Antibacterial carboxymethyl cellulose hemostatic gauze and preparation method thereof

Abstract

The invention provides antibacterial carboxymethyl cellulose hemostatic gauze and a preparation method thereof.The preparation method comprises the steps that carboxymethyl cellulose gauze is soaked in a PBS buffer solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added into the PBS buffer solution, and a soaking solution is obtained after uniform mixing; adding the graft modified alginate oligosaccharide into the soaking solution, mixing and stirring to generate grafting reaction, taking out the carboxymethyl cellulose gauze after the reaction is finished, washing and drying to obtain the antibacterial carboxymethyl cellulose hemostatic gauze. According to the preparation method disclosed by the invention, the catechol-grafted modified alginate oligosaccharide and partially carboxymethylated carboxymethyl cellulose gauze are subjected to amidation grafting compounding, so that the hemostatic gauze with excellent antibacterial and hemostatic properties is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of medical supplies and relates to an antibacterial carboxymethyl cellulose hemostatic gauze and a preparation method thereof. Background Art

[0002] Whether in war or peacetime, uncontrolled bleeding is the main cause of death before receiving emergency treatment. Deaths due to untimely hemostasis account for about 30% to 40% of trauma deaths. Excessive bleeding also increases the risk of hemorrhagic shock, coagulopathy, infection, and multiple organ failure. Therefore, the development of safe and fast hemostatic functional materials has become the focus of hemostasis research at this stage. The ideal hemostatic material should have the following characteristics: high hemostasis efficiency, high safety, low cost, and light weight.

[0003] Traditional medical gauze is widely used in the field of hemostasis because of its softness, breathability, cheap and easy-to-obtain characteristics, and almost no irritation to human skin. However, common medical gauze in clinical applications, due to its strong hydrophilicity, will inevitably absorb too much blood in wound treatment, increase blood loss, cause wound recovery to become slow or even hinder wound recovery, and is not conducive to rapid hemostasis. Excessive residual biological fluid also leads to skin immersion and bacterial growth. In addition, cotton gauze has poor tissue adhesion, neither starts nor promotes platelet activation or clot formation. It concentrates platelets, red blood cells and coagulation factors by absorbing water in the blood, thereby achieving coagulation, and the hemostatic effect is not obvious, lacking a strong physiological hemostatic effect. Therefore, it is urgent to design a hemostatic gauze with physiological hemostasis function that can achieve rapid hemostasis and antibacterial. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an antibacterial carboxymethyl cellulose hemostatic gauze and a preparation method thereof. The present invention firstly performs hydroxylation and amino modification on brown algae oligosaccharide, and adopts 3,4-dihydroxybenzoic acid containing catechol groups as a modification reagent, and grafts catechol on amino-modified brown algae oligosaccharide to prepare grafted modified brown algae oligosaccharide with hemostatic and antibacterial properties, tissue adhesion and biocompatibility; then, the grafted modified brown algae oligosaccharide grafted with catechol and partially carboxymethylated carboxymethyl cellulose gauze are amidated and grafted to prepare hemostatic gauze with excellent antibacterial and hemostatic properties.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, the preparation method comprising:

[0007] (I) soaking the degreased gauze in a sodium hydroxide solution for alkalization treatment, taking out the degreased gauze after the treatment, washing and drying to obtain alkalized gauze; soaking the alkalized gauze in a chloroacetic acid solution, performing an etherification reaction under heating conditions, taking out the alkalized gauze after the reaction, washing and drying to obtain carboxymethyl cellulose gauze;

[0008] (II) dispersing brown algae oligosaccharide in a sodium hydroxide solution, mixing, stirring and heating to carry out a hydroxylation reaction, filtering, washing and drying after the reaction is completed to obtain hydroxylated brown algae oligosaccharide; dispersing the hydroxylated brown algae oligosaccharide and an amino modifier in deionized water to obtain a mixed solution, adding epichlorohydrin to the mixed solution, mixing, stirring and heating to carry out a reaction, filtering, washing and drying after the reaction is completed to obtain amino-modified brown algae oligosaccharide;

[0009] (III) dissolving 3,4-dihydroxybenzoic acid in PBS buffer to obtain a grafting precursor solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the grafting precursor solution under a nitrogen atmosphere, and mixing them evenly to obtain a reaction solution; then adding the amino-modified brown algae oligosaccharide obtained in step (II) to the reaction solution, mixing and stirring to cause a grafting reaction, and after the reaction is completed, dialyzing and freeze-drying are performed to obtain a grafted modified brown algae oligosaccharide with catechol grafted on the surface;

[0010] (IV) soaking the carboxymethyl cellulose gauze in the PBS buffer solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the PBS buffer solution under a nitrogen atmosphere, and mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (III) to the soaking solution, mixing and stirring to cause a grafting reaction, and after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0011] Brown algae oligosaccharide is a degradation product of alginate, has a small molecular weight and good water solubility, and has biological activities such as anti-oxidation, anti-tumor, anti-inflammatory, and growth promotion. In addition, brown algae oligosaccharide has a certain film-forming property, and the formed film has a physical barrier function, which can maintain a local moist environment on the wound surface and promote wound healing. However, the antibacterial and antimicrobial ability of brown algae oligosaccharide is insufficient. For this reason, the present invention first performs hydroxylation and amino modification on brown algae oligosaccharide, and then uses 3,4-dihydroxybenzoic acid containing a catechol group as a modification reagent. By means of an amidation reaction between the amino group on the amino-modified brown algae oligosaccharide molecular chain and the carboxyl group on the 3,4-dihydroxybenzoic acid molecular chain, catechol is grafted on the amino-modified brown algae oligosaccharide, thereby preparing a grafted modified brown algae oligosaccharide with excellent hemostatic and coagulative properties, tissue adhesion, and antibacterial and antimicrobial properties.

[0012] On the one hand, the grafted modified brown algae oligosaccharide after catechol grafting has excellent tissue adhesion and hemostatic and coagulation properties. This is because the grafted catechol on the grafted modified brown algae oligosaccharide can be quickly anchored on the skin tissue through non-covalent bonds such as hydrogen bonds and π-π interactions, and has excellent tissue adhesion. It can form a physical barrier around the wound, thereby forming a closed microenvironment on the open wound, which plays a role in hemostasis, coagulation and prevention of bacterial infection. On the other hand, the grafted modified brown algae oligosaccharide after catechol grafting has excellent antibacterial and antibacterial properties, which makes up for the defect of insufficient antibacterial and antibacterial properties of brown algae oligosaccharide. Catechol can chelate metal ions and some special heteropolymeric proteins that affect the normal physiological activities of bacteria, thereby affecting the cell membrane, disrupting the normal physiological metabolism of bacteria, and achieving bactericidal and antibacterial effects; at the same time, the phenolic functional groups on catechol can enhance the antioxidant properties of the grafted modified brown algae oligosaccharide, remove reactive oxygen, and thus significantly improve the wound healing rate.

[0013] Cellulose and other polysaccharide compounds have many advantages such as abundant sources, biodegradability, biocompatibility, and non-immunoreactivity, and are important raw materials for biomedical materials. Carboxymethyl cellulose is a derivative of cellulose with high water absorption. When carboxymethyl cellulose is completely infiltrated by blood, it will swell and appear as a light brown or black block colloid. The block colloid helps the blood to coagulate into blocks, which can not only fill the gaps in the wound surface by volume expansion, but also compress blood vessels, which helps to accelerate blood coagulation and achieve the purpose of hemostasis.

