Low molecular weight chitosan nicotinamide derivative and application thereof

Abstract

The application discloses a low-molecular-weight chitosan nicotinamide derivative and application thereof, relates to the field of bacteriostatic agents, and grafts nicotinic acid (VPP), 2-chloronicotinic acid (2-CNA) and 2-aminonicotinic acid (2-ANA) and other nicotinic acid compounds with bacteriostatic activity onto low-molecular-weight chitosan to prepare a novel low-molecular-weight chitosan nicotinamide derivative; the inhibiting effect of the derivative on phytophthora capsici, gaeumannomyces graminis, thielaviopsis basicola and alternaria solani is determined by using a mycelium growth rate method, and the result shows that the fungistatic effect of the derivative is obviously higher than that of chitosan raw materials.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agents, and in particular to a low molecular weight chitosan nicotinamide derivative and its applications. Background Technology

[0002] Plant diseases are a major cause of crop losses, and fungi are among the most important pathogens causing various plant diseases. In recent years, the direct application of natural products or structural modifications has become an effective way to discover and create new pesticides. Chitosan is a natural high-molecular-weight polysaccharide with biocompatibility, safety, non-toxicity, antioxidant, and antibacterial properties, making it widely used in food, medicine, and cosmetics. However, its biological activity is relatively weak, resulting in poor efficacy as an antibacterial agent. Summary of the Invention

[0003] The purpose of this invention is to provide a low molecular weight chitosan nicotinamide derivative and its application. Nicotinic acid compounds with antibacterial activity, such as nicotinic acid (VPP), 2-chloronicotinic acid (2-CNA), and 2-aminonicotinic acid (2-ANA), are grafted onto low molecular weight chitosan to obtain a novel low molecular weight chitosan nicotinamide derivative. The inhibitory effects of the derivative on *Phytophthora capsici*, *Fusarium graminearum*, *Rhizoctonia solani*, and *Early blight* of tomato were determined using the mycelial growth rate method. The results showed that the antifungal effect of the derivative of this invention was significantly higher than that of the chitosan raw material.

[0004] To solve the above problems, the present invention adopts the following technical solution:

[0005] According to a first aspect of the present invention, a low molecular weight chitosan nicotinamide derivative is provided, the general structural formula of which is shown in Formula I:

[0006]

[0007]

[0008] Where n is an integer selected from 13 to 23, and R is any of the following structural formulas:

[0009]

[0010] Furthermore, the preparation method of the above-mentioned low molecular weight chitosan nicotinamide derivatives is as follows: the carboxyl group on the nicotinic acid compound is reacted with the amino group at the C2 position of the low molecular weight chitosan by an acylation reaction.

[0011] Furthermore, the preparation method of the above-mentioned low molecular weight chitosan nicotinamide derivatives includes the following steps: dissolving nicotinic acid compounds in 2-morpholine ethanesulfonic acid buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide and stirring to activate; then adding low molecular weight chitosan and stirring to react, the amino groups of low molecular weight chitosan react with the carboxyl groups of nicotinic acid compounds to form amides; then dialyzing with distilled water using a dialysis bag, and finally concentrating and freeze-drying the solution in the dialysis bag to obtain the low molecular weight chitosan nicotinamide derivatives. The purpose of using distilled water dialysis in this invention is to remove small molecule impurities more thoroughly; the concentration process can remove excess water and reduce volume; because the compounds obtained in this invention are easily decomposed at high temperatures, low-temperature freeze-drying is used to completely remove water, turning the liquid into a solid powder, which is the finished product.

[0012] Furthermore, the nicotinic acid compound is any one of nicotinic acid, 2-chloronicotinic acid, and 2-aminonicotinic acid.

[0013] Furthermore, chitosan is soluble in dilute acids with a pH < 6.5, but insoluble in water and alkaline solutions; therefore, the preferred 2-morpholine ethanesulfonic acid buffer solution used in this invention has a pH of 5.5-6.5 and a concentration of 0.1 mol / L.

[0014] Furthermore, the theoretical feed ratio of nicotinic acid compounds: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide is 1:3:3. In actual practice, the ratio needs to be slightly higher than the theoretical value, but to prevent waste, the maximum value is generally 1:5:5. Therefore, the preferred molar ratio of nicotinic acid compounds: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide is 1:3:3-1:5:5.

[0015] Furthermore, the molecular weight of the low molecular weight chitosan used is n = 13-23, and the molar ratio of the amino group of the low molecular weight chitosan to the carboxyl group of the nicotinic acid compound is slightly less than 1:1, so that the carboxyl group is slightly in excess in the reaction system; this is conducive to the full amide reaction of the amino group of the low molecular weight chitosan.

