High liquid absorption rate porous hemostatic sponge and its preparation method

Abstract

The present invention discloses a highly absorbent porous hemostatic sponge and a preparation method thereof, comprising the following steps: (1) chopping seabuckthorn pulp into small pieces, then mixing the small pieces, lentil powder and calcium gluconate evenly, adding pectinase and neutral protease, and then adding deionized water, carrying out enzymatic hydrolysis, solid-liquid separation, enzyme inactivation treatment, and vacuum concentration to obtain a composite extract; (2) dividing the composite extract into two parts, adding arginine to one part of the extract under stirring, adjusting the pH to 8, heating in a water bath, stirring at a constant temperature, cooling naturally, and mixing with the other part of the composite extract, and stirring evenly to obtain an extract component; (3) mixing a carboxymethyl chitosan solution, a sodium alginate solution and the extract component, stirring under an ultrasonic environment, standing at 4-5°C for 24-30 h after stirring is completed, and then freeze-drying to obtain the hemostatic sponge. The hemostatic sponge prepared by the method of the present invention has good hemostatic function and high liquid absorption rate.

Description

Technical Field

[0001] The present invention relates to the technical field of hemostatic sponge materials, and particularly relates to a highly absorbent porous hemostatic sponge and a preparation method thereof. Background Art

[0002] Hemostasis is a crucial step in trauma treatment. Rapid and effective hemostasis can significantly reduce the mortality rate and the risk of complications. Traditional hemostatic materials such as gauze and bandages have deficiencies such as slow hemostasis speed and easy adhesion to wounds. In recent years, hemostatic sponge materials have become a research hotspot due to their advantages such as rapid hemostasis and good biocompatibility. Hemostatic sponge materials are mainly divided into two categories: natural and synthetic. Natural hemostatic sponge materials include cellulose, chitosan, gelatin, starch, etc. Synthetic hemostatic sponge materials include polyvinyl alcohol, polyurethane, polyethylene glycol, etc. Among them, chitosan is a natural polysaccharide with good biocompatibility, biodegradability, antibacterial property and hemostatic performance, so it is widely used in the research of hemostatic materials. In order to further improve the performance of chitosan-based hemostatic sponge materials, related modification processes are often required, such as doping other hemostatic materials, antibacterial materials, etc., or introducing new functional groups through grafting crosslinking and other processes to improve the liquid absorption, hemostatic performance and biocompatibility of the sponge materials. Summary of the Invention

[0003] Therefore, the present invention provides a preparation method of a highly absorbent porous hemostatic sponge, which comprises the following steps:

[0004] (1) Chop seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Enzymatic hydrolysis extraction is carried out at a constant temperature of 50±3°C for more than 40h, then solid-liquid separation is carried out, enzyme inactivation treatment is carried out, and the liquid phase is concentrated under reduced pressure to obtain a composite extraction solution;

[0005] (2) Divide the composite extraction solution into two parts. Add arginine to one part of the extraction solution under stirring. After the addition is completed, add sodium acetate to the solution to adjust the pH of the solution to 8, then heat it in a water bath to 80±3°C, and keep stirring at a constant temperature of 80±3°C for more than 8h, with condensation reflux during the constant temperature process; after the constant temperature stirring is completed, cool it naturally to room temperature, mix it with the other part of the composite extraction solution, and stir evenly to obtain an extract component;

[0006] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component, stir for more than 30min under ultrasonic environment, after the stirring is completed, stand still at 4-5°C for 24-30h, and then freeze-dry to obtain the hemostatic sponge.

[0007] Further, in the step (1), the enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the diced pieces, lentil powder and calcium gluconate is diced pieces: lentil powder: calcium gluconate = 20:3 - 5:1 - 2; the material-to-water ratio is 1:3.

[0008] Further, in the step (1), the liquid phase is concentrated under reduced pressure to 1 / 2 of the volume before concentration.

[0009] Further, in the step (2), the composite extraction solution is divided into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, arginine is added to the part II extraction solution under stirring, and the feeding mass ratio is part II extraction solution: arginine = 10:2 - 3.

[0010] Further, in the step (3), the mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1.

[0011] Further, in the step (3), the power of the ultrasonic wave is 200 - 300 W, and the frequency is 30 kHz.

