Phenolic acid modified chitosan, phenolic acid modified chitosan hydrogel and application thereof

Abstract

The present invention provides a phenolic acid modified chitosan, a phenolic acid modified chitosan hydrogel and an application thereof, and specifically relates to the technical field of medical materials. The preparation method of the phenolic acid modified chitosan is to add an amino activator to a chitosan solution and mix in the dark, then add a phenolic acid solution and mix evenly, and finally drop a carboxyl activator to react in the dark to obtain the phenolic acid modified chitosan; wherein the phenolic acid includes syringic acid, gentisic acid, vanillic acid or protocatechuic acid. The amino activation process is carried out under light-proof conditions, which helps to improve the selectivity of the reaction, so that the amino activator and chitosan can react more effectively instead of causing unnecessary side reactions; at the same time, the grafting reaction is carried out in the dark, which can also improve the grafting rate, that is, the proportion of phenolic acid successfully grafted onto chitosan. The preparation method is beneficial to protecting reactants and intermediates, improving the selectivity of the reaction and the quality of the final product, and the process is continuous, which is conducive to industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, in particular to phenolic acid modified chitosan, phenolic acid modified chitosan hydrogel and applications thereof. Background Art

[0002] Chitosan is a natural polymer material with many excellent properties. It is a product of chitin deacetylation and has extremely high safety. Chitosan has strong hydrogen bonds between and within molecules and high crystallinity, which makes it insoluble in water and common organic solvents. This solubility limitation affects the direct use of chitosan in certain applications, such as water treatment and drug delivery systems, which require chitosan to be soluble in order to play its role.

[0003] In order to overcome the solubility problem, researchers have developed a variety of chemical modification technologies to improve the solubility, biocompatibility and bioactivity of chitosan by introducing new functional groups. For example, charged or hydrophilic groups can be introduced to the surface of chitosan through carboxymethylation, alkylation, sulfonation and quaternization reactions, thereby improving its solubility. However, these modification methods will introduce new toxicity problems and affect the original bioactivity of chitosan, making it unsuitable for direct use in drugs or organisms.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for preparing phenolic acid modified chitosan, aiming to solve at least one of the above technical problems in the prior art.

[0006] The second object of the present invention is to provide a phenolic acid modified chitosan.

[0007] The third object of the present invention is to provide a phenolic acid modified chitosan hydrogel.

[0008] A fourth object of the present invention is to provide an application of phenolic acid modified chitosan hydrogel.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] The first aspect of the present invention provides a method for preparing phenolic acid modified chitosan, which comprises adding an amino activator to a chitosan solution and mixing in the dark, then adding a phenolic acid solution and mixing evenly, and finally dropping a carboxyl activator and reacting in the dark to obtain phenolic acid modified chitosan.

[0011] Wherein, the phenolic acid includes syringic acid, gentisic acid, vanillic acid or protocatechuic acid.

[0012] Furthermore, the amino activator includes 1-hydroxybenzotriazole and N-hydroxysuccinimide.

[0013] Preferably, the carboxyl activating agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0014] Furthermore, the molar ratio of the amino activator to chitosan is 1:1-3.

[0015] Preferably, the molar ratio of phenolic acid to chitosan is 1:1-3.

[0016] Preferably, the molar ratio of the carboxyl activator to chitosan is 1:1-3.

[0017] Furthermore, the light-proof mixing time is 2 to 10 hours.

[0018] Preferably, the reaction time in the dark is 12 to 48 hours.

[0019] Furthermore, in the chitosan solution, the solvent includes an acetic acid aqueous solution.

[0020] Preferably, the volume percentage of acetic acid in the acetic acid aqueous solution is 1 to 5%, preferably 1 to 3%.

[0021] Preferably, in the chitosan solution, the concentration of chitosan is 10-15 mg / mL.

[0022] Furthermore, the method for preparing phenolic acid modified chitosan also includes a separation process after the reaction in the dark.

[0023] Preferably, the separation is carried out by dialysis.

[0024] Preferably, deionized water is used as the dialysate, and a dialysis bag is used to dialyze the reaction solution to obtain the phenolic acid-modified chitosan.

[0025] Preferably, the dialysis time is 2 to 5 days.

[0026] Preferably, during the dialysis process, the dialysate is replaced every 8 to 16 hours.

[0027] The second aspect of the present invention provides a phenolic acid modified chitosan, which is prepared by the preparation method.

[0028] The third aspect of the present invention provides a phenolic acid modified chitosan hydrogel, which is obtained by mixing an aqueous solution of phenolic acid modified chitosan with an aqueous solution of vanillin for gelation.

[0029] Wherein, the aqueous solution of phenolic acid modified chitosan includes the phenolic acid modified chitosan.

[0030] Furthermore, the gelling time is 20 to 60 minutes.

[0031] Preferably, the concentration of the aqueous solution of phenolic acid modified chitosan is 20-50 mg / mL.

[0032] Preferably, the concentration of the vanillin aqueous solution is 20-50 mg / mL.

[0033] Preferably, the volume ratio of the aqueous solution of phenolic acid modified chitosan to the aqueous solution of vanillin is 1:0.8-1.2.

[0034] The fourth aspect of the present invention provides the use of the phenolic acid modified chitosan hydrogel in the preparation of a medicine for promoting wound healing.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] In the preparation method of phenolic acid modified chitosan provided by the present invention, the amino activation process is carried out under light-proof conditions, which helps to improve the selectivity of the reaction, so that the amino activator and chitosan can react more effectively instead of causing unnecessary side reactions; at the same time, the grafting reaction is carried out in the dark, which can also improve the grafting rate, that is, the proportion of phenolic acid successfully grafted onto chitosan. The preparation method is conducive to protecting reactants and intermediates, improving the selectivity of the reaction and the quality of the final product, and the process is continuous, which is conducive to industrial large-scale production.

[0037] The phenolic acid modified chitosan provided by the present invention can not only change the structure and physicochemical properties (solubility, thermal stability, crystallinity and rheological properties) of chitosan by introducing phenolic groups on the chitosan skeleton, but also significantly improve the biological activity of chitosan (antioxidant, antibacterial, antitumor, antiallergic, anti-inflammatory, etc.). The phenolic acid modified chitosan has different degrees of improvement in the size of the inhibition zone of S. aureus, S. epidermidis, P. aeruginosa, B. subtilis and E. coli, and effectively enhances the antibacterial ability against bacteria; at the same time, the phenolic acid modified chitosan has no cytotoxicity when co-cultured with HaCaT cells at a dosage concentration of up to 800 μg / mL, and has an extremely high safety concentration. In addition, the phenolic acid modified chitosan has antioxidant capacity against DPPH free radicals, ABTS free radicals and FRAP.

[0038] The phenolic acid modified chitosan hydrogel provided by the present invention is formed based on Schiff base bond and hydrogen bond. Schiff base bond is a reversible chemical bond obtained by the reaction of the amino group of modified chitosan and the aldehyde group of vanillin. The hydrogen bond comes from the fact that the modified chitosan macromolecule contains many primary amino groups, alcohol hydroxyl groups and phenolic hydroxyl groups of grafted phenolic acid. Scanning electron microscopy shows that the phenolic acid modified chitosan hydrogel has a loose and porous structure, which is conducive to simulating the cell environment. The phenolic acid modified chitosan hydrogel has good antibacterial ability against a variety of bacteria, and has antibacterial effects on both Gram-positive bacteria and Gram-negative bacteria. Cell experiments have verified that the phenolic modified chitosan hydrogel has high cell safety. In vivo wound healing experiments show that different drug administration groups can promote wound healing in rats.

