Hemostatic granules for diffuse bleeding and preparation method and application thereof

Abstract

The present invention provides hemostatic particles for diffuse bleeding, and a preparation method and application thereof, wherein the raw materials of the hemostatic particles include: gelatin, silk fibroin, xylooligosaccharide and auxiliary hemostatic materials. The present invention effectively matches gelatin, silk fibroin, xylooligosaccharide and auxiliary hemostatic materials, and the prepared hemostatic particles maintain high swelling capacity and good adhesion while effectively stopping bleeding and accelerating wound healing; wherein, silk fibroin and xylooligosaccharide have a synergistic effect, and jointly enhance the anti-inflammatory, antibacterial and growth-promoting effects of the hemostatic particles; in the preparation method provided by the present invention, gelatin, silk fibroin, xylooligosaccharide and auxiliary hemostatic materials are processed into rehydrated substances, which have good biocompatibility, excellent water absorption and swelling properties, are not easy to cause allergic or rejection reactions, and can improve the phenomenon of ensuring that the hemostatic material does not collapse in a high-humidity environment.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and in particular relates to hemostatic particles for diffuse bleeding and a preparation method and application thereof. Background Art

[0002] Diffuse bleeding is a common phenomenon in clinical surgery. It refers to the symptoms of bleeding in one or more tissues and organs. It is generally tissue damage caused by trauma. Especially in clinical surgery to remove diseased tissues, diffuse bleeding is prone to occur at the anastomosis due to the gaps in the arrangement of staples. Commonly used methods for treating diffuse bleeding in clinical practice are ultrasonic scalpels and electric scalpels. It is worth noting that improper operation of ultrasonic scalpels can cause tissue rupture. Electric scalpel treatment can burn tissue and may cause tissue contracture. By applying medical materials such as hemostatic particles, bleeding can be stopped accurately and secondary damage caused by surgical instruments can be reduced.

[0003] Bioactive materials have broad application prospects in the medical field. Currently, the materials used as hemostatic particles are mainly classified into polysaccharides and proteins. Polysaccharide materials include starch, chitosan and cellulose. These products have poor biocompatibility and are prone to cause tissue wound hardening.

[0004] CN113975455A discloses a composite hemostatic powder and its preparation method and application, the absorbable hemostatic powder comprises: raw material A and raw material B; the raw material A is oxidized regenerated cellulose; the raw material B is at least one of oxidized regenerated cellulose sodium salt, cross-linked sodium carboxymethyl starch, and sodium alginate; the mass percentage of the raw material A in the absorbable hemostatic powder is 30%-99%; the absorbable hemostatic powder has the advantages of high water absorption rate, fast water absorption rate, and high gel adhesion, which solves the problem of single function of hemostatic materials, but the addition of a cross-linking agent in the preparation process will ultimately affect the biocompatibility and safety of the product.

[0005] CN106963976A discloses a method for preparing a microporous starch composite hemostatic powder, which comprises hydrolyzing starch with α-amylase and saccharifying enzyme, and then washing, drying, sieving and other steps to obtain microporous starch, and then fully mixing and granulating the microporous starch with 10-50 parts of sodium carboxymethyl starch according to a weight ratio of 50-90 parts, and finally obtaining the microporous starch composite hemostatic powder. The obtained material has a high-efficiency hemostatic function, forms a hydrogel on the wound surface, promotes wound healing, and has good fluidity, can adapt to irregular wounds, but its adhesion is poor and easy to fall off.

[0006] Protein materials are degradable and have excellent biocompatibility. Among them, gelatin, as a representative, has good biocompatibility and degradability, can promote wound healing, penetrate drugs, and control drug release rate, and is an excellent hemostatic granule material. CN113134113A discloses a preparation method of absorbable hemostatic fluid gelatin and absorbable hemostatic fluid gelatin, which alleviates the defects of poor adhesion and long degradation cycle of existing paste hemostatic products, and alleviates the problem of diffuse bleeding on uneven surfaces or deeper or narrower areas, but the multiple grinding and crushing in the powder making process is prone to overheating, causing thermal degradation of the material, resulting in decreased material stability.