[0012] The beneficial effects of the present invention are as follows: The hemostatic sponge prepared by the method of the present invention has a low in vitro whole blood coagulation index (BCI) value and a high liquid absorption ratio at 10 s. The smaller the BCI index, the stronger the ability to promote thrombus formation, indicating better in vitro hemostatic ability of the material. Therefore, the hemostatic sponge prepared by the method of the present invention has good hemostatic function and high liquid absorption rate, and its performance is significantly improved, showing good application prospects in medical hemostatic devices. By introducing the composite enzymatic hydrolysate of seabuckthorn fruit and lentil powder into the traditional chitosan-based hemostatic sponge, the present invention can significantly improve the hemostatic performance and liquid absorption of the hemostatic sponge. This may be because: The polysaccharides and polyphenols obtained after enzymatic hydrolysis of seabuckthorn fruit. Among them, the polysaccharide in seabuckthorn is a natural degradable polysaccharide, which has functions such as promoting wound healing, moisturizing and moisture absorption, improving the liquid absorption performance of the sponge. At the same time, incorporating large molecules of seabuckthorn polysaccharide can form a cross-linked three-dimensional network structure with the amino groups of chitosan in the sponge and the polypeptide structure formed by enzymatic hydrolysis of lentil powder, increasing the porosity of the sponge and thus improving the liquid absorption. Seabuckthorn polysaccharides and polyphenols can also form hydrogen bonds with chitosan molecules to form hydrogels, improving the hemostatic effect of chitosan sponges. In addition, polysaccharide polyphenols also have antibacterial and antioxidant effects, promoting the rapid healing of wounds. In the traditional method of directly adding calcium gluconate or calcium alginate to the hemostatic sponge, due to the easy loss of calcium gluconate and the weak ability of calcium alginate to release calcium ions in water. Therefore, the present invention uses lentil peptides obtained by enzymatic hydrolysis of lentil powder to form chelates with calcium ions, reducing calcium loss caused by the binding of calcium ions to other macromolecules during the preparation process, and also playing an effective role in releasing calcium ions during the hemostasis process, increasing the calcium ion concentration in the blood. In addition, the calcium chelated by peptides can utilize the absorption channels of peptides to quickly promote platelet aggregation, improve the adhesion of platelets, and increase their activity, which can accelerate the formation of thrombus and effectively stop bleeding for patients. Adding arginine can promote the formation of the above hydrogel, and at the same time, arginine is a semi-essential amino acid that can promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Comparison chart of in vitro whole blood coagulation index (BCI) of hemostatic sponges prepared in each example and comparative example;

[0014] Figure 2 Comparison chart of liquid absorption ratio at 10 s of hemostatic sponges prepared in each example and comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be further described below in conjunction with examples.

[0016] Example 1

[0017] A preparation method of a high liquid absorption rate porous hemostatic sponge, comprising the following steps:

[0018] (1) Chop the seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:3:1; the solid-liquid ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and concentrate the liquid phase under reduced pressure to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0019] (2) Divide the composite extraction solution into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, arginine is added to part II extraction solution under stirring, and the mass ratio of the added material is part II extraction solution: arginine = 10:2; after the addition is completed, sodium acetate is added to the solution to adjust the pH of the solution to 8, and then the solution is heated in a water bath to 80°C. After reaching the temperature, stir at a constant temperature of 80°C for 8 h, and carry out condensation reflux during the constant temperature process; after the constant temperature stirring is completed, naturally cool to room temperature and mix with the other part of the composite extraction solution, part I extraction solution, and stir evenly to obtain an extract component;

[0020] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for 30 min under an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, let it stand at 5°C for 24 h, and then carry out freeze-drying to obtain the hemostatic sponge.

[0021] Example 2

[0022] A preparation method of a highly absorbent porous hemostatic sponge, comprising the following steps:

[0023] (1) Chop the seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:4:1; the solid-liquid ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and concentrate the liquid phase under reduced pressure to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0024] (2) Divide the composite extraction solution into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, arginine is added to part II extraction solution under stirring, and the feeding mass ratio is part II extraction solution: arginine = 10:2; after the feeding is completed, sodium acetate is added to the solution to adjust the pH of the solution to 8, and then the solution is heated in a water bath to 80°C. After reaching the temperature, stir at a constant temperature of 80°C for 8 h, and carry out condensation reflux during the constant temperature process; after the constant temperature stirring is completed, naturally cool to room temperature and mix with the other part of the composite extraction solution, part I extraction solution, and stir evenly to obtain the extract component;

[0025] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for 30 min in an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, let it stand in a 5°C environment for 24 h, and then carry out freeze-drying to obtain the hemostatic sponge.

