Composite hemostatic sponge and preparation method thereof

Abstract

The invention discloses a composite hemostatic sponge and a preparation method thereof, and belongs to the technical field of hemostatic materials, the preparation method of the composite hemostatic sponge comprises the following steps: preparing cationic starch, preparing an adduct, and cross-linking; the preparation of the cationic starch comprises the following steps: preparing a sorbitol calcium ion chelate, and cationizing; the preparation of the adduct comprises the following steps: mixing beta-cyclodextrin, calcium chloride and deionized water, stirring at 60-65 DEG C, and evaporating all water in vacuum to obtain the adduct; the cationic starch is prepared through a water solvent method and then mixed with the chitosan, ionic crosslinking is carried out under the action of metal ions, the composite hemostatic sponge with the excellent hemostatic effect and antibacterial effect is obtained, the substitution speed is high when the cationic starch is prepared, and the prepared composite hemostatic sponge is high in mechanical strength and good in antibacterial effect. The mechanical strength and hygroscopicity are uniform.

Description

Technical Field

[0001] The invention relates to the technical field of hemostatic materials, and in particular to a composite hemostatic sponge and a preparation method thereof. Background Art

[0002] In military conflicts, natural disasters, and other events, uncontrolled massive bleeding causes countless casualties. Furthermore, massive bleeding in traffic accidents, civil injuries, or surgery can also be life-threatening. Therefore, the ability to quickly and effectively control bleeding is crucial for improving survival rates and saving lives. Currently, common methods for controlling bleeding rely on the body's own coagulation system and external hemostatic materials. On the one hand, after a bleeding injury, the body's normal coagulation system forms stable and insoluble fibrin at the wound site through coagulation mechanisms. Fibrin interacts with other blood components to form a clot, ultimately achieving hemostasis. On the other hand, for certain urgent and severe bleeding situations, relying solely on the body's own coagulation system often fails to achieve hemostasis, necessitating the use of hemostatic materials.

[0003] Currently, commonly used hemostatic materials include gauze, bandages, and hemostatic sponges. Although gauze and bandages are simple to manufacture and inexpensive, they are prone to adhesion to the wound surface, causing secondary damage during dressing changes, tearing the wound, causing pain to the patient, and prolonging wound healing time. Hemostatic sponges are convenient, efficient, green, environmentally friendly, and safe. They can undergo repeated hydration when in contact with the wound surface, absorbing tissue fluid exuded from the wound. In addition, hemostatic sponges have good biocompatibility, high mechanical strength, good moisture retention, and are not prone to adhesion to the wound surface. As a new wound repair material, hemostatic sponges have been widely used in the medical field.

[0004] Cationic starch is a derivative of starch. Starch molecules are polymerized from glucose monomers. The active hydroxyl groups in starch molecules react with cationic monomers to form cationic starch, which is widely used in papermaking, printing and dyeing, textiles, water treatment, and other fields. Because cationic starch carries a cationic charge, it can generate electrostatic interactions with anionic substances in the blood, thereby promoting blood coagulation. Furthermore, the cationic charge can generate electrostatic interactions with bacterial cell membranes, disrupting bacterial structure and thus exerting an antibacterial effect. Furthermore, because natural starch is inexpensive and readily available, cationic starch is also frequently used in the preparation of hemostatic materials, particularly hemostatic sponges.

[0005] When applying cationic starch to the preparation of hemostatic sponges, the most commonly used preparation method is to cationize starch using a quaternary ammonium cationic etherifying agent to obtain cationic starch, and then use a crosslinking agent to crosslink the cationic starch to make a hemostatic sponge.

[0006] However, when preparing hemostatic sponges according to the above method, the following problems exist: First, when cationizing starch, the most commonly used method is the water solvent method, that is, using water as a solvent, dispersing the starch, and then under the action of an alkaline catalyst, reacting with a quaternary ammonium cationic etherifying agent to generate cationic starch. However, since starch is prone to gelatinization in water, the quaternary ammonium cationic etherifying agent cannot diffuse and penetrate well in the starch and react, further resulting in a slow substitution rate and a low degree of substitution of the cationic starch prepared by the water solvent method, leading to poor hemostatic and antibacterial effects of the prepared hemostatic sponge; Second, during crosslinking, the crosslinking agent reacts with the hydroxyl groups in the cationic starch and other water-soluble high-molecular polymers to form a crosslinked structure. The crosslinking agents mainly used are epichlorohydrin, sodium tripolyphosphate, glutaraldehyde, etc. However, in the formed crosslinked structure, there are unreacted crosslinking agents, that is, crosslinking agent residues. Since the above crosslinking agents are all irritating to the skin, the prepared hemostatic sponge is irritating to the skin; Third, when the crosslinking agent reacts with the hydroxyl groups in the cationic starch and other water-soluble high-molecular polymers, it mainly reacts through the reaction between the crosslinking agent and the hydroxyl groups of the cationic starch. For cationic starch with a high degree of substitution, the hydroxyl group content is low, which will affect the crosslinking between cationic starches, resulting in a decrease in crosslinking density and further leading to a decrease in the mechanical strength of the prepared hemostatic sponge.