[0014] The present invention carries out amidation grafting and compounding of grafted modified brown algae oligosaccharide grafted with catechol and partially carboxymethylated carboxymethyl cellulose gauze to prepare a hemostatic gauze with excellent antibacterial hemostatic performance. When in use, the grafted modified brown algae oligosaccharide directly contacts the bleeding wound. Since the grafted modified brown algae oligosaccharide contains positively charged amino groups, it can interact with the negatively charged blood cell membrane, thereby significantly enhancing the adhesion of the hemostatic gauze to red blood cells and platelets. It can effectively filter blood and promote the aggregation, adhesion and activation of red blood cells, platelets and plasma proteins, thereby playing a role in promoting hemostasis and coagulation. As a non-contact substrate for bleeding wounds, carboxymethyl cellulose gauze can effectively absorb the plasma filtered by the grafted modified brown algae oligosaccharide, create a relatively dry environment for the wound, and make the hemostatic gauze physically block the bleeding wound and stabilize the blood clot by tissue adhesion to prevent blood from seeping out from the bleeding point, and achieve the purpose of hemostasis through the synergistic effect between the carboxymethyl cellulose gauze and the grafted modified brown algae oligosaccharide.

[0015] As a preferred technical solution of the present invention, in step (I), the mass fraction of the sodium hydroxide solution is 20-30wt%, for example, it can be 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt% or 30wt%, but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0016] The present invention particularly limits the mass fraction of the sodium hydroxide solution to 20-30wt%. When the mass fraction of the sodium hydroxide solution is lower than 20wt%, the alkalization degree of the absorbent gauze is low, the generated alkali cellulose is less, and it is not conducive to the subsequent etherification reaction. As the mass fraction of the sodium hydroxide solution increases, the alkali cellulose content increases, which is conducive to promoting the etherification reaction. When the mass fraction of the sodium hydroxide solution is higher than 30wt%, the number of alkalizable hydroxyl groups on the cellulose macromolecular chain decreases, the difficulty of alkalization gradually increases, and the free alkali in the system increases, resulting in a large number of side reactions, generating byproducts such as sodium glycolate, causing the chloroacetic acid concentration in the reaction system to drop rapidly, so that the carboxymethyl cellulose generated by the etherification reaction is reduced, and finally affecting the hemostatic and coagulation function of the carboxymethyl cellulose gauze.

[0017] In some optional examples, the temperature of the sodium hydroxide solution is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0018] In some optional examples, the soaking time of the absorbent gauze in the sodium hydroxide solution is 40 to 50 min, for example, it can be 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min or 50 min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0019] As a preferred technical solution of the present invention, in step (I), the mass fraction of chloroacetic acid in the chloroacetic acid solution is 65-75wt%, for example, it can be 65wt%, 66wt%, 67wt%, 68wt%, 69wt%, 70wt%, 71wt%, 72wt%, 73wt%, 74wt% or 75wt%, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0020] The present invention particularly limits the mass fraction of chloroacetic acid in the chloroacetic acid solution to 65-75wt%. When the mass fraction of chloroacetic acid is lower than 65wt%, the amount of carboxymethyl cellulose generated by the reaction of alkali cellulose generated after alkalization of the degreased gauze with chloroacetic acid is low. With the increase of the mass fraction of chloroacetic acid, the probability of molecular collision between alkali cellulose and chloroacetic acid increases, and the content of carboxymethyl cellulose generated by the reaction increases. When the mass fraction of chloroacetic acid exceeds 75wt%, the pH value of the reaction system will be rapidly decreased, and the solution will be weakly alkaline, which is not conducive to the nucleophilic substitution etherification reaction, thereby reducing the amount of carboxymethyl cellulose generated, and finally affecting the hemostatic and coagulation function of the carboxymethyl cellulose gauze.

[0021] In some optional examples, the ratio of the mass of chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is (0.4-0.5):1, for example, it can be 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1, 0.49:1 or 0.5:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0022] In some optional examples, the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of ethanol to isopropanol is (3.5-4.5):1, for example, it can be 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1 or 4.5:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0023] In some optional examples, the heating temperature of the etherification reaction is 70-80°C, for example, it can be 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0024] The present invention specifically limits the heating temperature of the etherification reaction to 70-80°C. When the etherification temperature is lower than 70°C, the molecular thermal motion is inactive, the degree of etherification reaction between molecules is low, and the amount of carboxymethyl cellulose generated is small. As the etherification temperature increases, the molecular kinetic energy increases, and the amount of carboxymethyl cellulose generated gradually increases. When the etherification temperature exceeds 80°C, the generated carboxymethyl cellulose will form a carboxymethyl cellulose gel layer on the fiber surface, hindering the further penetration and diffusion of chloroacetic acid into alkali cellulose, resulting in a decrease in the amount of carboxymethyl cellulose generated and uneven distribution; in addition, too high a temperature also increases the occurrence of side reactions, generating byproducts such as sodium glycolate, resulting in a decrease in the concentration of chloroacetic acid in the reaction system, reducing the amount of carboxymethyl cellulose generated by the etherification reaction, and ultimately affecting the hemostatic and coagulation function of the carboxymethyl cellulose gauze.

[0025] In some optional examples, the alkalized gauze is immersed in the ethanol solution of chloroacetic acid for 2 to 3 hours, for example, 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours or 3.0 hours, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0026] As a preferred technical solution of the present invention, in step (II), the concentration of the sodium hydroxide solution is 5 to 10 mol / L, for example, it can be 5.0 mol / L, 5.5 mol / L, 6.0 mol / L, 6.5 mol / L, 7.0 mol / L, 7.5 mol / L, 8.0 mol / L, 8.5 mol / L, 9.0 mol / L, 9.5 mol / L or 10.0 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0027] In some optional examples, the mass ratio of the brown algae oligosaccharide to the sodium hydroxide solution is 1:(3-4), for example, it can be 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9 or 1:4.0, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0028] In some optional examples, the heating temperature of the hydroxylation reaction is 130-140°C, for example, it can be 130°C, 131°C, 132°C, 133°C, 134°C, 135°C, 136°C, 137°C, 138°C, 139°C or 140°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0029] In some optional examples, the stirring time of the hydroxylation reaction is 8 to 10 hours, for example, it can be 8.0 hours, 8.2 hours, 8.4 hours, 8.6 hours, 8.8 hours, 9.0 hours, 9.2 hours, 9.4 hours, 9.6 hours, 9.8 hours or 10.0 hours, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0030] As a preferred technical solution of the present invention, in step (II), the mass ratio of the hydroxylated brown algae oligosaccharide to the amino modifier is (3-4):1, for example, it can be 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1 or 4.0:1, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0031] In some optional examples, the ratio of the total mass of the hydroxylated brown algae oligosaccharides and the amino modifier to the mass of the deionized water is 1:(10-20), for example, it can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0032] In some optional examples, the amino modifier includes any one of ethylenediaminetetraacetic acid, triethylenetetramine, triethylenetetramine or polyetherimide, or a combination of at least two thereof.

[0033] In some optional examples, the ratio of the total mass of hydroxylated brown algae oligosaccharides and amino modifiers in the mixed solution to the volume of the epichlorohydrin is (12-15) g:1 mL, for example, it can be 12 g:1 mL, 12.5 g:1 mL, 13 g:1 mL, 13.5 g:1 mL, 14 g:1 mL, 14.5 g:1 mL or 15 g:1 mL, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0034] In some optional examples, the heating temperature when the mixed solution is mixed with epichlorohydrin is 60-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0035] In some optional examples, the stirring time when the mixed solution is mixed with epichlorohydrin is 3 to 5 hours, for example, it can be 3.0 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, 4.0 hours, 4.2 hours, 4.4 hours, 4.6 hours, 4.8 hours or 5.0 hours, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0036] As a preferred technical solution of the present invention, in step (III), the PBS buffer is prepared by the following method:

[0037] Sodium dihydrogen phosphate monohydrate, anhydrous disodium hydrogen phosphate and sodium chloride are dissolved in deionized water, mixed evenly to obtain a precursor buffer, and an alkali solution is added dropwise to the precursor buffer to adjust its pH value to obtain the PBS buffer.