[0007] The main component of gelatin is protein, and its molecules contain many amino acid chains. These amino acid chains can form a cross-linked structure through hydrogen bonds and charge interactions, which makes gelatin have good swelling properties. CN108187129A discloses an absorbable gelatin hemostatic powder and a preparation method thereof. The BET specific surface area of ​​the gelatin hemostatic powder is 30 m 2 / g-70m 2 / g, and its preparation process includes the steps of cross-linking gelatin, water swelling, mechanical crushing, freeze drying and secondary crushing. The hemostatic powder can be well attached to the wound surface, quickly absorb moisture in the blood, and achieve a good hemostatic effect. However, in a high humidity environment, gelatin easily absorbs moisture and swells, resulting in reduced adhesion with the substrate, especially under long-term immersion or high humidity conditions, gelatin may lose adhesion and cannot effectively maintain bonding with the substrate. Therefore, other biomaterials can be compounded to improve the adhesion ability of gelatin.

[0008] Therefore, developing a hemostatic particle that can effectively stop bleeding and accelerate wound healing while maintaining high swelling capacity, good adhesion and good degradation ability is a research focus in this field. Summary of the invention

[0009] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a hemostatic granule for diffuse bleeding and a preparation method and application thereof, which can effectively stop bleeding and accelerate wound healing while maintaining high swelling ability, good adhesion and good degradation ability.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides hemostatic particles for diffuse bleeding, wherein the raw materials of the hemostatic particles include gelatin, silk fibroin, xylo-oligosaccharide and auxiliary hemostatic materials.

[0012] Compared with synthetic materials, animal-derived materials are closer to natural components in the body, are more easily accepted by human tissues and have good degradation functions. In the present invention, animal-derived materials are used and degraded after they have played a role, reducing the potential risks caused by long-term implantation. Gelatin is an animal-derived protein with good hydration ability. When gelatin comes into contact with water, it can form a viscous gel with strong adhesion. It can be applied to tissues for up to 24 hours, which helps to stop bleeding and control bleeding.

[0013] Silk fibroin has good swelling, biocompatibility and degradability. At the same time, silk fibroin has sufficient strength and flexibility, and can also be used in combination with other materials or technologies with antibacterial properties to achieve the effect of inhibiting the growth of bacteria. In particular, silk fibroin still has good adhesion in a wet environment. However, silk fibroin has excellent water solubility and is difficult to block blood from forming gels. It is not suitable for the preparation of hemostatic materials when used alone.

[0014] Among the polymeric sugars, oligoxylose is one of the most powerful varieties in promoting the proliferation of bifidobacteria. Its efficacy is nearly 20 times that of other polymeric sugars. There is no enzyme in the human gastrointestinal tract that can hydrolyze oligoxylose, so it can directly enter the large intestine and be preferentially used by bifidobacteria, promoting the proliferation of bifidobacteria while producing a variety of organic acids. It can reduce the pH value of the intestine and inhibit the growth of harmful bacteria. Therefore, oligoxylose can activate or promote the immune function of the body's cells and body fluids, which is beneficial for the body to protect itself from the invasion and damage of pathogenic microorganisms.

[0015] The present invention combines gelatin, silk fibroin, xylooligosaccharides and auxiliary hemostatic materials to prepare hemostatic particles which can effectively stop bleeding and accelerate wound healing while maintaining high swelling capacity, good adhesion and good degradation capacity. Silk fibroin and xylooligosaccharides have a synergistic effect, which together enhance the anti-inflammatory, antibacterial and growth-promoting effects of the hemostatic particles.

[0016] Preferably, the auxiliary hemostatic material includes any one of sodium alginate, calcium alginate, gelatin, carrageenan or casein, or a combination of at least two of them.

[0017] Preferably, the auxiliary hemostatic material comprises sodium alginate.

[0018] Preferably, the auxiliary hemostatic material is a combination of sodium alginate and carrageenan.

[0019] Preferably, the auxiliary hemostatic material is a combination of sodium alginate and casein.

[0020] When sodium alginate and carrageenan or casein are used in combination, the prepared hemostatic particles have stronger adhesion ability.