[0007] For the first above-mentioned problem, the commonly used solution is to prepare cationic starch by the dry method. Specifically, after wetting the starch with a small amount of organic solvent or water, it is mixed with an alkaline catalyst and a quaternary ammonium cationic etherifying agent, dried to a substantially sewage-free state, and then reacted to obtain cationic starch. The dry method can avoid the gelatinization problem of starch, thereby obtaining cationic starch with a high degree of substitution. However, the dry method lacks a dispersing solvent, resulting in uneven heating, leading to a large difference in the degree of substitution among the same batch of cationic starch prepared. Generally speaking, after considering the third above-mentioned problem at the same time, the commonly used solution is also to combine the cationic starch prepared by the water solvent method with other components having hemostatic and antibacterial effects to jointly prepare a composite hemostatic sponge. For components having hemostatic and antibacterial effects, inorganic nanoparticles, metal nanoparticles, metal oxide nanoparticles, and chitosan are commonly used. However, inorganic nanoparticles are prone to generate heat when absorbing the tissue fluid exuded from the wound surface, causing further damage to the wound surface; both metal nanoparticles and metal oxide nanoparticles have the problem of poor binding force with cationic starch, resulting in the problem of shedding of metal nanoparticles or metal oxide nanoparticles during long-term storage, further leading to poor storage resistance of the prepared composite hemostatic sponge; although using chitosan can avoid the problems existing in inorganic nanoparticles, metal nanoparticles, and metal oxide nanoparticles, and can also play a synergistic hemostatic and antibacterial role with cationic starch, it cannot solve the problem of slow substitution rate during the preparation of cationic starch, and the cross-linking between chitosan and cationic starch still requires the use of a cross-linking agent, and there are more cross-linking agent residues in the composite hemostatic sponge obtained after cross-linking chitosan and cationic starch, that is, it will cause the second above-mentioned problem to be more serious. For this problem, the commonly used solution is to use metal ions for ionic cross-linking. However, the composite hemostatic sponge obtained by cross-linking with metal ions has low mechanical strength, and when using metal ions for ionic cross-linking, it is necessary to first add chitosan and cationic starch into a mold and mix them, and then soak them with an aqueous solution containing calcium ions. During the soaking process, the diffusion rate of calcium ions is uneven, resulting in the problem that the cross-linking rate is difficult to control during the preparation, resulting in poor uniformity of the prepared composite hemostatic sponge, and further leading to uneven mechanical strength and hygroscopicity of the prepared composite hemostatic sponge. Summary of the Invention