[0016] Furthermore, the dialysis bag used has a molecular weight cutoff of 1500 Da to remove unreacted nicotinic acid compounds, EDC·HCl, and NHS. Low molecular weight chitosan itself is a macromolecule with Mn>2000 Da, so the molecular weight of the target low molecular weight chitosan derivative will only increase and not decrease after the reaction.

[0017] According to a second aspect of the present invention, the present invention provides an application of the above-mentioned low molecular weight chitosan nicotinamide derivatives as an antibacterial agent for *Phytophthora capsici*, *Fusarium graminearum*, *Rhizoctonia solani*, and *Early blight* of tomato.

[0018] According to a third aspect of the present invention, the present invention provides an application of the above-mentioned low molecular weight chitosan nicotinamide derivative for the preservation of cherry tomatoes.

[0019] The following is a supplementary explanation of some of the process principles of this invention:

[0020] In this invention, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, abbreviated as EDC·HCl, is a water-soluble carbodiimide with a linear molecular structure, used in the condensation reaction of carboxyl groups with primary amines. EDC·HCl reacts with an amino group to form an O-acylurea intermediate that is reactive with the amino group. If this intermediate does not react with the amino group, it will quickly hydrolyze and release the carboxyl group again. In the presence of N-hydroxysuccinimide (NHS), EDC·HCl can convert the carboxyl group into an amino-reactive NHS ester. This reaction can be achieved by mixing EDC·HCl, carboxyl-containing molecules, and NHS. In this invention, EDC·HCl acts as a condensing agent, enabling rapid dehydration condensation.

[0021] N-hydroxysuccinimide, abbreviated as NHS, is synthesized as an NHS ester through a dehydration reaction in a carboxylic acid in the presence of EDC. The presence of NHS improves the efficiency of EDC-mediated coupling, and amine-reactive NHS esters can be prepared using any carboxyl-containing molecule. In this invention, NHS controls the carbodiimide crosslinking reaction activated by the carboxylate coupled to the amino group of chitosan oligosaccharide.

[0022] The beneficial effects of this invention are as follows: This invention provides a low molecular weight chitosan nicotinamide derivative and its application. This invention grafts nicotinic acid compounds with antibacterial activity, such as nicotinic acid (VPP), 2-chloronicotinic acid (2-CNA), and 2-aminonicotinic acid (2-ANA), onto low molecular weight chitosan to generate novel low molecular weight chitosan nicotinamide derivatives. Using the mycelial growth rate method, the inhibitory effects of the derivatives on *Phytophthora capsici*, *Fusarium graminearum*, *Rhizoctonia solani*, and *Early Blight of tomato* were determined. The results showed that the antifungal effects of the derivatives were significantly higher than those of the chitosan raw material. Furthermore, the cytotoxicity of several derivatives was determined, and the results showed that the derivatives have good cell compatibility. Finally, the prepared compounds were applied to the preservation of cherry tomatoes. Studies have shown that treatment with chitosan nicotinamide derivatives can significantly reduce the weight loss rate of cherry tomatoes, delay the decrease in vitamin C content and titratable acid content, and effectively extend the shelf life of cherry tomatoes. Attached Figure Description

[0023] Figure 1 This is the structural formula of the low molecular weight chitosan nicotinamide derivative of the present invention.

[0024] Figure 2This is the infrared spectrum of existing low molecular weight chitosan (n=13-23).

[0025] Figure 3 This is the infrared spectrum of pyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with nicotinic acid in Example 1 of the present invention.

[0026] Figure 4 This is the infrared spectrum of 2-chloropyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with 2-chloronicotinic acid in Example 2 of the present invention.

[0027] Figure 5 This is the infrared spectrum of 2-aminopyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with 2-aminonicotinic acid in Example 3 of the present invention.

[0028] Figure 6 This invention relates to pyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with nicotinic acid. 13 C10 NMR spectrum.

[0029] Figure 7 This invention relates to 2-chloropyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with 2-chloronicotinic acid. 13 C10 NMR spectrum.

[0030] Figure 8 This invention relates to 2-aminopyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with 2-aminonicotinic acid. 13 C10 NMR spectrum.

[0031] Figure 9 This invention relates to the reaction formula for preparing pyridine-3-formyl chitosan by reacting low molecular weight chitosan (n=13-23) with nicotinic acid.

[0032] Figure 10 This invention relates to the reaction formula for preparing 2-chloropyridine-3-formylchitosan by reacting low molecular weight chitosan (n=13-23) with 2-chloronicotinic acid.

