is an infrared spectrum diagram of 4-azido-6-chlorocoumarin provided by Example 2 of the present invention.

[0032] Figure 8 It is an infrared spectrum diagram of 4-azido-7-methylcoumarin provided by Example 3 of the present invention.

[0033] Figure 9 It is an infrared spectrum diagram of 4-azido-6-methylcoumarin provided by Example 4 of the present invention.

[0034] Figure 10 It is an infrared spectrum diagram of the chitosan derivative containing bifunctional groups provided by Example 1 of the present invention.

[0035] Figure 11This is the infrared spectrum of the chitosan derivative containing dual functional groups provided in Example 2 of the present invention.

[0036] Figure 12 This is the infrared spectrum of the chitosan derivative containing dual functional groups provided in Example 3 of the present invention.

[0037] Figure 13 This is the infrared spectrum of the chitosan derivative containing dual functional groups provided in Example 4 of the present invention. Detailed implementation mode

[0038] The present invention will be further described below in conjunction with examples. It should be noted that the specific implementation modes described herein are only for explaining and interpreting the present invention and do not limit the present invention. Example 1

[0039] As Figure 1 shown, prepare the chitosan derivative containing dual functional groups (Formula (1)):

[0040] (1) Preparation of 6-bromo chitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) of chitosan and 2.8 g (19.7 mmol) of sodium iodide, add 5.2 mL of iodomethane and 4.48 mL of 15% sodium hydroxide solution, add them to 5.6 mL of 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 of chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64 g (2.2 mmol) of chitosan quaternary ammonium salt and 1.7 g (9.5 mmol) N N-bromosuccinimide, add 7 mL of 1-methyl-2-pyrrolidone, mix well and then slowly add 2.6 g (9.9 mmol) of triphenylphosphine in batches. After reacting at 0 °C for 15 minutes, react at 80 °C for 2 hours. After the reaction, precipitate the reaction solution with absolute ethanol, wash it twice with absolute ethanol, and dry it at 65 °C to obtain 6-bromo chitosan quaternary ammonium salt (see Figure 3 ).

[0041] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.17 g (0.48 mmol) of 6-bromo chitosan quaternary ammonium salt and add it to 1 mL (15 mmol) of ethylenediamine. Under nitrogen protection, react at 80 °C for 16 hours. After the reaction, pour the reaction solution into acetone for precipitation, wash it twice with acetone, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-chitosan quaternary ammonium salt (see Figure 4 ).

[0042] (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 an excess of absolute ethanol for precipitation, wash twice with absolute ethanol, filter by suction, and dry at 65 °C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ).

[0043] (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 of N, N -dimethylformamide, and react at 100 °C for 3 hours. After the reaction, extract the reaction solution three times with 20 mL of toluene, collect the extraction solution and perform vacuum distillation to obtain 4-bromocoumarin.

[0044] (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 of N, N -dimethylformamide, and react at 90 °C for 6.5 hours. After the reaction, pour the reaction solution into 65 mL of deionized water to precipitate. Wash the precipitate with petroleum ether and dry at 65 °C to obtain 4-azidocoumarin (see Figure 6 ).

[0045] (6) Add 0.1 g (5 mmol) of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 0.08 g (0.42 mmol) of the 4-azidocoumarin obtained in step (5) and 0.05 mg (10 mmol) of copper(I) iodide to 2.8 mL of dimethyl sulfoxide and 0.61 mL (45 mmol) of triethylamine, heat to 80 °C and react for 20 hours. After the reaction, pour the reaction solution into 20 mL of acetone to precipitate. Filter the precipitate by suction, wash the precipitate with acetone, and dry at 65 °C to obtain a chitosan derivative containing bifunctional groups (see Figure 10 ), where R1 is hydrogen and R2 is hydrogen (see Figure 10 ).

