A water-soluble conjugate, and a method of preparing and using the same

Abstract

The application belongs to the field of agriculture and medicine, and can be applied to the fields of crop disease prevention, green agriculture and medical instrument coating, and specifically relates to a water-soluble conjugate (carboxymethyl chitosan-carbendazim conjugate), a preparation method and application thereof. The water-soluble conjugate is a water-soluble carboxymethyl chitosan-carbendazim conjugate. The application successfully breaks through the solubility limitation of traditional chitosan and carbendazim through chemical modification means, so that it can remain in a stable dissolved state in a neutral and weak alkaline environment, greatly widens the pH range of application, solves technical problems such as that conventional chitosan can only be dissolved in an acidic medium and carbendazim is easy to precipitate under alkaline conditions, and provides technical support for practical application.

Description

TECHNICAL FIELD

[0001] The application belongs to the fields of agriculture and medicine, and can be applied to the fields of crop disease prevention and treatment, green agriculture and medical device coating, and specifically relates to a water-soluble conjugate (carboxymethyl chitosan-carbendazim conjugate) and a preparation method and application thereof. BACKGROUND

[0002] Carbendazim is a broad-spectrum and high-efficiency benzimidazole fungicide, which plays an inhibitory role by inhibiting the synthesis of fungal beta-tubulin, and is widely used in the fields of agriculture, industry and medicine, and is mainly used for preventing and treating fungal diseases. The fungicide has the advantages of strong systemicity and remarkable control effect, but has inherent defects such as poor water solubility, easy development of drug resistance and toxicity to aquatic organisms. In view of these problems, currently, strategies such as compounding fungicides with different mechanisms of action and developing slow-release nano drug delivery systems are mainly adopted, and these methods can effectively delay the development of drug resistance and reduce environmental risk, but still face challenges such as high production cost and insufficient stability of biological pesticides.

[0003] Chitosan is a natural polymer polymerized by acetyl amino-D-glucose through beta-(1, 4)-glycosidic bond, and has the characteristics of antibacterial, antioxidant, biocompatible and biodegradable, and the combination of chitosan and carbendazim is expected to provide new ideas for inhibiting bacteria, but due to the weak water solubility and biological activity of chitosan, the modification of the combined system becomes a key and difficult problem. SUMMARY

[0004] The purpose of the present application is to provide a water-soluble conjugate (carboxymethyl chitosan-carbendazim conjugate) and a preparation method and application thereof.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A water-soluble conjugate, the water-soluble conjugate is a water-soluble carboxymethyl chitosan-carbendazim conjugate.

[0007] The water-soluble conjugate is shown in the following structural formula a or b or c;

[0008]

[0009] Wherein a is N,O-carboxymethyl chitosan-carbendazim conjugate, b is N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate, and c is N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate, and the average value range of n is 5-12000.

[0010] ​A method for preparing a water-soluble conjugate, uniformly dispersing carbendazim in a hydrochloric acid solution to obtain carbendazim hydrochloride; introducing the carbendazim hydrochloride into a chitosan intermediate through an ion exchange reaction to prepare a water-soluble carboxymethyl chitosan-carbendazim conjugate.

[0011] The chitosan intermediate is any one of the following:

[0012] 1) reacting a chitosan raw material with chloroacetic acid to obtain an N,O-carboxymethyl chitosan intermediate;

[0013] 2) reacting a chitosan raw material with a nucleophile, methyl iodide, to obtain N,N,N-trimethyl chitosan, and then reacting the N,N,N-trimethyl chitosan with chloroacetic acid to obtain an N,N,N-trimethyl-O-carboxymethyl chitosan intermediate;

[0014] 3) reacting a chitosan raw material with 2,3-epoxypropyltrimethylammonium chloride to obtain N-hydroxypropyltrimethyl chitosan, and then reacting the N-hydroxypropyltrimethyl chitosan with chloroacetic acid to obtain an N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate.

[0015] The preparation of the carbendazim hydrochloride: uniformly dispersing carbendazim in a hydrochloric acid solution, stirring at room temperature until completely dissolved to obtain a carbendazim hydrochloride solution; the molar ratio of the carbendazim to the hydrochloric acid is 1:20.

[0016] The preparation of the N,O-carboxymethyl chitosan-carbendazim conjugate: uniformly dispersing chitosan in isopropyl alcohol, slowly adding a NaOH solution, stirring until uniform, then adding an aqueous chloroacetic acid solution, stirring at 70-80°C for 4-6h, after the reaction, alcohol precipitation, freeze-drying to obtain the N,O-carboxymethyl chitosan intermediate, then dissolving the N,O-carboxymethyl chitosan intermediate in deionized water, adding a carbendazim hydrochloride solution, stirring at room temperature for 12-14h, dialyzing in deionized water for 48h, then centrifuging to collect the upper clear liquid, and freeze-drying to obtain the N,O-carboxymethyl chitosan-carbendazim conjugate.

[0017] The amount of isopropyl alcohol used is 10mL per 1g of chitosan, the amount of the NaOH solution (40% NaOH solution) used is 3mL per 1g of chitosan, the molar ratio of the chloroacetic acid to the chitosan is (2-4):1, the alcohol used for alcohol precipitation is anhydrous ethanol, the amount of anhydrous ethanol used is 150mL per 1g of chitosan; the molar ratio of the N,O-carboxymethyl chitosan intermediate to the carbendazim hydrochloride is 1:2-4.