[0038] In some optional examples, the concentration of sodium dihydrogen phosphate monohydrate in the precursor buffer is 6-7 mmol / L, for example, it can be 6.0 mmol / L, 6.1 mmol / L, 6.2 mmol / L, 6.3 mmol / L, 6.4 mmol / L, 6.5 mmol / L, 6.6 mmol / L, 6.7 mmol / L, 6.8 mmol / L, 6.9 mmol / L or 7.0 mmol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0039] In some optional examples, the concentration of anhydrous disodium hydrogen phosphate in the precursor buffer is 3-4 mmol / L, for example, 3.0 mmol / L, 3.1 mmol / L, 3.2 mmol / L, 3.3 mmol / L, 3.4 mmol / L, 3.5 mmol / L, 3.6 mmol / L, 3.7 mmol / L, 3.8 mmol / L, 3.9 mmol / L or 4.0 mmol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0040] In some optional examples, the concentration of sodium chloride in the precursor buffer is 0.1-0.2 mol / L, for example, it can be 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L or 0.2 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0041] In some optional examples, the concentration of the alkali solution is 0.1-0.5 mol / L, for example, it can be 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L or 0.5 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0042] In some optional examples, alkaline solution is added to the precursor buffer to adjust its pH value to 5-6, for example, it can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0043] In some optional examples, the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution is 0.5-1.5 mol / L, for example, it can be 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L or 1.5 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0044] In some optional examples, the concentration of N-hydroxysuccinimide in the reaction solution is 0.2-0.3 mol / L, for example, it can be 0.2 mol / L, 0.21 mol / L, 0.22 mol / L, 0.23 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, 0.27 mol / L, 0.28 mol / L, 0.29 mol / L or 0.3 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0045] In some optional examples, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the reaction solution is 0.2-0.3 mol / L, for example, 0.2 mol / L, 0.21 mol / L, 0.22 mol / L, 0.23 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, 0.27 mol / L, 0.28 mol / L, 0.29 mol / L or 0.3 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0046] In some optional examples, the grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are mixed by magnetic stirring, and the rotation speed of the magnetic stirring is 800-1000r / min, for example, it can be 800r / min, 820r / min, 840r / min, 860r / min, 880r / min, 900r / min, 920r / min, 940r / min, 960r / min, 980r / min or 1000r / min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0047] In some optional examples, the mixing time of the grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 30 to 40 minutes, for example, it can be 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0048] In some optional examples, the mixing temperature of the grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 20-30°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0049] As a preferred technical solution of the present invention, in step (III), the molar ratio of 3,4-dihydroxybenzoic acid to the amino-modified brown algae oligosaccharide in the reaction solution is (250-350):1, for example, it can be 250:1, 260:1, 270:1, 280:1, 290:1, 300:1, 310:1, 320:1, 330:1, 340:1 or 350:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0050] The present invention particularly limits the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution to (250-350):1. The molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide directly affects the concentration of 3,4-dihydroxybenzoic acid in the reaction system. The higher the concentration of 3,4-dihydroxybenzoic acid, the greater the probability of amidation reaction between 3,4-dihydroxybenzoic acid and the amino group on the amino-modified brown algae oligosaccharide, and the higher the grafting rate.

[0051] In some optional examples, the temperature of the grafting reaction is 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0052] In some optional examples, the stirring rate of the grafting reaction is 800-1000 r / min, for example, it can be 800 r / min, 820 r / min, 840 r / min, 860 r / min, 880 r / min, 900 r / min, 920 r / min, 940 r / min, 960 r / min, 980 r / min or 1000 r / min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0053] In some optional examples, the stirring time of the grafting reaction is 5 to 6 hours, for example, it can be 5.0h, 5.1h, 5.2h, 5.3h, 5.4h, 5.5h, 5.6h, 5.7h, 5.8h, 5.9h or 6.0h, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0054] In some optional examples, the molecular weight cutoff of the dialysis bag used in the dialysis operation is 3000-4000Da, for example, it can be 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3500Da, 3600Da, 3700Da, 3800Da, 3900Da or 4000Da, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0055] In some optional examples, the freeze-drying temperature is -30 to -40°C, for example, it can be -30°C, -31°C, -32°C, -33°C, -34°C, -35°C, -36°C, -37°C, -38°C, -39°C or -40°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0056] In some optional examples, the freeze-drying time is 24 to 48 hours, for example, it can be 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours or 48 hours, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0057] As a preferred technical solution of the present invention, in step (IV), the immersion ratio of the carboxymethyl cellulose gauze in the PBS buffer is 1g:(15-20)mL, for example, it can be 1g:15mL, 1g:15.5mL, 1g:16mL, 1g:16.5mL, 1g:17mL, 1g:17.5mL, 1g:18mL, 1g:18.5mL, 1g:19mL, 1g:19.5mL or 1g:20mL, but it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0058] In some optional examples, the mass ratio of the carboxymethyl cellulose gauze, the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:(0.5-0.8):(1.5-2.5), for example, it can be 1:0.5:1.5, 1:0.55:1.6, 1:0.6:1.7, 1:0.65:1.8, 1:0.7:1.9, 1:0.75:2.0, 1:0.8:2.1, 1:0.5:2.2, 1:0.6:2.3, 1:0.7:2.4 or 1:0.8:2.5, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0059] As a preferred technical solution of the present invention, in step (IV), the mass ratio of the carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide is 1:(0.5-1), for example, it can be 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:0.95 or 1:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0060] In some optional examples, the temperature of the grafting reaction is 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0061] In some optional examples, the stirring rate of the grafting reaction is 800-1000 r / min, for example, it can be 800 r / min, 820 r / min, 840 r / min, 860 r / min, 880 r / min, 900 r / min, 920 r / min, 940 r / min, 960 r / min, 980 r / min or 1000 r / min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0062] In some optional examples, the stirring time of the grafting reaction is 5 to 6 hours, for example, it can be 5.0h, 5.1h, 5.2h, 5.3h, 5.4h, 5.5h, 5.6h, 5.7h, 5.8h, 5.9h or 6.0h, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0063] In a second aspect, the present invention provides an antibacterial carboxymethyl cellulose hemostatic gauze prepared by the preparation method described in the first aspect.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] Brown algae oligosaccharide is a degradation product of alginate, has a small molecular weight and good water solubility, and has biological activities such as anti-oxidation, anti-tumor, anti-inflammatory, and growth promotion. In addition, brown algae oligosaccharide has a certain film-forming property, and the formed film has a physical barrier function, which can maintain a local moist environment on the wound surface and promote wound healing. However, the antibacterial and antimicrobial ability of brown algae oligosaccharide is insufficient. For this reason, the present invention first performs hydroxylation and amino modification on brown algae oligosaccharide, and then uses 3,4-dihydroxybenzoic acid containing a catechol group as a modification reagent. By means of an amidation reaction between the amino group on the amino-modified brown algae oligosaccharide molecular chain and the carboxyl group on the 3,4-dihydroxybenzoic acid molecular chain, catechol is grafted on the amino-modified brown algae oligosaccharide, thereby preparing a grafted modified brown algae oligosaccharide with excellent hemostatic and coagulative properties, tissue adhesion, and antibacterial and antimicrobial properties.