[0026] Example 3

[0027] A preparation method of a highly absorbent porous hemostatic sponge, comprising the following steps:

[0028] (1) Chop the seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:4:2; the material-to-water ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and concentrate the liquid phase under reduced pressure to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0029] (2) Divide the composite extraction solution into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, add arginine to part II extraction solution under stirring, and the mass ratio of feeding is part II extraction solution: arginine = 10:3; after the feeding is completed, add sodium acetate to the solution to adjust the pH of the solution to 8, then heat it in a water bath to 80°C, and stir at 80°C for 8 h after reaching the temperature, and carry out condensation reflux during the constant temperature process; after the constant temperature stirring is completed, cool it naturally to room temperature, and mix it with the other part of the composite extraction solution, part I extraction solution, and stir evenly to obtain the extract component;

[0030] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for 30 min in an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, let it stand for 24 h at 5°C, and then carry out freeze-drying to obtain the hemostatic sponge.

[0031] Example 4

[0032] A preparation method of a highly absorbent porous hemostatic sponge, comprising the following steps:

[0033] (1) Chop the seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:5:2; the material-to-water ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and concentrate the liquid phase under reduced pressure to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0034] (2) Divide the composite extraction solution into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, arginine is added to part II extraction solution under stirring, and the mass ratio of feeding is part II extraction solution: arginine = 10:3; after the feeding is completed, sodium acetate is added to the solution to adjust the pH of the solution to 8, and then the solution is heated in a water bath to 80°C. After reaching the temperature, stir at 80°C for 8 h, and carry out condensation reflux during the constant temperature process; after the constant temperature stirring is completed, naturally cool to room temperature and mix with the other part of the composite extraction solution, part I extraction solution, and stir evenly to obtain an extract component;

[0035] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for 30 min under an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, stand at 5°C for 24 h, and then carry out freeze-drying to obtain the hemostatic sponge.

[0036] Comparative Example 1

[0037] A preparation method of a hemostatic sponge for comparison includes the following steps:

[0038] (1) Chop the seabuckthorn pulp into small pieces, then mix the small pieces evenly with calcium gluconate, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the small pieces to calcium gluconate is small pieces:calcium gluconate = 20:2; the material-water ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and reduce the pressure of the liquid phase to concentrate it to 1 / 2 of the volume before concentration to obtain an extraction solution;

[0039] (2) Divide the extraction solution into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, add arginine to part II extraction solution under stirring, and the feeding mass ratio is part II extraction solution:arginine = 10:3; after the feeding is completed, add sodium acetate to the solution to adjust the pH of the solution to 8, and then heat it in a water bath to 80°C. After reaching the temperature, stir at a constant temperature of 80°C for 8 h, and carry out condensation reflux during the constant temperature process; after the constant temperature stirring is completed, naturally cool it to room temperature and mix it with the other part of the extraction solution, part I extraction solution, and stir evenly to obtain an extract component;

[0040] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution:sodium alginate solution:extract component = 2:1:1; stir for 30 min under an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, let it stand in a 5°C environment for 24 h, and then carry out freeze-drying to obtain the hemostatic sponge of this comparative example.

[0041] Comparative Example 2

[0042] A preparation method of a hemostatic sponge for comparison includes the following steps:

[0043] (1) Mix lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50±3°C for 40 h. The enzymatic hydrolysis process is as follows: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mixing mass ratio of the lentil powder to calcium gluconate is lentil powder:calcium gluconate = 4:2; the material-water ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and reduce the pressure of the liquid phase to concentrate it to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0044] (2) The composite extraction solution is divided into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, arginine is added to part II extraction solution under stirring, and the mass ratio of the added material is part II extraction solution: arginine = 10:3. After the addition is completed, sodium acetate is added to the solution to adjust the pH of the solution to 8, and then the solution is heated in a water bath to 80 °C. After reaching the temperature, it is stirred at a constant temperature of 80 °C for 8 h, and condensation reflux is carried out during the constant temperature process. After the constant temperature stirring is completed, it is naturally cooled to room temperature and mixed with the other part of the composite extraction solution, part I extraction solution, and stirred evenly to obtain the extract component;

[0045] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for 30 min under an ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after the stirring is completed, let it stand in an environment of 5 °C for 24 h, and then freeze-dry to obtain the hemostatic sponge of this comparative example.