[0018] Preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: chitosan was uniformly dispersed in 1-methyl-2-pyrrolidone, and sodium iodide, sodium hydroxide solution and methyl iodide were added in turn under stirring at room temperature, and then refluxed at 60-70°C for 2-3h, and then precipitated and washed with excess ethanol to obtain N,N,N-trimethyl chitosan, which was then dispersed in isopropanol, and NaOH solution was slowly added and stirred, and then chloroacetic acid aqueous solution was added, and stirred at 50-60°C for 4-6h, and then alcohol precipitation, freeze-drying to obtain N,N,N-trimethyl-O-carboxymethyl chitosan intermediate, which was then dissolved in deionized water, and carbendazim hydrochloride solution was added, and stirred at room temperature for 12-14h, and then dialyzed in deionized water for 48h, and then centrifuged to collect the supernatant, and freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0019] The amount of 1-methyl-2-pyrrolidone is 30-40mL per 1g of chitosan, the amount of sodium iodide, sodium hydroxide and methyl iodide is 1.5-2g of sodium iodide, 20-30mmol of sodium hydroxide and 5-7mL of methyl iodide per 1g of chitosan, the amount of isopropanol is 10mL per 1g of N,N,N-trimethyl chitosan, the amount of NaOH solution (40% NaOH solution) is 3mL per 1g of N,N,N-trimethyl chitosan, the molar ratio of chloroacetic acid to N,N,N-trimethyl chitosan is (1-2):1, the alcohol precipitation uses anhydrous ethanol, and the amount of anhydrous ethanol is 150mL per 1g of chitosan, and the molar ratio of N,N,N-trimethyl-O-carboxymethyl chitosan intermediate to carbendazim hydrochloride is 1:2-4.

[0020] Preparation of N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: chitosan was dispersed in isopropanol and stirred at 50-60°C for 2h, then 2,3-epoxypropyl trimethyl ammonium chloride aqueous solution was added, and refluxed at 70-80°C, and then precipitated and washed with anhydrous ethanol to obtain N-hydroxypropyl trimethyl chitosan, which was then dispersed in isopropanol, and NaOH solution was slowly added and stirred, and then chloroacetic acid aqueous solution was added, and stirred at 50-60°C for 4-6h, and then alcohol precipitation, freeze-drying to obtain N-hydroxypropyl trimethyl-O-carboxymethyl chitosan intermediate, which was then dissolved in deionized water, and carbendazim hydrochloride solution was added, and stirred at room temperature for 12-14h, and then dialyzed in deionized water for 48h, and then centrifuged to collect the supernatant, and freeze-dried to obtain N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0021] The isopropanol is used in an amount of 25-30 mL per 1 g of chitosan, the molar ratio of 2,3-epoxypropyl trimethyl ammonium chloride to chitosan is (2-4):1, the isopropanol is used in an amount of 10 mL per 1 g of N-hydroxypropyl trimethyl chitosan, the NaOH solution is used in an amount of 3 mL per 1 g of N-hydroxypropyl trimethyl chitosan, the molar ratio of chloroacetic acid to N-hydroxypropyl trimethyl chitosan is (1-2):1, the molar ratio of N-hydroxypropyl trimethyl-O-carboxymethyl chitosan intermediate to carbendazim hydrochloride is 1:2-4, and the anhydrous ethanol is used in an amount of 150 mL per 1 g of chitosan.

[0022] The dialysis operation is as follows: the solution is added into a dialysis bag with a molecular weight cut-off of 10000, the dialysis bag is hung in a beaker containing deionized water after ensuring that the dialysis bag is well closed, and stirring dialysis is performed for 48 h to remove residual reagents, and the dialysis is completed.

[0023] The application of a water-soluble conjugate, and the application of the water-soluble conjugate in preparing a fungicide.

[0024] Compared with the prior art, the application has the following beneficial technical features:

[0025] (1) The application successfully breaks through the solubility limitation of traditional chitosan and carbendazim by means of chemical modification, so that the chitosan and carbendazim can be stably dissolved in a neutral and weak alkaline environment, the pH range of application is greatly widened, technical problems such as the fact that conventional chitosan can only be dissolved in an acidic medium and carbendazim is easy to precipitate under alkaline conditions are solved, and technical support is provided for practical application.

[0026] (2) The combination of the prepared chitosan derivative and carbendazim exhibits a significant synergistic effect, a double sterilization mechanism is formed through the membrane damage mechanism of the chitosan derivative and the inhibition of beta-tubulin by carbendazim, and the synergistic mechanism not only improves the antibacterial activity of the product, but also reduces the risk of pathogenic bacteria developing drug resistance, thereby providing an important reference for developing a new type of sterilizing agent with high efficiency and low resistance.

[0027] (3) The water-soluble conjugate prepared by the application significantly improves the convenience of applying the sterilizing agent, so that the water-soluble conjugate can be directly used for foliar spraying, can be applied to roots through an irrigation system, and can be stably compounded with commonly used pesticide adjuvants, the dosage form formula can be flexibly adjusted according to actual needs, the field operation process is greatly simplified, and a more convenient and efficient pesticide application scheme is provided for agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The conjugate synthesis route map provided for the embodiments of the application;

[0029] Figure 2 The infrared spectrum of chitosan;

[0030] Figure 3 is the infrared spectrum of carbendazim;

[0031] Figure 4 is the infrared spectrum of the N, O-carboxymethyl chitosan intermediate prepared in Example 1 of the present invention;

[0032] Figure 5 is the infrared spectrum of the N, O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 1 of the present invention;

[0033] Figure 6 is the hydrogen nuclear magnetic spectrum of the N, O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 1 of the present invention;