[0039] The application of the phenolic acid modified chitosan hydrogel provided by the present invention, in view of the advantages of the above-mentioned phenolic acid modified chitosan hydrogel, enables the prepared medicine for promoting wound healing to have more comprehensive antibacterial properties, free radical scavenging ability, higher safety and good blood compatibility, and can better promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 A in it is the infrared spectrum of raw materials (CS, SA, GTA, VA, PCA); Figure 1 B in the figure is the infrared spectrum of the product obtained in Comparative Examples 1-4; Figure 1 C in FIG. 1 is the infrared spectrum of the product obtained in Example 1-4;

[0042] Figure 2 is the infrared spectrum of the product obtained in Examples 13-16;

[0043] Figure 3 XRD patterns of CS, SA, GTA, VA and PCA;

[0044] Figure 4 The XRD patterns of the products obtained in Examples 1-4;

[0045] Figure 5 is the XRD pattern of vanillin (VL);

[0046] Figure 6 is the XRD pattern of modified chitosan hydrogel;

[0047] Figure 7 It is the differential thermal scanning analysis diagram of CS, SA, GTA, VA and PCA;

[0048] Figure 8 The differential thermal scanning analysis diagram of the product obtained in Example 1-4;

[0049] Fig. 9 is a differential thermal scanning analysis diagram of the product obtained in Examples 13-16;

[0050] Fig.10 A in the figure is the scanning electron microscope (SEM) image of CS-SA / VL hydrogel; Fig.10 B in the figure is the scanning electron microscope (SEM) image of CS-GTA / VL hydrogel; Fig.10 C in the figure is the scanning electron microscope (SEM) image of CS-VA / VL hydrogel; Fig.10 D in the figure is the scanning electron microscope (SEM) image of CS-PCA / VL hydrogel;

[0051] Fig.11 The figure shows the scavenging ability of phenolic acid modified chitosan and vitamin C on DPPH free radicals (n=3);

[0052] Fig.12 The result diagram of the scavenging ability of phenolic acid modified chitosan and vitamin C on ABTS free radicals (n=3);

[0053] Fig.13 The total antioxidant capacity of phenolic acid modified chitosan on FRAP results (n = 3);

[0054] Fig.14 Cell survival rate (%) after incubation of HaCaT cells with phenolic acid modified chitosan

[0055] (n=5);

[0056] Fig.15 This is the result of the cell scratch test (100 μg / mL);

[0057] Fig.16 is the cell migration rate analysis graph (100 μg / mL);

[0058] Fig.17 This is the result of the cell scratch test (200 μg / mL);

[0059] Fig.18 is the cell migration rate analysis graph (200 μg / mL);

[0060] Fig.19 A in the figure is the result of hemolysis experiment of negative control (normal saline); Fig.19B in the figure is the result of hemolysis experiment of positive control (deionized water); Fig.19 C in the figure is the result of hemolysis experiment of CS-SA / VL hydrogel; Fig.19 D in the figure is the result of hemolysis experiment of CS-GTA / VL hydrogel; Fig.19 Figure E is the result of hemolysis experiment of CS-VA / VL hydrogel; Fig.19 Figure F is the result of the CS-PCA / VL hydrogel hemolysis experiment;

[0061] Fig. 20 is the hemolysis experiment analysis diagram (n=3);

[0062] Fig.21 This is a graph showing the trend of wound changes in experimental rats during treatment;

[0063] Fig. 22 is the weight change curve of experimental rats (n=6);

[0064] Fig.23 A in the figure is the tissue morphology of the wound of the trauma rats in the blank group; Fig.23 B in the figure is the tissue morphology of the wound of the model group trauma rats; Fig.23 C in the figure is the tissue morphology of the wound of rats with trauma in the CS-SA / VL hydrogel group; Fig.23 D in the figure is the tissue morphology of the wound of rats with trauma in the CS-GTA / VL hydrogel group; Fig.23 E in the figure is the tissue morphology of the wound of rats with trauma in the CS-VA / VL hydrogel group; Fig.23 F in the figure is the tissue morphology of the wound of traumatic rats in the CS-PCA / VL hydrogel group. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0066] Hereinafter, the terms "including", "having" and their cognates, which may be used in various embodiments of the present invention, are intended only to indicate specific features, numbers, steps, operations, elements, components or combinations of the foregoing items, and should not be understood as first excluding the existence of one or more other features, numbers, steps, operations, elements, components or combinations of the foregoing items or adding the possibility of one or more features, numbers, steps, operations, elements, components or combinations of the foregoing items.

[0067] The first aspect of the present invention provides a method for preparing phenolic acid modified chitosan, which comprises adding an amino activator to a chitosan solution and mixing in the dark, then adding a phenolic acid solution and mixing evenly, and finally dropping a carboxyl activator and reacting in the dark to obtain phenolic acid modified chitosan.

[0068] Wherein, the phenolic acid includes syringic acid, gentisic acid, vanillic acid or protocatechuic acid.

[0069] In the preparation method of phenolic acid modified chitosan provided by the present invention, the amino activation process is carried out under light-proof conditions, which helps to improve the selectivity of the reaction, so that the amino activator and chitosan can react more effectively instead of causing unnecessary side reactions; at the same time, the grafting reaction is carried out in the dark, which can also improve the grafting rate, that is, the proportion of phenolic acid successfully grafted onto chitosan. The preparation method is conducive to protecting reactants and intermediates, improving the selectivity of the reaction and the quality of the final product, and the process is continuous, which is conducive to industrial large-scale production.

[0070] Furthermore, the amino activator includes 1-hydroxybenzotriazole and N-hydroxysuccinimide.

[0071] Preferably, the carboxyl activating agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0072] The molar ratio of the amino activator to chitosan is 1:1 to 3. Typically but not limiting, the molar ratio of the amino activator to chitosan can be, for example, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, or any value within the range of 1:1 to 3.

[0073] Preferably, the molar ratio of phenolic acid to chitosan is 1:1 to 3. Typically but not limiting, the molar ratio of phenolic acid to chitosan may be, for example, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, or any value within the range of 1:1 to 3.

[0074] Preferably, the molar ratio of the carboxyl activator to chitosan is 1:1 to 3. Typically but not limiting, the molar ratio of the carboxyl activator to chitosan may be, for example, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, or any value within the range of 1:1 to 3.

[0075] Furthermore, the time of mixing in the dark is 2 to 10 hours. Typically but not limiting, the time of mixing in the dark can be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours, or any value within the range of 2 to 10 hours.

[0076] Preferably, the reaction time in the dark is 12 to 48 hours. Typically but not limiting, the reaction time in the dark can be 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours or 48 hours, or any value within the range of 12 to 48 hours.

[0077] Furthermore, in the chitosan solution, the solvent includes an acetic acid aqueous solution.

[0078] Preferably, the volume percentage of acetic acid in the acetic acid aqueous solution is 1-5%, preferably 1-3%. Typically but not limiting, the volume percentage of acetic acid in the acetic acid aqueous solution may be 1%, 2%, 3%, or any value within the range of 1% to 5%; more preferably, the volume percentage of acetic acid may be 1%, 2% or 3%, or any value within the range of 1% to 3%.

[0079] Preferably, in the chitosan solution, the concentration of chitosan is 10-15 mg / mL. Typically but not limiting, the concentration of chitosan can be 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL or 15 mg / mL, or any value within the range of 10-15 mg / mL.

[0080] Furthermore, the method for preparing phenolic acid modified chitosan also includes a separation process after the reaction in the dark.

[0081] Preferably, the separation is carried out by dialysis.

[0082] Preferably, deionized water is used as the dialysate, and a dialysis bag is used to dialyze the reaction solution to obtain the phenolic acid-modified chitosan.

[0083] Preferably, the dialysis time is 2 to 5 days. Typically but not limiting, the dialysis time can be 2 days, 3 days, 4 days or 5 days, or any value within the range of 2 to 5 days.

[0084] Preferably, during dialysis, the dialysate is replaced every 8 to 16 hours. Typically, but not limiting, the dialysate is replaced every 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or 16 hours.

[0085] The second aspect of the present invention provides a phenolic acid modified chitosan, which is prepared by the preparation method.

[0086] The phenolic acid modified chitosan provided by the present invention can not only change the structure and physicochemical properties (solubility, thermal stability, crystallinity and rheological properties) of chitosan by introducing phenolic groups on the chitosan skeleton, but also significantly improve the biological activity of chitosan (antioxidant, antibacterial, antitumor, antiallergic, anti-inflammatory, etc.). The phenolic acid modified chitosan has different degrees of improvement in the size of the inhibition zone of S. aureus, S. epidermidis, P. aeruginosa, B. subtilis and E. coli, and effectively enhances the antibacterial ability against bacteria; at the same time, the phenolic acid modified chitosan has no cytotoxicity when co-cultured with HaCaT cells at a dosage concentration of up to 800 μg / mL, and has an extremely high safety concentration. In addition, the phenolic acid modified chitosan has antioxidant capacity against DPPH free radicals, ABTS free radicals and FRAP.