[0046] The infrared spectrum of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt provided in Example 1 of the present invention (see Figure 5 ) compared with that of chitosan quaternary ammonium salt (see Figure 2 ), 2125.02 cm -1The peak at is the stretching vibration peak of the alkyne bond, indicating the successful synthesis of 6-aminoethyl-propynyl chitosan quaternary ammonium salt. Figure 6 This is the infrared spectrum of 4-azidocoumarin provided in Example 1 of the present invention. From Figure 6 it can be seen that at 1724.78 cm -1 is the stretching vibration peak of the C=O bond on the coumarin, and at 1610.33 cm -1 is the stretching vibration peak of the C=C bond on the coumarin, and at 2128.02 cm -1 is the peak of the azide group, indicating the successful synthesis of 4-azidocoumarin. Figure 10 This is the infrared spectrum of the chitosan derivative containing dual functional groups provided in Example 1 of the present invention. From Figure 10 it can be seen that compared with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ), at 1723.10 cm -1 is the stretching vibration peak of C=O on the coumarin, at 1608.68 cm -1 is the stretching vibration peak of C=C on the benzene ring, at 764.73 cm -1 is the deformation vibration peak of C-H on the benzene ring, and at 2125.02 cm -1 the peak of the alkyne bond disappears. The above analysis data prove the successful synthesis of the chitosan derivative containing dual functional groups with R1 and R2 being hydrogen. Example 2

[0047] In this example, a chitosan derivative containing dual functional groups with R1 being chlorine and R2 being hydrogen was synthesized according to the above synthesis route. The difference from Example 1 is that:

[0048] (1) Preparation of 6-bromochitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) of chitosan and 2.8 g (19.7 mmol) of sodium iodide, add 5.2 mL of iodomethane and 4.48 mL of a 15% sodium hydroxide solution, add them to 5.6 mL of 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 of chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64 g (2.2 mmol) of chitosan quaternary ammonium salt and 0.78 g (4.4 mmol) of N N-bromosuccinimide, add 7 mL of 1-methyl-2-pyrrolidone, mix well and then slowly add 1.2 g (4.4 mmol) of triphenylphosphine in batches. After reacting at 0 °C for 30 minutes, react at 90 °C for 5 hours. After the reaction, precipitate the reaction solution with absolute ethanol, wash it twice with absolute ethanol, and dry it at 65 °C to obtain 6-bromochitosan quaternary ammonium salt.

[0049] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.15 g (0.42 mmol) of 6-bromo-chitosan quaternary ammonium salt and add it to 0.56 mL (8.4 mmol) of ethylenediamine. Under nitrogen protection, react at 70 °C for 12 hours. After the reaction, pour the reaction solution into acetone for precipitation, wash it twice with acetone, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0050] (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, pour the reaction solution into excess absolute ethanol for precipitation, wash it twice with absolute ethanol, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-propynyl-chitosan quaternary ammonium salt.

[0051] (4) Weigh 1.5 g (8 mmol) of 4-hydroxy-6-chlorocoumarin, 5.1 g (16 mmol) of tetrabutylammonium bromide, and 3.4 g (24 mmol) of phosphorus pentoxide, and add them to 20 mL of N, N -dimethylformamide. React at 100 °C for 3 hours. After the reaction, extract the reaction solution three times with 20 mL of toluene, collect the extraction solution, and perform vacuum distillation to obtain 4-bromo-6-chlorocoumarin.

[0052] (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, and add them to 12 mL of N, N -dimethylformamide. React at 90 °C for 6.5 hours. After the reaction, pour the reaction solution into 65 mL of deionized water to precipitate. Wash the precipitate with petroleum ether and dry it at 65 °C to obtain 4-azido-6-chlorocoumarin (see Figure 7 ).

[0053] (6) Add 0.1 g (5 mmol) of the 6-aminoethyl-propynyl-chitosan quaternary ammonium salt obtained in step (1), 4 g (20 mmol) of the 4-azido-6-chlorocoumarin obtained in step (5), and 0.08 mg (10 mmol) of cuprous iodide to 2.8 mL of dimethyl sulfoxide and 0.68 mL (50 mmol) of triethylamine, heat to 100 °C and react for 30 hours. After the reaction, pour the reaction solution into 20 mL of acetone to precipitate. Filter the precipitate by suction, wash the precipitate with acetone, and dry it at 65 °C to obtain a chitosan derivative containing bifunctional groups (see Figure 11 ), where R1 is chlorine and R2 is hydrogen.