[0034] Figure 7 is the infrared spectrum of the N, N, N-trimethyl-O-carboxymethyl chitosan intermediate prepared in Example 2 of the present invention;

[0035] Figure 8 is the infrared spectrum of the N, N, N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 2 of the present invention;

[0036] Figure 9 is the hydrogen nuclear magnetic spectrum of the N, N, N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 2 of the present invention;

[0037] Figure 10 is the infrared spectrum of the N-hydroxypropyl trimethyl-O-carboxymethyl chitosan intermediate prepared in Example 3 of the present invention;

[0038] Figure 11 is the infrared spectrum of the N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 3 of the present invention;

[0039] Figure 12 is the hydrogen nuclear magnetic spectrum of the N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate prepared in Example 3 of the present invention;

[0040] Figure 13 is the dissolution state picture of chitosan, carbendazim, the prepared carboxymethyl chitosan intermediate and the carboxymethyl chitosan-carbendazim conjugate at a concentration of 1 mg / mL;

[0041] Figure 14 is the inhibition effect picture of carbendazim and the prepared N, O-carboxymethyl chitosan-carbendazim conjugate on Fusarium oxysporum, Colletotrichum gloeosporioides and Apiosporina morbosa at different test concentrations. DETAILED DESCRIPTION

[0042] The application will be further described in connection with the embodiments and the accompanying drawings. However, the application is not limited to the embodiments, and any equivalent replacement in the art according to the disclosure of the application shall fall within the protection scope of the application.

[0043] The application significantly improves the water solubility and antibacterial performance of chitosan by introducing a carboxymethyl group, a quaternary ammonium salt group and a protonated carbendazim. The modification method not only expands the research direction of chitosan derivatives as antibacterial agents, but also provides a theoretical basis and technical support for the industrial application of chitosan derivatives in crop disease control, green agriculture, pharmaceutical preparations and other fields.

[0044] The application utilizes chitosan raw materials to modify N,O-carboxymethyl chitosan or N-quaternary ammonium-O-carboxymethyl chitosan intermediates, and then under acidic conditions, the carboxylate group can be combined with protonated carbendazim through electrostatic interaction. At the same time, the benzimidazole ring of carbendazim and the chitosan skeleton form hydrogen bonds and hydrophobic interactions, and finally a stable water-soluble carboxymethyl chitosan-carbendazim conjugate is constructed. The system has high solubility and synergistic antibacterial effect, providing a new idea for the structural optimization of carbendazim and the development of green antibacterial agents, and is expected to achieve efficient, low-residue application in agricultural disease control. Example 1

[0045] The synthesis route of the carboxymethyl chitosan-carbendazim conjugate is as shown in Figure 1

[0046] (1) Preparation of carbendazim hydrochloride: 0.382 g of carbendazim was uniformly dispersed in 300 mL of an aqueous solution containing 40 mmol of HCl, and stirred at room temperature until completely dissolved to obtain a carbendazim hydrochloride solution.

[0047] (2) Preparation of N,O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was uniformly dispersed in 10 mL of isopropanol, 3 mL of 40% NaOH solution was slowly added, and the mixture was stirred at 60°C for 1 h. Then, 2.35 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added to the above chitosan isopropanol solution, and the mixture was heated and stirred at 80°C for 4 h. After the reaction was completed, the mixture was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain the N,O-carboxymethyl chitosan intermediate. Then, 0.161 g of N,O-carboxymethyl chitosan was dissolved in 10 mL of deionized water, and the mixture was stirred at room temperature for 12 h with the carbendazim hydrochloride solution prepared in step (1). Then, the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at a speed of 3000 rpm, and the precipitate was discarded. The upper clear liquid was freeze-dried to obtain the N,O-carboxymethyl chitosan-carbendazim conjugate.

[0048] ​(3) Preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was uniformly dispersed in 30 mL of 1-methyl-2-pyrrolidone, and 1.5 g of sodium iodide, 20 mL of 15% sodium hydroxide solution and 5 mL of iodomethane were added in turn under stirring at room temperature, and then the mixture was refluxed at 60 °C for 2 h, and then poured into excess ethanol for precipitation. Then the obtained precipitate was dispersed in 10 mL of isopropanol, and 3 mL of 40% NaOH solution was slowly added dropwise, and then the mixture was stirred at 60 °C for 1 h, and then 1.17 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added dropwise into the above N,N,N-trimethyl chitosan isopropanol solution, and then the mixture was continuously heated and stirred at 60 °C for 4 h. After the reaction was completed, the mixture was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.411 g of N,N,N-trimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water, and then the mixture was stirred at room temperature for 12 h with the carbendazim hydrochloride solution prepared in step (1). Then the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at 3000 rpm, and the precipitate was discarded. The supernatant was freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0049] (4) Preparation of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was dispersed in 25 mL of isopropyl alcohol, stirred at 60°C for 2 h, 3.752 g of 2,3-epoxypropyltrimethylammonium chloride was dissolved in deionized water and slowly added to the above chitosan isopropyl alcohol solution, and refluxed at 80°C for 10 h. After the reaction was completed, it was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl chitosan. Then the obtained precipitate was dispersed in 10 mL of isopropyl alcohol, 3 mL of 40% NaOH solution was slowly added, and stirred at 60°C for 1 h. Then 1.17 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added to the above N-hydroxypropyltrimethyl chitosan isopropyl alcohol solution, and heated and stirred at 60°C for 4 h. After the reaction was completed, it was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.393 g of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water, and stirred at room temperature for 12 h with the carbendazim hydrochloride solution prepared in step (1). Then the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at 3000 rpm, and the precipitate was discarded. The supernatant was freeze-dried to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate, wherein the average value of n is 5-12000.