[0087] The third aspect of the present invention provides a phenolic acid modified chitosan hydrogel, which is obtained by mixing an aqueous solution of phenolic acid modified chitosan with an aqueous solution of vanillin for gelation.

[0088] Wherein, the aqueous solution of phenolic acid modified chitosan includes the phenolic acid modified chitosan.

[0089] The phenolic acid modified chitosan hydrogel provided by the present invention is formed based on Schiff base bond and hydrogen bond. Schiff base bond is a reversible chemical bond obtained by the reaction of the amino group of modified chitosan and the aldehyde group of vanillin. The hydrogen bond comes from the fact that the modified chitosan macromolecule contains many primary amino groups, alcohol hydroxyl groups and phenolic hydroxyl groups of grafted phenolic acid. Scanning electron microscopy shows that the phenolic acid modified chitosan hydrogel has a loose and porous structure, which is conducive to simulating the cell environment. The phenolic acid modified chitosan hydrogel has good antibacterial ability against a variety of bacteria, and has antibacterial effects on both Gram-positive bacteria and Gram-negative bacteria. Cell experiments have verified that the phenolic modified chitosan hydrogel has high cell safety. In vivo wound healing experiments show that different drug administration groups can promote wound healing in rats.

[0090] Furthermore, the gelling time is 20 to 60 minutes. Typically but not limiting, the gelling time can be 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, or any value within the range of 20 to 60 minutes.

[0091] Preferably, the concentration of the aqueous solution of phenolic acid modified chitosan is 20-50 mg / mL. Typically but not limiting, the concentration of the aqueous solution of phenolic acid modified chitosan can be 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL, or any value within the range of 20-50 mg / mL.

[0092] Preferably, the concentration of the vanillin aqueous solution is 20 to 50 mg / mL. Typically but not limiting, the concentration of the vanillin aqueous solution may be 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL, or any value within the range of 20 to 50 mg / mL.

[0093] Preferably, the volume ratio of the aqueous solution of phenolic acid modified chitosan to the aqueous solution of vanillin is 1:0.8 to 1.2. Typically but not limiting, the volume ratio of the aqueous solution of phenolic acid modified chitosan to the aqueous solution of vanillin can be 1:0.8, 1:0.9, 1:1.0, 1:1.1 or 1:1.2, or any ratio within the range of 1:0.8 to 1.2.

[0094] The fourth aspect of the present invention provides the use of the phenolic acid modified chitosan hydrogel in the preparation of a medicine for promoting wound healing.

[0095] The application of the phenolic acid modified chitosan hydrogel provided by the present invention, in view of the advantages of the above-mentioned phenolic acid modified chitosan hydrogel, enables the prepared medicine for promoting wound healing to have more comprehensive antibacterial properties, free radical scavenging ability, higher safety and good blood compatibility, and can better promote wound healing.

[0096] The present invention is further described below by specific examples and comparative examples, but it should be understood that these examples are only used for more detailed description and should not be understood as limiting the present invention in any form. The raw materials used in the examples and comparative examples of the present invention, if no specific conditions are specified, are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0097] Example 1

[0098] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0099] 1. Add chitosan (CS, 500 mg, 2.61 mmol) and 1-hydroxybenzotriazole (HOBt, 270 mg, 2 mmol) to 40 mL of acetic acid solution (2%, v / v) and stir for 2 h in the dark to activate the amine group of CS with HOBt.

[0100] 2. 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol was introduced into the CS solution. Then, 5 mL of ethanol containing EDCI (382 mg, 2 mmol) was added dropwise and reacted in the dark at room temperature for 12 h.

[0101] 3. The reaction solution was dialyzed using a dialysis bag (MWCO 8000Da) with deionized water for a total of 72 hours, with the water replaced every 12 hours. Finally, the dialyzed reaction solution was freeze-dried to obtain CS-SA.

[0102] Example 2

[0103] This embodiment provides a gentisic acid modified chitosan (CS-GTA). The difference from Example 1 is that gentisic acid (GTA, 308 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in Example 1 and are not described again.

[0104] Example 3

[0105] This embodiment provides a protocatechuic acid modified chitosan (CS-PCA). The difference from Example 1 is that protocatechuic acid (PCA, 308 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in Example 1 and are not described again.

[0106] Example 4

[0107] This embodiment provides a vanillic acid modified chitosan (CS-VA). The difference from Example 1 is that vanillic acid (VA, 336 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in Example 1 and are not described again.

[0108] Example 5

[0109] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0110] 1. Same as the step in Example 1.

[0111] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 2 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 24 h.

[0112] 3. Same as the step in Example 1.

[0113] Example 6

[0114] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0115] 1. Same as the step in Example 1.

[0116] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 4 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 48 h.

[0117] 3. Same as the step in Example 1.

[0118] Example 7

[0119] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0120] 1. Add chitosan (CS, 500 mg, 2.61 mmol) and 1-hydroxybenzotriazole (HOBt, 270 mg, 2 mmol) to 40 mL of acetic acid solution (2%, v / v) and stir for 6 h in the dark to activate the amine group of CS with HOBt.

[0121] 2. 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol was introduced into the CS solution. Then, 5 mL of ethanol containing EDCI (382 mg, 2 mmol) was added dropwise and reacted in the dark at room temperature for 24 h.

[0122] 3. Same as the step in Example 1.

[0123] Example 8

[0124] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0125] 1. Same as the step in Example 7.

[0126] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 2 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 48 h.

[0127] 3. Same as the step in Example 1.

[0128] Example 9

[0129] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0130] 1. Same as the step in Example 7.

[0131] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 4 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 12 h.

[0132] 3. Same as the step in Example 1.

[0133] Example 10

[0134] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0135] 1. Add chitosan (CS, 500 mg, 2.61 mmol) and 1-hydroxybenzotriazole (HOBt, 270 mg, 2 mmol) to 40 mL of acetic acid solution (2%, v / v) and stir for 10 h in the dark to activate the amine group of CS with HOBt.

[0136] 2. 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol was introduced into the CS solution. Then, 5 mL of ethanol containing EDCI (382 mg, 2 mmol) was added dropwise and reacted in the dark at room temperature for 48 h.

[0137] 3. Same as the step in Example 1.

[0138] Embodiment 11

[0139] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0140] 1. Same as the step in Example 10.

[0141] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 2 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 12 h.

[0142] 3. Same as the step in Example 1.

[0143] Example 12

[0144] This embodiment provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0145] 1. Same as the step in Example 10.

[0146] 2. Add 5 mL of ethanol containing EDCI (382 mg, 2 mmol) to 2 mmol of syringic acid (SA, 396 mg) dissolved in an appropriate amount of ethanol and activate for 4 h. Finally, add the above mixed solution dropwise into the CS solution and react in the dark at room temperature for 24 h.

[0147] 3. Same as the step in Example 1.

[0148] Example 13

[0149] This embodiment provides a syringic acid modified chitosan hydrogel (CS-SA / VL hydrogel), and the preparation process is as follows:

[0150] 1. Prepare the syringic acid-modified chitosan obtained in Example 1 into an aqueous solution with a concentration of 20 mg / mL. Prepare vanillin into an aqueous solution with a concentration of 20 mg / mL.

[0151] 2. The above two solutions were mixed and gelled in a volume ratio of 1:1 to obtain syringic acid-modified chitosan hydrogel.

[0152] Examples 14-16

[0153] This embodiment provides a modified chitosan hydrogel, wherein the gentisic acid / protocatechuic acid / vanillic acid modified chitosan obtained in Examples 2-4 is used to replace the syringic acid modified chitosan, and the remaining raw materials and preparation methods are the same as those in Example 13, which will not be repeated here.

[0154] Comparative Example 1

[0155] This comparative example provides a syringic acid modified chitosan (CS-SA), and the preparation process is as follows:

[0156] 1. Add 1-hydroxybenzotriazole (HOBt, 270 mg, 2 mmol) and CS (500 mg, 2.61 mmol) to 40 mL of acetic acid solution (2%, v / v) and stir overnight.