[0033] Figure 11 This invention relates to the reaction formula for preparing 2-aminopyridine-3-formyl chitosan by reacting low molecular weight chitosan (n=13-23) with 2-aminonicotinic acid.

[0034] Figure 12 This is a statistical chart showing the antibacterial activity of chitosan and chitosan derivatives of different concentrations against Phytophthora capsici, the pathogen of the present invention.

[0035] Figure 13This is a statistical graph showing the antibacterial activity of different concentrations of chitosan and chitosan derivatives against Fusarium graminearum, the causal agent of wheat blight.

[0036] Figure 14 This is a statistical graph showing the antibacterial activity of different concentrations of chitosan and chitosan derivatives against *Rhizoctonia solani* of sweet potato.

[0037] Figure 15 This is a statistical chart showing the antibacterial activity of different concentrations of chitosan and chitosan derivatives against *Phytophthora blight*, the pathogen of this invention.

[0038] Figure 16 This is a statistical graph showing the cytotoxicity of different concentrations of chitosan and chitosan derivatives on L929 cells according to the present invention.

[0039] Figure 17 This is a statistical graph showing the effect of different concentrations of chitosan and chitosan derivatives on the weight loss rate of cherry tomatoes.

[0040] Figure 18 This is a statistical graph showing the effect of different concentrations of chitosan and chitosan derivatives on the vitamin C content of cherry tomatoes.

[0041] Figure 19 This is a statistical graph showing the effect of different concentrations of chitosan and chitosan derivatives on the titratable acid content of cherry tomatoes. Detailed Implementation

[0042] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments. The following description is only used to illustrate the technical solutions of the present invention and not to limit them; unless otherwise specified, the experimental methods used in the embodiments are conventional methods; unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels.

[0043] Example 1

[0044] A method for preparing pyridine-3-formyl chitosan by reacting low molecular weight chitosan (n=13-23) with nicotinic acid includes the following steps:

[0045] Step 1: Activation of nicotinic acid carboxyl groups. Dissolve nicotinic acid in 2-morpholine ethanesulfonic acid buffer solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, stir until completely dissolved, and continue stirring for 3 hours to activate the carboxyl groups. The 2-morpholine ethanesulfonic acid buffer solution used has a pH of 5.5-6.5, a concentration of 0.1 mol / L, and a volume of 100 mL; the molar ratio of other reagents is nicotinic acid: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide = 1:3:3-1:5:5.

[0046] Step 2: Low molecular weight chitosan reacts with activated nicotinic acid to form an amide. 1.0 g of low molecular weight chitosan (n = 13-23) is added to the mixture from Step 1, and the mixture is stirred at room temperature for 24 h to allow the amino groups of chitosan to react with the carboxyl groups of nicotinic acid to form an amide. The molar ratio of nicotinic acid to chitosan (n = 13-23) is 13:1-23:1.

[0047] Step 3: Separation and purification of the target compound. The product from the reaction in Step 2 was dialyzed against distilled water for 4 days using a dialysis bag. The solution in the dialysis bag was then concentrated to approximately 30 mL, followed by freeze-drying for 48 hours to obtain pyridine-3-formyl chitosan. The molecular weight cutoff of the dialysis bag used was 1500 Da.

[0048] The pyridine-3-formyl chitosan prepared in Example 1 has the following structural formula: [See attached diagram]. Figure 1 , where R = n = 13 - 23.

[0049] Infrared spectroscopy analysis was performed as follows: Figure 2 The image shown is the infrared spectrum of existing low molecular weight chitosan (n = 13-23), and its characteristic infrared (cm²) spectrum is shown in the figure. -1 ): 3271, 2879, 1571.3, 1514.8, 1373.7, 1066.4, 1038.2. (e.g.) Figure 3 The image shown is the infrared spectrum of pyridine-3-formyl chitosan obtained from the reaction of low molecular weight chitosan (n = 13-23) with nicotinic acid in Example 1. Its characteristic infrared (cm²) spectrum is shown in the image. -1 ): 3286.6, 2872.6, 1643.4, 1555.6, 1376.8, 1069.5, 1028.7. Figure 3 and Figure 2 In comparison, those located at 3500-3200cm -1 The characteristic broad peaks of OH and NH at 2872.6 cm⁻¹ show a significant shift, indicating that NH₄⁺ has reacted; -1 A distinct absorption peak for the saturated -CH stretching vibration was observed at this location; Figure 3 The middle is 1643.4cm -1 The characteristic absorption peak of C=O appears at 1555.6 cm⁻¹. -1 The absorption peak at 1376.8 cm⁻¹ is due to the bending vibration of NH₂ (amide II band), indicating that the NH₂ of chitosan has undergone an acylation reaction with the -COOH group of nicotinic acid; -1 An absorption peak for the saturated CH deformation vibration appears at 1069.5 cm⁻¹. -1 The strong absorption peak at that point is due to the symmetrical stretching vibration of COC.