[0050] By Figure 2 In the infrared spectrum of chitosan, the absorption peak at 3419 cm -1 is the stretching vibration peak of -OH on the sugar ring of chitosan, the absorption peaks at 2919 cm -1 and 2874 cm -1 are the vibration absorption peaks of methylene and methyl, the absorption peak at 1651 cm -1 is attributed to the bending vibration of N-H bond at C2 position, and the relatively strong absorption peak at 1092 cm -1 corresponds to the stretching vibration absorption peak of C-O bond in the sugar ring and on the sugar ring.

[0051] By Figure 3 In the infrared spectrum of carbendazim, the absorption peaks at 1712 cm -1 , 1630 cm -1 and 1596 cm -1 are the characteristic absorption peaks of amide carbonyl (-CONH-), the absorption peak at 3322 cm -1 is the stretching vibration absorption peak of N-H bond, and the characteristic absorption peaks of C-H bond and C-N bond in the benzimidazole ring are at 3060 cm -1 , 1467 cm -1 , 1443 cm-1 , 731 cm -1 and 1329 cm -1 .

[0052] The infrared spectrum of the N, O-carboxymethyl chitosan intermediate prepared by Figure 4 shows a characteristic peak at 1600 cm -1 , which is the asymmetric stretching vibration absorption peak of COO-, and a characteristic peak at 1415 cm -1 , which is the symmetric stretching absorption peak of COO-, thus proving the success of the synthesis of N, O-carboxymethyl chitosan.

[0053] The infrared spectrum of the N, O-carboxymethyl chitosan-carbendazim conjugate prepared by Figure 5 shows that, after the addition of carbendazim, the product has a characteristic absorption peak of C=O at 1751 cm -1 , and characteristic absorption peaks of the benzimidazole ring of carbendazim at 1638 cm -1 , 1476 cm -1 , 754 cm -1 , etc., thus proving the successful preparation of the N, O-carboxymethyl chitosan-carbendazim conjugate.

[0054] The 1H NMR spectrum of the N, O-carboxymethyl chitosan-carbendazim conjugate prepared by Figure 6 shows that the hydrogen signals of the polysaccharide backbone in the chitosan molecule are mainly distributed in the range of δ 3-5 ppm, in which the characteristic peaks at δ 3.3-3.5 ppm and δ 2.7 ppm correspond to [H3]-[H6] and [H2] protons on the sugar ring, respectively; the signal at δ 3.86 ppm in the spectrum is attributed to the methylene protons in the carboxymethyl group; it is worth noting that, after the addition of carbendazim, the newly appearing characteristic peaks at δ 7.0-7.5 ppm and δ 3.2 ppm in the spectrum of the product correspond to the hydrogen protons on the benzene ring and the methyl group of carbendazim, respectively, and the explicit attribution of these characteristic signals fully confirms the successful preparation of the N, O-carboxymethyl chitosan-carbendazim conjugate. Example 2

[0055] The difference from Example 1 is that:

[0056] (1) Preparation of carbendazim hydrochloride: 0.382 g of carbendazim was uniformly dispersed in 300 mL of an aqueous solution containing 40 mmol of HCl, and stirred at room temperature until completely dissolved to obtain a carbendazim hydrochloride solution.

[0057] (2) Preparation of N, O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was uniformly dispersed in 10 mL of isopropyl alcohol, 3 mL of 40% NaOH solution was slowly added dropwise, and the mixture was stirred at 60°C for 1 h. Then, 1.76 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added dropwise to the above chitosan isopropyl alcohol solution, and the mixture was heated and stirred at 70°C for 5 h. After the reaction was completed, the mixture was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N, O-carboxymethyl chitosan intermediate. Then, 0.241 g of N, O-carboxymethyl chitosan was dissolved in 10 mL of deionized water, and the solution was stirred at room temperature for 13 h. Then, the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at a speed of 3000 rpm, and the precipitate was discarded. The supernatant was freeze-dried to obtain N, O-carboxymethyl chitosan-carbendazim conjugate.

[0058] (3) Preparation of N, N, N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was uniformly dispersed in 40 mL of 1-methyl-2-pyrrolidone, and 1.75 g of sodium iodide, 25 mL of 15% NaOH solution, and 6 mL of iodomethane were sequentially added dropwise under stirring at room temperature. The mixture was refluxed at 65°C for 2.5 h, and then poured into excess ethanol for precipitation. Then, the obtained precipitate was dispersed in 10 mL of isopropyl alcohol, 3 mL of 40% NaOH solution was slowly added dropwise, and the mixture was stirred at 60°C for 1 h. Then, 1.76 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added dropwise to the above N, N, N-trimethyl chitosan isopropyl alcohol solution, and the mixture was heated and stirred at 60°C for 5 h. After the reaction was completed, the mixture was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N, N, N-trimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.274 g of N, N, N-trimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water, and the solution was stirred at room temperature for 13 h. Then, the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at a speed of 3000 rpm, and the precipitate was discarded. The supernatant was freeze-dried to obtain N, N, N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0059] (4) Preparation of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was dispersed in 30 mL of isopropyl alcohol, stirred at 50°C for 2 h, 1.876 g of 2,3-epoxypropyltrimethylammonium chloride was dissolved in deionized water and slowly added to the above chitosan isopropyl alcohol solution, and refluxed at 70°C for 10 h. After the reaction was completed, it was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl chitosan. Then the obtained precipitate was dispersed in 10 mL of isopropyl alcohol, 3 mL of 40% NaOH solution was slowly added, and stirred at 60°C for 1 h. Then 1.76 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added to the above N-hydroxypropyltrimethyl chitosan isopropyl alcohol solution, and heated and stirred at 60°C for 5 h. After the reaction was completed, it was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.262 g of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water, and stirred at room temperature for 13 h with the carbendazim hydrochloride solution prepared in step (1). Then the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at 3000 rpm, and the precipitate was discarded. The upper clear liquid was freeze-dried to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0060] Prepared by Figure 7 The infrared spectrum of the N,N,N-trimethyl-O-carboxymethyl chitosan intermediate prepared in the above step (3) showed characteristic peaks at 1598 cm -1 -1 for asymmetric stretching vibration absorption peak of COO-, at 1411 cm -1 -1 for symmetric stretching absorption peak of COO-, and at 1479 cm -1 -1 for characteristic absorption peak of trimethyl group. It can be proved from the above that the synthesis of N,N,N-trimethyl-O-carboxymethyl chitosan is successful.