[0054] From Figure 11 it can be seen that, compared with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ), for 1666.14 cm -1 it is the stretching vibration peak of C=O on coumarin, and for 1620.74 cm -1 it is the stretching vibration peak of C=C on the benzene ring, and for 798.86 cm -1 it is the deformation vibration peak of C-H on the benzene ring. The peak of the alkyne bond at 2125.02 cm -1 disappears. The above analysis data prove that the synthesis of the chitosan derivative containing dual functional groups with R1 being chlorine and R2 being hydrogen is successful. Example 3

[0055] In this example, a chitosan derivative containing dual functional groups with R1 being hydrogen and R2 being methyl is synthesized according to the above synthesis route. The difference from Example 1 is that:

[0056] (1) Preparation of 6-bromo chitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) of chitosan and 2.8 g (19.7 mmol) of sodium iodide, add 5.2 mL of iodomethane and 4.48 mL of 15% sodium hydroxide solution, add them to 5.6 mL of 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 of chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64 g (2.2 mmol) of chitosan quaternary ammonium salt and 3.92 g (22 mmol) of N-bromosuccinimide, add 10 mL of 1-methyl-2-pyrrolidone, mix well and then slowly add 5.8 g (22 mmol) of triphenylphosphine in batches. After reacting at 0 °C for 30 minutes, react at 100 °C for 4 hours. After the reaction, precipitate the reaction solution with absolute ethanol, wash it twice with absolute ethanol, and dry it at 65 °C to obtain 6-bromo chitosan quaternary ammonium salt.

[0057] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.2 g (0.56 mmol) of 6-bromo chitosan quaternary ammonium salt and add it to 1.1 mL (22.4 mmol) of ethylenediamine. Under nitrogen protection, react at 90 °C for 20 hours. After the reaction, pour the reaction solution into acetone for precipitation, wash it twice with acetone, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0058] (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, the reaction was carried out at 70 °C for 18 hours. After the reaction, the reaction solution was poured into an excess of anhydrous ethanol for precipitation, washed twice with anhydrous ethanol, filtered by suction, and dried at 65 °C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0059] (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, and react at 100 °C for 3 hours. After the reaction, the reaction solution was extracted three times with 20 mL of toluene, and the collected extraction solution was subjected to vacuum distillation to obtain 4-bromo-7-methylcoumarin.

[0060] (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 and add them to 12 mL N, N -Dimethylformamide, and react at 90 °C for 6.5 hours. After the reaction, the reaction solution was poured into 65 mL of deionized water to precipitate. The precipitate was washed with petroleum ether and dried at 65 °C to obtain 4-azido-7-methylcoumarin (see Figure 8 ).

[0061] (6) Add 0.1 g (5 mmol) of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 1.5 g (7.5 mmol) of the 4-azido-7-methylcoumarin obtained in step (5), and 0.3 mg (5 mmol) of copper iodide to 2.8 mL of dimethyl sulfoxide and 0.34 mL (25 mmol) of triethylamine, and raise the temperature to 50 °C for reaction for 20 hours. After the reaction, the reaction solution was poured into 20 mL of acetone to precipitate. The precipitate was filtered by suction, washed with acetone, and dried at 65 °C to obtain a chitosan derivative containing bifunctional groups (see Figure 12 ), where R1 is hydrogen and R2 is methyl.