[0008] In view of the deficiencies of the prior art, the present invention provides a composite hemostatic sponge and its preparation method. Cationic starch is prepared by the water solvent method, and then mixed with chitosan, and ionic cross-linking is carried out under the action of metal ions to obtain a composite hemostatic sponge with excellent hemostatic and antibacterial effects. Moreover, when preparing cationic starch, the substitution rate is fast, the mechanical strength of the prepared composite hemostatic sponge is high, and the mechanical strength and hygroscopicity are uniform.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A preparation method of a composite hemostatic sponge, comprising: preparing cationic starch, preparing an adduct, and crosslinking; The preparation of the cationic starch includes: preparing a sorbitol calcium ion chelate and cationization; In the preparation of the sorbitol calcium ion chelate, sorbitol, calcium acetate, and deionized water are mixed, and then stirred at a stirring speed of 50 - 200 rpm for 10 - 20 min at room temperature, the pH is adjusted to 7 - 7.5, the temperature is raised to 75 - 85 °C, and stirred at a stirring speed of 50 - 200 rpm for 50 - 60 min at 75 - 85 °C, left standing for 14 - 16 h, centrifuged at a centrifugation speed of 8000 - 9000 rpm for 30 - 35 min, the precipitate is taken and dried to obtain the sorbitol calcium ion chelate; In the preparation of the sorbitol calcium ion chelate, the dosage ratio of sorbitol, calcium acetate, and deionized water is 180 g: 48 - 50 g: 1800 - 2000 mL; For the cationization, tapioca starch and deionized water are mixed, and then stirred at a stirring speed of 50 - 200 rpm for 30 - 40 min at room temperature, sodium chloride, sorbitol, and the sorbitol calcium ion chelate are added, stirred for 30 - 40 min, the temperature is raised to 50 - 55 °C, the pH is adjusted to 11 - 12 using an aqueous sodium hydroxide solution, 2,3 - epoxypropyltrimethylammonium chloride is added, stirred for 5 - 5.5 h, the pH is adjusted to 6.5 - 7 using dilute hydrochloric acid, and then washed, dehydrated, and dried to obtain the cationic starch; In the cationization, the dosage ratio of tapioca starch, deionized water, sodium chloride, sorbitol, the sorbitol calcium ion chelate, and 2,3 - epoxypropyltrimethylammonium chloride is 100 g: 1000 - 1200 mL: 3 - 4 g: 6.2 - 6.5 g: 9.5 - 10 g: 4.5 - 5 g; The concentration of the aqueous sodium hydroxide solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; For the preparation of the adduct, β - cyclodextrin, calcium chloride, and deionized water are mixed, and then stirred at a stirring speed of 50 - 200 rpm for 6 - 7 h at 60 - 65 °C, and all the water is evaporated under vacuum at 50 °C to obtain the adduct; In the preparation of the adduct, the dosage ratio of β - cyclodextrin, calcium chloride, and deionized water is 11.3 g: 1.1 - 1.3 g: 950 - 1050 mL; For the crosslinking, after mixing chitosan and an aqueous acetic acid solution, stir at room temperature for 20 - 30 min, add an aqueous genipin solution, heat up to 50 - 55 °C, stir for 50 - 60 min, add cationic starch and an adduct, stir for 30 - 40 min, pour into a mold, let stand at room temperature for 47 - 50 h, then take out from the mold, fully immerse in an aqueous calcium chloride solution, let stand at room temperature for 30 - 40 min, carry out water washing, freeze-drying, and sterilization to obtain a composite hemostatic sponge; In the crosslinking, the dosage ratio of chitosan, aqueous acetic acid solution, aqueous genipin solution, cationic starch, adduct, and aqueous calcium chloride solution is 20 g: 950 - 1050 mL: 100 - 130 mL: 140 - 160 g: 20 - 25 g: 1900 - 2000 mL; The degree of deacetylation of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the aqueous acetic acid solution is 2 wt%; The concentration of the aqueous genipin solution is 1 wt%; The concentration of the aqueous calcium chloride solution is 0.1 mol / L.

[0010] A composite hemostatic sponge prepared by the aforementioned preparation method.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the preparation method of the composite hemostatic sponge of the present invention, when preparing cationic starch, sorbitol and sorbitol calcium ion chelate are added. During cationization, sorbitol can interact with starch, increasing the gelatinization temperature and difficulty of starch, and can also promote the mixing of alkaline catalyst and quaternary ammonium cationic etherifying agent with starch. Sorbitol calcium ion chelate can combine with part of the starch to form a gel. During cationization, gel particles can be formed. The gel particles contain un-cationized hydroxyl groups, which can act as crosslinking points during crosslinking. Through the molecular chain entanglement between the gel particles, chitosan, and cationic starch, the mechanical strength and water absorption multiple of the obtained composite hemostatic sponge are improved; In addition, in the crosslinking of the present invention, an adduct of β-cyclodextrin and calcium chloride is added. Through the molecular-ion interaction between β-cyclodextrin and calcium ions in calcium chloride, calcium ions are bound to the surface of β-cyclodextrin. During crosslinking, β-cyclodextrin in the adduct can promote the uniform dispersion of calcium ions, thus playing a role in pre-crosslinking. Moreover, when calcium ions crosslink with chitosan and cationic starch, it plays a regulatory role, thereby obtaining a hydrogel with uniform pores, avoiding the problem of poor uniformity of the composite hemostatic sponge caused by uneven calcium ion penetration when directly mixing chitosan and cationic starch and then soaking in an aqueous calcium chloride solution; (2) The preparation method of the composite hemostatic sponge of the present invention also performs double crosslinking with genipin and calcium ions. Among them, genipin can crosslink with the amino groups of chitosan, and calcium ions can promote the crosslinking between cationic starch and chitosan, thereby ensuring that a composite hemostatic sponge with excellent mechanical properties can be obtained; (3) In the preparation method of the composite hemostatic sponge of the present invention, the sorbitol calcium chelate, sorbitol, and adduct used in the preparation of cationic starch all have certain hemostatic and bactericidal effects, thereby further improving the hemostatic performance and antibacterial performance of the prepared composite hemostatic sponge; (4) The composite hemostatic sponge prepared by the present invention has a good hemostatic effect, and the whole blood coagulation index (BCI) is 19.32 - 19.35%; in the mouse liver hemostasis test, the bleeding volume of the blank control is 403.5 mg, and the bleeding time is 117.2 s. The bleeding volume of the composite hemostatic sponge of the present invention is 96.5 - 97.8 mg, and the bleeding time is 40.5 - 41.3 s; in the mouse tail hemostasis test, the bleeding volume of the blank control is 157.1 mg, and the bleeding time is 254.3 s. The bleeding volume of the composite hemostatic sponge of the present invention is 44.0 - 45.7 mg, and the bleeding time is 79.1 - 81.5 s; (5) The composite hemostatic sponge prepared by the present invention has a good antibacterial effect. According to GB / T 20944.3 - 2008 "Evaluation of Antibacterial Properties of Textiles - Part 3: Oscillation Method" for testing, the antibacterial rate of the composite hemostatic sponge of the present invention against Staphylococcus aureus is 95.31 - 95.75%, the antibacterial rate against Staphylococcus aureus is 99.05 - 99.18%, and the antibacterial rate against Candida albicans is 98.24 - 98.52%; (6) The composite hemostatic sponge prepared by the present invention has high mechanical strength, the tensile strength is 98.05 - 100.26 kPa, and the elongation at break is 21.38 - 23.17; (7) The composite hemostatic sponge prepared by the present invention has a large water absorption multiple, the water absorption multiple is 31.58 - 34.06, and the difference between the maximum water absorption multiple and the minimum water absorption multiple is 0.40 - 0.43; (8) The composite hemostatic sponge prepared by the present invention has uniform mechanical strength and hygroscopicity. Randomly take 5 samples from the prepared composite hemostatic sponge. The difference between the maximum tensile strength and the minimum tensile strength is 1.20 - 1.54 kPa, the difference between the maximum elongation at break and the minimum elongation at break is 0.35 - 0.51%, and the difference between the maximum water absorption multiple and the minimum water absorption multiple is 0.50 - 0.62; (9)The preparation method of the composite hemostatic sponge of the present invention. When preparing, cationic starch is first prepared by the aqueous solvent method. When the substitution time in cationization (i.e., the stirring time after adding 2,3-epoxypropyltrimethylammonium chloride) is 5 h, the degree of substitution of the prepared cationic starch is 0.034. When the substitution time in cationization (i.e., the stirring time after adding 2,3-epoxypropyltrimethylammonium chloride) is 5.5 h, the degree of substitution of the prepared cationic starch is 0.037. Detailed implementation mode