[0061] Prepared by Figure 8 The infrared spectrum of the N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate prepared in the above step (4) showed characteristic absorption peaks of N,N,N-trimethyl-O-carboxymethyl chitosan, and new characteristic absorption peaks of C=O at 1719 cm -1 -1, characteristic absorption peaks of carbendazim benzimidazole ring at 1634 cm -1 -1, 719 cm -1 -1, and 1480 cm -1The overlapped absorption peak of trimethyl and carbendazim was obviously enhanced, which proved the successful preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0062] Again by Figure 9 In the 1H NMR spectrum of the prepared N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate, the hydrogen proton signals of chitosan polysaccharide skeleton were mainly distributed in the range of δ3-5ppm, in which the signal peaks at δ3.3-3.6ppm and δ2.7ppm corresponded to [H3]-[H6] and [H2] hydrogen protons on the sugar ring respectively; among the signals of the modification group, the methylene protons of carboxymethyl appeared at δ3.74ppm, while the hydrogen proton signals of trimethyl were located at δ3.15ppm; it is particularly noteworthy that after the introduction of carbendazim, new signal peaks appeared in the spectrum at δ7.0-7.5ppm, which belonged to the absorption peaks of hydrogen protons on the benzene ring of carbendazim, in addition, the methyl proton signals of carbendazim and the trimethyl signals overlapped at δ3.15ppm, the clear attribution of these characteristic signals fully confirmed the successful preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate. Example 3

[0063] The difference from Example 1 is that:

[0064] (1) Preparation of carbendazim hydrochloride: 0.382g of carbendazim was uniformly dispersed in 300mL of aqueous solution containing 40mmol of HCl, and stirred at room temperature until completely dissolved to obtain a carbendazim hydrochloride solution.

[0065] (2) Preparation of N,O-carboxymethyl chitosan-carbendazim conjugate: 1g of chitosan was uniformly dispersed in 10mL of isopropanol, 3mL of 40% NaOH solution was slowly added dropwise, and stirred at 60°C for 1h, then 1.17g of chloroacetic acid was dissolved in 10mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added dropwise to the above chitosan isopropanol solution, and heated and stirred at 80°C for 6h, after the reaction was completed, it was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N,O-carboxymethyl chitosan intermediate; then, 0.161g of N,O-carboxymethyl chitosan was dissolved in 10mL of deionized water, and reacted with the carbendazim hydrochloride solution prepared in step (1) at room temperature for 14h, then the reaction system was placed in a dialysis bag and dialyzed in deionized water for 48h, the obtained solution was centrifuged at a speed of 3000rpm, the precipitate was discarded, and the upper clear liquid was freeze-dried to obtain N,O-carboxymethyl chitosan-carbendazim conjugate.

[0066] (3) Preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: 1 g of chitosan was uniformly dispersed in 30 mL of 1-methyl-2-pyrrolidone, and 2 g of sodium iodide, 30 mL of 15% sodium hydroxide solution and 7 mL of iodomethane were sequentially added under stirring at room temperature. The mixture was refluxed at 70 °C for 3 h, and then poured into excess ethanol for precipitation. The obtained precipitate was then dispersed in 10 mL of isopropanol, and 3 mL of 40% NaOH solution was slowly added dropwise. The mixture was stirred at 60 °C for 1 h, and then 2.35 g of chloroacetic acid was dissolved in 10 mL of water to obtain a chloroacetic acid aqueous solution, which was slowly added dropwise to the above N,N,N-trimethyl chitosan isopropanol solution. The mixture was continuously heated and stirred at 60 °C for 6 h. After the reaction was completed, the mixture was poured into excess ethanol, and the obtained precipitate was freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.411 g of N,N,N-trimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water, and reacted with the carbendazim hydrochloride solution prepared in step (1) at room temperature for 14 h. The reaction system was then placed in a dialysis bag and dialyzed in deionized water for 48 h. The obtained solution was centrifuged at 3000 rpm, and the precipitate was discarded. The supernatant was freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0067] (4) Preparation of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate: Weigh 1 g of chitosan and disperse it in 25 mL of isopropanol. Stir at 60 °C for 2 h. Weigh 2.814 g of the conjugate. 2,3-Epoxypropyltrimethylammonium chloride was dissolved in deionized water and slowly added to the above chitosan isopropanol solution. The mixture was refluxed at 80°C for 10 hours. After the reaction, the solution was poured into excess ethanol. The resulting precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl chitosan. The precipitate was then dispersed in 10 mL of isopropanol, and 3 mL of 40% NaOH solution was slowly added dropwise. The mixture was stirred at 60°C for 1 hour. Then, 2.35 g of chloroacetic acid was dissolved in 10 mL of water to obtain an aqueous chloroacetic acid solution, which was slowly added dropwise to the above N-hydroxypropyltrimethyl chitosan isopropanol solution. The mixture was heated and stirred at 60°C for another 6 hours. After the reaction, the solution was poured into excess ethanol. The resulting precipitate was freeze-dried to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate. Finally, 0.197 g of... N-hydroxypropyltrimethyl-O-carboxymethyl chitosan was dissolved in 20 mL of deionized water and reacted with the carbendazim hydrochloride solution prepared in step (1) at room temperature for 14 h. The reaction system was then placed in a dialysis bag and dialyzed in deionized water for 48 h. The resulting solution was centrifuged at 3000 rpm, the precipitate was discarded, and the supernatant was lyophilized to obtain the N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0068] Depend on Figure 10 In the infrared spectrum of the prepared N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate, 1602 cm⁻¹ -1 The characteristic peak at 1413 cm⁻¹ is the absorption peak of the asymmetric stretching vibration of COO⁻. -1 The characteristic peak at 1480 cm⁻¹ is a symmetric stretching absorption peak of COO⁻. Additionally, there is also a peak at 1480 cm⁻¹. -1 The presence of a characteristic absorption peak for trimethyl at this location proves that N-hydroxypropyltrimethyl-O-carboxymethyl chitosan was successfully synthesized.