[0066] On the one hand, the grafted modified brown algae oligosaccharide after catechol grafting has excellent tissue adhesion and hemostatic and coagulation properties. This is because the grafted catechol on the grafted modified brown algae oligosaccharide can be quickly anchored on the skin tissue through non-covalent bonds such as hydrogen bonds and π-π interactions, and has excellent tissue adhesion. It can form a physical barrier around the wound, thereby forming a closed microenvironment on the open wound, which plays a role in hemostasis, coagulation and prevention of bacterial infection. On the other hand, the grafted modified brown algae oligosaccharide after catechol grafting has excellent antibacterial and antibacterial properties, which makes up for the defect of insufficient antibacterial and antibacterial properties of brown algae oligosaccharide. Catechol can chelate metal ions and some special heteropolymeric proteins that affect the normal physiological activities of bacteria, thereby affecting the cell membrane, disrupting the normal physiological metabolism of bacteria, and achieving bactericidal and antibacterial effects; at the same time, the phenolic functional groups on catechol can enhance the antioxidant properties of the grafted modified brown algae oligosaccharide, remove reactive oxygen, and thus significantly improve the wound healing rate.

[0067] Cellulose and other polysaccharide compounds have many advantages such as abundant sources, biodegradability, biocompatibility, and non-immunoreactivity, and are important raw materials for biomedical materials. Carboxymethyl cellulose is a derivative of cellulose with high water absorption. When carboxymethyl cellulose is completely infiltrated by blood, it will swell and appear as a light brown or black block colloid. The block colloid helps the blood to coagulate into blocks, which can not only fill the gaps in the wound surface by volume expansion, but also compress blood vessels, which helps to accelerate blood coagulation and achieve the purpose of hemostasis.

[0068] The present invention carries out amidation grafting and compounding of grafted modified brown algae oligosaccharide grafted with catechol and partially carboxymethylated carboxymethyl cellulose gauze to prepare a hemostatic gauze with excellent antibacterial hemostatic performance. When in use, the grafted modified brown algae oligosaccharide directly contacts the bleeding wound. Since the grafted modified brown algae oligosaccharide contains positively charged amino groups, it can interact with the negatively charged blood cell membrane, thereby significantly enhancing the adhesion of the hemostatic gauze to red blood cells and platelets. It can effectively filter blood and promote the aggregation, adhesion and activation of red blood cells, platelets and plasma proteins, thereby playing a role in promoting hemostasis and coagulation. As a non-contact substrate for bleeding wounds, carboxymethyl cellulose gauze can effectively absorb the plasma filtered by the grafted modified brown algae oligosaccharide, create a relatively dry environment for the wound, and make the hemostatic gauze physically block the bleeding wound and stabilize the blood clot by tissue adhesion to prevent blood from seeping out from the bleeding point, and achieve the purpose of hemostasis through the synergistic effect between the carboxymethyl cellulose gauze and the grafted modified brown algae oligosaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 A process flow chart of the preparation of the antibacterial carboxymethyl cellulose hemostatic gauze provided in Examples 1-5 of the present invention;

[0070] Figure 2 This is a scanning electron microscope image of the absorbent gauze used in Example 1 of the present invention;

[0071] Figure 3 This is a scanning electron microscope image of the carboxymethyl cellulose gauze prepared in Example 1 of the present invention;

[0072] Figure 4 The infrared spectra of the carboxymethyl cellulose gauze, grafted modified brown algae oligosaccharide and antibacterial carboxymethyl cellulose hemostatic gauze prepared in Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0073] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and their accompanying drawings. The embodiments recorded herein are specific embodiments of the present invention, which are used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary and should not be construed as limitations on the embodiments of the present invention and the scope of protection of the present invention. In addition to the embodiments recorded herein, those skilled in the art can also adopt other obvious technical solutions based on the contents disclosed in the claims of this application and its specification, including technical solutions that adopt any obvious replacements and modifications to the embodiments recorded herein.

[0074] Example 1

[0075] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, such as Figure 1 As shown, the preparation method specifically comprises the following steps:

[0076] (1) soaking the absorbent gauze in a 20 wt % sodium hydroxide solution for alkalization treatment, wherein the temperature of the sodium hydroxide solution is 20° C. and the soaking time is 50 min. After the treatment, the absorbent gauze is taken out, washed and dried to obtain alkalized gauze;

[0077] The alkalized gauze is immersed in a 65wt% chloroacetic acid solution, wherein the mass ratio of the chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is 0.4:1, and the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of ethanol to isopropanol is 3.5:1; the mixture is mixed and stirred for 3h under a heating condition of 70°C to perform an etherification reaction, and after the reaction is completed, the alkalized gauze is taken out, washed and dried to obtain a carboxymethyl cellulose gauze;

[0078] (2) dispersing brown algae oligosaccharide in a 5 mol / L sodium hydroxide solution, with the mass ratio of brown algae oligosaccharide to the sodium hydroxide solution being 1:4, and mixing and stirring at 130° C. for 10 h to carry out a hydroxylation reaction. After the reaction is completed, filtering, washing and drying are performed to obtain hydroxylated brown algae oligosaccharide;

[0079] Dispersing hydroxylated brown algae oligosaccharide and ethylenediaminetetraacetic acid in deionized water to obtain a mixed solution, wherein the mass ratio of hydroxylated brown algae oligosaccharide to ethylenediaminetetraacetic acid is 3:1, and the mass ratio of the total mass of hydroxylated brown algae oligosaccharide and ethylenediaminetetraacetic acid to the mass of deionized water is 1:10, adding epichlorohydrin to the mixed solution, wherein the volume ratio of the total mass of hydroxylated brown algae oligosaccharide and ethylenediaminetetraacetic acid to epichlorohydrin is 12g:1mL, mixing and stirring for 5h under heating conditions of 60°C to react, filtering, washing and drying after the reaction is completed to obtain amino-modified brown algae oligosaccharide;

[0080] (3) dissolving sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate anhydrous and sodium chloride in deionized water and mixing them evenly to obtain a precursor buffer, wherein the concentration of sodium dihydrogen phosphate monohydrate in the precursor buffer is 6 mmol / L, the concentration of disodium hydrogen phosphate anhydrous is 3 mmol / L, and the concentration of sodium chloride is 0.1 mol / L; adding 0.1 mol / L of sodium hydroxide solution to the precursor buffer to adjust its pH value to 5 to obtain a PBS buffer;

[0081] 3,4-dihydroxybenzoic acid was dissolved in the PBS buffer prepared above to obtain a grafting precursor solution, wherein the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution was 0.5 mol / L; N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to the grafting precursor solution under a nitrogen atmosphere, wherein the concentration of NHS in the reaction solution was 0.2 mol / L, and the concentration of EDC was 0.2 mol / L. l / L, magnetically stirred at a speed of 800r / min and 20°C for 40min to obtain a reaction solution; then, the amino-modified brown algae oligosaccharide obtained in step (2) is added to the reaction solution, the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution is 250:1, mixed and stirred at a speed of 800r / min and a heating condition of 30°C for 6h to cause a grafting reaction, and dialyzed and freeze-dried after the reaction, wherein the dialysis bag used in the dialysis operation has a molecular weight cutoff of 3000Da, the freeze-drying temperature is -30°C, and the time is 48h, and after freeze-drying, a grafted modified brown algae oligosaccharide with catechol grafted on the surface is obtained;

[0082] (4) Soaking carboxymethyl cellulose gauze in PBS buffer solution, the soaking ratio of carboxymethyl cellulose gauze in PBS buffer solution is 1 g:15 mL; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the PBS buffer solution, the mass ratio of carboxymethyl cellulose gauze, NHS and EDC being 1:0.5:1.5; mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (3) to the soaking solution, the mass ratio of carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide being 1:0.5; mixing and stirring at a speed of 800 r / min and heating at 30° C. for 6 h to cause a grafting reaction; after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it, to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0083] Figure 2 The scanning electron microscope image of the absorbent gauze used in this embodiment is shown in FIG. Figure 3This is a scanning electron microscope image of the carboxymethyl cellulose gauze prepared in this example. The fiber surface in the degreased gauze has more hairiness, is rough and not smooth, and the fibers are arranged more neatly. The fiber diameter of the carboxymethyl cellulose gauze increases. This is because after the fiber is modified by carboxymethylation, part of the hydroxyl groups on the glucose ring are replaced by carboxymethyl groups, and the steric hindrance increases, resulting in a decrease in hydrogen bonds between cellulose macromolecules, increasing the disorder of the macromolecular chain arrangement, and water easily enters, causing the fiber to swell and increase its diameter.

