Carboxymethyl chitosan-guanidyl copolymer as well as preparation method and application thereof

Abstract

The invention discloses a carboxymethyl chitosan-guanidyl copolymer as well as a preparation method and application thereof. The carboxymethyl chitosan-guanidyl copolymer solves the problem that when a chitosan-based antioxidant is prepared at present, good oxidation resistance can be realized only by relying on a complex chemical modification process or utilizing compounding of multiple functional aids. The method has the advantages of simplicity and convenience in operation, easiness in implementation of the reaction process, lower overall cost and the like. The method comprises the following steps: designing a two-step preparation process of the carboxymethyl chitosan-guanidyl copolymer, firstly swelling and alkalifying chitosan in isopropanol, dropwise adding a chloroacetic acid aqueous solution according to a specific molar ratio, reacting to prepare carboxymethyl chitosan, carrying out ion exchange reaction on the carboxymethyl chitosan and guanidine hydrochloride at room temperature to obtain the carboxymethyl chitosan-guanidyl copolymer. The carboxymethyl chitosan-guanidyl copolymer with good antioxidant activity and biocompatibility can be prepared only by using a conventional reaction container and simple stirring and centrifugal equipment. The method is suitable for the fields of food, medicine, agriculture and the like.

Description

Technical Field

[0001] This invention relates to the fields of food, medicine and agriculture, and specifically to a carboxymethyl chitosan-guanidine copolymer, its preparation method and application. Background Technology

[0002] Chitosan is the only alkaline natural polysaccharide found in nature. Due to the presence of hydroxyl and amino groups, chitosan possesses certain antioxidant activity and is non-toxic and pollution-free, allowing for the derivation of secondary lead compounds through structural modification. However, chitosan's poor water solubility and weak antioxidant activity severely limit its application value. Carboxymethyl chitosan, as an important derivative of chitosan, significantly improves water solubility and antioxidant activity compared to chitosan, while retaining good biocompatibility and biodegradability; however, it still falls short of fully meeting the application needs in food, pharmaceutical, and agricultural fields. Creatine, a representative drug with antioxidant activity, belongs to the guanidine class of compounds. It can scavenge free radicals in the body, reduce oxidative stress damage, and protect cells (especially muscle and nerve cells) from oxidative damage. Therefore, introducing the guanidine group, the antioxidant pharmacological group of creatine, into the chitosan molecule is expected to enhance the antioxidant activity of chitosan. Chitosan contains -OH and -NH2 active groups. By modifying the structure of chitosan and introducing other active groups, derivatives with good antioxidant activity can be obtained. Therefore, a carboxymethyl chitosan-guanidine copolymer, its preparation method and application are provided. Summary of the Invention

[0003] The purpose of this invention is to address the deficiencies of the prior art by providing a carboxymethyl chitosan-guanidine copolymer, its preparation method, and its application, thereby solving the problems raised in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a carboxymethyl chitosan-guanidine copolymer, wherein the structure of the carboxymethyl chitosan-guanidine copolymer is shown in formula (1). Equation (1), where the average degree of polymerization n ranges from 100 to 3000.

[0005] A method for preparing a carboxymethyl chitosan-guanidine copolymer involves first carboxylating chitosan, and then reacting it directly with guanidine hydrochloride via ion exchange at room temperature without the aid of a catalyst to obtain the product shown in formula (1), the carboxymethyl chitosan-guanidine copolymer. The synthetic route is as follows:

[0006] A method for preparing the carboxymethyl chitosan-guanidine copolymer:

[0007] S1: After chitosan is swollen and homogenized in isopropanol, 5-7 mL (40%, w / v) of sodium hydroxide solution is added. The reaction solution is alkalized in a water bath at 55-60℃. After chloroacetic acid is completely dissolved in water, the aqueous chloroacetic acid solution is added dropwise to the mixture at a rate of 1-2 drops per second. The reaction is stirred at 60-70℃ for 6-12 h. After the reaction, the mixture is precipitated with ethanol, washed, centrifuged, filtered, and freeze-dried to constant weight to obtain carboxymethyl chitosan.

[0008] S2: After completely dissolving carboxymethyl chitosan in water, react it with guanidine hydrochloride at room temperature for 6-12 h to obtain the carboxymethyl chitosan-guanidine copolymer shown in formula (1).

[0009] The molar ratio of chloroacetic acid to chitosan is 4-6:1; the molar ratio of guanidine hydrochloride to carboxymethyl chitosan is 2-4:1.

[0010] An application of the aforementioned carboxymethyl chitosan-guanidine copolymer, specifically its application in the preparation of antioxidants.