[0050] The following analysis was performed using carbon nuclear magnetic resonance spectroscopy: Figure 6 The image shows pyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n = 13-23) with nicotinic acid. 13 C NMR spectrum; its characteristic chemical shifts (ppm): 178.60, 174.69, 172.72, 160.62, 150.30, 148.96, 143.00, 137.96, 124.56, 101.35, 98.37, 76.90, 74.86, 72.64, 69.65, 65.03, 61.85, 55.38, 52.17, 42.71, 35.68, 31.28, 25.04, 13.42 ppm.

[0051] Analysis showed that: 174.69 ppm was the chemical shift of the C=O carbon in the amide bond; 101.35-98.37 (C1), 76.90 (C4), 74.86 (C5), 72.64-69.65 (C3), 61.85 (C6), and 55.38-52.17 (C2) ppm were the chemical shifts of the low molecular weight chitosan glycocycle; and 160.62, 150.30, 148.96, 143.00, 137.96, and 124.56 ppm were the chemical shifts of the nicotinic acid carbon chain. These results confirm the formation of the target compound.

[0052] Example 2

[0053] A method for preparing 2-chloropyridine-3-formyl chitosan by reacting low molecular weight chitosan (n=13-23) with 2-chloronicotinic acid is provided. The preparation process is the same as in Example 1, except that 2-chloronicotinic acid is used instead of nicotinic acid.

[0054] The 2-chloropyridine-3-formyl chitosan prepared in Example 2 has the following structural formula: [See attached diagram]. Figure 1 , where R = n = 13 - 23.

[0055] Infrared spectroscopy analysis was performed as follows: Figure 4 The image shown is the infrared spectrum of 2-chloropyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n = 13-23) with 2-chloronicotinic acid in Example 2; its characteristic infrared spectrum (cm²) is shown in the image. -1 ): 3314.8, 2882, 1649.7, 1561.8, 1376.8, 1066.4, 1031.9. Figure 4 and Figure 2 In comparison, those located at 3500-3200cm -1 The characteristic broad peaks of OH and NH at 2882 cm⁻¹ show a significant shift, indicating that NH reacts; -1 A distinct absorption peak for the saturated -CH stretching vibration was observed at this location; Figure 4The middle is 1649.7cm -1 The characteristic absorption peak of C=O appears at 1561.8 cm⁻¹. -1 The absorption peak at 1376.8 cm⁻¹ is due to the bending vibration of NH₂ (amide II band), indicating that the NH₂ of chitosan has undergone an acylation reaction with the -COOH group of nicotinic acid; -1 An absorption peak for the saturated CH deformation vibration appears at 1066.4 cm⁻¹. -1 The strong absorption peak at that point is due to the symmetrical stretching vibration of COC.

[0056] The following analysis was performed using carbon nuclear magnetic resonance spectroscopy: Figure 7 The image shows 2-chloropyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n = 13-23) with 2-chloronicotinic acid. 13 C NMR spectrum; its characteristic chemical shifts (ppm): 178.61, 174.68, 159.82, 150.74, 148.79, 145.47, 137.68, 135.55, 123.62, 102.50, 101.38, 98.84, 76.41, 74.43, 72.86, 71.61, 69.61, 61.86, 55.76, 35.69, 13.42 ppm.

[0057] Analysis showed that: 174.68 ppm was the chemical shift of the C=O carbon in the amide bond; 102.50-98.84 (C1), 76.41 (C4), 74.43 (C5), 72.86-69.61 (C3), 61.86 (C6), and 55.76 (C2) ppm were the chemical shifts of the low molecular weight chitosan glycocycle; and 159.82, 150.74, 148.79, 145.47, 137.68, 135.55, and 123.62 ppm were the chemical shifts of the 2-chloronicotinic acid carbon chain. These results confirm the formation of the target compound.

[0058] Example 3

[0059] A method for preparing 2-aminopyridine-3-formyl chitosan by reacting low molecular weight chitosan (n=13-23) with 2-aminonicotinic acid is provided. The preparation process is the same as in Example 1, except that 2-aminonicotinic acid is used instead of nicotinic acid.

[0060] The 2-aminopyridine-3-formyl chitosan prepared in Example 3 has the following structural formula: [See attached diagram]. Figure 1 , where R = n = 13 - 23.