[0084] Figure 4 The infrared spectra of carboxymethyl cellulose gauze, grafted modified brown algae oligosaccharide and antibacterial carboxymethyl cellulose hemostatic gauze are shown in the figure. As can be seen from the figure, in the infrared spectrum of carboxymethyl cellulose gauze, 2917cm -1 The stretching vibration absorption peak of the CH single bond is 1600-1370cm -1 The asymmetric stretching vibration peak of the carboxyl group (COO-) (1607 cm -1 ) and the symmetrical stretching vibration peak (1414cm -1 and 1377cm -1 ), 1323cm -1 The peak at 3435cm is the stretching vibration peak of C-OH. -1 The stretching vibration peaks of OH and NH are at 1065 cm -1 The peak at 1598cm is the stretching vibration peak of CO. -1 The stretching vibration peak at 1510 cm -1 The peak at 1510 cm is the stretching vibration peak of the C=C double bond of the benzene ring in catechol. -1 The stretching vibration peak of the C=C double bond of the catechol benzene ring of the grafted modified brown algae oligosaccharide appeared at the center, indicating that the grafted modified brown algae oligosaccharide was successfully grafted onto the carboxymethyl cellulose gauze.

[0085] Example 2

[0086] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, such as Figure 1 As shown, the preparation method specifically comprises the following steps:

[0087] (1) soaking the absorbent gauze in a 22t% sodium hydroxide solution for alkalization treatment, wherein the temperature of the sodium hydroxide solution is 22 and the soaking time is 48 min. After the treatment, the absorbent gauze is taken out, washed and dried to obtain alkalized gauze;

[0088] The alkalized gauze is immersed in a 68wt% chloroacetic acid solution, wherein the ratio of the mass of the chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is 0.42:1, and the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of ethanol to isopropanol is 3.8:1; the mixture is mixed and stirred for 2.8h under a heating condition of 72°C to perform an etherification reaction, and after the reaction is completed, the alkalized gauze is taken out, washed and dried to obtain a carboxymethyl cellulose gauze;

[0089] (2) dispersing brown algae oligosaccharide in a 6 mol / L sodium hydroxide solution, with the mass ratio of brown algae oligosaccharide to the sodium hydroxide solution being 1:3.8, and mixing and stirring at 132° C. for 9.5 h to perform a hydroxylation reaction. After the reaction is completed, filtering, washing and drying are performed to obtain hydroxylated brown algae oligosaccharide;

[0090] Dispersing hydroxylated brown algae oligosaccharide and triethylenetetramine in deionized water to obtain a mixed solution, wherein the mass ratio of hydroxylated brown algae oligosaccharide and triethylenetetramine is 3.2:1, and the ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to the mass of deionized water is 1:12, adding epichlorohydrin to the mixed solution, wherein the volume ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to epichlorohydrin is 13g:1mL, mixing and stirring for 4.5h under heating conditions of 62°C to react, filtering, washing and drying after the reaction to obtain amino-modified brown algae oligosaccharide;

[0091] (3) Dissolve monosodium dihydrogen phosphate monohydrate, anhydrous disodium hydrogen phosphate and sodium chloride in deionized water, mix well to obtain a precursor buffer, wherein the concentration of monosodium dihydrogen phosphate monohydrate in the precursor buffer is 6.2 mmol / L, the concentration of anhydrous disodium hydrogen phosphate is 3.2 mmol / L, and the concentration of sodium chloride is 0.12 mol / L; add 0.2 mol / L of sodium hydroxide solution to the precursor buffer to adjust its pH value to 5.2 to obtain a PBS buffer; dissolve 3,4-dihydroxybenzoic acid in the PBS buffer prepared above to obtain a grafting precursor solution, wherein the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution is 0.8 mol / L; under a nitrogen atmosphere, add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the grafting precursor solution, and the concentration of NHS in the reaction solution is 0.22 mol / L. l / L, the concentration of EDC is 0.22 mol / L, and magnetic stirring is performed for 38 minutes at a speed of 850r / min and 22°C to obtain a reaction solution; then, the amino-modified brown algae oligosaccharide obtained in step (2) is added to the reaction solution, the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution is 280:1, and the mixture is mixed and stirred for 5.8 hours at a speed of 850r / min and a heating condition of 32°C to cause a grafting reaction, and after the reaction is completed, dialysis and freeze-drying are performed, wherein the dialysis bag used in the dialysis operation has a molecular weight cutoff of 3200Da, the freeze-drying temperature is -32°C, and the time is 42 hours, and after freeze-drying, a grafted modified brown algae oligosaccharide with catechol grafted on the surface is obtained;

[0092] (4) Soaking carboxymethyl cellulose gauze in PBS buffer solution, the soaking ratio of carboxymethyl cellulose gauze in PBS buffer solution is 1 g:16 mL; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the PBS buffer solution, the mass ratio of carboxymethyl cellulose gauze, NHS and EDC being 1:0.6:1.8; mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (3) to the soaking solution, the mass ratio of carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide being 1:0.6; mixing and stirring at a speed of 850 r / min and heating at 32° C. for 5.8 h to cause a grafting reaction; after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it, to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0093] Example 3

[0094] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, such as Figure 1 As shown, the preparation method specifically comprises the following steps:

[0095] (1) soaking the absorbent gauze in a 25wt% sodium hydroxide solution for alkalization treatment, wherein the temperature of the sodium hydroxide solution is 25°C and the soaking time is 45 minutes. After the treatment, the absorbent gauze is taken out, washed and dried to obtain alkalized gauze;

[0096] The alkalized gauze is immersed in a 70wt% chloroacetic acid solution, wherein the ratio of the mass of the chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is 0.45:1, and the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of the ethanol to the isopropanol is 4:1; the mixture is mixed and stirred for 2.5h under a heating condition of 75°C to perform an etherification reaction, and after the reaction is completed, the alkalized gauze is taken out, washed and dried to obtain a carboxymethyl cellulose gauze;

[0097] (2) dispersing brown algae oligosaccharide in 7 mol / L sodium hydroxide solution, with the mass ratio of brown algae oligosaccharide to sodium hydroxide solution being 1:3.5, mixing and stirring at 135° C. for 9 h to carry out a hydroxylation reaction, and filtering, washing and drying after the reaction to obtain hydroxylated brown algae oligosaccharide;