[0012] For a clearer understanding of the technical features, objectives and effects of the present invention, the specific implementation mode of the present invention is now described. The normal temperature in the examples is 23 °C.

[0013] Example 1 A preparation method of a composite hemostatic sponge is as follows: 1. Preparation of cationic starch: (1) Preparation of sorbitol calcium ion chelate: Mix 180 g of sorbitol, 48 g of calcium acetate, and 1800 mL of deionized water, stir at a stirring speed of 50 rpm at normal temperature for 10 min, adjust the pH to 7, heat up to 75 °C, stir at a stirring speed of 50 rpm at 75 °C for 50 min, let stand for 14 h, centrifuge at a centrifugation speed of 8000 rpm for 30 min, take the precipitate, and dry it to obtain sorbitol calcium ion chelate; (2) Cationization: Mix 100 g of cassava starch and 1000 mL of deionized water, stir at a stirring speed of 50 rpm at normal temperature for 30 min, add 3 g of sodium chloride, 6.2 g of sorbitol, and 9.5 g of sorbitol calcium ion chelate, stir for 30 min, heat up to 50 °C, use an aqueous sodium hydroxide solution to adjust the pH to 11, add 4.5 g of 2,3-epoxypropyltrimethylammonium chloride, stir for 5 h, use dilute hydrochloric acid to adjust the pH to 6.5, wash, dehydrate, and dry to obtain cationic starch; The concentration of the aqueous sodium hydroxide solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; The degree of substitution of the prepared cationic starch is tested by the Kjeldahl method, and the measured degree of substitution of the cationic starch is 0.034; 2. Preparation of the adduct: Mix 11.3 g of β-cyclodextrin, 1.1 g of calcium chloride, and 950 mL of deionized water, stir at a stirring speed of 50 rpm at 60 °C for 6 h, and vacuum evaporate all the water at 50 °C to obtain the adduct; 3. Crosslinking: After mixing 20 g of chitosan and 950 mL of acetic acid aqueous solution, stir at room temperature for 20 min, add 100 mL of genipin aqueous solution, heat up to 50 °C, stir for 50 min, add 140 g of cationic starch and 20 g of adduct, stir for 30 min, pour into a mold, place at room temperature for 47 h, then take out from the mold, completely immerse in 1900 mL of calcium chloride aqueous solution, stand still at room temperature for 30 min, carry out water washing, freeze-drying, and sterilization to obtain a composite hemostatic sponge; The deacetylation degree of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the calcium chloride aqueous solution is 0.1 mol / L.