[0011] The present invention has the following beneficial effects:

[0012] (1) After the carboxymethyl chitosan-guanidine copolymer is prepared according to the present invention, its antioxidant activity is significantly improved.

[0013] (2) The carboxymethyl chitosan-guanidine copolymer prepared by the present invention has good biocompatibility.

[0014] (3) The present invention has the advantages of simple synthesis steps, mild reaction conditions, readily available raw materials and equipment, and low overall cost; the products obtained by the present invention can be widely used in food, medicine and agriculture. Attached Figure Description

[0015] Figure 1 This is the infrared spectrum of chitosan.

[0016] Figure 2 This is the infrared spectrum of carboxymethyl chitosan from an embodiment of the present invention. Compared with the chitosan raw material, it has a wavelength of 3504.95 cm⁻¹. -1 The peak width at that point is due to the superposition effect of -COOH and -OH, indicating that carboxymethyl chitosan has been successfully synthesized.

[0017] Figure 3 The infrared spectrum of the carboxymethyl chitosan-guanidine copolymer in an embodiment of the present invention is shown at 1588.12 cm⁻¹. -1 and 1321.96 cm -1 Corresponding to the NH deformation vibration and CN tensile vibration of the guanidinyl group, respectively, 1646.91 cm -1The band at that point corresponds to the C=NH tensile vibration, indicating that the carboxymethyl chitosan-guanidine copolymer has been successfully synthesized. Detailed Implementation

[0018] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0019] This invention first carboxymethylates chitosan to improve its solubility. Then, the resulting carboxymethyl chitosan is directly reacted with the guanidinium cation of guanidine hydrochloride through an ion exchange reaction at room temperature to prepare a carboxymethyl chitosan-guanidinium copolymer. The scavenging ability of this copolymer against superoxide anion radicals and DPPH radicals and its cytotoxicity to L929 cells were investigated.

[0020] The synthetic route for carboxymethyl chitosan-guanidine copolymer is as follows:

[0021] The average degree of polymerization n ranges from 100 to 3000.

[0022] Example 1: In this example, the target compound, carboxymethyl chitosan-guanidine copolymer, was synthesized according to the above synthetic route:

[0023] 1) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.61 g (10 mmol) of chitosan (see... Figure 1 After swelling uniformly in 40 mL of isopropanol, 5 mL (40%, w / v) of sodium hydroxide solution was added, and the reaction solution was alkalized in a water bath at 55 °C. After 4 h, 4.8 g (50 mmol) of chloroacetic acid was dissolved in 13 mL of water, and the solution was added dropwise to the mixture at a rate of 1 drop per second. The reaction was stirred at 60 °C for 6 h. After the reaction, the mixture was precipitated with ethanol, washed, centrifuged, filtered, and freeze-dried to constant weight to obtain 1.67 g of carboxymethyl chitosan (see [link to product description]). Figure 2 ),spare;

[0024] 2) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.3 g (5 mmol) of carboxymethyl chitosan (see... Figure 2 After being completely dissolved in water, it was reacted with 1.44 g (15 mmol) of guanidine hydrochloride at room temperature for 12 h. After the reaction was completed, it was dialyzed for 3 days and then freeze-dried under vacuum to obtain the carboxymethyl chitosan-guanidine copolymer (see [link to product]). Figure 3 ), where n ranges from 100 to 3000.

[0025] Example 2: The difference from Example 1 is that:

[0026] 1) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.61 g (10 mmol) of chitosan (see... Figure 1 After swelling uniformly in 50 mL of isopropanol, 7 mL (40%, w / v) of sodium hydroxide solution was added, and the reaction solution was alkalized in a water bath at 60 °C. After 6 h, 5.76 g (60 mmol) of chloroacetic acid was dissolved in 15 mL of water, and the solution was added dropwise to the mixture at a rate of 2 drops per second. The reaction was stirred at 70 °C for 10 h. After the reaction, the mixture was precipitated with ethanol, washed, centrifuged, filtered, and freeze-dried to constant weight to obtain 1.89 g of carboxymethyl chitosan (see [link to product description]). Figure 2 ),spare;

[0027] 2) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.3 g (5 mmol) of carboxymethyl chitosan (see... Figure 2 After being completely dissolved in water, it was reacted with 1.92 g (20 mmol) of guanidine hydrochloride at room temperature for 14 h. After the reaction was completed, it was dialyzed for 4 days and then freeze-dried under vacuum to obtain the carboxymethyl chitosan-guanidine copolymer (see [link to product]). Figure 3 ), where n ranges from 100 to 3000.