[0098] Dispersing hydroxylated brown algae oligosaccharide and triethylenetetramine in deionized water to obtain a mixed solution, wherein the mass ratio of hydroxylated brown algae oligosaccharide to triethylenetetramine is 3.5:1, and the ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to the mass of deionized water is 1:15, adding epichlorohydrin to the mixed solution, wherein the volume ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to epichlorohydrin is 13g:1mL, mixing and stirring for 4h under heating conditions of 65°C to react, filtering, washing and drying after the reaction is completed to obtain amino-modified brown algae oligosaccharide;

[0099] (3) dissolving monosodium dihydrogen phosphate monohydrate, anhydrous disodium hydrogen phosphate and sodium chloride in deionized water, and mixing them evenly to obtain a precursor buffer, wherein the concentration of monosodium dihydrogen phosphate monohydrate in the precursor buffer is 6.5 mmol / L, the concentration of anhydrous disodium hydrogen phosphate is 3.5 mmol / L, and the concentration of sodium chloride is 0.15 mol / L; adding 0.3 mol / L of sodium hydroxide solution to the precursor buffer to adjust its pH value to 5.5 to obtain a PBS buffer; dissolving 3,4-dihydroxybenzoic acid in the PBS buffer prepared above to obtain a grafting precursor solution, wherein the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution is 1 mol / L; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the grafting precursor solution, wherein the concentration of NHS in the reaction solution is 0.25 mol / L. l / L, the concentration of EDC is 0.25 mol / L, and magnetic stirring is performed for 35 minutes at a speed of 900 r / min and 25°C to obtain a reaction solution; then, the amino-modified brown algae oligosaccharide obtained in step (2) is added to the reaction solution, and the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution is 300:1, and the mixture is mixed and stirred for 5.5 hours at a speed of 900 r / min and a heating condition of 35°C to produce a grafting reaction, and after the reaction is completed, dialysis and freeze-drying are performed, wherein the dialysis bag used in the dialysis operation has a molecular weight cutoff of 3500Da, the freeze-drying temperature is -35°C, and the time is 36 hours. After freeze-drying, grafted modified brown algae oligosaccharide with catechol grafted on the surface is obtained;

[0100] (4) Soaking carboxymethyl cellulose gauze in PBS buffer solution, the soaking ratio of carboxymethyl cellulose gauze in PBS buffer solution is 1 g:17 mL; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the PBS buffer solution, the mass ratio of carboxymethyl cellulose gauze, NHS and EDC being 1:0.7:2; mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (3) to the soaking solution, the mass ratio of carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide being 1:0.7; mixing and stirring at a speed of 900 r / min and heating at 35° C. for 5.5 h to cause a grafting reaction; after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it, to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0101] Example 4

[0102] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, such as Figure 1 As shown, the preparation method specifically comprises the following steps:

[0103] (1) soaking the absorbent gauze in a 28 wt % sodium hydroxide solution for alkalization treatment, wherein the temperature of the sodium hydroxide solution is 28° C. and the soaking time is 42 min. After the treatment, the absorbent gauze is taken out, washed and dried to obtain alkalized gauze;

[0104] The alkalized gauze is immersed in a 72wt% chloroacetic acid solution, wherein the mass ratio of the chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is 0.48:1, and the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of ethanol to isopropanol is 4.2:1; the mixture is mixed and stirred for 2.2h under a heating condition of 78°C to perform an etherification reaction, and after the reaction is completed, the alkalized gauze is taken out, washed and dried to obtain a carboxymethyl cellulose gauze;

[0105] (2) dispersing brown algae oligosaccharide in 8 mol / L sodium hydroxide solution, with the mass ratio of brown algae oligosaccharide to sodium hydroxide solution being 1:3.2, mixing and stirring at 138° C. for 8.5 h to carry out a hydroxylation reaction, and filtering, washing and drying after the reaction to obtain hydroxylated brown algae oligosaccharide;

[0106] Dispersing hydroxylated brown algae oligosaccharide and triethylenetetramine in deionized water to obtain a mixed solution, wherein the mass ratio of hydroxylated brown algae oligosaccharide and triethylenetetramine is 3.8:1, and the ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to the mass of deionized water is 1:18, adding epichlorohydrin to the mixed solution, wherein the volume ratio of the total mass of hydroxylated brown algae oligosaccharide and triethylenetetramine to epichlorohydrin is 14g:1mL, mixing and stirring for 3.5h under heating conditions of 68°C to react, filtering, washing and drying after the reaction to obtain amino-modified brown algae oligosaccharide;

[0107] (3) dissolving sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate anhydrous and sodium chloride in deionized water and mixing them evenly to obtain a precursor buffer, wherein the concentration of sodium dihydrogen phosphate monohydrate in the precursor buffer is 6.8 mmol / L, the concentration of disodium hydrogen phosphate anhydrous is 3.8 mmol / L, and the concentration of sodium chloride is 0.18 mol / L; adding 0.4 mol / L of sodium hydroxide solution to the precursor buffer to adjust its pH value to 5.8 to obtain a PBS buffer;

[0108] 3,4-dihydroxybenzoic acid was dissolved in the PBS buffer prepared above to obtain a grafting precursor solution, wherein the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution was 1.2 mol / L; N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to the grafting precursor solution under a nitrogen atmosphere, wherein the concentration of NHS in the reaction solution was 0.28 mol / L, and the concentration of EDC was 0.28 mol / L. l / L, magnetically stirred for 32 minutes at a speed of 950r / min and 28°C to obtain a reaction solution; then, the amino-modified brown algae oligosaccharide obtained in step (2) was added to the reaction solution, the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution was 320:1, and mixed and stirred for 5.2 hours at a speed of 950r / min and a heating condition of 38°C to cause a grafting reaction, and after the reaction was completed, dialyzed and freeze-dried, wherein the dialysis bag used in the dialysis operation had a molecular weight cutoff of 3800Da, the freeze-drying temperature was -38°C, and the time was 30 hours, and after freeze-drying, a grafted modified brown algae oligosaccharide with catechol grafted on the surface was obtained;

[0109] (4) Soaking carboxymethyl cellulose gauze in PBS buffer solution, the soaking ratio of carboxymethyl cellulose gauze in PBS buffer solution is 1 g:18 mL; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the PBS buffer solution, the mass ratio of carboxymethyl cellulose gauze, NHS and EDC being 1:0.7:2.2; mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (3) to the soaking solution, the mass ratio of carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide being 1:0.8; mixing and stirring at a speed of 950 r / min and heating at 38° C. for 5.2 h to cause a grafting reaction; after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it, to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0110] Example 5

[0111] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, such as Figure 1 As shown, the preparation method specifically comprises the following steps:

[0112] (1) soaking the absorbent gauze in a 30 wt % sodium hydroxide solution for alkalization treatment, wherein the temperature of the sodium hydroxide solution is 30° C. and the soaking time is 40 min. After the treatment, the absorbent gauze is taken out, washed and dried to obtain alkalized gauze;

[0113] The alkalized gauze is immersed in a 75wt% chloroacetic acid solution, wherein the mass ratio of the chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is 0.5:1, and the chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of ethanol to isopropanol is 4.5:1; the mixture is mixed and stirred for 2h under a heating condition of 80°C to perform an etherification reaction, and after the reaction is completed, the alkalized gauze is taken out, washed and dried to obtain a carboxymethyl cellulose gauze;

[0114] (2) dispersing brown algae oligosaccharide in a 10 mol / L sodium hydroxide solution, with the mass ratio of brown algae oligosaccharide to the sodium hydroxide solution being 1:3, and mixing and stirring at 140° C. for 8 h to perform a hydroxylation reaction. After the reaction is completed, filtering, washing and drying are performed to obtain hydroxylated brown algae oligosaccharide;