[0014] This example also provides a composite hemostatic sponge prepared by the aforementioned preparation method.

[0015] Example 2 A preparation method of a composite hemostatic sponge is as follows: 1. Preparation of cationic starch: (1) Preparation of sorbitol calcium ion chelate: After mixing 180 g of sorbitol, 50 g of calcium acetate, and 2000 mL of deionized water, stir at a stirring speed of 200 rpm at room temperature for 20 min, adjust the pH to 7.5, heat up to 85 °C, stir at a stirring speed of 200 rpm at 85 °C for 60 min, stand still for 16 h, centrifuge at a centrifugal speed of 9000 rpm for 35 min, take the precipitate, and dry to obtain sorbitol calcium ion chelate; (2) Cationization: After mixing 100 g of cassava starch and 1200 mL of deionized water, stir at a stirring speed of 200 rpm at room temperature for 40 min, add 4 g of sodium chloride, 6.5 g of sorbitol, and 10 g of sorbitol calcium ion chelate, stir for 40 min, heat up to 55 °C, use sodium hydroxide aqueous solution to adjust the pH to 12, add 5 g of 2,3-epoxypropyltrimethylammonium chloride, stir for 5.5 h, use dilute hydrochloric acid to adjust the pH to 7, carry out washing, dehydration, and drying to obtain cationic starch; The concentration of the sodium hydroxide aqueous solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; The degree of substitution of the prepared cationic starch is tested by the Kjeldahl method, and the degree of substitution of the obtained cationic starch is 0.037; 2. Preparation of the adduct: Mix 11.3 g of β-cyclodextrin, 1.3 g of calcium chloride, and 1050 mL of deionized water, stir at a speed of 200 rpm at 65 °C for 7 h, and evaporate all the water under vacuum at 50 °C to obtain the adduct; 3. Crosslinking: Mix 20 g of chitosan and 1050 mL of acetic acid aqueous solution, stir at room temperature for 30 min, add 130 mL of genipin aqueous solution, raise the temperature to 55 °C, stir for 60 min, add 160 g of cationic starch and 25 g of the adduct, stir for 40 min, pour into a mold, place at room temperature for 50 h, then take out from the mold, completely immerse in 2000 mL of calcium chloride aqueous solution, stand still at room temperature for 40 min, wash with water, freeze-dry, and sterilize to obtain the composite hemostatic sponge; The deacetylation degree of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the calcium chloride aqueous solution is 0.1 mol / L.

[0016] This example also provides a composite hemostatic sponge prepared by the foregoing preparation method.

[0017] Examples 3-5 are for single-factor variable analysis based on Example 2.

[0018] Example 3 This example is for the variable analysis of sorbitol calcium chelate on the basis of Example 2. Specifically, in the preparation of cationic starch in Step 1 of Example 2, Step (1) for preparing sorbitol calcium chelate is omitted; and in Step (2) of cationization, the addition of sorbitol calcium chelate is omitted. Specifically: A preparation method of a composite hemostatic sponge is as follows: 1. Preparation of cationic starch: Mix 100 g of tapioca starch and 1200 mL of deionized water, stir at a speed of 200 rpm at room temperature for 40 min, add 4 g of sodium chloride and 6.5 g of sorbitol, stir for 40 min, raise the temperature to 55 °C, adjust the pH to 12 with sodium hydroxide aqueous solution, add 5 g of 2,3-epoxypropyltrimethylammonium chloride, stir for 5.5 h, adjust the pH to 7 with dilute hydrochloric acid, wash, dehydrate, and dry to obtain cationic starch; The concentration of the sodium hydroxide aqueous solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; The degree of substitution of the prepared cationic starch is tested by the Kjeldahl method, and the degree of substitution of the obtained cationic starch is 0.034; 2. Preparation of the adduct: Mix 11.3 g of β-cyclodextrin, 1.3 g of calcium chloride, and 1050 mL of deionized water, then stir at a speed of 200 rpm at 65 °C for 7 h, and evaporate all the water under vacuum at 50 °C to obtain the adduct; 3. Crosslinking: Mix 20 g of chitosan and 1050 mL of acetic acid aqueous solution, stir at room temperature for 30 min, add 130 mL of genipin aqueous solution, raise the temperature to 55 °C, stir for 60 min, add 160 g of cationic starch and 25 g of the adduct, stir for 40 min, pour into a mold, place at room temperature for 50 h, then take out from the mold, fully immerse in calcium chloride aqueous solution, stand still at room temperature for 40 min, carry out water washing, freeze-drying, and sterilization to obtain the composite hemostatic sponge; The deacetylation degree of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the calcium chloride aqueous solution is 0.1 mol / L.