[0046] Comparative Example 3

[0047] A preparation method of a hemostatic sponge for comparison includes the following steps:

[0048] (1) Cut the seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly, add pectinase and neutral protease, and then add deionized water. Carry out enzymatic hydrolysis extraction at a constant temperature of 50 ± 3 °C for 40 h. The enzymatic hydrolysis process is: the addition amount of pectinase is 4500 U / g, the addition amount of neutral protease is 5000 U / g, and the mass ratio of the mixture of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:4:2; the solid-liquid ratio is 1:4; then carry out solid-liquid separation and enzyme inactivation treatment, and concentrate the liquid phase under reduced pressure to 1 / 2 of the volume before concentration to obtain a composite extraction solution;

[0049] (2) The composite extraction solution is divided into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:3. Among them, sodium acetate is added to part II extraction solution to adjust the pH of the solution to 8, and then the solution is heated in a water bath to 80 °C. After reaching the temperature, it is stirred at a constant temperature of 80 °C for 8 h, and condensation reflux is carried out during the constant temperature process. After the constant temperature stirring is completed, it is naturally cooled to room temperature and mixed with the other part of the composite extraction solution, part I extraction solution, and stirred evenly to obtain the extract component;

[0050] (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; in the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; the mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution:sodium alginate solution:extract component = 2:1:1; stir for 30 min under ultrasonic environment, the power of the ultrasonic wave is 200 W, and the frequency is 30 kHz; after stirring, let it stand at 5 °C for 24 h, and then freeze-dry to obtain the hemostatic sponge of this comparative example.

[0051] Example 5

[0052] Test the in vitro whole blood coagulation index (BCI) and 10 s liquid absorption ratio of the hemostatic sponges prepared by the methods of the above examples and comparative examples respectively. The test method for the in vitro whole blood coagulation index (BCI) is: add 5 mg of the hemostatic sponge to be tested into a test centrifuge tube; take 5 μL of 0.2 mol / L calcium chloride aqueous solution, add 50 μL of sodium citrate anticoagulated blood taken from the heart of SD rats to the calcium chloride aqueous solution, aspirate and blow until the blood recovers its coagulation ability, and then add it into the test centrifuge tube to mix with the hemostatic sponge to be tested. Incubate at 37 °C for 80 s. After incubation, pour the mixture into 5 mL of deionized water, incubate for 3 min, lyse the uncoagulated blood, take the supernatant to a 96-well plate, and place it in an enzyme-labeled instrument to measure the absorbance OD at 545 nm s , the one without the hemostatic sponge to be tested is the blank group, measure its absorbance OD0 at 545 nm, in vitro whole blood coagulation index (BCI) = OD s / OD0×100%. The test method for the 10 s liquid absorption ratio is: weigh the original weight M0 of the hemostatic sponge to be tested, put the sponge into deionized water for 10 s and then take it out, let it stand in the air until no more liquid drips, and weigh the weight M t , 10 s liquid absorption ratio = (M t - M0) / M0, and the results are as shown in Figure 1 and Figure 2 shown.