[0115] Dispersing hydroxylated brown algae oligosaccharide and polyetherimide in deionized water to obtain a mixed solution, wherein the mass ratio of hydroxylated brown algae oligosaccharide to polyetherimide is 4:1, and the mass ratio of the total mass of hydroxylated brown algae oligosaccharide and polyetherimide to the mass of deionized water is 1:20, adding epichlorohydrin to the mixed solution, wherein the volume ratio of the total mass of hydroxylated brown algae oligosaccharide and polyetherimide to epichlorohydrin is 15 g:1 mL, mixing and stirring for 3 hours under heating conditions of 70° C. to react, filtering, washing and drying after the reaction is completed to obtain amino-modified brown algae oligosaccharide;

[0116] (3) dissolving sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate anhydrous and sodium chloride in deionized water and mixing them evenly to obtain a precursor buffer, wherein the concentration of sodium dihydrogen phosphate monohydrate in the precursor buffer is 7 mmol / L, the concentration of disodium hydrogen phosphate anhydrous is 4 mmol / L, and the concentration of sodium chloride is 0.2 mol / L; adding 0.5 mol / L of sodium hydroxide solution to the precursor buffer to adjust its pH value to 6 to obtain a PBS buffer;

[0117] 3,4-dihydroxybenzoic acid was dissolved in the PBS buffer prepared above to obtain a grafting precursor solution, wherein the concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution was 1.5 mol / L; N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to the grafting precursor solution under a nitrogen atmosphere, wherein the concentration of NHS in the reaction solution was 0.3 mol / L, and the concentration of EDC was 0.3 mol / L. l / L, magnetically stirred for 30 min at a speed of 1000 r / min and 30°C to obtain a reaction solution; then, the amino-modified brown algae oligosaccharide obtained in step (2) is added to the reaction solution, the molar ratio of 3,4-dihydroxybenzoic acid to amino-modified brown algae oligosaccharide in the reaction solution is 350:1, mixed and stirred for 5 h at a speed of 1000 r / min and a heating condition of 40°C to cause a grafting reaction, and dialyzed and freeze-dried after the reaction, wherein the dialysis bag used in the dialysis operation has a molecular weight cutoff of 4000 Da, the freeze-drying temperature is -40°C, and the time is 24 h, and after freeze-drying, a grafted modified brown algae oligosaccharide with catechol grafted on the surface is obtained;

[0118] (4) Soaking carboxymethyl cellulose gauze in PBS buffer solution, the soaking ratio of carboxymethyl cellulose gauze in PBS buffer solution is 1 g:20 mL; under a nitrogen atmosphere, adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to the PBS buffer solution, the mass ratio of carboxymethyl cellulose gauze, NHS and EDC being 1:0.8:2.5; mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (3) to the soaking solution, the mass ratio of carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide being 1:1; mixing and stirring at a speed of 1000 r / min and heating at 40° C. for 5 h to cause a grafting reaction; after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it, to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

[0119] Example 6

[0120] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which is different from Embodiment 1 in that in step (1), the mass fraction of the sodium hydroxide solution is adjusted to 15wt%, and other process parameters and operating conditions are exactly the same as those in Embodiment 1.

[0121] Example 7

[0122] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which differs from Embodiment 1 in that, in step (1), the mass fraction of the sodium hydroxide solution is adjusted to 35 wt %, and other process parameters and operating conditions are exactly the same as those in Embodiment 1.

[0123] Example 8

[0124] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which differs from Embodiment 1 in that, in step (1), the mass fraction of chloroacetic acid in the chloroacetic acid solution is adjusted to 60 wt %, and other process parameters and operating conditions are exactly the same as those in Embodiment 1.

[0125] Example 9

[0126] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which differs from Embodiment 1 in that, in step (1), the mass fraction of chloroacetic acid in the chloroacetic acid solution is adjusted to 80 wt %, and other process parameters and operating conditions are exactly the same as those in Embodiment 1.

[0127] Example 10

[0128] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which is different from Example 1 in that in step (1), the heating temperature of the etherification reaction is 60° C., and other process parameters and operating conditions are exactly the same as those in Example 1.

[0129] Embodiment 11

[0130] This embodiment provides a method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, which is different from Example 1 in that in step (1), the heating temperature of the etherification reaction is 90° C., and other process parameters and operating conditions are exactly the same as those in Example 1.

[0131] The coagulation index and cytotoxicity of the antibacterial carboxymethyl cellulose hemostatic gauze prepared in Example 1-11 were tested, and the specific test steps were as follows:

[0132] (1) Coagulation index test:

[0133] The antibacterial carboxymethyl cellulose hemostatic gauze prepared in Example 1-11 was cut into gauze samples of 1 cm × 1 cm, and the procoagulant activity of different gauze samples was evaluated by the coagulation index. The lower the coagulation index, the better the procoagulant performance. First, fresh citrated rabbit whole blood (100 μL) and CaCl2 (0.2M, 10 μL) were added dropwise to the surface of the gauze sample. Then, incubated at 37°C for 5 min, deionized water (25 mL) was added, and incubated in a constant temperature oscillator at 37°C for 5 min. Finally, a UV-visible spectrophotometer was used to measure the absorbance (A) of the supernatant at 540 nm. s The group without sample was used as negative control (A n ). Each sample was tested 3 times, and the coagulation index BCI (%) was calculated by the following formula:

[0134]

[0135] (2) Cytotoxicity test:

[0136] The antibacterial carboxymethyl cellulose hemostatic gauze prepared in Example 1-11 was cut into 1 cm × 1 cm gauze samples, and the cytotoxicity of different gauze samples was evaluated by cell survival rate. The higher the cell survival rate, the lower the cytotoxicity. The gauze samples were soaked in 5 mL of DMEM culture medium for 24 h to obtain an extract. Mouse skin fibroblasts (1×10 4 Cells / well) were cultured in 200 μL DMEM containing 10% fetal bovine serum for 24 h (37°C, 5% CO2 in air and 100% humidity). Subsequently, the cells were treated with the extract and incubated for 72 h (37°C, 5% CO2 in air and 100% humidity) as the experimental group, and the absorbance at 450 nm (OD s ).

[0137] Replace the gauze sample with absorbent gauze and repeat the above steps as the control group to obtain the absorbance at 450nm (OD c ).

[0138] Mouse skin fibroblasts treated with medium only were used as blank group, and the absorbance at 450 nm (OD b ).

[0139] The cell survival rate (%) was calculated using the following formula:

[0140]

[0141] The test results are shown in Table 1.

[0142] Table 1

[0143]

[0144]

[0145] It can be seen from the test data provided in Table 1 that the coagulation index of the antibacterial carboxymethyl cellulose hemostatic gauze prepared in Examples 1-5 of the present invention is much lower than that of Examples 6-11, and the cell survival rate is much higher than that of Examples 6-11, which indicates that the hemostatic gauze prepared by the preparation method provided by the present invention has excellent hemostatic and coagulation ability and low cytotoxicity.

[0146] It can be seen from the test data of Example 1, Example 6 and Example 7 that the coagulation index of the hemostatic gauze prepared in Example 6 and Example 7 is higher than that in Example 1, and the cell survival rate is lower than that in Example 1. This is because the mass fraction of the sodium hydroxide solution in Example 6 is too low, while the mass fraction of the sodium hydroxide solution in Example 7 is too high, which in turn affects the hemostatic and coagulation ability and cytotoxicity of the hemostatic gauze.