[0061] Infrared spectroscopy analysis was performed as follows: Figure 5The image shown is the infrared spectrum of 2-aminopyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n=13-23) with 2-aminonicotinic acid in Example 3; its characteristic infrared spectrum (cm²) is shown in the image. -1 ): 3336.8, 2875.8, 1725, 1621.4, 1568.1, 1376.8, 1072.6, 1031.9. Figure 5 and Figure 2 In comparison, those located at 3500-3200cm -1 The characteristic broad peaks of OH and NH at 2875.8 cm⁻¹ show a significant shift, indicating that NH reacts; -1 A distinct absorption peak for the saturated -CH stretching vibration was observed at this location; Figure 5 The middle is 1725 and 1621.4cm. -1 The characteristic absorption peak of C=O appears at 1568.1 cm⁻¹. -1 The absorption peak at 1376.8 cm⁻¹ is due to the bending vibration of NH₂ (amide II band), indicating that the NH₂ of chitosan has undergone an acylation reaction with the -COOH group of nicotinic acid; -1 An absorption peak for the saturated CH deformation vibration appears at 1072.6 cm⁻¹. -1 The strong absorption peak at that point is due to the symmetrical stretching vibration of COC.

[0062] The following analysis was performed using carbon nuclear magnetic resonance spectroscopy: Figure 11 The image shows 2-aminopyridine-3-formyl chitosan obtained by reacting low molecular weight chitosan (n = 13-23) with 2-aminonicotinic acid. 13 C NMR spectrum; its characteristic chemical shifts (ppm): 174.74, 160.03, 144.57, 116.04, 113.41, 101.37, 98.93, 76.91, 74.87, 69.67, 61.83, 55.40, 52.55, 42.74, 35.00, 25.07, 13.41 ppm.

[0063] Analysis showed that: 174.74 ppm was the chemical shift of the C=O carbon in the amide bond; 101.37–98.93 (C1), 76.91 (C4), 74.87 (C5), 69.67 (C3), 61.83 (C6), and 55.40 (C2) ppm were the chemical shifts of the low molecular weight chitosan glycocycle; and 160.03, 144.57, 116.04, and 113.41 ppm were the chemical shifts of the 2-aminonicotinic acid carbon chain. These results confirm the formation of the target compound.

[0064] Furthermore, the present invention uses the mycelial growth rate method to determine the inhibitory effects of the above derivatives on *Phytophthora capsici*, *Fusarium graminearum*, *Rhizoctonia solani*, and *Early blight* of tomato, as detailed below.

[0065] Mycelial growth rate method: In a sterile workbench, the sample was dissolved in sterile water to obtain a sample solution with an initial concentration of 10.0 mg / mL. The sample solution was filtered through a 0.22 μm sterile filter (to remove impurities present in the sample), and the filtered sample solution was diluted to a series of concentrations. In a laminar flow hood, 4.5 mL of the sample solution was taken and 40.5 mL of melted liquid PDA medium was added to make the final concentrations of the test sample 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL. After thorough mixing, the mixture was evenly poured into three petri dishes and placed in a laminar flow hood to cool and solidify. Sterile water was used as a control. After inoculating the test strain in the center of the solidified medium, the dishes were inverted and incubated at 25°C in a mold incubator. After 48 hours, the colony diameter was measured using the cross-cross method. The inhibition rate was calculated using the following formula:

[0066] Colony diameter = Average of two diameters - Diameter of the mycelial cake

[0067]

[0068] Each experiment was performed in triplicate, and the data are expressed as mean ± standard deviation (SD, n = 3). One-way ANOVA was used to determine statistical significance. A p-value < 0.05 was considered statistically significant.

[0069] The results of the antibacterial experiment are analyzed as follows: Figure 12The figure shows the antibacterial activity of three low molecular weight chitosan derivatives (VPPLCS, 2-CNALCS, and 2-ANALCS), as well as the low molecular weight chitosan raw material (LCS) and the positive control (Wuyi mycin) against *Phytophthora capsici*. With increasing concentration, the inhibition rate of the derivatives against *Phytophthora capsici* gradually increased. The inhibition rate of VPPLCS against *Phytophthora capsici* showed a more significant change with concentration, while the change of 2-CNALCS was not significant. The inhibition rates of the three derivatives against *Phytophthora capsici* were significantly higher than those of the chitosan raw material. At a concentration of 1.0 mg / mL, the inhibition rates of VPPLCS, 2-CNALCS, 2-ANALCS, LCS, and Wuyi mycin were (84.54±2.13)%, (77.34±3.35)%, (71.93±2.5)%, (56.26±1.88)%, and (93.01±2.69)%, respectively. At a tested concentration of 0.0625 mg / mL, the inhibition rates of VPPLCS, 2-CNALCS, 2-ANALCS, LCS, and Wuyi mycin were (50.53±3.18)%, (68.82±2.39)%, (56.64±2.6)%, (18.2±4.87)%, and (87.4±4.25)%, respectively. At 0.0625 mg / mL, the inhibitory rates of the derivatives against *Phytophthora capsici* were significantly higher than those of the chitosan raw material. Among the three derivatives, 2-CNALCS showed the best inhibitory effect against *Phytophthora capsici*.