[0069] Depend on Figure 11 The infrared spectrum of the prepared N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate shows that after inoculation with carbendazim, the characteristic absorption peak of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan is still present, and the product reaches a peak at 1737 cm⁻¹. -1 A new characteristic absorption peak for C=O appears at 1638 cm⁻¹. -1 704cm -1 The characteristic absorption peak of the benzimidazole ring of carbendazim appeared in [various locations], and was observed at 1480 cm⁻¹. -1 The significantly enhanced overlap of the trimethyl and carbendazim absorption peaks at the site of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate demonstrates the successful preparation of the conjugate.

[0070] Again by Figure 12 In the1H-NMR spectrum of the prepared N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate, the hydrogen proton signals of the chitosan polysaccharide skeleton are mainly distributed in the range of δ3-5ppm, in which the signal peaks at δ3.3-3.5ppm and δ2.8ppm correspond to the [H3]-[H6] and [H2] hydrogen protons on the sugar ring, respectively; among the signals of the modification groups, the methylene protons of the carboxymethyl group appear at δ3.74ppm, while the hydrogen proton signals of the methyl and methylene groups in the hydroxypropyltrimethyl group appear at δ4.05ppm, δ3.15ppm, δ2.69ppm and δ2.36ppm, respectively; it is particularly noteworthy that after the introduction of carbendazim, new signal peaks appear at δ7.0-7.5ppm in the spectrum, which belong to the absorption peaks of the hydrogen protons on the benzene ring of carbendazim, in addition, the methyl proton signal of carbendazim overlaps with the trimethyl signal at δ3.15ppm, and the clear attribution of these characteristic signals fully confirms the successful preparation of the N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

[0071] Application Example

[0072] Solubility determination

[0073] The solubilities of the chitosan, carbendazim, carboxymethyl chitosan intermediate and carboxymethyl chitosan-carbendazim conjugate used in Examples 1-3 were determined by the gravimetric method and compared (Table 1): first, 10 mL of deionized water was added as a solvent in a centrifuge tube, and then 10 mg of each sample, which was freeze-dried to a constant weight, was accurately weighed. At room temperature (25±2℃), the sample was added to the solvent in several portions and continuously stirred by a magnetic stirrer until the system reached a supersaturated state. After filtration through a 0.45μm microporous filter, the mass of the undissolved residue was accurately weighed, and the solubility value of the sample in deionized water was calculated according to the formula: solubility (mg / mL) = 10-undissolved mass. Three parallel samples were set for each group, and the average value was calculated, the higher the solubility value, the better the water solubility of the sample, and the test results are shown in Table 1 and Figure 13

[0074] Table 1 Solubility of chitosan, carbendazim, carboxymethyl chitosan intermediate and carboxymethyl chitosan-carbendazim conjugate

[0075]

[0076] ​Solubility test results: Experimental data showed that the water solubility of chitosan was significantly improved after carboxymethylation and quaternization modification, among which N,N,N-trimethyl-O-carboxymethyl chitosan showed the best solubility performance, with a solubility of 8.79±0.06 mg / mL. After conjugation with carbendazim, the quaternized derivatives still maintained high water solubility, especially N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate, which had a solubility as high as 9.17±0.03 mg / mL. This phenomenon confirmed that the synergistic effect of carboxymethylation and quaternization not only significantly improved the solubility of chitosan, but also effectively neutralized the hydrophobicity of carbendazim, making the resulting conjugate have excellent solubility and potential antibacterial activity. In addition, N-hydroxypropyl trimethyl-O-carboxymethyl chitosan-carbendazim conjugate (8.84±0.09 mg / mL) and N,O-carboxymethyl chitosan-carbendazim conjugate (4.77±0.03 mg / mL) also showed good water solubility, which was significantly improved compared with unmodified chitosan (0.58±0.10 mg / mL) and carbendazim raw material (0.63±0.05 mg / mL). As shown in FIG. 8, at a concentration of 1 mg / mL, the solutions of carboxymethyl chitosan intermediates and their carbendazim conjugates all showed clear and transparent state, which was in sharp contrast to the turbid solution of unmodified chitosan and carbendazim raw material, directly verifying the excellent water solubility of the conjugate. The above research results provide important experimental basis for the development of new chitosan derivatives with excellent solubility and antibacterial activity. Figure 13