[0147] It can be seen from the test data of Example 1, Example 8 and Example 9 that the coagulation index of the hemostatic gauze prepared in Example 8 and Example 9 is higher than that in Example 1, and the cell survival rate is lower than that in Example 1. This is because the mass fraction of chloroacetic acid in the chloroacetic acid solution in Example 8 is too low, while the mass fraction of chloroacetic acid in the chloroacetic acid solution in Example 9 is too high, which in turn affects the hemostatic and coagulation ability and cytotoxicity of the hemostatic gauze.

[0148] It can be seen from the test data of Example 1, Example 10 and Example 11 that the coagulation index of the hemostatic gauze prepared in Example 10 and Example 11 is higher than that in Example 1, and the cell survival rate is lower than that in Example 1. This is because the etherification reaction in Example 10 is too low, while the etherification reaction in Example 11 is too high, which in turn affects the hemostatic and coagulation ability and cytotoxicity of the hemostatic gauze.

[0149] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing an antibacterial carboxymethyl cellulose hemostatic gauze, characterized in that: The preparation method comprises: (I) soaking the degreased gauze in a sodium hydroxide solution for alkalization treatment, taking out the degreased gauze after the treatment, washing and drying to obtain alkalized gauze; soaking the alkalized gauze in a chloroacetic acid solution, performing an etherification reaction under heating conditions, taking out the alkalized gauze after the reaction, washing and drying to obtain carboxymethyl cellulose gauze; (II) dispersing brown algae oligosaccharide in a sodium hydroxide solution, mixing, stirring and heating to carry out a hydroxylation reaction, filtering, washing and drying after the reaction is completed to obtain hydroxylated brown algae oligosaccharide; dispersing the hydroxylated brown algae oligosaccharide and an amino modifier in deionized water to obtain a mixed solution, adding epichlorohydrin to the mixed solution, mixing, stirring and heating to carry out a reaction, filtering, washing and drying after the reaction is completed to obtain amino-modified brown algae oligosaccharide; (III) dissolving 3,4-dihydroxybenzoic acid in PBS buffer to obtain a grafting precursor solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the grafting precursor solution under a nitrogen atmosphere, and mixing them evenly to obtain a reaction solution; then adding the amino-modified brown algae oligosaccharide obtained in step (II) to the reaction solution, mixing and stirring to cause a grafting reaction, and after the reaction is completed, dialyzing and freeze-drying are performed to obtain a grafted modified brown algae oligosaccharide with catechol grafted on the surface; (IV) soaking the carboxymethyl cellulose gauze in the PBS buffer solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the PBS buffer solution under a nitrogen atmosphere, and mixing them evenly to obtain a soaking solution; adding the grafted modified brown algae oligosaccharide obtained in step (III) to the soaking solution, mixing and stirring to cause a grafting reaction, and after the reaction is completed, taking out the carboxymethyl cellulose gauze, washing and drying it to obtain the antibacterial carboxymethyl cellulose hemostatic gauze.

2. The preparation method according to claim 1, characterized in that: In step (I), the mass fraction of the sodium hydroxide solution is 20-30wt%; The temperature of the sodium hydroxide solution is 20-30° C. The absorbent gauze is immersed in the sodium hydroxide solution for 40 to 50 minutes.

3. The preparation method according to claim 1, characterized in that: In step (I), the mass fraction of chloroacetic acid in the chloroacetic acid solution is 65-75wt%; The ratio of the mass of chloroacetic acid in the chloroacetic acid solution to the mass of the alkalized gauze is (0.4-0.5):1; The chloroacetic acid solution is composed of chloroacetic acid and a composite organic solvent, wherein the composite organic solvent is ethanol and isopropanol, and the volume ratio of the ethanol to the isopropanol is (3.5-4.5):1; The heating temperature of the etherification reaction is 70-80°C; The alkalized gauze is immersed in the chloroacetic acid ethanol solution for 2 to 3 hours.

4. The preparation method according to claim 1, characterized in that: In step (II), the concentration of the sodium hydroxide solution is 5 to 10 mol / L; The mass ratio of the brown algae oligosaccharide to the sodium hydroxide solution is 1:(3-4); The heating temperature of the hydroxylation reaction is 130-140°C; The stirring time of the hydroxylation reaction is 8 to 10 hours.

5. The preparation method according to claim 1, characterized in that: In step (II), the mass ratio of the hydroxylated brown algae oligosaccharide to the amino modifier is (3-4):1; The ratio of the total mass of the hydroxylated brown algae oligosaccharide and the amino modifier to the mass of the deionized water is 1:(10-20); The amino modifier includes any one of ethylenediaminetetraacetic acid, triethylenetetramine, triethylenetetramine or polyetherimide or a combination of at least two thereof; The ratio of the total mass of the hydroxylated brown algae oligosaccharide and the amino modifier in the mixed solution to the volume of the epichlorohydrin is (12-15) g:1 mL; The heating temperature when the mixed solution is mixed with epichlorohydrin is 60-70°C; The stirring time when the mixed solution is mixed with epichlorohydrin is 3 to 5 hours.

6. The preparation method according to claim 1, characterized in that: In step (III), the PBS buffer is prepared by the following method: Dissolving sodium dihydrogen phosphate monohydrate, anhydrous disodium hydrogen phosphate and sodium chloride in deionized water, mixing them evenly to obtain a precursor buffer, and adding alkaline solution dropwise to the precursor buffer to adjust the pH value thereof to obtain the PBS buffer; The concentration of sodium dihydrogen phosphate monohydrate in the precursor buffer is 6-7 mmol / L; The concentration of anhydrous disodium hydrogen phosphate in the precursor buffer is 3-4 mmol / L; The concentration of sodium chloride in the precursor buffer is 0.1-0.2 mol / L; The concentration of the alkali solution is 0.1-0.5 mol / L; Adding alkaline solution dropwise to the precursor buffer to adjust the pH value thereof to 5-6; The concentration of 3,4-dihydroxybenzoic acid in the grafting precursor solution is 0.5 to 1.5 mol / L; The concentration of N-hydroxysuccinimide in the reaction solution is 0.2-0.3 mol / L; The concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the reaction solution is 0.2-0.3 mol / L; The grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are mixed by magnetic stirring at a speed of 800 to 1000 r / min; The grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are mixed for 30 to 40 minutes; The mixing temperature of the grafting precursor solution, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 20-30°C.

7. The preparation method according to claim 1, characterized in that: In step (III), the molar ratio of 3,4-dihydroxybenzoic acid to the amino-modified brown algae oligosaccharide in the reaction solution is (250-350):1; The temperature of the grafting reaction is 30 to 40°C; The stirring rate of the grafting reaction is 800-1000 r / min; The stirring time of the grafting reaction is 5 to 6 hours; The molecular weight cut-off of the dialysis bag used in the dialysis operation is 3000-4000Da; The freeze drying temperature is -30 to -40°C; The freeze-drying time is 24 to 48 hours.

8. The preparation method according to claim 1, characterized in that: In step (IV), the carboxymethyl cellulose gauze is immersed in the PBS buffer at a ratio of 1 g: (15-20) mL; The mass ratio of the carboxymethyl cellulose gauze, the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:(0.5-0.8):(1.5-2.5).

9. The preparation method according to claim 1, characterized in that: In step (IV), the mass ratio of the carboxymethyl cellulose gauze to the grafted modified brown algae oligosaccharide is 1:(0.5-1); The temperature of the grafting reaction is 30 to 40°C; The stirring rate of the grafting reaction is 800-1000 r / min; The stirring time of the grafting reaction is 5 to 6 hours.

10. An antibacterial carboxymethyl cellulose hemostatic gauze prepared by the preparation method according to any one of claims 1 to 9.

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