[0157] 2. 2 mmol of syringic acid (SA, 396 mg) / gentisic acid (GTA, 308 mg) / vanillic acid (VA, 336 mg) / protocatechuic acid (PCA, 308 mg) dissolved in ethanol were introduced into the CS solution respectively. Then, 5 mL of ethanol containing EDCI (382 mg, 2 mmol) was added dropwise and reacted at room temperature in the dark for 24 hours.

[0158] 3. The final product was dialyzed against deionized water using a dialysis tube (MWCO 8000Da) for 72 hours, with the water replaced every 8 hours. Finally, the dialysate was freeze-dried to obtain CS-SA.

[0159] Comparative Example 2

[0160] This comparative example provides a syringic acid modified chitosan (CS-GTA). The difference from comparative example 1 is that gentisic acid (GTA, 308 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in comparative example 1 and will not be repeated here.

[0161] Comparative Example 3

[0162] This comparative example provides a syringic acid modified chitosan (CS-PCA). The difference from comparative example 1 is that protocatechuic acid (PCA, 308 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in comparative example 1 and are not described again.

[0163] Comparative Example 4

[0164] This comparative example provides a syringic acid modified chitosan (CS-VA). The difference from comparative example 1 is that vanillic acid (VA, 336 mg) is used to replace syringic acid (SA, 396 mg). The remaining raw materials and preparation methods are the same as those in comparative example 1 and are not described again.

[0165] Characterization Example 1

[0166] The raw materials (CS, SA, GTA, VA and PCA), the products obtained in Examples 1-4 and the products obtained in Comparative Examples 1-4 were subjected to Fourier transform infrared spectroscopy (FT-IR), and the obtained spectra were as follows: Figure 1 shown.

[0167] Figure 1 A in it is the infrared spectrum of raw materials (CS, SA, GTA, VA, PCA); Figure 1 B in the figure is the infrared spectrum of the product obtained in Comparative Examples 1-4; Figure 1 C in FIG. 1 is the infrared spectrum of the product obtained in Example 1-4.

[0168] Infrared spectrum analysis: Figure 1 As shown in A, the raw material CS is at 3442.92cm -1 The stretching vibration peaks of OH and NH appear superimposed, and the peak is relatively broad, at 2926.05 cm -1 and 2877.05cm -1 The CH stretching vibration peak of -CH2- appears at 1652.50 cm -1 and 1601.72cm -1 is the stretching vibration peak of CN, at 1423.23cm -1 and 1383.83cm -1 It is the bending vibration peak of CH. SA is at 3376.99cm -1 and 3240.79cm-1 The stretching vibration peaks of hydroxyl and carboxyl OH are at 2967.18 cm -1 and 2940.26cm -1 The stretching vibration peak of CH in -OCH3 appears, which is due to the presence of two methoxy groups in the SA structure. GTA is at 3125.95cm -1 The stretching vibration peaks of hydroxyl and carboxyl OH appear superimposed. VA is at 3483.41 cm -1 The stretching vibration peaks of hydroxyl and carboxyl OH are shown at 2954.99 cm -1 It is the stretching vibration peak of CH in -OCH3 in its structure. PCA is at 3331.30cm -1 A strong broad peak appears, which is derived from the superposition of the stretching vibration peaks of the hydroxyl group and the carboxyl group OH. -1 is the stretching vibration peak of the carbonyl C=O on the carboxyl group of each phenolic acid, at 1600 cm -1 -1450cm -1 It is the stretching vibration absorption of the benzene ring skeleton of each phenolic acid, producing 2-4 absorption peaks.

[0169] like Figure 1 As shown in B, since the chitosan skeleton is the main chain structure of these products, the FT-IR spectra of the products are also affected by the chitosan main chain. -1 It is the superposition of the stretching vibration peaks of the hydroxyl group OH on the benzene ring, the amino group NH on the chitosan skeleton, and the NH on the amide bond, 2875 cm -1 It is the superposition of the stretching vibration peaks of CH of -CH3 and -CH2- in the structure, 1635cm -1 is the C=O stretching vibration of amide I band, 1540 cm -1 The bending vibration peak of amide II with NH is 1378 cm -1 It is the stretching vibration peak of CN of amide III band. Figure 1 As shown in C, 3250cm -1 It is the superposition of the stretching vibration peaks of the hydroxyl group OH on the benzene ring, the amino group NH on the chitosan skeleton, and the NH on the amide bond, 2875 cm -1 It is the superposition of the stretching vibration peaks of CH of -CH3 and -CH2- in the structure, 1635cm -1 is the C=O stretching vibration of amide I band, 1535 cm -1 The bending vibration peak of amide II with NH is 1375 cm -1 It is the stretching vibration peak of CN of amide III band.

[0170] The hydrogels obtained in Examples 13-16 were subjected to Fourier transform infrared spectroscopy (FT-IR), and the obtained spectra were as follows: Figure 2As shown. Due to the influence of the main chain structure of chitosan skeleton, the FT-IR spectrum of the product is consistent with that of modified chitosan. -1 The peaks of hydroxyl OH on the benzene ring of chitosan grafted with phenolic acid and vanillin, NH on the chitosan backbone amino group, and NH on the amide bond are superimposed. -1 It is the superposition of the stretching vibration peaks of CH in the structure -CH3 and -CH2-, 1662cm -1 It is the stretching vibration of the carbon-nitrogen double bond -C=N-, which is a medium-intensity peak with a sharp peak shape, 1593cm -1 is the bending vibration peak of amide NH, 1291 cm -1 It is the stretching vibration peak of CN, with strong intensity and sharp peak shape. The above three peaks can be attributed to the absorption of the special chemical bond of Schiff base bond.

[0171] Characterization Example 2

[0172] The raw materials (CS, SA, GTA, VA and PCA) were characterized by X-ray diffraction (XRD), and the obtained spectra were as follows: Figure 3 The products obtained in Example 1-4 were characterized by X-ray diffraction (XRD), and the obtained spectrum was as shown Figure 4 shown.

[0173] The raw material vanillin was characterized by X-ray diffraction (XRD), and the spectrum obtained was as follows Figure 5 The products obtained in Examples 13-16 were subjected to X-ray diffraction characterization (XRD), and the obtained spectra were as shown Figure 6 shown.

[0174] from Figure 3 It can be seen that the four phenolic acids (SA, GTA, VA and PCA) have obvious sharp diffraction peaks, indicating that the four phenolic acids are in a stable and uniform highly crystalline state.

[0175] from Figure 4 It can be seen that when the four phenolic acids reacted with chitosan to obtain modified chitosan, no obvious sharp diffraction peaks were observed, but a halo peak appeared, and the peak of the conjugate was broad and weak. This result shows that after grafting, the peak of the chitosan polymer moved to the lower direction and the intensity decreased. On the other hand, the crystallinity index of the modified chitosan decreased, indicating that the grafted phenolic acid partially reduced the crystallinity of CS. This may be due to the destruction of the intermolecular and intramolecular hydrogen bonds of CS when the phenolic acid was introduced.

[0176] Figure 5 This is the XRD result of vanillin, the raw material for hydrogel synthesis. Figure 6The figure shows the XRD pattern of modified chitosan hydrogel. When the modified chitosan and vanillin are co-assembled to form a hydrogel, the peak is sharper and more obvious than that of the modified chitosan. This result shows that after the formation of the hydrogel, the peak of the chitosan polymer moves to a higher direction and the intensity increases. On the other hand, compared with the modified chitosan, the crystallinity index of the hydrogel increases, indicating that after co-assembly with VL, the modified chitosan hydrogel has a higher crystallinity than the modified chitosan. This may be due to the formation of a new chemical bond, the Schiff base bond, and the formation of hydrogen bonds between chitosan macromolecules and hydrogen bonds within the molecules. This result is consistent with the FT-IR results.

[0177] Characterization Example 3

[0178] The raw materials (CS, SA, GTA, VA and PCA) were subjected to differential scanning calorimetry (DSC) and the obtained spectra were as follows: Figure 7 The product obtained in Example 1-4 was subjected to differential scanning calorimetry (DSC), and the spectrum obtained was as shown Figure 8 shown.