[0021] Sodium alginate is a natural high molecular weight polysaccharide compound extracted from seaweed, which has good gelation and high viscosity, water absorption and stability, and biocompatibility. Adding an appropriate amount of sodium alginate to the hemostatic granules can increase the viscosity of the product, improve stability, maintain its functions and properties in various environments, and increase the moisture retention and water retention of the product.

[0022] Preferably, the raw materials of the hemostatic particles include, by weight: 20-60 parts of gelatin (for example, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, etc.), 2.5-2.9 parts of silk fibroin (for example, 2.6 parts, 2.7 parts, 2.8 parts, etc.), 0.1-0.5 parts of xylo-oligosaccharide (for example, 0.2 parts, 0.3 parts, 0.4 parts, etc.), and 2-6 parts of auxiliary hemostatic material (for example, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, 5.5 parts, etc.).

[0023] Preferably, the raw materials of the hemostatic particles include, by weight: 30-50 parts of gelatin, 2.5-2.9 parts of silk fibroin, 0.1-0.5 parts of xylo-oligosaccharide, and 3-6 parts of auxiliary hemostatic material.

[0024] Preferably, the molecular weight of the silk fibroin is 100-10000 Da, for example, 200 Da, 500 Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, etc.

[0025] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0026] In a second aspect, the present invention provides a method for preparing the hemostatic granules for diffuse bleeding as described in the first aspect, the preparation method comprising the following steps:

[0027] (1) Evenly mixing the gelatin aqueous solution, the auxiliary hemostatic material and the xylo-oligosaccharide, and then adding the silk fibroin to obtain a mixed solution;

[0028] (2) subjecting the mixed solution to an oxidation reaction, and freeze-drying the mixed solution after the reaction is completed to obtain a porous sponge;

[0029] (3) Cross-linking the porous sponge under ultraviolet light, and after the cross-linking is completed, deodorizing, crushing, and sterilizing to obtain the hemostatic particles.

[0030] When gelatin comes into contact with water, a viscous gel can be formed. The rehydrated substances sodium alginate and silk fibroin have good water absorption and can be used in hemostatic particles to quickly absorb the surrounding water and form a viscous gel state through hydration reaction. The rehydrated substances have certain strength and viscosity and can provide a small pressure, which is very beneficial for controlling the bleeding environment, accelerating wound healing and protecting the wound surface. The rehydrated substances also have good biocompatibility and are not easy to cause allergic or rejection reactions. The prepared hemostatic granule hemostatic material has excellent water absorption and swelling, which can ensure that the hemostatic material does not collapse in a high humidity environment.

[0031] During the preparation process, oxidants are added to oxidize all the raw materials, changing the state of the substances and preparing a porous structure. The color of the oxidized gelatin becomes lighter and the solubility decreases; the molecular structure of the oxidized sodium alginate changes, with higher viscosity, improving the gel formation ability, strength and stability.

[0032] Preferably, in step (1), the liquid ratio of gelatin to water in the gelatin aqueous solution is 1:(10-15), for example, it can be 1:11, 1:12, 1:13, 1:14, etc.

[0033] Preferably, in step (1), the silk fibroin is added at a temperature of 15-30°C, for example, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, etc.

[0034] Preferably, in step (2), the freeze-drying time is 36-48 h, for example, 38 h, 40 h, 42 h, 44 h, 46 h, etc.

[0035] Preferably, the oxidant used in the oxidation reaction includes any one of hydrogen peroxide, sodium percarbonate or peracetic acid.

[0036] Preferably, the amount of the oxidant added in the oxidation reaction system is 0.1-0.5 mol / L, for example, 0.15 mol / L, 0.25 mol / L, 0.35 mol / L, 0.45 mol / L, etc. Based on 1 L of the mixed solution, the amount of the oxidant added is 0.1-0.5 mol.

[0037] Preferably, the oxidation reaction temperature is 2-6°C (for example, 3°C, 4°C, 5°C, etc.), and the time is 10-30 min, for example, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, etc.

[0038] Preferably, the oxidation reaction further includes a step of adjusting the pH value to neutral.