[0070] like Figure 13 The figure shows the antibacterial activity of three low molecular weight chitosan derivatives (VPPLCS, 2-CNALCS, and 2-ANALCS), as well as the low molecular weight chitosan raw material (LCS) and the positive control (Wuyi mycin) against *Fusarium graminearum*, the causal agent of wheat scab. With increasing concentration, the inhibition rate of the derivatives against *Fusarium graminearum* gradually increased, and the change in inhibition rate with concentration was significant. The inhibition rates of the three derivatives against *Fusarium graminearum* were significantly higher than those of the chitosan raw material. At a concentration of 0.5 mg / mL, the inhibition rates of VPPLCS, 2-CNALCS, 2-ANALCS, LCS, and Wuyi mycin were (47.13±3.84)%, (49.96±5.44)%, (43.82±4.02)%, (22.28±1.61)%, and (78.27±6.52)%, respectively. Among the three derivatives, VPPLCS and 2-CNALCS showed better inhibitory effects against Fusarium graminearum, followed by 2-ANALCS.

[0071] like Figure 14The figure shows the antibacterial activity of three low molecular weight chitosan derivatives (LVPPCS, 2-CNALCS, and 2-ANALCS), as well as the low molecular weight chitosan raw material (LCS) and the positive control (Wuyi mycin) against *Rhizoctonia solani*. With increasing concentration, the inhibition rate of the derivatives against *Rhizoctonia solani* gradually increased, and the variation in inhibition rate with concentration was significant. The inhibition rates of the three derivatives against *Rhizoctonia solani* were significantly higher than those of the chitosan raw material. At a concentration of 1.0 mg / mL, the inhibition rates of LVPPCS, 2-CNALCS, 2-ANALCS, LCS, and Wuyi mycin were (73.73±3.45)%, (70.35±1.88)%, (60.45±6.02)%, (48.4±2.48)%, and (84.58±4.44)%, respectively. Among the three derivatives, VPPLCS showed the best inhibitory effect on Rhizoctonia solani, followed by 2-CNALCS.

[0072] like Figure 15 The figure shows the antibacterial activity of three low molecular weight chitosan derivatives (VPPLCS, 2-CNALCS, and 2-ANALCS), as well as low molecular weight chitosan raw material (LCS) and a positive control (Wuyi mycin) against *Bacillus cereus*, the pathogen of early blight of tomato. With increasing concentration, the inhibition rate of the derivatives against *Bacillus cereus* gradually increased. The inhibition rates of VPPLCS and 2-CNALCS against *Bacillus cereus* changed significantly with concentration, while the inhibition rate of 2-ANALCS against *Bacillus cereus* did not change significantly with concentration. The three derivatives showed significantly higher inhibition rates against *Early Blight of Tomato* than the chitosan raw material. The chitosan raw material had no inhibitory effect on *Early Blight of Tomato* at concentrations of 0.0625-0.25 mg / mL. At a concentration of 0.5 mg / mL, the inhibition rates of VPPLCS, 2-CNALCS, 2-ANALCS, LCS, and Wuyi mycin were (67.07±1.62)%, (64.11±0.78)%, (75.73±1.51)%, (25.06±3.53)%, and (86.93±0.57)%, respectively. Among the three derivatives, 2-ANALCS showed the best inhibitory effect against *Early Blight of Tomato*, with an inhibition rate of (74.48±1.17)% at a concentration of 0.0625 mg / mL, significantly higher than that of VPPLCS and 2-CNALCS.

[0073] Furthermore, this invention also investigated the effects of chitosan and its derivatives on the viability of L929 cells; the cytotoxicity of chitosan derivatives, such as... Figure 16As shown, the cell viability of all three derivatives was greater than 90%, with 2-CNALCS exhibiting significantly higher cell viability than 2-CNA. At 2 mg / mL, the cell viability of all three derivatives exceeded 100%, indicating that the samples did not inhibit cell growth and demonstrating good biocompatibility. The figure also reveals that the cell viability of the derivatives was generally greater than that of the raw materials, suggesting that chitosan reduced the toxicity of nicotinic acid compounds, resulting in good biocompatibility of the obtained derivatives. Furthermore, the combination of chitosan and nicotinic acid compounds can reduce the toxicity of nicotinic acid compounds, expanding their application range.