[0179] The products obtained in Examples 13-16 were subjected to differential scanning calorimetry (DSC) and the obtained spectra were as follows: Fig. 9 shown.

[0180] from Figure 7 It can be seen that syringic acid monomer, gentisic acid monomer, vanillic acid monomer and protocatechuic acid monomer have an obvious endothermic peak at 210.58°C, 208.48°C, 213.61°C and 204.84°C, respectively.

[0181] from Figure 8 It can be seen that CS has a broad endothermic peak at 109.19°C. When phenolic acid is grafted into chitosan to obtain modified chitosan, the endothermic peak of phenolic acid monomer disappears, and new endothermic peaks appear at 92.36°C, 111.83°C, 91.67°C and 91.48°C, respectively, indicating that the modified chitosan has a similar crystal state to chitosan. This shows that the four phenolic acids are grafted onto chitosan, and the chitosan macromolecular skeleton structure also affects the absorption peak shape of the modified chitosan. This result is consistent with the XRD results.

[0182] from Fig. 9It can be seen that as the temperature rises, the four phenolic acid modified chitosan hydrogels (CS-SA / VL, CS-GTA / VL, CS-VA / VL, CS-PCA / VL) have endothermic peaks at 90.97°C, 82.78°C, 78.84°C and 79.43°C, respectively. The peaks are relatively wide, indicating that the material continues to absorb heat during this temperature period, but it is slightly narrower than that of modified chitosan. This indicates that the four hydrogels have molecular structures similar to chitosan and modified chitosan, which is consistent with the results of FT-IR and XRD. After the modified chitosan and vanillin are co-assembled to form a hydrogel, the endothermic peak temperature point decreases.

[0183] Characterization Example 4

[0184] The products obtained in Examples 13-16 were subjected to scanning electron microscopy, and the obtained electron microscopy images are as follows: Fig.10 shown.

[0185] from Fig.10 It can be seen that the four hydrogels are loose and porous honeycomb structures in the microscopic state. The pore sizes of the freeze-dried products of different modified chitosan hydrogels are different, which may be related to the different grafting rates of different phenolic acids and the different degrees of co-assembly and cross-linking with vanillin.

[0186] Test Example 1

[0187] The grafting rate of the products obtained in Examples 1-12 and Comparative Examples 1-4 was tested by the Folin phenol method. The results are shown in Table 1 below.

[0188] Table 1

[0189]

[0190] As can be seen from Table 1, the activation time of phenolic acid is directly related to the grafting rate, while the activation time and reaction time of chitosan have little effect on the grafting rate of the product. Therefore, the reaction conditions are selected as follows: chitosan activation time is 2h, reaction time is 12h, and phenolic acid is not activated separately.

[0191] Test Example 2

[0192] The products obtained in Examples 1-4 were subjected to minimum inhibitory concentration experiments.

[0193] The products (CS-SA, CS-GTA, CS-VA, CS-PCA) obtained in Examples 1-4 were set to 8 concentrations (concentration gradient: 10000 μg / mL, 5000 μg / mL, 2500 μg / mL, 1250 μg / mL, 625 μg / mL, 312.5 μg / mL, 156.3 μg / mL, 0 μg / mL) with chitosan (CS). First, 1 mL of MH broth medium was added to each 2 mL centrifuge tube. 1 mL of drug (concentration of 20 mg / mL) was added to the first culture medium, and after mixing evenly with the culture medium, 1 mL was sucked out and added to the next centrifuge tube, and the drug-culture medium mixture of different concentrations was obtained by analogy.

[0194] The cultured bacteria: Staphylococcus aureus (S.aureus), Staphylococcus epidermidis (S.epidermidis), Pseudomonas aeruginosa (P.aeruginosa), Bacillus subtilis (B.subtilis) and Escherichia coli (E.coli) were made into bacterial suspension with physiological saline, dispersed evenly, and the turbidity was 1 by McFarland turbidimetric tube. 10 μL was added to each of the above centrifuge tubes, placed in an incubator with a temperature of 37°C and a humidity of 60%, and the growth of various bacteria in the centrifuge tubes was observed at 24h and 48h. The 24h minimum inhibitory concentration (MIC) results are shown in Table 2 below.

[0195] Table 2

[0196]

[0197] The 48h minimum inhibitory concentration (MIC) results are shown in Table 3 below.

[0198] Table 3

[0199]

[0200]

[0201] Table 2 and Table 3 are the experimental results of the minimum inhibitory concentration of chitosan and modified chitosan at 24h and 48h, respectively. Combining the results of the two tables, it can be seen that CS itself has a certain antibacterial ability. At 24h, the MICs of S.aureus, S.epidermidis, P.aeruginosa, B.subtilis and E.coli were 625μg / mL, 625μg / mL, 1250μg / mL, 625μg / mL and 625μg / mL, respectively. However, after another 24h, the MICs of S.aureus, S.epidermidis and B.subtilis all increased to 1250μg / mL. This shows that the antibacterial ability of CS is not stable, and a higher concentration is required to effectively inhibit bacterial growth and kill bacteria.

[0202] For these five bacteria, the MIC of the four modified chitosans did not change at 24h and 48h, indicating that the modified chitosan has a more stable and lasting antibacterial ability and can effectively kill a variety of bacteria. The MIC of CS-SA against the five bacteria in this experiment was 625μg / mL, among which the ability to fight S.aureus, S.epidermidis, P.aeruginosa, and B.subtilis was significantly better than CS. The MIC of CS-GTA against S.aureus, S.epidermidis, P.aeruginosa, B.subtilis and E.coli were 312.5μg / mL, 156.3μg / mL, 625μg / mL, 625μg / mL and 312.5μg / mL, respectively. It is worth mentioning that compared with CS, CS-GTA has extremely significant antibacterial properties against the five bacteria, especially for S.aureus and S.epidermidis, the antibacterial efficiency is 4 times and 8 times that of CS. The MIC of CS-VA against five fungi was 625μg / mL. Compared with CS, the MIC of CS-VA against S. aureus, S. epidermidis, P. aeruginosa, and B. subtilis was significantly lower. The MIC of CS-PCA against S. epidermidis was 312.5μg / mL, and the MIC of the other four fungi was 625μg / mL. Compared with CS, except for E. coli, the MIC of CS-PCA against S. epidermidis was significantly reduced, and the MIC of CS-PCA against S. epidermidis was reduced to one-fourth of the original.

[0203] Test Example 3

[0204] The products obtained in Examples 1-4 were subjected to an inhibition zone test.

[0205] The products obtained in Examples 1-4 (CS-SA, CS-GTA, CS-VA, CS-PCA) and chitosan (CS) were diluted to 2.5 mg / mL, 1.25 mg / mL and 0.625 mg / mL and used for later use. The solvent was 1% acetic acid solution, and 1% acetic acid solution was used as a blank control. Different bacteria: Staphylococcus aureus, (S. aureus), Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli were inoculated into a culture dish by streaking coating method, and each bacterium was repeated three times (n=3). Use tweezers to place the paper on the culture dish to ensure that the paper is evenly placed to avoid overlapping of the inhibition zones. Then, 10 μL of different modified chitosan 1% acetic acid solutions and 1% acetic acid solutions were added to the paper sheets, and the culture dishes were placed in an incubator at 37°C and 60% humidity. After 24 hours, the inhibition zone in the culture dishes was observed and the size of the inhibition zone was measured. The results are shown in Table 4 below.

[0206] Table 4

[0207]

[0208] Test Example 4

[0209] The products obtained in Examples 1-4 were evaluated for their antioxidant properties.

[0210] 1. DPPH free radical scavenging experiment

[0211] Preparation of sample solution and reference solution:

[0212] The products obtained in Examples 1-4 were weighed and prepared into solutions with a concentration of 10 mg / mL for later use, and diluted to 1000, 500, 250, 125, 62.5, 31.25 and 15.63 μg / mL. In the same manner, vitamin C (positive control) was prepared into solutions with the above concentrations for later use.

[0213] Preparation of DPPH working solution:

[0214] Weigh 3.05 mg of DPPH (Mw: 394.32) and prepare a 154.7 μmol / L DPPH working solution with anhydrous ethanol for later use.