[0039] Preferably, the wavelength of the ultraviolet light is 250-260 nm, for example, 252 nm, 254 nm, 256 nm, 258 nm, etc., preferably 254 nm.

[0040] Preferably, the cross-linking time is 3.5-4.5 h, for example, 3.8 h, 4 h, 4.3 h, etc.

[0041] Preferably, the strength of the porous sponge after cross-linking is 2-10 J / cm 2 , for example, 3 J / cm 2 , 5 J / cm 2 , 7 J / cm 2 , 9 J / cm 2 wait.

[0042] Preferably, the deodorization temperature is 30-50°C, for example, 32°C, 34°C, 36°C, 38°C, etc., and the deodorization time is 24-72 h, for example, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, 60 h, 65 h, 70 h, etc.

[0043] Preferably, the deodorization is performed by alternating nitrogen filling and vacuuming.

[0044] Preferably, the time for nitrogen filling and vacuum flushing is independently 0.8-1.5 h at each location, for example, it can be 1 h, 1.2 h, 1.3 h, 1.4 h, etc.

[0045] Preferably, the particle size of the crushed hemostatic particles is 50-300 mesh, for example, 60 mesh, 80 mesh, 100 mesh, 150 mesh, 200 mesh, 250 mesh, etc.

[0046] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0047] Preferably, the preparation method comprises the following steps:

[0048] (1) dissolving gelatin in water at a material-liquid ratio of 1:(10-15) to obtain a gelatin aqueous solution, mixing the gelatin aqueous solution, an auxiliary hemostatic material and xylo-oligosaccharide, dissolving the solution, cooling the solution to 15-30° C., adding silk fibroin, and mixing well to obtain a mixed solution;

[0049] (2) adding any one of hydrogen peroxide, sodium percarbonate or peracetic acid to the mixed solution and performing an oxidation reaction at 2-6° C. for 10-30 min. After the reaction is completed, adjusting the pH value to neutral and freeze-drying for 36-48 h to obtain a porous sponge;

[0050] (3) The porous sponge is cross-linked under ultraviolet light with a wavelength of 250-260 nm for 3.5-4.5 h, and the strength after cross-linking is 2-10 J / cm 2 Then, the mixture is deodorized at 30-50°C by alternately filling with nitrogen and evacuating with vacuum for 24-72 hours, and then crushed to 50-300 meshes and sterilized to obtain the hemostatic particles.

[0051] In a third aspect, the present invention provides a use of the hemostatic granules for diffuse bleeding as described in the first aspect in the preparation of a drug and / or hemostatic material for treating diffuse visceral bleeding.

[0052] The internal organs include any one of the gastrointestinal tract, liver, heart, lung, pancreas or spleen.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention effectively combines gelatin, silk fibroin, xylooligosaccharide and auxiliary hemostatic materials, and the prepared hemostatic particles can effectively stop bleeding and accelerate wound healing while maintaining high swelling capacity and good adhesion; wherein, silk fibroin and xylooligosaccharide have a synergistic effect, and together enhance the anti-inflammatory, antibacterial and growth-promoting effects of the hemostatic particles;

[0055] In the preparation method provided by the present invention, gelatin, silk fibroin, xylo-oligosaccharide and auxiliary hemostatic material are processed into a rehydrated material, which has good biocompatibility, excellent water absorption and swelling properties, is not easy to cause allergic or rejection reactions, and can improve the guarantee that the hemostatic material does not disintegrate in a high humidity environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is an electron microscope image of the hemostatic particles prepared in Example 1;

[0057] Figure 2 This is a diagram showing the wound surface condition when the hemostatic particles prepared in Example 1 are applied;

[0058] Figure 3 This is a diagram showing the wound surface after the hemostatic particles prepared in Example 1 were applied for testing;

[0059] Figure 4 This is a tensile diagram of the adhesion ability of the hemostatic particles prepared in Example 1;

[0060] Figure 5Schematic diagram of the swelling ability of the hemostatic particles prepared in Example 1. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0062] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0063] "Optional" or "any one" means that the subsequently described matter or event can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0064] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0065] The terms "one embodiment", "some embodiments", "exemplarily", "specific examples" or "some examples" described in the present invention mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this document, the schematic representation of the above terms does not necessarily refer to the same embodiment or example.