[0074] Furthermore, this invention also investigated the preservative effect of the derivative on cherry tomatoes, as detailed below.

[0075] Fresh cherry tomatoes free from pests and diseases, without mechanical damage, of similar maturity, size, and color were selected. The cherry tomatoes were washed with distilled water and allowed to air dry at room temperature. Each test group contained 27 cherry tomatoes. The cherry tomatoes were immersed in the test solution (0, 0.5, 1.0, 1.5, 2.0 g / L) for 3 minutes, and then the coated fruits were allowed to air dry at room temperature. They were then packaged in 0.1 mm thick ordinary PE film bags and stored in a 25°C artificial climate chamber. The cherry tomatoes from each group were stored at 25°C for 0, 4, 8, 12, 16, 20, 24, 28, and 32 days. On days 0, 4, 8, 12, 16, 20, 24, 28, and 32 of the storage period, three cherry tomatoes were randomly selected from each group for testing. Each treatment was repeated three times.

[0076] Weight loss rate determination: The weight loss rate of cherry tomatoes was determined by weighing. Three fruits were used per group, and the result was repeated three times. The weight loss rate was calculated using the following formula:

[0077]

[0078] In the formula: m0 is the initial weight of the cherry tomatoes (g), and m1 is the weight of the cherry tomatoes after a certain number of days of storage (g).

[0079] Weight loss is a significant factor affecting the commercial value of fruits and vegetables. After harvesting, fruits and vegetables continue to undergo transpiration and respiration, leading to water loss and nutrient depletion, directly impacting fruit quality. The weight loss of cherry tomatoes during storage is shown in the following figure. Figure 17As shown in the figure, the effect of different concentrations of chitosan and its derivatives on the weight loss rate of cherry tomatoes is illustrated. Different letters in the figure indicate significant differences (p < 0.05). With the extension of storage time, the weight loss rate of each group of samples gradually increased, and the weight loss rate of the blank treatment cherry tomatoes was significantly higher than that of the sample treatment. On the 32nd day of storage, the weight loss rate of cherry tomatoes treated with 2 g / L PLCS was (4.0 ± 0.52)%, which was significantly lower than that of the chitosan raw material (5.5 ± 1.33)% and the blank treatment (5.94 ± 1.02)%. This indicates that the derivative treatment formed a protective film on the surface of the cherry tomatoes, preventing oxygen from penetrating into the external environment, thereby reducing transpiration and respiration, effectively alleviating water loss, and mitigating the decrease in the weight loss rate of the cherry tomatoes.

[0080] Furthermore, this invention also investigated the effect of derivatives on the change of vitamin C content during the preservation of cherry tomatoes.

[0081] First, accurately weigh V C 20.0 mg of the standard was added to 4 mL of 10% hydrochloric acid, and the volume was adjusted to 100 mL with distilled water. (V) C Plot a standard curve with content on the x-axis and absorbance on the y-axis, and determine the equation of the standard curve. Place 5.0 g of cherry tomatoes in a mortar, add 5 mL of 1% hydrochloric acid, grind into a homogenate, transfer to a 25 mL volumetric flask, and dilute to the mark. Then centrifuge at 1000 rpm for 10 min. Measure the absorbance values ​​of the sample to be tested and the alkali-treated sample to be tested. From the difference in absorbance values ​​between the sample to be tested and the alkali-treated sample to be tested, and the standard curve, the content of V in the sample can be calculated. C The content of.

[0082] V C The content is calculated using the following formula:

[0083]

[0084] In the formula: μ is the VC content (μg) obtained from the standard curve, V1 is the volume of sample solution taken during absorbance measurement (mL), and V 总 W is the total volume (mL) used to bring the sample to volume. 总 The mass of cherry tomatoes is (g).

[0085] Cherry tomatoes are high in vitamin C, but the vitamin C content can easily decrease due to oxidation during storage. Figure 18 Treatment of cherry tomatoes with different concentrations of chitosan and its derivatives CEffects on content. Different letters a and e indicate significant differences (p < 0.05). The figure shows the changes in VC content of cherry tomatoes during storage. The VC content of cherry tomatoes gradually decreased with the extension of storage time, and the decrease was most significant in the control group. The VC content of the three derivative treatment groups was higher than that of the control group at the same time point, with VPPLCS showing the most significant effect. From 4 to 28 days of storage, the VC content of cherry tomatoes in the VPPLCS treatment group was significantly higher than that in the control group. This further illustrates that all three derivatives can effectively alleviate the oxidative decomposition of cherry tomatoes, thereby extending their shelf life.