[0215] Determination of samples:

[0216] Accurately pipette 150μL DPPH solution into a 96-well plate, and add 50μL of each sample solution with a concentration of 1000, 500, 250, 125, and 62.5μg / mL, respectively. Repeat 3 times for each concentration. Slightly shake and mix, and let it stand at room temperature in the dark for 30 minutes. Detect the absorbance of the reaction solution at a wavelength of 517nm. Use deionized water instead of the sample solution to be tested as a blank control, and use ascorbic acid (vitamin C) as a positive control. The calculation method of the DPPH clearance rate of each sample is as follows.

[0217] CR DPPH ·(%)=(1-A 样品 / A 空白对照 )×100%

[0218] Where A 样品 is the sample absorbance, A 空白对照 is the absorbance of blank control.

[0219] The experimental results are plotted as a bar graph, such as Fig.11 As shown, Vit C is the positive control. After 30 minutes of incubation, different modified chitosans in the 96-well plate showed different colors, indicating that different modified chitosans had different abilities to scavenge DPPH free radicals. Under the experimental conditions, the results in the figure show that within the concentration range of 15.63μg / mL-1000μg / mL, the scavenging rate of CS-SA at the highest concentration reached 88.08±0.44%, and the lowest concentration reached 43.83±1.22%. Although it is not as good as Vit C overall, it still shows good DPPH free radical scavenging ability. The scavenging ability of CS-GTA is not significantly different from that of Vit C, with a maximum of 88.18±0.08%. Even if the administration concentration is reduced to 15.63μg / mL and 31.25μg / mL, it still shows excellent DPPH free radical scavenging ability (78.16±1.09% and 87.70±0.14%), and is significantly stronger than Vit C (41.34% and 74.2%). In this experiment, the antioxidant capacity of CS-VA was significantly weaker than that of other modified chitosans, and the scavenging rate was only 39.72±6.43% even at the highest concentration. The scavenging rate of CS-PCA reached 84.74±3.26% at the highest concentration and 37.01±1.30% at the lowest concentration, showing a good DPPH free radical scavenging ability.

[0220] 2. ABTS free radical antioxidant test

[0221] Preparation of sample solution:

[0222] The products obtained in Examples 1-4 were weighed and prepared into 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL and 62.5 μg / mL solutions for later use. Following the same procedure, vitamin C (positive control) was prepared into solutions of the above concentrations for later use.

[0223] Preparation of ABTS reaction solution:

[0224] Prepare the working solution according to the instructions. Before use, take a bottle of reagent 2, add 11mL of reagent 1, shake and mix for 20 minutes, let it stand, and take the supernatant for use. (Note: prepare it and use it immediately).

[0225] Determination of samples:

[0226] Add 50 μL of sample solution to a 96-well plate, repeat 3 times for each concentration, add 150 μL of ABTS working solution, mix thoroughly, and measure the absorbance at 734 nm within 10 min.

[0227] CR ABTS (%) = (1-A 样品 / A 空白对照 )×100%

[0228] Where A 样品 is the sample absorbance, A 空白对照 is the absorbance of blank control.

[0229] The experimental results are plotted as a bar graph, such as Fig.12 As shown, Vit C is the positive control. Fig.12 It can be seen from the results that the ABTS free radical scavenging ability of the four modified chitosans is weaker than that of Vit C. At a concentration of 1000 μg / mL, the scavenging rate of CS-SA reached 63.29±2.68%, which is quite impressive. However, after reducing the concentration, the scavenging rate dropped significantly, and the scavenging rate was 40.16±7.02% at 500 μg / mL, and dropped to below 20% at a dosing concentration of 250 μg / mL. The scavenging ability of CS-GTA is slightly weaker than that of Vit C, reaching 90.52±0.48% at the maximum concentration. When the concentration dropped to 250 μg / mL, the scavenging rate was still at a high level of 77.11±6.94%. In this experiment, the ABTS free radical scavenging rate of CS-VA reached 76.04±2.40% at the highest concentration, but as the dosing concentration decreased, the scavenging rate dropped significantly, and the scavenging rate was only 26.07±1.34% at 250 μg / mL. The scavenging rate of CS-PCA at the highest concentration reached 87.04±0.33%, and at 500μg / mL it still maintained a high ABTS free radical scavenging ability of 59.63±1.63%.

[0230] 3. FRAP antioxidant assay

[0231] Preparation of sample solution:

[0232] The products obtained in Examples 1-4 were weighed and prepared into 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL and 125 μg / mL solutions for later use, and the FRAP reducing power of the sample solutions was determined using a FRAP total antioxidant capacity detection kit.

[0233] Preparation of FRAP reaction solution: Prepare the working solution according to the instructions.

[0234] Mix 10 μL of the sample solution prepared above with 190 μL of FRAP reaction solution in a 96-well plate, repeat 3 times for each concentration, and measure the absorbance of each well at 593 nm after 20 minutes of reaction. Kit standard curve: y = 1.2416x + 0.0134R 2 =0.9996.

[0235] Where x is the Trolox concentration (μmol / mL), y is the absorbance difference △A, that is, A 测定 -A 空 white.

[0236] That is, by calculating the ΔA after the reaction of different modified chitosans, the standard curve is used to obtain the antioxidant Trolox equivalent to express the FRAP total antioxidant capacity of different modified chitosans.

[0237] The above results are plotted into a bar graph, and the results are as follows Fig.13 As shown. Fig.13 It can be seen that within the concentration range of 125μg / mL-2000μg / mL, the total antioxidant capacity of CS-SA at the highest concentration of FRAP was only 0.58±0.01μmol / mL Trolox, while CS-GTA showed a stronger total antioxidant capacity of FRAP, which was equivalent to 2.38±0.21μmol / mL Trolox when the administration concentration was 2000μg / mL. Even when the concentration was reduced to 500μg / mL, it still showed a higher total antioxidant capacity of FRAP. In this experiment, the antioxidant capacity of CS-VA was significantly weaker than that of other modified chitosans, and its total antioxidant capacity for FRAP was the weakest. CS-PCA reached 0.86±0.06μmol / mL Trolox at the highest concentration, and its overall total antioxidant capacity of FRAP was higher than that of CS-SA.

[0238] Test Example 5

[0239] The products obtained in Examples 1-4 were subjected to cytotoxicity experiments.

[0240] Pre-treatment of cytotoxicity experiments:

[0241] The operation was performed in a clean bench. Cells with good growth were taken out. All the culture medium in the culture bottle was aspirated. 1.5 mL of trypsin was added and placed in an incubator for incubation for 3 minutes to digest the adherent HaCaT cells as much as possible until they were spherical. Then 5 mL of DMEM high-glucose complete medium was added to terminate the digestion, and all were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes. The supernatant was aspirated, 5 mL of PBS was added and blown evenly, and then centrifuged again. After the supernatant was aspirated, 5 mL of DMEM high-glucose complete medium was added and blown evenly. 1 drop of cell suspension was taken to calculate the number of cells, and 100 μL of cell suspension was aspirated per well and dropped on a 96-well plate, 5000 cells / well. There were 4 drugs in total, 5 different drug concentrations were set, and 5 repeated experiments were performed for each drug concentration. In addition, there were 5 blank groups. After the addition, the 96-well plate was placed in an incubator for culture (37°C, 5% CO2).

[0242] Cytotoxicity assay:

[0243] Observe the cells in the incubated 96-well plate and observe that most of the cells have attached to the wall, so as to carry out cytotoxicity experiments. Use DMEM high-glucose complete medium to dissolve the products of Examples 1-4 (CS-SA, CS-GTA, CS-VA, CS-PCA) to obtain solutions, and dilute the concentrations to 800μg / mL, 400μg / mL, 200μg / mL, 100μg / mL and 50μg / mL for standby use. All the culture medium in the 96-well plate for culturing HaCaT cells is aspirated, and the above-mentioned drugs of different concentrations are added respectively, and 5 repeated experiments are set up. The 96-well plate is then placed in an incubator (37°C, 5% CO2) and incubated for 24h.

[0244] After incubation for 24 h, the culture medium in the 96-well plate was removed and washed with PBS buffer, and then 100 μL of CCK-8 solution (concentration of 10%) diluted with complete culture medium was added to each well. The 96-well plate was incubated in an incubator for 2 h. The absorbance OD value at 450 nm was then measured with an ELISA instrument, and the cell survival rate was calculated according to the following formula.