[0028] Example 3: The difference from Example 1 is that:

[0029] 1) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.61 g (10 mmol) of chitosan (see... Figure 1 After swelling uniformly in 45 mL of isopropanol, 6 mL (40%, w / v) of sodium hydroxide solution was added, and the reaction solution was alkalized in a water bath at 55 °C. After 5 h, 3.84 g (40 mmol) of chloroacetic acid was dissolved in 12 mL of water, and the solution was added dropwise to the mixture at a rate of 1 drop per second. The reaction was stirred at 65 °C for 8 h. After the reaction, the mixture was precipitated with ethanol, washed, centrifuged, filtered, and freeze-dried to constant weight to obtain 1.51 g of carboxymethyl chitosan (see [link to product description]). Figure 2 ),spare;

[0030] 2) Preparation of carboxymethyl chitosan-guanidine copolymer: Take 1.3 g (5 mmol) of carboxymethyl chitosan (see... Figure 2 After being completely dissolved in water, it was reacted with 0.96 g (10 mmol) of guanidine hydrochloride at room temperature for 10 h. After the reaction was completed, it was dialyzed for 2 days and then freeze-dried under vacuum to obtain the carboxymethyl chitosan-guanidine copolymer (see [link to product]). Figure 3 ), where n ranges from 100 to 3000.

[0031] Application Example 1: Determination of Antioxidant Activity

[0032] Superoxide anion radical scavenging rate determination: The superoxide anion radical scavenging ability of chitosan and the carboxymethyl chitosan and carboxymethyl chitosan-guanidine copolymer prepared in the above examples was determined and compared (Table 1): The carboxymethyl chitosan, carboxymethyl chitosan-guanidine copolymer and experimental chitosan prepared in the above examples were prepared into a stock solution with a concentration of 10 mg / mL. Different volumes of the stock solution were taken to prepare test solutions with concentration gradients of 0.1, 0.2, 0.4, 0.8 and 1.6 mg / mL. 0.5 mL of reducing coenzyme I (NADH, 0.2453 mg / mL), 0.5 mL of nitrotetrazole blue (NBT, 0.3657 mg / mL), and 0.5 mL of methyl phenazine sulfate (PMS, 0.01838 mg / mL) were added to the experimental group in sequence. In the control group, the NADH solution was replaced with Tris-HCl buffer (1.9697 mg / mL); the blank sample solution was replaced with Tris-HCl buffer. After mixing all solutions, allow them to stand for 5 min. Measure the absorbance of each sample at 560 nm, three times for each sample. The formula for calculating the superoxide anion radical scavenging capacity of the test samples is as follows:

[0033] Clearance rate (%) = [1 - (A)] 样品 -A 对照 ) / A 空白 ]×100

[0034] Among them, A 样品 A represents the absorbance of the experimental group. 对照 Absorbance of the control group; A 空白 The absorbance is for the blank group.

[0035] The results of the superoxide anion free radical scavenging capacity determination are shown in Table 1:

[0036] Table 1. Superoxide anion radical scavenging capacity of chitosan and chitosan derivatives (%)

[0037] Application Example 2: Determination of Antioxidant Activity

[0038] DPPH radical scavenging rate determination: The DPPH radical scavenging ability of chitosan and the carboxymethyl chitosan and carboxymethyl chitosan-guanidine copolymer prepared in the above examples was determined and compared (Table 2): The carboxymethyl chitosan, carboxymethyl chitosan-guanidine copolymer, and experimental chitosan prepared in the above examples were used to prepare a stock solution with a concentration of 10 mg / mL. The sample solution was mixed with deionized water, and 2.0 mL of DPPH / ethanol (180 μmol / L) was added, with concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL, respectively. 2.0 mL of DPPH / ethanol was replaced by 2.0 mL of ethanol as a control. 1.0 mL of deionized water was mixed with 2.0 mL of DPPH / ethanol as a blank. The mixture was incubated in the dark at room temperature for 20 min, and the absorbance of each group of samples at 517 nm was measured. Each sample was measured three times. The DPPH radical scavenging ability was calculated by the following formula:

[0039] Clearance rate (%) = [1 - (A)] 样品 -A 对照 ) / A 空白 ]×100

[0040] Among them, A 样品 A represents the absorbance of the experimental group. 对照 Absorbance of the control group; A 空白 The absorbance is for the blank group.