[0066] The sources of reagents or instruments in the following examples are as follows:

[0067] Gelatin: The quality standard is Part II of the Pharmacopoeia, and it is extracted from cowhide;

[0068] Silk fibroin: 1000-10000 Da, purchased from Hanzhong Hansuyuan Biotechnology Co., Ltd.;

[0069] Xylo-oligosaccharide: molecular weight 300-1000, purchased from Shandong Longli Biotechnology Co., Ltd.;

[0070] Sodium alginate: The quality standard is Pharmacopoeia Part IV;

[0071] Casein: purchased from Henan Wanbang Industrial Co., Ltd.;

[0072] Carrageenan: purchased from Henan Wanbang Industrial Co., Ltd.

[0073] Example 1

[0074] This embodiment provides a hemostatic granule for diffuse bleeding, wherein the raw materials of the hemostatic granule include, by weight: 40 parts of gelatin, 2.7 parts of silk fibroin, 0.3 parts of xylo-oligosaccharide, 3 parts of sodium alginate, and 1 part of casein;

[0075] The preparation method comprises the following steps:

[0076] (1) Dissolve gelatin in water at 80°C at a material-liquid ratio of 1:12.5, add sodium alginate, casein and xylo-oligosaccharide while hot, dissolve, mix, cool to room temperature, add silk fibroin, mix, and obtain a mixed solution;

[0077] (2) adding 0.15 mol / L hydrogen peroxide (the concentration of hydrogen peroxide is 30%) to the mixed solution for oxidation reaction for 20 min. After the reaction, adjusting the pH value to neutral with NaOH / HCl and freeze-drying for 40 h to obtain a porous sponge;

[0078] (3) The porous sponge was cross-linked under 254 nm ultraviolet light for 4 h. After the cross-linking was completed, the intensity was 6 J / cm 2 Then, the mixture was deodorized at 40° C. by alternately filling with nitrogen and evacuating with vacuum for 60 h, and then crushed to 200 meshes and sterilized to obtain the hemostatic granules.

[0079] The obtained hemostatic particles were subjected to electron microscopy test and the obtained electron microscopy images are shown in FIG. Figure 1 shown.

[0080] Example 2

[0081] This embodiment provides a hemostatic granule for diffuse bleeding, wherein the raw materials of the hemostatic granule include, by weight: 30 parts of gelatin, 2.9 parts of silk fibroin, 0.1 parts of xylo-oligosaccharide, 2 parts of sodium alginate, and 2 parts of carrageenan;

[0082] The preparation method comprises the following steps:

[0083] (1) Dissolve gelatin in 90°C water at a material-liquid ratio of 1:10, add sodium alginate, carrageenan and xylo-oligosaccharide while hot, dissolve, mix, cool to room temperature, add silk fibroin, mix, and obtain a mixed solution;

[0084] (2) adding 0.1 mol / L hydrogen peroxide (the concentration of hydrogen peroxide is 30%) to the mixed solution for oxidation reaction for 30 min. After the reaction, adjusting the pH value to neutral with NaOH / HCl and freeze-drying for 36 h to obtain a porous sponge;

[0085] (3) The porous sponge was cross-linked under ultraviolet light at a wavelength of 254 nm for 3.5 h. After the cross-linking was completed, the strength was 2 J / cm 2 Then, the mixture was deodorized at 50° C. by alternately filling with nitrogen and evacuating with vacuum for 24 h, and then crushed to 100 mesh and sterilized to obtain the hemostatic granules.