[0062] From Figure 12 It can be seen that compared with 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ), 1720.25 cm -1 is the stretching vibration peak of C=O on the coumarin, 1625.43 cm -1 is the stretching vibration peak of C=C on the benzene ring, 815.90 cm -1 is the deformation vibration peak of C-H on the benzene ring, and the peak of the triple bond at 2125.02 cm -1 disappears. The above analysis data prove that the synthesis of the chitosan derivative containing bifunctional groups with R1 being hydrogen and R2 being methyl is successful. Example 4

[0063] In this example, a chitosan derivative with dual functional groups where R1 is methyl and R2 is hydrogen is synthesized according to the above synthetic route. The difference from Example 1 is as follows:

[0064] (1) Preparation of 6-bromo chitosan quaternary ammonium salt: Weigh 1 g (6.2 mmol) of chitosan and 2.8 g (19.7 mmol) of sodium iodide, add 5.2 mL of iodomethane and 4.48 mL of 15% sodium hydroxide solution, add them to 5.6 mL of 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 of chitosan quaternary ammonium salt (see Figure 2 ). Weigh 0.64 g (2.2 mmol) of chitosan quaternary ammonium salt and 2.0 g (11 mmol) of N-bromosuccinimide, add 7 mL of 1-methyl-2-pyrrolidone, mix well, and then slowly add 2.9 g (11 mmol) of triphenylphosphine in batches. After reacting at 0 °C for 20 minutes, react at 85 °C for 3 hours. After the reaction, precipitate the reaction solution with absolute ethanol, wash it twice with absolute ethanol, and dry it at 65 °C to obtain 6-bromo chitosan quaternary ammonium salt.

[0065] (2) Preparation of 6-aminoethyl-chitosan quaternary ammonium salt: Weigh 0.17 g (0.48 mmol) of 6-bromo chitosan quaternary ammonium salt and add it to 0.96 mL (14.4 mmol) of ethylenediamine. Under nitrogen protection, react at 85 °C for 18 hours. After the reaction, pour the reaction solution into acetone for precipitation, wash it twice with acetone, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-chitosan quaternary ammonium salt.

[0066] (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, pour the reaction solution into an excess of absolute ethanol for precipitation, wash it twice with absolute ethanol, filter by suction, and dry it at 65 °C to obtain 6-aminoethyl-propynyl chitosan quaternary ammonium salt.

[0067] (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, add them to 20 mL of N, N -dimethylformamide, and react at 100 °C for 3 hours. After the reaction, extract the reaction solution three times with 20 mL of toluene, collect the extraction solution, and perform vacuum distillation to obtain 4-bromo-6-methylcoumarin.

[0068] (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 and add them to 1 mL N, N -dimethylformamide. React at 90 °C for 6.5 hours. After the reaction is complete, pour the reaction solution 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 Figure 9 ).

[0069] (6) Add 0.1 g (5 mmol) of the 6-aminoethyl-propynyl chitosan quaternary ammonium salt obtained in step (1), 0.08 g (0.38 mmol) of the 4-azido-6-methylcoumarin obtained in step (5), and 0.05 mg (10 mmol) of copper(I) iodide to 2.8 mL of dimethyl sulfoxide and 0.61 mL of triethylamine. Heat up to 80 °C and react for 20 hours. After the reaction is complete, pour the reaction solution into 20 mL of acetone to precipitate. The precipitate is filtered by suction, washed with acetone, and dried at 65 °C to obtain a chitosan derivative containing dual functional groups (see Figure 13 ), where R1 is hydrogen and R2 is methyl.

[0070] From Figure 13 it can be seen that compared with the 6-aminoethyl-propynyl chitosan quaternary ammonium salt (see Figure 5 ), the stretching vibration peak of C=O on the coumarin is at 1720.48 cm -1 , the stretching vibration peak of C=C on the benzene ring is at 1607.21 cm -1 , the deformation vibration peak of C-H on the benzene ring is at 820.08 cm -1 , and the peak of the triple bond at 2125.02 cm -1 disappears. The above analysis data prove that the synthesis of the chitosan derivative containing dual functional groups with R1 being methyl and R2 being hydrogen is successful.