[0245]

[0246] Among them, OD 给药组 is the absorbance value of the drug-treated group, OD 对照组 is the absorbance of the control group.

[0247] The obtained cell survival rate data were plotted into a bar graph, such as Fig.14As shown. The survival rates of the four modified chitosans on HaCaT cells were all above 100% in the concentration range of 50μg / mL-800μg / mL. The survival rates of cells treated with CS-SA were all above 140%, with the highest survival rate at 100μg / mL, reaching 203.54±6.71%, and 194.86±12.85% at 200μg / mL. Even the lowest reached 148.22±2.92% (at 400μg / mL). The overall trend of CS-GTA was different from that of CS-SA. In the range of 50μg / mL-800μg / mL, the higher the administration concentration, the higher the cell survival rate, which was 141.04±7.89% at 50μg / mL and 168.97±9.90% at 800μg / mL. The survival rate of CS-VA cells was the lowest at 400 μg / mL, but it also reached 99.12±2.11%, and the highest was 140.45±6.96% (concentration was 800 μg / mL). The survival rate of CS-PCA cells was the lowest at 50 μg / mL, but it also reached 99.81±2.96%, and the highest was 134.37±6.68% at 800 μg / mL.

[0248] Test Example 6

[0249] The products obtained in Examples 1-4 were subjected to a cell scratch test.

[0250] Pre-treatment of cell scratch assay:

[0251] First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, and then use a ruler to compare. The horizontal lines are approximately 0.5 to 1 cm apart, and cross the holes horizontally. Each hole should have at least 3 lines. Digest the cells in the logarithmic growth phase in the culture flask with trypsin to make them into freely suspended spherical cells. After counting with a cell counting plate, the number of cells is calculated according to 1×10 6 cells / well, inoculate the cell suspension in a 6-well culture plate, and the final total volume of culture medium in each well is 2 mL. Place the 6-well plate in an incubator (37°C, 5% CO2) and culture until the cells completely cover the bottom of the well plate, which takes about 48 hours.

[0252] Cell scratch assay:

[0253] The drug concentrations were selected as 100 μg / mL and 200 μg / mL. The drug administration group was prepared by dissolving the products of Examples 1-4 (CS-SA, CS-GTA, CS-VA, CS-PCA) in DMEM high-glucose serum-free medium, and the blank group was prepared by dissolving the products in DMEM high-glucose serum-free medium.

[0254] After the cells have covered the bottom of the six-well plate, use a 200μL pipette tip to scratch the bottom of the well plate parallel or perpendicular to the horizontal line on the back with the 6-well plate cover or ruler. After scratching, rinse the cells with PBS buffer 3 times, wash away the scratched cells, and add them to the drug group and blank group.

[0255] Wipe off the horizontal scratches on the back of the 6-well plate, take a picture of the scratch shape under a microscope at 4x magnification, try to ensure that the scratch is centered and vertical, and pay attention to the consistency of the background, add the sample to be tested, set the concentration gradient, and take pictures and samples at 0, 6, 12, and 24 hours. After opening the picture with Image J software, randomly draw 6 to 8 horizontal lines and calculate the mean of the distance between cells or the mean of the scratch area. Calculate the cell migration rate according to the following formula:

[0256]

[0257] Where W0 is the scratch width at 0h, W t is the scratch width at time t.

[0258] Cell migration plays an important role in the wound healing process. If the cell migration at the wound site is slow, it will also affect the wound healing process. Fig.15 The results of the cell migration experiment (100 μg / mL) are shown. It can be seen intuitively that over time, the cells in the drug group gradually migrated to the center of the scratch, while the cells in the blank control group migrated slowly. Fig.16 The figure is the cell migration analysis chart (100μg / mL). At 6h after administration, the migration rate of the blank control group was 12.33±2.23%. At this time, the migration rate of the CS-SA group and the CS-VA group was significantly higher than that of the blank control (p<0.01, p<0.05), reaching 20.25±0.04% and 19.61±1.44%, respectively. The cell migration rates of the CS-GTA group and the CS-PCA group were lower than those of the blank control, only 10.35±3.14% and 5.89±0.79%. At 12h, the cell migration rates of CS-SA and CS-VA reached 27.68±3.14% and 28.59±1.75%, significantly higher than those of the blank control (p<0.0001). At this time, the cell migration rate of CS-GTA also reached 19.19±2.43%, significantly higher than that of the blank control (p<0.01), but there was no significant difference between CS-PCA and the blank control. At 24h, the cell migration rate of the blank control was 24.37±0.94%, and the four modified chitosans were significantly higher than the blank control (p<0.0001), among which CS-SA was 46.99±1.59%, CS-GTA was the highest, reaching 48.22±3.27%, CS-VA and CS-PCA were 47.49±3.31% and 39.02±1.51%, respectively.

[0259] like Fig.17 The figure shows the results of the cell migration experiment (200 μg / mL). As time goes by, the cells in the drug-treated group gradually migrate to the center of the scratch and migrate rapidly, while the cells in the blank control group migrate slowly. Fig.18 The figure is a cell migration analysis chart (200μg / mL). At 6h after administration, the migration rate of the blank control group was 6.27±1.64%, while the migration rates of the CS-SA group and CS-VA group were no different from the blank control, which were 7.81±0.16% and 5.98±1.25%, respectively. At this time, the CS-GTA group and CS-PCA group were significantly higher than the blank control (p<0.001, p<0.0001), reaching 14.62±2.49% and 17.72±2.56%, respectively. At 12h, the cell migration rate of the blank control was 9.40±1.66%, and the CS-SA group had no significant difference from the blank control at this time, which was only 10.97±0.57%. There were significant differences between CS-GTA and CS-VA (p < 0.01), 16.55 ± 1.12% and 16.32 ± 0.58%, respectively, and CS-PCA reached 26.49 ± 2.20% (p < 0.0001). At 24h, the cell migration rate of the blank control was 18.65 ± 1.14%, and the four modified chitosans were significantly higher than the blank control (p < 0.0001), among which CS-SA was 48.76 ± 1.42%, CS-GTA was the highest, reaching 52.20 ± 0.85%, CS-VA and CS-PCA were 49.10 ± 3.67%, 40.92 ± 2.32%, respectively.

[0260] Test Example 7

[0261] The products obtained in Examples 13-16 were subjected to in vivo efficacy experiments.

[0262] 1. Hemolysis test

[0263] 1.1 Preparation of diluted blood samples

[0264] Blood was collected from the abdominal aorta of SD rats and collected in anticoagulation tubes. After standing for 30 minutes, an equal amount of saline was added to the anticoagulation tube with a syringe, mixed and centrifuged at 2500rpm for 5 minutes, and the supernatant was discarded. The precipitated red blood cells were repeatedly washed with saline 3 to 4 times according to the above method until the supernatant was no longer red. The washed red blood cells were prepared into a 2% cell suspension (v / v) in a centrifuge tube with saline and set aside.

[0265] 1.2 Preparation of test samples

[0266] Prepare a 400 μg / mL sample solution of the modified chitosan hydrogel with physiological saline for later use, with 1 mL of deionized water as the positive control and 1 mL of physiological saline as the negative control, and keep the solution in a 37° C. water bath for 30 min.

[0267] 1.3 Sample testing

[0268] 1 mL of diluted blood sample was added to each test tube and incubated at 37°C for 60 min. All samples were then centrifuged at 1000 rpm for 15 min at room temperature, and the supernatant was measured for absorbance at 545 nm using a UV-visible spectrophotometer to calculate the hemolysis rate.

[0269] The hemolysis rate was calculated as follows:

[0270] Hemolysis rate (%) = [(OD t -OD n ) / (OD p -OD n )]×100%

[0271] OD t OD n and OD p are the absorbance values ​​of the sample, negative control, and positive control, respectively.

[0272] The hemolysis results were as follows Fig.19 As shown, it can be observed that the negative control has basically no hemolysis, and the supernatant is colorless and clear, while the positive control is severely hemolyzed, and the supernatant is bright red. Compared with the positive control, the four modified chitosan hydrogel groups did not show obvious hemolysis, and the hemolysis rate was further analyzed by UV absorbance.