[0041] The results of the DPPH free radical scavenging capacity determination are shown in Table 2:

[0042] Table 2. DPPH free radical scavenging capacity of chitosan and chitosan derivatives (%)

[0043] Application Example 3: Biocompatibility Determination

[0044] The cytotoxicity of the samples to L929 cells was determined using the MTT assay: Well-cultured cell suspensions were seeded into 96-well plates and cultured until reaching 10 × 10⁶ cells / well. 4 ~ 5 Cells / well. L929 cells were treated with different concentrations of 1000, 500, 250, 125, and 62.5 μg / mL. After incubation in a cell culture incubator for 48 h, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and incubation continued for 4 h. After separating the MTT solution, 150 μL of DMSO was added to each well, and the cells were shaken for 15 min in the dark. The absorbance was measured at 490 nm, and cell viability was calculated using the following formula:

[0045] Cell viability (%) = (A 样品 -A 空白 ) / (A对照 -A 空白 )×100

[0046] Among them, A 样品 A represents the absorbance of the experimental group. 对照 Absorbance of the control group; A 空白 The absorbance is for the blank group.

[0047] The results of cell viability assays are shown in Table 3:

[0048] Table 3. Cell viability (%) of chitosan and chitosan derivatives

[0049] Experimental Results: The ability of the chitosan derivatives synthesized in this invention to inhibit superoxide anion radicals and DPPH radicals is shown in Tables 1 and 2. Chitosan itself has weak antioxidant activity, while the antioxidant capacity of the synthesized carboxymethyl chitosan is stronger than that of chitosan, proving that the presence of carboxymethyl groups can improve the free radical scavenging ability of chitosan. The antioxidant capacity of the carboxymethyl chitosan-guanidin copolymer prepared by further introducing guanidin groups is significantly better than that of carboxymethyl chitosan. The free radical scavenging rate is more than 50% at the test concentration of 0.8 mg / mL, and the scavenging rate can reach more than 70% at the highest test concentration of 1.0 mg / mL, indicating that the introduction of carboxymethyl and guanidin groups can enhance the antioxidant activity of chitosan.

[0050] The cytotoxicity tests of the chitosan derivatives and chitosan synthesized in this invention on L929 cells are shown in Table 3. After treatment with chitosan samples at different test concentrations, the cell viability of L929 cells was consistently above 100%, demonstrating that chitosan is non-toxic. At a concentration of 1000 μg / mL, the cell viability of both carboxymethyl chitosan-guanidine copolymer and carboxymethyl chitosan was above 80%, indicating that both carboxymethyl chitosan-guanidine copolymer and carboxymethyl chitosan are non-toxic at this concentration and possess good biocompatibility.

[0051] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make several improvements without departing from the concept of the present invention, such as replacing the type of guanidine compound, changing the molecular weight of chitosan, changing the concentration of reactants, etc. These improvements should also be considered within the scope of protection of the present invention.

Claims

1. A carboxymethyl chitosan-guanidine copolymer, characterized in that: The structure of the carboxymethyl chitosan-guanidine copolymer is shown in formula (1): Equation (1), The average degree of polymerization n ranges from 100 to 3000.

2. A method for preparing the carboxymethyl chitosan-guanidine copolymer as described in claim 1, characterized in that: First, chitosan is carboxymethylated, and then reacted directly with guanidine hydrochloride at room temperature without the aid of a catalyst through ion exchange to obtain the product shown in formula (1), which is a carboxymethyl chitosan-guanidine copolymer. The specific steps are as follows: S1: After chitosan is swollen and homogenized in isopropanol, 5-7 mL (40%, w / v) of sodium hydroxide solution is added. The reaction solution is alkalized in a water bath at 55-60℃. After chloroacetic acid is completely dissolved in water, the aqueous chloroacetic acid solution is added dropwise to the mixture at a rate of 1-2 drops per second. The reaction is stirred at 60-70℃ for 6-12 h. After the reaction, the mixture is precipitated with ethanol, washed, centrifuged, filtered, and freeze-dried to constant weight to obtain carboxymethyl chitosan. S2: After completely dissolving carboxymethyl chitosan in water, react it with guanidine hydrochloride at room temperature for 6-12 h to obtain the carboxymethyl chitosan-guanidine copolymer shown in formula (1).

3. A method for preparing the carboxymethyl chitosan-guanidine copolymer as described in claim 2, characterized in that: The molar ratio of chloroacetic acid to chitosan is 4-6:1; the molar ratio of guanidine hydrochloride to carboxymethyl chitosan is 2-4:

1.

4. An application of the carboxymethyl chitosan-guanidine copolymer as described in claim 1, characterized in that: Application of the carboxymethyl chitosan-guanidine copolymer in the preparation of antioxidants.

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