[0086] Furthermore, this invention also investigated the effect of derivatives on the titratable acid content during the preservation of cherry tomatoes.

[0087] First, grind 10.0g of cherry tomato sample in a mortar and pestle, transfer to a 100mL volumetric flask, dilute to the mark with distilled water, and shake thoroughly. Allow to stand at room temperature for 30 minutes, filter, and take 20mL of the filtrate. Add two drops of phenolphthalein indicator and titrate with standardized NaOH solution. Titrate until the solution turns pink and remains pink for 30 seconds; this is the titration endpoint. Record the volume of NaOH solution consumed at this point. Repeat the measurement three times and take the average value. Then, titrate with distilled water instead of the filtrate as a blank control.

[0088] The formula for calculating the content of titratable acid is as follows:

[0089]

[0090] In the formula: V 总 V is the total volume (mL) of the cherry tomato grinding liquid; S V0 is the volume of cherry tomato filtrate consumed during titration (mL); V1 is the volume of NaOH solution consumed during titration (mL); m is the mass of the sample (g); F is the acid conversion factor; and C is the concentration of NaOH solution (mol / L).

[0091] Figure 19 The effects of different concentrations of chitosan and its derivatives on the titratable acid content of cherry tomatoes were demonstrated, with different letters ae indicating significant differences (p < 0.05).

[0092] The figure shows the changes in titratable acid content of cherry tomatoes during storage. With prolonged storage, the titratable acid content in each group gradually decreased, with the control group showing the most significant decrease. The titratable acid content of cherry tomatoes treated with the three derivatives was significantly higher than that of the blank control group (p < 0.05). On day 32 of treatment, the titratable acid contents of cherry tomatoes treated with 1 g / L LCS, VPPLCS, 2-CNALCS, and 2-ANALCS were (0.30 ± 0.02)%, (0.38 ± 0.02)%, (0.31 ± 0.02)%, and (0.35 ± 0.03)%, respectively, while the titratable acid content of the blank control group was (0.25 ± 0.03)%. The results indicate that the use of chitosan derivatives can alleviate the decrease in titratable acid content during cherry tomato storage.

[0093] The above embodiments are merely illustrative examples to clearly illustrate the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations can be made based on the above description. All obvious variations derived therefrom fall within the scope of protection of this invention. Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

Claims

1. A low molecular weight chitosan nicotinamide derivative, characterized in that, The general structural formula of the low molecular weight chitosan nicotinamide derivatives is shown in Formula I: Formula I, Where n is an integer selected from 13 to 23, and R is any of the following structural formulas: Formula II Formula III Formula IV.

2. The low molecular weight chitosan nicotinamide derivative according to claim 1, characterized by: The preparation method is as follows: it is prepared by acylation reaction of the carboxyl group on the nicotinic acid compound with the amino group at the C2 position of low molecular weight chitosan.

3. The low molecular weight chitosan nicotinamide derivative according to claim 2, characterized in that: The preparation method includes the following steps: dissolving nicotinic acid compounds in 2-morpholine ethanesulfonic acid buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide and stirring to activate; then adding low molecular weight chitosan and stirring to react; then dialyzing with distilled water using a dialysis bag, and finally concentrating and freeze-drying the solution in the dialysis bag to obtain low molecular weight chitosan nicotinamide derivatives.

4. The low molecular weight chitosan nicotinamide derivative according to claim 3, characterized by: The nicotinic acid compounds are any one of nicotinic acid, 2-chloronicotinic acid, and 2-aminonicotinic acid.

5. The low molecular weight chitosan nicotinamide derivative according to claim 3, characterized by: The 2-morpholinoethanesulfonic acid buffer solution used had a pH of 5.5-6.5 and a concentration of 0.1 mol / L.

6. The low molecular weight chitosan nicotinamide derivative according to claim 3, characterized by: Nicotinic acid compounds: The molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:3:3-1:5:

5.

7. The low molecular weight chitosan nicotinamide derivative according to claim 3, characterized by: The average molecular weight cutoff of the dialysis bags used was 1500 Da.

8. Use of the low molecular weight chitosan nicotinamide derivative according to any one of claims 1 to 7, characterized in that: It is used as an inhibitory agent for Phytophthora capsici, Fusarium graminearum, Rhizoctonia solani, and Phytophthora blight of tomato.

9. The application of a low molecular weight chitosan nicotinamide derivative according to any one of claims 1-7, characterized in that: Used to preserve cherry tomatoes.

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