[0019] This example also provides a composite hemostatic sponge prepared by the foregoing preparation method.

[0020] Example 4 This example is based on Example 2 and conducts a variable analysis of sorbitol. Specifically, on the basis of Example 2, in step (2) of cationization in the preparation of cationic starch in step 1, the addition of sorbitol is omitted. Specifically: A preparation method of a composite hemostatic sponge is as follows: 1. Preparation of cationic starch: (1) Preparation of sorbitol calcium ion chelate: Mix 180 g of sorbitol, 50 g of calcium acetate, and 2000 mL of deionized water, then stir at a speed of 200 rpm at room temperature for 20 min, adjust the pH to 7.5, raise the temperature to 85 °C, stir at a speed of 200 rpm at 85 °C for 60 min, stand still for 16 h, centrifuge at a speed of 9000 rpm for 35 min, take the precipitate, and dry to obtain the sorbitol calcium ion chelate; (2) Cationization: Mix 100 g of tapioca starch and 1200 mL of deionized water, then stir at a speed of 200 rpm at room temperature for 40 min, add 4 g of sodium chloride and 10 g of sorbitol calcium ion chelate, stir for 40 min, raise the temperature to 55 °C, use sodium hydroxide aqueous solution to adjust the pH to 12, add 5 g of 2,3-epoxypropyltrimethylammonium chloride, stir for 5.5 h, use dilute hydrochloric acid to adjust the pH to 7, carry out washing, dehydration, and drying to obtain the cationic starch; The concentration of the sodium hydroxide aqueous solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; The degree of substitution of the prepared cationic starch was measured by the Kjeldahl method, and the degree of substitution of the cationic starch was 0.027; 2. Preparation of the adduct: Mix 11.3 g of β-cyclodextrin, 1.3 g of calcium chloride, and 1050 mL of deionized water, stir at a stirring speed of 200 rpm at 65 °C for 7 h, and vacuum distill all the water at 50 °C to obtain the adduct; 3. Crosslinking: Mix 20 g of chitosan and 1050 mL of acetic acid aqueous solution, stir at room temperature for 30 min, add 130 mL of genipin aqueous solution, raise the temperature to 55 °C, stir for 60 min, add 160 g of cationic starch and 25 g of the adduct, stir for 40 min, pour into a mold, place at room temperature for 50 h, then take out from the mold, completely immerse in the calcium chloride aqueous solution, stand still at room temperature for 40 min, wash with water, freeze-dry, and sterilize to obtain the composite hemostatic sponge; The degree of deacetylation of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the calcium chloride aqueous solution is 0.1 mol / L.

[0021] This example also provides a composite hemostatic sponge prepared by the foregoing preparation method.

[0022] Example 5 This example is based on Example 2, and variable analysis of the adduct is carried out. Specifically, on the basis of Example 2, the second step of preparing the adduct is omitted; and in the third step of crosslinking, the addition of the adduct is omitted. Specifically: A preparation method of a composite hemostatic sponge is as follows: 1. Preparation of cationic starch: (1) Preparation of sorbitol calcium chelate: Mix 180 g of sorbitol, 50 g of calcium acetate, and 2000 mL of deionized water, stir at a stirring speed of 200 rpm at room temperature for 20 min, adjust the pH to 7.5, raise the temperature to 85 °C, stir at a stirring speed of 200 rpm at 85 °C for 60 min, stand still for 16 h, centrifuge at a centrifugal speed of 9000 rpm for 35 min, take the precipitate, and dry to obtain sorbitol calcium chelate; (2)Cationization: After mixing 100 g of cassava starch and 1200 mL of deionized water, stir at a stirring speed of 200 rpm for 40 min at room temperature. Add 4 g of sodium chloride, 6.5 g of sorbitol, and 10 g of calcium ion chelate of sorbitol, stir for 40 min, heat up to 55 °C, adjust the pH to 12 using an aqueous sodium hydroxide solution, add 5 g of 2,3-epoxypropyltrimethylammonium chloride, stir for 5.5 h, adjust the pH to 7 using dilute hydrochloric acid, and perform washing, dehydration, and drying to obtain cationic starch; The concentration of the aqueous sodium hydroxide solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L; The degree of substitution of the prepared cationic starch was tested by the Kjeldahl method, and the degree of substitution of the obtained cationic starch was 0.037; 2. Crosslinking: After mixing 20 g of chitosan and 1050 mL of acetic acid aqueous solution, stir at room temperature for 30 min, add 130 mL of genipin aqueous solution, heat up to 55 °C, stir for 60 min, add 160 g of cationic starch, stir for 40 min, pour into a mold, place at room temperature for 50 h, then take out from the mold, completely immerse in an aqueous calcium chloride solution, stand still at room temperature for 40 min, perform water washing, freeze-drying, and sterilization to obtain a composite hemostatic sponge; The deacetylation degree of the chitosan is 90%, and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the aqueous calcium chloride solution is 0.1 mol / L.