[0053] From Figure 1 and Figure 2It can be seen that the hemostatic sponge prepared by the method of the present invention has a lower in vitro whole blood coagulation index (BCI) value and a higher liquid absorption ratio at 10 s. The smaller the BCI index, the stronger the ability to promote thrombus formation, indicating better in vitro hemostatic ability of the material. Therefore, the hemostatic sponge prepared by the method of the present invention has good hemostatic function and high liquid absorption rate, and its performance is significantly improved, showing good application prospects in medical hemostatic devices. By comparing Example 3 with each comparative example, it can be seen that by introducing the composite enzymatic hydrolysate of seabuckthorn fruit and lentil powder into the traditional chitosan-based hemostatic sponge, the hemostatic performance and liquid absorption of the hemostatic sponge can be significantly improved. This may be because: the polysaccharides and polyphenols obtained after enzymatic hydrolysis of seabuckthorn fruit, among which the polysaccharide in seabuckthorn is a natural biodegradable polysaccharide with functions such as promoting wound healing, moisturizing and moisture absorption, which can improve the liquid absorption performance of the sponge. At the same time, incorporating large molecules of seabuckthorn polysaccharide can form a cross-linked three-dimensional network structure with the amino groups of chitosan in the sponge and the polypeptide structure formed by enzymatic hydrolysis of lentil powder, increasing the porosity of the sponge and thus improving the liquid absorption. Seabuckthorn polysaccharides and polyphenols can also form hydrogen bonds with chitosan molecules to form hydrogels, improving the hemostatic effect of chitosan sponges. In addition, polysaccharide polyphenols also have antibacterial and antioxidant effects, promoting the rapid healing of wounds. In the traditional method of directly adding calcium gluconate or calcium alginate to the hemostatic sponge, calcium gluconate is easily lost, and the ability of calcium alginate to release calcium ions in water is weak. Therefore, in the present invention, lentil peptides obtained by enzymatic hydrolysis of lentil powder are combined with calcium ions to form chelates, reducing calcium loss caused by the combination of calcium ions with other macromolecules during the preparation process, and can also effectively release calcium ions during the hemostasis process, increasing the calcium ion concentration in the blood. In addition, calcium chelated by peptides can utilize the absorption channels of peptides to quickly promote platelet aggregation, improve platelet adhesion, and increase their activity, which can accelerate thrombus formation and effectively stop bleeding for patients. Adding arginine can promote the formation of the above hydrogel, and at the same time, arginine is a semi-essential amino acid that can promote wound healing.

[0054] The technical solutions provided by the present invention have been introduced in detail above. For those of ordinary skill in the art, according to the idea of the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A preparation method of a highly absorbent porous hemostatic sponge, characterized in that, It includes the following steps: (1) Chop seabuckthorn pulp into small pieces, then mix the small pieces, lentil powder and calcium gluconate evenly. The mixing mass ratio of the small pieces, lentil powder and calcium gluconate is small pieces: lentil powder: calcium gluconate = 20:3 - 5:1 - 2; the material - water ratio is 1:3; add pectinase and neutral protease, then add deionized water, carry out enzymatic hydrolysis extraction at a constant temperature of 50 ± 3°C for more than 40 h, then carry out solid - liquid separation, enzyme inactivation treatment, and reduce the pressure of the liquid phase for concentration to obtain a composite extraction solution; (2) Divide the composite extraction solution into two parts. Add arginine to one part of the extraction solution under stirring. The composite extraction solution is divided into two parts according to the mass ratio of part I extraction solution: part II extraction solution = 7:

3. Add arginine to part II extraction solution under stirring. The feeding mass ratio is part II extraction solution: arginine = 10:2 - 3; after feeding, add sodium acetate to the solution to adjust the pH of the solution to 8, then heat it in a water bath to 80 ± 3°C, and keep stirring at a constant temperature of 80 ± 3°C for more than 8 h, with condensation reflux during the constant - temperature process; after the constant - temperature stirring is completed, cool it naturally to room temperature, mix it with the other part of the composite extraction solution, and stir evenly to obtain an extract component; (3) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; dissolve sodium alginate in deionized water to prepare a sodium alginate solution; the mass percentage of carboxymethyl chitosan in the carboxymethyl chitosan solution is 3%, and the solvent is water; In the sodium alginate solution, the mass percentage of sodium alginate is 2.5%, and the solvent is water; Mix the carboxymethyl chitosan solution, the sodium alginate solution and the extract component. The mixing mass ratio of the carboxymethyl chitosan solution, the sodium alginate solution and the extract component is carboxymethyl chitosan solution: sodium alginate solution: extract component = 2:1:1; stir for more than 30 min under an ultrasonic environment, after the stirring is completed, let it stand in an environment of 4 - 5°C for 24 - 30 h, and then carry out freeze - drying to obtain the hemostatic sponge.

2. The preparation method of a highly absorbent porous hemostatic sponge according to claim 1, characterized in that, In the step (1), the enzymatic hydrolysis process is: the addition amount of pectinase is 4500 U / g, and the addition amount of neutral protease is 5000 U / g.

3. The preparation method of a highly absorbent porous hemostatic sponge according to claim 1, characterized in that, In the step (1), the liquid phase is concentrated under reduced pressure to 1 / 2 of the volume before concentration.

4. A highly absorbent porous hemostatic sponge, characterized in that, It is prepared by using the preparation method described in any one of claims 1 - 3.

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