[0086] Example 3

[0087] This embodiment provides a hemostatic granule for diffuse bleeding, wherein the raw materials of the hemostatic granule include, by weight: 50 parts of gelatin, 2.5 parts of silk fibroin, 0.5 parts of xylo-oligosaccharide, 1.5 parts of sodium alginate, and 1.5 parts of casein;

[0088] The preparation method comprises the following steps:

[0089] (1) Dissolve gelatin in boiling water at a material-liquid ratio of 1:15, add sodium alginate, casein and xylo-oligosaccharide while hot, dissolve, mix, cool to room temperature, add silk fibroin, mix, and obtain a mixed solution;

[0090] (2) Add 0.25 mol / L hydrogen peroxide (the concentration of hydrogen peroxide is 30%) to the hemostatic sponge for oxidation reaction for 15 min. After the reaction, adjust the pH value to neutral with NaOH / HCl and freeze-dry for 48 h to obtain a porous sponge.

[0091] (3) The porous sponge was cross-linked under ultraviolet light at a wavelength of 254 nm for 4.5 h. After the cross-linking was completed, the strength was 10 J / cm 2 Then, the mixture was deodorized at 30° C. by alternately filling with nitrogen and evacuating with vacuum for 72 h, and then crushed to 300 mesh and sterilized to obtain the hemostatic granules.

[0092] Example 4

[0093] This embodiment provides hemostatic particles for diffuse bleeding. The difference between this embodiment and embodiment 1 is that the sodium alginate is adjusted to 4 parts and casein is not added. Other raw materials, dosages and preparation methods are the same as those in embodiment 1.

[0094] Example 5

[0095] This embodiment provides hemostatic particles for diffuse bleeding, which differ from Example 1 only in that the total amount of silk fibroin and xylo-oligosaccharide is kept unchanged, the silk fibroin is adjusted to 2 parts and the xylo-oligosaccharide is adjusted to 1 part, and other raw materials, amounts and preparation methods are the same as those in Example 1.

[0096] Example 6

[0097] This embodiment provides hemostatic particles for diffuse bleeding, which differ from Example 1 only in that the silk fibroin is adjusted to 1.8 parts and the oligoxylose is adjusted to 0.2 parts, and other raw materials, dosages and preparation methods are the same as those in Example 1.

[0098] Example 7

[0099] This embodiment provides hemostatic particles for diffuse bleeding. The difference between this embodiment and embodiment 1 is that the silk fibroin is adjusted to 3.6 parts and the oligoxylose is adjusted to 0.4 parts. Other raw materials, dosages and preparation methods are the same as those in embodiment 1.

[0100] Comparative Example 1

[0101] This comparative example provides a hemostatic granule for diffuse bleeding, which differs from Example 1 only in that the silk fibroin is adjusted to 3 parts and oligoxylose is not added. Other raw materials, dosages and preparation methods are the same as those in Example 1.

[0102] Comparative Example 2

[0103] This comparative example provides a hemostatic granule for diffuse bleeding, which is different from Example 1 only in that the amount of xylo-oligosaccharide is adjusted to 3 parts, and silk fibroin is not added. Other raw materials, dosages and preparation methods are the same as those in Example 1.

[0104] Comparative Example 3

[0105] This comparative example provides hemostatic particles for diffuse bleeding, which differ from Example 1 only in the preparation method. In the preparation method of this comparative example, the oxidation reaction of step (2) is not performed, and after completing step (1), the mixed solution is directly subjected to step (3) cross-linking. Other raw materials, amounts and preparation methods are the same as those in Example 1.

[0106] Comparative Example 4

[0107] This comparative example provides a hemostatic granule for diffuse bleeding, which differs from Example 1 only in step (3) of the preparation method. Step (3) of this comparative example is:

[0108] The porous sponge is crushed to 200 meshes and sterilized to obtain the hemostatic particles.

[0109] Test Example 1

[0110] Adhesion

[0111] The determination of adhesion refers to YY / T 1477.4-2017 Standard test model for performance evaluation of contact wound dressings Part 4: In vitro model for evaluating the potential adhesion of wound dressings;

[0112] The hemostatic granule samples prepared in the examples and comparative examples were evenly spread on two tinplates, and then the wound model was placed between the two samples. The sample and wound model assembly was placed on a horizontal tray, a pressing block was applied on the assembly and incubated at room temperature for 10 min. The assembly of the sample and wound model was placed in a drying oven at (37±2)°C and dried for 24 h. After the sample surface was dry, it was taken out and placed at room temperature.