[0023] This example also provides a composite hemostatic sponge prepared by the aforementioned preparation method.

[0024] It can be seen from the degree of substitution of the cationic starch prepared in Examples 3 - 5 and Example 2 that the degree of substitution of the cationic starch prepared in Examples 3 and 4 is less than that of the cationic starch prepared in Example 2.

[0025] Performance Test 1 The whole blood dynamic coagulation index of the composite hemostatic sponges prepared in Examples 1 - 5 was tested, and the test method and results are as follows: Use blood collection tubes containing 3.8% sodium citrate anticoagulant to draw the blood of SD rats respectively. Take 6 centrifuge tubes with a specification of 10 mL and label them as No. 1 - 6 respectively. Add 25 μL of anticoagulant - containing blood to each of the centrifuge tubes No. 1 - 6. Take the composite hemostatic sponges prepared in Examples 1 - 5 with a specification of 1 cm×1 cm×0.2 cm and add them to the surface of the blood in centrifuge tubes No. 1 - 5 respectively. Take a medical gelatin dressing with a specification of 1 cm×1 cm×0.2 cm and add it to the surface of the blood in centrifuge tube No. 6. Let the centrifuge tubes No. 1 - 6 stand for 10 min, then add 10 mL of deionized water to each of the centrifuge tubes No. 1 - 6 and vortex for 2 min. Take 200 μL of the solution from each of the centrifuge tubes No. 1 - 6 and add it to a 96 - well plate, and then measure the absorbance at 414 nm using an enzyme - linked immunosorbent assay (ELISA) reader respectively, which is used as the absorbance value of the experimental group; then dissolve 25 μL of anticoagulant - containing blood in 10 mL of deionized water, and measure the absorbance at 414 nm using an enzyme - linked immunosorbent assay (ELISA) reader, which is used as the absorbance value of the control group; Calculate the whole - blood coagulation index (BCI), and the calculation formula and calculation results are as follows: BCI = absorbance value of the experimental group / absorbance value of the control group × 100%;

[0026] The whole - blood coagulation index (BCI) can be used to characterize the in - vitro coagulation ability. The smaller the BCI value, the stronger the ability to promote thrombus formation.

[0027] Performance test 2 Anesthetize 7 female SD rats with a body weight of 200 - 220 g and label them as No. 1 - 7 respectively. After anesthetizing the female SD rats No. 1 - 7, expose the liver of the rats through an abdominal incision, remove the tissue fluid around the liver, place a filter paper under the liver, and use a 16 - gauge needle to pierce 1 cm into the liver surface of the female SD rats No. 1 - 7 respectively. Then use gauze and the composite hemostatic sponges prepared in Examples 1 - 5 to stop bleeding for the female SD rats No. 1 - 6 respectively, and use the female SD rat No. 7 as a blank control. Record the blood loss and bleeding time of each female SD rat, and the recording results are as follows:

[0028] Performance test 3 Anesthetize 7 female SD rats with a body weight of 200 - 220 g and label them as No. 1 - 7 respectively. Cut half of the length of the tails of the female SD rats No. 1 - 7, and then use gauze and the composite hemostatic sponges prepared in Examples 1 - 5 to stop bleeding for the female SD rats No. 1 - 6 respectively, and use the female SD rat No. 7 as a blank control. Record the blood loss and bleeding time of each female SD rat, and the recording results are as follows:

[0029] Performance test 4 According to GB / T 20944.3-2008 "Evaluation of antibacterial properties of textiles - Part 3: Oscillation method", the antibacterial rates of the composite hemostatic sponges prepared in Examples 1-5 against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC6538), and Candida albicans (ATCC 10231) were tested, and the test results are as follows:

[0030] Performance tests 1-4 were tests on hemostatic performance and antibacterial performance. From the results of performance tests 1-4, it can be seen that the hemostatic performance and antibacterial performance of the composite hemostatic sponges prepared in Examples 3-5 are lower than those of the composite hemostatic sponge prepared in Example 2, indicating that the addition of sorbitol calcium ion chelate, sorbitol, and adduct can play a certain role in improving the hemostatic performance and antibacterial performance of the composite hemostatic sponge.