[0071] Application Example

[0072] Determination of Antioxidant Activity

[0073] (1) Determination of the ability to scavenge superoxide anions: The ability of chitosan and the chitosan derivative containing bifunctional groups obtained in the above examples to scavenge superoxide anions was measured and compared (Table 1): The chitosan and the chitosan derivative containing bifunctional groups used in the experiments in the examples were prepared into solutions with a concentration of 1 mg / mL. Tris-HCl buffer solution, 0.969 g of Tris and 0.4 mL of concentrated HCl were diluted with water to 500 mL. Different volumes of the sample solution were taken according to the preset ratio, 0.5 mL of NBT solution (300 μM), 0.5 mL of NADH (550 μM), and then 0.5 mL of 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 mixture was allowed to stand at room temperature for 5 minutes, and the absorbance was measured at 560 nm. In the blank group, 0.5 mL of Tris-HCl buffer solution was used instead of NADH (Note: Each sample to be measured was measured three times and the average value was taken).

[0074] Scavenging ability of superoxide anions (%) = [(A 空白 - A 样品 ) / A 空白 × 100

[0075] The results of the determination of the antioxidant ability to scavenge superoxide anions are shown in Table 1.

[0076] Table 1 Scavenging ability of chitosan and chitosan derivatives containing bifunctional groups on superoxide anions (%)

[0077]

[0078] (2) Determination of the ability to scavenge hydroxyl radicals: The ability of chitosan and the chitosan derivative containing bifunctional groups to scavenge hydroxyl radicals was measured and compared (Table 2): The chitosan and the chitosan derivative containing bifunctional groups used in the experiments in the examples were prepared into a sample solution with a concentration of 5 mg / mL. Prepared with phosphate buffer solution (pH 7.4), 20.79 g of disodium hydrogen phosphate dodecahydrate and 2.644 g of sodium dihydrogen phosphate dihydrate were diluted with water to 500 mL. Different volumes of the sample solution were taken according to the preset ratio, 0.5 mL of phosphate buffer solution, 0.25 mL of EDTA-Fe 2+Solution (2 mmol / L, prepared with phosphate buffer solution), 0.5 ml of H2O2 solution (3%, prepared with phosphate buffer solution), then add 0.5 mL of safranine T solution (360 μg / ml, prepared with phosphate buffer solution) to the reaction solution. After mixing well in the test tube, the final concentrations of the samples are 0.1, 0.2, 0.4, 0.6, 0.8 mg / mL. Let it stand at 40 °C for 30 minutes, and measure the absorbance at 520 nm. In the blank group, 0.5 mL of phosphate buffer solution is used instead of the sample solution. The control group is 1 mL of phosphate buffer solution, 0.5 mL of safranine T solution and 0.5 mL of H2O2 solution mixed well (Note: Each tested sample is measured three times and the average value is taken).

[0079] Scavenging ability of hydroxyl radicals (%) = [(A 样品 - A 空白 ) / (A 对照 - A 空白 )] × 100

[0080] The measurement results of the scavenging ability of hydroxyl radicals are shown in Table 2.

[0081] Table 2 Scavenging ability of chitosan and chitosan derivatives containing bifunctional groups on hydroxyl radicals (%)

[0082]

[0083] (3) Measurement of the scavenging ability of DPPH radicals: Measure and compare the scavenging ability of DPPH radicals of chitosan and chitosan derivatives containing bifunctional groups (Table 3): Prepare the sample solutions of chitosan and chitosan derivatives containing bifunctional groups used in the examples into 1 mg / mL. Then, mix the DPPH ethanol solution, the sample solution and distilled water in proportion and let it stand at 25 °C for 30 minutes. The final concentrations of the samples are 0.01, 0.05, 0.1, 0.2 and 0.3 mg / mL respectively. The sample solution is replaced with an equal volume of distilled water as the blank group. In addition, an equal volume of ethanol is used to replace the DPPH ethanol solution as the control group. Measure the absorbance of each sample at different concentrations at a wavelength of 517 nm. (Note: Each tested sample is measured three times and the average value is taken).