[0113] The combination of the sample and the wound model was mounted on the clamp of the tensile testing machine, so that the upper and lower clamps clamped two samples respectively. The initial spacing between the two clamps was about 50 mm, and the clamping distance was 2.5 cm. The sample was stretched at a rate of 100 mm / min until one of the samples was completely separated from the fibrin clot. Each test was repeated three times. The test results are shown in Table 1. For example, the adhesion tensile force diagram of the hemostatic particles prepared in Example 1 is shown in Table 1. Figure 4 shown.

[0114] Test Example 2

[0115] Swelling capacity

[0116] The determination of swelling capacity refers to GB / T 20405.5-2006 / ISO 17190-5:2001 Urine absorbents for incontinence: Test methods for properties of polymer matrix absorbent materials - Part 5: Determination of water absorption by weighing in physiological saline;

[0117] At room temperature, weigh the hemostatic granule samples prepared in the examples and comparative examples and record them as m1, accurate to 0.001 g. Immerse the gauze and rubber band completely in the physiological saline solution and take them out. Wipe the surface of the gauze with absorbent cotton and weigh the physiological saline solution accurately. The mass of the gauze is recorded as m2. The mass of the rubber band is recorded as m3. After wrapping the sample in gauze, place it in a beaker, add physiological saline solution, make the product completely immersed in the physiological saline solution, and shake the gauze slightly to make the inside of the product fully contact with water.

[0118] After soaking for 24 minutes, take the gauze out of the beaker and let it stand for 4 minutes. Then use absorbent paper to gently wipe off the saline solution on the surface of the gauze, and accurately weigh and record m4. The swelling capacity (X) is calculated as follows:

[0119] Each test was repeated three times. The results are shown in Table 1. For example, the swelling capacity of the hemostatic particles prepared in Example 1 is shown in Table 1. Figure 5 shown.

[0120] Test Example 3

[0121] In vitro blood clotting time

[0122] Blood clotting time method: Weigh 2.5 mL of sterile defibrinated sheep blood into a 15 mL centrifuge tube, add 0.2 g of the hemostatic particles prepared in the example and the comparative example respectively, and vibrate on a vortex shaker at 3000 rmp / min, and record the blood clotting time. Each sample is tested 3 times in parallel. The measurement results are shown in Table 1.

[0123] Test Example 4

[0124] In vivo hemostasis test

[0125] Test method: (1) SD rats (male, 0.2-0.25 kg) were selected and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.03 mL / kg); (2) three cuts were made on the liver with a scalpel, with a length of about 1 cm and a depth of about 2-4 mm; (3) the wound was covered with the hemostatic granule samples prepared in the embodiment and the comparative example with minimal pressure; (4) after 60 s, the wound was rinsed with physiological saline and the hemostasis condition was recorded. For example, the wound condition when the hemostatic granules prepared in Example 1 were applied was as follows: Figure 2 As shown, the wound surface condition after applying the hemostatic particles prepared in Example 1 is as follows Figure 3 As shown in the figure, it can be seen that the hemostatic particles prepared in Example 1 are used to treat the wound, which can effectively stop bleeding, reduce the wound surface, and the wound does not show symptoms such as inflammation and swelling, indicating that the hemostatic particles prepared in Example 1 have good hemostatic, anti-inflammatory, antibacterial and growth-promoting effects.

[0126] Table 1

[0127]