[0273] The obtained hemolysis rate data is plotted Fig. 20 .from Fig. 20 It can be seen that the data analysis was performed with the positive control hemolysis rate as 100%, and no obvious hemolysis was observed for CS-SA / VL hydrogel, CS-GTA / VL hydrogel, CS-VA / VL hydrogel and CS-PCA / VL hydrogel, and the hemolysis rates were all below 4%, which were 0.29±1.37%, 3.17±0.44%, 1.98±0.19% and 2.87±0.41%, respectively. Therefore, the modified chitosan hydrogel has good blood compatibility and is suitable for use as a wound dressing.

[0274] 2. Animal experiments

[0275] 2.1 Grouping and modeling

[0276] Male SD rats weighing about 220 g were randomly divided into 6 groups, namely, blank group, model group (control group), syringic acid modified chitosan hydrogel (CS-SA / VL) group, gentisic acid modified chitosan hydrogel (CS-GTA / VL) group, vanillic acid modified chitosan hydrogel (CS-VA / VL) group and protocatechuic acid modified chitosan hydrogel (CS-PCA / VL) group, with 6 rats in each group.

[0277] After 2 days of adaptive feeding in a suitable environment, the back of SD rats was depilated and adaptive feeding was carried out for 1 day after depilation. Then, a shear wound model was established: after the rats were anesthetized with sodium pentobarbital (3%), surgical scissors were used to shear the depilated skin on the back of the rats, except for the blank group, to create a wound with a diameter of 15 mm.

[0278] 2.2 Animal Dosing

[0279] Dosage:

[0280] On the second day after the modeling, after anesthetizing each group of rats, the hydrogel of Examples 13-16 was applied to the wound site of each treatment group at 1 mL / 250 g body weight, the blank group was fed normally without treatment, and the model group was only anesthetized. The modeling cycle lasted for 12 days. The drug was administered every other day, and the SD rats were weighed each time the drug was administered. The back of the rat was photographed to record the wound diameter, and the changes in the wound were observed. The wound healing rate (WR) was calculated by the following formula:

[0281] A0=πD0 2 At=πDt 2

[0282]

[0283] Among them, D0 represents the wound diameter on the day of modeling (mm), Dt represents the wound diameter at time t (mm), A0 is the wound area on the day of modeling, and At is the wound area at time t after modeling.

[0284] The change trend of the wounds of experimental rats during treatment is as follows Fig.21 The weight change curve of experimental rats (n=6) is shown in Fig. 22 shown.

[0285] The calculated wound healing rates are shown in Table 5 below.

[0286] Table 5

[0287] Model Group CS-SA / VL CS-GTA / VL CS-VA / VL CS-PCA / VL Day 2 2.43±1.98 43.99±6.70 43.82±11.88 30.51±8.77 33.65±10.20 Day 4 6.58±4.29 57.50±3.58 64.09±4.66 49.89±9.25 53.16±11.20 Day 6 16.28±10.52 72.74±5.28 70.46±4.72 63.49±5.01 66.65±5.16 Day 8 46.14±11.18 83.98±3.42 82.30±5.18 80.85±2.50 80.58±3.57 Day 10 65.86±10.94 92.48±0.97 91.23±2.07 92.92±1.59 91.64±1.17 Day 12 82.43±3.42 98.52±0.86 97.69±2.62 99.04±0.81 96.85±1.71

[0288] As can be seen from Table 5, during the administration cycle, the wound healing rate of the hydrogel administration group was significantly higher than that of the model group (****p<0.0001). On the second day of modeling, the hydrogel administration group was significantly higher than the model group (****p<0.0001), and the healing rates of the CS-SA / VL and CS-GTA / VL groups reached more than 43%, and the CS-VA / VL and CS-PCA / VL groups also reached more than 30%, while the model group was only 2.43±1.98%. On the fourth day of modeling, the wound healing rates of the hydrogel administration group reached 50% or more, while the model group was only 6.58±4.29%. On the last day of the administration cycle, that is, on the 12th day of modeling, the wounds in the hydrogel administration group had basically healed, reaching 98.52±0.86%, 97.69±2.62%, 99.04±0.81% and 96.85±1.71%, respectively, while the model group was only 82.43±3.42%.

[0289] 2.3 Preparation of skin pathology sections

[0290] After the medication cycle, the SD rats were anesthetized and the skin of the rats was peeled and collected. The site of the previous modeling was found, the skin was cut at the edge, and it was completely cut off. The skin was wrapped in tin foil and fixed in 4% paraformaldehyde. After dehydration, wax immersion and embedding, it was cut into 4μm thick slices, the tissue sections were stained with hematoxylin and eosin, and the sections were sealed with neutral gum. After fixation, the sections were scanned with a fully automatic digital pathology section scanner.

[0291] The tissue morphology of the wound of the trauma rat is shown in the figure Fig.23 As shown. By observing the tissue morphology of the healed wound, it can be seen that the subcutaneous tissue structure of the model group is relatively disordered, and there are no clear and orderly structures of skin appendages such as hair follicles and sebaceous glands, accompanied by inflammatory infiltration and a small amount of subcutaneous bleeding; in addition, the skin of the unhealed wound is severely damaged, and no skin structure is seen. After treatment with modified chitosan hydrogel, significant changes occurred in the wounds of different drug-administered groups. New blood vessels, hair follicles, sebaceous glands and other cell structures can be seen in the hydrogel-administered group, and the skin appendages are arranged neatly and orderly, without obvious inflammatory infiltration, and the wound healing process is significantly faster than that of the model group.

[0292] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing phenolic acid modified chitosan, characterized in that: An amino activator is added to the chitosan solution and mixed in the dark, then a phenolic acid solution is added and mixed evenly, and finally a carboxyl activator is added dropwise to react in the dark to obtain phenolic acid-modified chitosan; Wherein, the phenolic acid includes syringic acid, gentisic acid, vanillic acid or protocatechuic acid.

2. The preparation method according to claim 1, characterized in that: The amino activator includes 1-hydroxybenzotriazole and N-hydroxysuccinimide; Preferably, the carboxyl activating agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the amino activator to chitosan is 1:1-3; Preferably, the molar ratio of phenolic acid to chitosan is 1:1 to 3; Preferably, the molar ratio of the carboxyl activator to chitosan is 1:1-3.

4. The preparation method according to claim 1, characterized in that: The light-proof mixing time is 2 to 10 hours; Preferably, the reaction time in the dark is 12 to 48 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In the chitosan solution, the solvent includes an acetic acid aqueous solution; Preferably, the volume percentage of acetic acid in the acetic acid aqueous solution is 1 to 5%, preferably 1 to 3%; Preferably, in the chitosan solution, the concentration of chitosan is 10-15 mg / mL.

6. The preparation method according to any one of claims 1 to 4, characterized in that: It also includes the separation process after the reaction in the dark; Preferably, the separation method is dialysis; Preferably, deionized water is used as the dialysate, and the reaction solution is dialyzed using a dialysis bag to obtain the phenolic acid-modified chitosan; Preferably, the dialysis time is 2 to 5 days; Preferably, during the dialysis process, the dialysate is replaced every 8 to 16 hours.

7. A phenolic acid modified chitosan, characterized in that: The preparation method is described in any one of claims 1 to 6.

8. A phenolic acid modified chitosan hydrogel, characterized in that: The aqueous solution of phenolic acid modified chitosan and the aqueous solution of vanillin are mixed for gelation to obtain phenolic acid modified chitosan hydrogel; Wherein, the aqueous solution of phenolic acid-modified chitosan includes the phenolic acid-modified chitosan according to claim 7.

9. The phenolic acid modified chitosan hydrogel according to claim 8, characterized in that: The gelling time is 20 to 60 minutes; Preferably, the concentration of the aqueous solution of phenolic acid modified chitosan is 20 to 50 mg / mL; Preferably, the concentration of the vanillin aqueous solution is 20 to 50 mg / mL; Preferably, the volume ratio of the aqueous solution of phenolic acid modified chitosan to the aqueous solution of vanillin is 1:

1.

10. Use of the phenolic acid modified chitosan hydrogel according to claim 8 or 9 in preparing a medicine for promoting wound healing.

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