[0077] Inhibition of F. oxysporum, C. gloeosporioides and N. litchii

[0078] First, the mycelial growth rate method was used to determine the ability of raw chitosan, carbendazim, and the carboxymethyl chitosan intermediates and carboxymethyl chitosan-carbendazim conjugates synthesized in each example to inhibit F. oxysporum, C. gloeosporioides and N. litchii, and comparisons were made: The carboxymethyl chitosan intermediates and carboxymethyl chitosan-carbendazim conjugates prepared in the examples were vacuum freeze-dried to a constant weight, and then fungal culture media with sample concentrations of 0.1 mg / mL, 0.5 mg / mL and 1.0 mg / mL were prepared, with an equal volume of sterile water instead of the sample as a blank control. The culture medium was poured into a petri dish with a diameter of 9 cm after shaking, and then a 5 mm diameter fungus cake was inoculated in each petri dish. After incubation at 27°C for 48-72 h, the colony diameter was measured by cross method, and the inhibition rate of the sample was calculated. All experiments were repeated three times to obtain the average value, and the test results are shown in Tables 2-4.

[0079] Inhibition rate (%) = 1-[(Dsample-5) / (Dblank-5)]x100

[0080] ​Table 2 Inhibition ability of water-soluble carboxymethyl chitosan-carbendazim conjugate on Fusarium oxysporum f. sp. cubense (%)

[0081]

[0082] Table 3 Inhibition ability of water-soluble carboxymethyl chitosan-carbendazim conjugate on Colletotrichum gloeosporioides (%)

[0083]

[0084] Table 4 Inhibition ability of water-soluble carboxymethyl chitosan-carbendazim conjugate on Leptosphaeria crassiasca (%)

[0085]

[0086] The results of the mycelial growth rate method for the antibacterial test show that the inhibition ability of the carboxymethyl chitosan intermediate, the carboxymethyl chitosan-carbendazim conjugate, and the raw materials chitosan and carbendazim on Fusarium oxysporum f. sp. cubense is shown in Table 2, the inhibition ability on Colletotrichum gloeosporioides is shown in Table 3, and the inhibition ability on Leptosphaeria crassiasca is shown in Table 4. The experimental results show that the carboxymethyl chitosan-carbendazim conjugate prepared in the present application exhibits excellent broad-spectrum antibacterial performance. In particular, the inhibition effect of the conjugate on Fusarium oxysporum f. sp. cubense, Colletotrichum gloeosporioides, and Leptosphaeria crassiasca is significantly better than that of the carboxymethyl chitosan intermediate and the raw material chitosan. Even at the lowest test concentration (0.1 mg / mL), they and carbendazim can achieve 100% inhibition rate. This phenomenon fully proves that the ionic bond between the carbendazim molecule and the carboxymethyl chitosan skeleton not only retains the original high-efficiency bactericidal characteristics of carbendazim, but also significantly improves the biological activity of the chitosan derivative. It is worth noting that while maintaining good water solubility, the chitosan component in the molecular structure of the conjugate can destroy the integrity of the pathogenic fungus cell membrane, and the carbendazim part can interfere with the synthesis of tubulin, and this dual-acting mechanism makes it exhibit a synergistic antibacterial effect. In addition, compared with free carbendazim, the conjugate can have more persistent efficacy and higher bioavailability. Therefore, the carboxymethyl chitosan-carbendazim conjugate, as a new type of antifungal agent with high-efficiency antibacterial activity, good water solubility, and potential sustained-release characteristics, has broad application prospects in the field of crop disease control.

[0087] Further, the ability of carbendazim and the synthesized carboxymethyl chitosan-carbendazim conjugate to inhibit F. oxysporum, C. gloeosporioides and D. dieback was tested by the inhibition zone method. The sterile medium was thoroughly shaken and poured into a 90 mm diameter petri dish. After complete solidification, the test bacterial solution was uniformly coated on the solid agar plate. Then, four 5 mm diameter holes were punched on the surface of the medium with a sterile puncher, and the sample solution was filled into the holes. After 48 hours of incubation in a 28°C constant temperature incubator, the diameters of the inhibition zones produced by different samples at different concentrations were accurately measured using a vernier caliper. All experiments were set up in triplicate, and the test results are shown in Tables 5-7, and the inhibition zone diameter photos are shown in Figures 5-7. Figure 14

[0088] Table 5 Inhibition zone diameter (mm) of water-soluble carboxymethyl chitosan-carbendazim conjugate on F. oxysporum

[0089]

[0090] Table 6 Inhibition zone diameter (mm) of water-soluble carboxymethyl chitosan-carbendazim conjugate on C. gloeosporioides

[0091]

[0092] Table 7 Inhibition zone diameter (mm) of water-soluble carboxymethyl chitosan-carbendazim conjugate on D. dieback

[0093]