[0084] Scavenging ability of DPPH radicals (%) = [(A 样品 - A 空白 ) / (A 对照 - A 空白 )] × 100

[0085] The measurement results of the scavenging ability of DPPH radicals are shown in Table 3.

[0086] Table 3 Scavenging ability of chitosan and chitosan derivatives containing bifunctional groups on DPPH radicals (%)

[0087]

[0088] As can be seen from the results, since triazole and coumarin have strong antioxidant capabilities, compared with the chitosan raw material, the antioxidant capacity of the bifunctional chitosan derivative synthesized in the present invention has been significantly improved.

[0089] Antifungal activity assay

[0090] (1) Activity against Fusarium oxysporum: The antifungal capabilities of chitosan and the bifunctional chitosan derivative were measured and compared respectively (Table 4): Pipette 200 μL of the Fusarium oxysporum bacterial solution onto the solidified solid medium and spread it evenly. Incubate in a mold incubator at 28 °C and 60% humidity for 48 h. Then, prepare a sample solution of 4 mg / mL from the chitosan and the bifunctional chitosan derivative used in the experiments in the examples. Next, mix the sample solution and the solid medium (28.5 g of fungal medium + 18 g of agar + 1000 ml of water) according to the preset concentration and pour it into the labeled sample petri dishes until it cools and solidifies. Use a sterile punch to punch out a 5-mm-diameter fungal cake from the periphery of the fresh colony, and carefully inoculate the fungal cake into the sample medium with sterile forceps. Incubate at 28 °C and 60% humidity. Replace the sample solution with an equal volume of solid medium as the blank group. After culturing for 48 hours, measure the diameter of the mycelial growth area by the cross method (Note: All the samples to be measured are measured three times and the average value is taken).

[0091] Table 4 Antifungal capabilities of chitosan and the bifunctional chitosan derivative (%)

[0092]

[0093] (2) Anti - Aspergillus asparagi activity: The anti - Aspergillus asparagi abilities of chitosan and chitosan derivatives containing bifunctional groups were measured and compared respectively (Table 5): Prepare 1000 mL of solid medium: 28.5 g of fungal medium plus 18 g of agar plus 1000 ml of water. After sterilization, pour it into petri dishes while it is still hot for standby. Use a pipette to transfer 200 μL of the Aspergillus asparagi bacterial solution obtained by the conventional method into the solidified solid medium, spread it evenly, and culture it in a mold incubator at 28 °C and 60% humidity for 48 h. Then, prepare the chitosan and chitosan derivatives containing bifunctional groups used in the examples into a sample solution with a concentration of 4 mg / mL. Then, mix the sample solution and the solid medium according to the preset concentration and pour it into the labeled sample petri dishes and wait for it to cool and solidify. Use a sterile punch to punch out a 5 - mm - diameter bacterial cake around the fresh colony, and carefully inoculate the bacterial cake into the sample medium with sterile forceps, and culture it at 28 °C and 60% humidity. The sample solution is replaced with an equal volume of solid medium as the blank group. After culturing for 48 hours, measure the diameter of the hyphal growth area by the cross - method (Note: All measured samples are measured three times and the average value is taken).

[0094] Table 5 Anti - Aspergillus asparagi abilities of chitosan and chitosan derivatives containing bifunctional groups (%)

[0095]

[0096] As can be seen from the results, due to the strong antioxidant ability of triazole and coumarin, compared with the chitosan raw material, the antifungal ability of the chitosan derivatives containing bifunctional groups synthesized in the present invention has been significantly improved.

[0097] The above - mentioned embodiments are the preferred embodiments of the present invention, but the present invention is not limited by the above - mentioned embodiments. Any changes, modifications, substitutions, and combinations made under the condition of being consistent with the essence and principle of the present invention are all 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), where 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; The preparation method of the chitosan derivative containing bifunctional groups is as follows: 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).

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 bifunctional chitosan derivative 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 is 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 amount of azidotrimethylsilane added is 10 times the molar amount of the bromocoumarin product, and the amount of hexamethylphosphoramide added 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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