[0031] Performance test 5 Randomly take 5 samples from the composite hemostatic sponges containing cationic starch prepared in Examples 1-5, test the tensile strength and elongation at break of the samples, calculate the average tensile strength and average elongation at break, and calculate the difference between the maximum tensile strength and the minimum tensile strength, and the difference between the maximum elongation at break and the minimum elongation at break. The calculation results are as follows:

[0032] Performance test 6 Randomly take 5 samples from the composite hemostatic sponges prepared in Examples 1-5, test the water absorption multiple of the samples, calculate the average water absorption multiple, and calculate the difference between the maximum water absorption multiple and the minimum water absorption multiple. The calculation results are as follows:

[0033] From the results of performance test 5 and performance test 6, it can be seen that the tensile strength, elongation at break, and water absorption multiple of the composite hemostatic sponges prepared in Examples 3-4 are much smaller than those of the composite hemostatic sponge prepared in Example 1. Moreover, compared with the composite hemostatic sponge prepared in Example 1, the differences in tensile strength, elongation at break, and water absorption multiple among different samples of the composite hemostatic sponges prepared in Examples 3-5 are larger.

[0034] Unless otherwise specified, the percentages used in the present invention are all mass percentages.

[0035] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a composite hemostatic sponge, characterized in that, Comprising: Preparing cationic starch, preparing an adduct, crosslinking; The preparation of the cationic starch includes: preparing a sorbitol calcium ion chelate, cationization; For the cationization, after mixing tapioca starch and deionized water, stirring at room temperature, adding sodium chloride, sorbitol, and sorbitol calcium ion chelate, stirring, heating to 50 - 55 °C, adjusting the pH to 11 - 12 using an aqueous sodium hydroxide solution, adding 2,3-epoxypropyltrimethylammonium chloride, stirring for 5 - 5.5 h, adjusting the pH to 6.5 - 7 using dilute hydrochloric acid, washing, dehydrating, and drying to obtain cationic starch; For the preparation of the adduct, after mixing β-cyclodextrin, calcium chloride, and deionized water, stirring at 60 - 65 °C, vacuum distilling off all the water to obtain the adduct.

2. The preparation method of the composite hemostatic sponge according to claim 1, wherein, For the preparation of the sorbitol calcium ion chelate, after mixing sorbitol, calcium acetate, and deionized water, stirring at room temperature, adjusting the pH to 7 - 7.5, heating to 75 - 85 °C, stirring at 75 - 85 °C, standing, centrifuging, taking the precipitate, and drying to obtain the sorbitol calcium ion chelate.

3. The preparation method of the composite hemostatic sponge according to claim 2, wherein, In the preparation of the sorbitol calcium ion chelate, the dosage ratio of sorbitol, calcium acetate, and deionized water is 180 g: 48 - 50 g: 1800 - 2000 mL.

4. The preparation method of the composite hemostatic sponge according to claim 1, characterized in that, In the cationization, the dosage ratio of tapioca starch, deionized water, sodium chloride, sorbitol, sorbitol calcium ion chelate, and 2,3-epoxypropyltrimethylammonium chloride is 100 g: 1000 - 1200 mL: 3 - 4 g: 6.2 - 6.5 g: 9.5 - 10 g: 4.5 - 5 g; The concentration of the aqueous sodium hydroxide solution is 1 mol / L; The concentration of the dilute hydrochloric acid is 1 mol / L.

5. The preparation method of the composite hemostatic sponge according to claim 1, characterized in that, In the preparation of the adduct, the dosage ratio of β-cyclodextrin, calcium chloride, and deionized water is 11.3 g: 1.1 - 1.3 g: 950 - 1050 mL; When vacuum distilling off all the water, the temperature is 50 °C.

6. The preparation method of the composite hemostatic sponge according to claim 1, wherein, For the crosslinking, after mixing chitosan and an acetic acid aqueous solution, stirring at room temperature, adding a genipin aqueous solution, heating to 50 - 55 °C, stirring, adding cationic starch and the adduct, stirring, pouring into a mold, placing at room temperature, then taking out from the mold, completely soaking in a calcium chloride aqueous solution, standing at room temperature, washing, freeze-drying, and sterilizing to obtain a composite hemostatic sponge.

7. The preparation method of the composite hemostatic sponge according to claim 6, characterized in that, In the crosslinking, the dosage ratio of chitosan, acetic acid aqueous solution, genipin aqueous solution, cationic starch, adduct, and calcium chloride aqueous solution is 20 g: 950 - 1050 mL: 100 - 130 mL: 140 - 160 g: 20 - 25 g: 1900 - 2000 mL.

8. The preparation method of the composite hemostatic sponge according to claim 6, wherein, In the crosslinking, the deacetylation degree of the chitosan is 90% and the molecular weight is 800,000; The concentration of the acetic acid aqueous solution is 2 wt%; The concentration of the genipin aqueous solution is 1 wt%; The concentration of the calcium chloride aqueous solution is 0.1 mol / L.

9. A composite hemostatic sponge prepared by the preparation method according to any one of claims 1 - 8.

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