[0094] Inhibition zone method test results: The results show that both the carboxymethyl chitosan-carbendazim conjugate and carbendazim exhibit significant antibacterial effects. Further, the N, O-carboxymethyl chitosan-carbendazim conjugate has the most outstanding inhibitory effect on the three types of pathogenic bacteria, and the inhibition zone diameter is significantly larger than that of carbendazim alone. This phenomenon may be related to the synergistic mechanism of the conjugate, that is, the introduction of the carboxymethyl group not only improves water solubility, but also may enhance the interaction with the fungal cell wall, promote the penetration and accumulation of carbendazim in the bacterial body, and thus improve the overall antibacterial activity. It can also be seen that the sensitivity of different pathogenic bacteria to the conjugate differs. For example, the response of F. oxysporum to the N, O-carboxymethyl chitosan-carbendazim conjugate is the most significant, with an inhibition zone diameter close to 15 mm, while the inhibition zone diameter for D. dieback is only about 9 mm. This result further verifies that the carboxymethyl chitosan-carbendazim conjugate has broad-spectrum and high-efficiency antibacterial properties, and its antibacterial effect is influenced by the degree of substitution and the target bacterial species. In summary, the inhibition zone experiment not only found that the antibacterial activity of the N, O-carboxymethyl chitosan-carbendazim conjugate is superior to that of free carbendazim, but also revealed its structure-activity relationship, providing an important basis for the development of new and efficient agricultural antifungal agents.​

Claims

1. A water-soluble conjugate, characterized in that: The water-soluble conjugate is shown in the following structural formula a, b, or c; ； Where a is N,O-carboxymethyl chitosan-carbendazim conjugate, b is N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate, c is N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate, and the average value of n ranges from 5 to 12000.

2. A method for preparing the water-soluble conjugate according to claim 1, characterized in that: Carbendazim was dissolved in hydrochloric acid solution to obtain carbendazim hydrochloride; the carbendazim hydrochloride was introduced into the chitosan intermediate through ion exchange reaction to obtain a water-soluble carboxymethyl chitosan-carbendazim conjugate.

3. The method for preparing the water-soluble conjugate according to claim 2, characterized in that: The chitosan intermediate is any one of the following: 1) Chitosan raw material is reacted with chloroacetic acid to obtain N,O-carboxymethyl chitosan intermediate; 2) Chitosan raw material reacts with nucleophilic reagent iodomethane to obtain N,N,N-trimethyl chitosan, which is then reacted with chloroacetic acid to obtain N,N,N-trimethyl-O-carboxymethyl chitosan intermediate; 3) Chitosan raw material reacts with 2,3-epoxypropyltrimethylammonium chloride to obtain N-hydroxypropyltrimethyl chitosan, which is then reacted with chloroacetic acid to obtain N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate.

4. The method for preparing the water-soluble conjugate according to claim 3, characterized in that: Preparation of N,O-carboxymethyl chitosan-carbendazim conjugate: Chitosan was uniformly dispersed in isopropanol, NaOH solution was slowly added dropwise and stirred, and then chloroacetic acid aqueous solution was added. The mixture was stirred and reacted at 70-80℃ for 4-6 hours. After the reaction, N,O-carboxymethyl chitosan intermediate was obtained by alcohol precipitation and freeze-drying. Then, it was dissolved in deionized water, carbendazim hydrochloride solution was added, and the mixture was stirred and reacted at room temperature for 12-14 hours. After dialyzing with deionized water for 48 hours, the supernatant was collected by centrifugation and freeze-dried to obtain N,O-carboxymethyl chitosan-carbendazim conjugate.

5. The method for preparing the water-soluble conjugate according to claim 3, characterized in that: Preparation of N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate: Chitosan was uniformly dispersed in 1-methyl-2-pyrrolidone. Sodium iodide, sodium hydroxide solution, and iodomethane were added sequentially under stirring at room temperature. The mixture was refluxed at 60-70℃ for 2-3 hours. After precipitation and washing with excess ethanol, N,N,N-trimethyl chitosan was obtained. It was then dispersed in isopropanol, and NaOH solution was slowly added dropwise while stirring. Then, chloroacetic acid aqueous solution was added and stirred at 50-60℃ for 4-6 hours. After alcohol precipitation and freeze-drying, N,N,N-trimethyl-O-carboxymethyl chitosan intermediate was obtained. It was then dissolved in deionized water, and carbendazim hydrochloride solution was added. The mixture was stirred at room temperature for 12-14 hours. After dialyzing with deionized water for 48 hours, the supernatant was collected by centrifugation and freeze-dried to obtain N,N,N-trimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

6. The method for preparing the water-soluble conjugate according to claim 3, characterized in that: Preparation of N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate: Chitosan was dispersed in isopropanol and stirred at 50-60℃ for 2 h. Then, 2,3-epoxypropyltrimethylammonium chloride aqueous solution was added, and the mixture was refluxed at 70-80℃. After the reaction, the mixture was precipitated and washed with anhydrous ethanol to obtain N-hydroxypropyltrimethyl chitosan. Then, it was dispersed in isopropanol, and NaOH solution was slowly added dropwise while stirring until homogeneous. Then, chloroacetic acid aqueous solution was added, and the mixture was stirred at 50-60℃ for 4-6 h. After the reaction, the mixture was precipitated with alcohol and freeze-dried to obtain the N-hydroxypropyltrimethyl-O-carboxymethyl chitosan intermediate. Then, it was dissolved in deionized water, and carbendazim hydrochloride solution was added. The mixture was stirred at room temperature for 12-14 h. After dialysis with deionized water for 48 h, the supernatant was collected by centrifugation and freeze-dried to obtain the N-hydroxypropyltrimethyl-O-carboxymethyl chitosan-carbendazim conjugate.

7. An application of the water-soluble conjugate according to claim 1, characterized in that: Application of the water-soluble conjugate in the preparation of antifungal agents.

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