[0128] According to the table data, it can be seen from Examples 1-4 that the auxiliary hemostatic material is used in combination with sodium alginate and casein or carrageenan, and the effect is optimal. When sodium alginate is used alone, the adhesion and in vitro coagulation time will decrease; it can be seen from Examples 1 and 5 that when the ratio of silk fibroin to oligoxylose is (2.5-2.9): (0.1-0.5), the prepared hemostatic particles have the best effect. When the amount of silk fibroin is low, the adhesion decreases and the coagulation time is prolonged; it can be seen from Examples 1 and 6-7 that when the total amount of silk fibroin and oligoxylose is too low, the proportion of other raw materials in the hemostatic particles increases, the swelling ability is higher, the adhesion ability is lower, and the coagulation time of the blood in vitro is longer; when the total amount of silk fibroin and oligoxylose is too high, the adhesion ability is higher, but the swelling ability decreases, and the coagulation time of the blood in vitro is prolonged; it can be seen from Example 1 and Comparative Examples 1-2 that silk fibroin and oligoxylose work synergistically. The hemostatic and anti-inflammatory effects of the hemostatic particles are jointly enhanced. When a single silk fibroin or a single oligoxylose is used, the adhesion ability and the swelling ability are reduced. Oligoxylose also has a certain immunomodulatory effect, which can enhance the body's immune function, reduce inflammatory reactions, and have a certain inhibitory effect on allergic reactions. Silk fibroin also has the effects of promoting wound healing, anti-oxidation and antibacterial, and has a positive effect on skin repair and protection. The combination of the two can promote anti-inflammation and increase adhesion ability; It can be seen from Example 1 and Comparative Example 3 that it is very necessary to carry out an oxidation reaction on the raw materials, which can increase the swelling rate, can be well attached to the wound surface, quickly absorb water in the blood, and achieve a good hemostatic effect; It can be seen from Example 1 and Comparative Example 4 that cross-linking can cause the protein in the absorbable hemostatic gel to form a network structure with considerable elasticity and resistance to mechanical pressure. If ultraviolet cross-linking is not carried out after the oxidation reaction, it will seriously affect the extension of the blood clotting time.

[0129] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A hemostatic granule for diffuse bleeding, characterized in that: The raw materials of the hemostatic granules include, by weight: 20-60 parts of gelatin, 2.5-2.9 parts of silk fibroin, 0.1-0.5 parts of xylo-oligosaccharide and 2-6 parts of auxiliary hemostatic material; The auxiliary hemostatic material includes sodium alginate; The hemostatic granules for diffuse bleeding are prepared by the following method, which comprises the following steps: (1) Evenly mixing the gelatin aqueous solution, the auxiliary hemostatic material and the xylo-oligosaccharide, and then adding the silk fibroin to obtain a mixed solution; (2) subjecting the mixed solution to an oxidation reaction, and freeze-drying the mixed solution after the reaction is completed to obtain a porous sponge; (3) Cross-linking the porous sponge under ultraviolet light, and after the cross-linking is completed, deodorizing, crushing, and sterilizing to obtain the hemostatic particles.

2. The hemostatic granules according to claim 1, characterized in that In parts by weight, the raw materials of the hemostatic granules include: 30-50 parts of gelatin, 2.5-2.9 parts of silk fibroin, 0.1-0.5 parts of xylo-oligosaccharide, and 3-6 parts of auxiliary hemostatic materials.

3. The hemostatic granules according to claim 1, characterized in that The molecular weight of the silk fibroin is 100-10000Da.

4. A method for preparing hemostatic granules for diffuse bleeding according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Evenly mixing the gelatin aqueous solution, the auxiliary hemostatic material and the xylo-oligosaccharide, and then adding the silk fibroin to obtain a mixed solution; (2) subjecting the mixed solution to an oxidation reaction, and freeze-drying the mixed solution after the reaction is completed to obtain a porous sponge; (3) Cross-linking the porous sponge under ultraviolet light, and after the cross-linking is completed, deodorizing, crushing, and sterilizing to obtain the hemostatic particles.

5. The preparation method according to claim 4, characterized in that: In step (1), the solid-liquid ratio of gelatin to water in the gelatin aqueous solution is 1:(10-15).

6. The preparation method according to claim 4, characterized in that: In step (1), the silk fibroin is added at a temperature of 15-30°C.

7. The preparation method according to claim 4, characterized in that: In step (2), the freeze-drying time is 36-48 h.

8. The preparation method according to claim 4, characterized in that: The oxidant used in the oxidation reaction includes any one of hydrogen peroxide, sodium percarbonate or peracetic acid.

9. The preparation method according to claim 4, characterized in that: The temperature of the oxidation reaction is 2-6°C and the time is 10-30 min.

10. The preparation method according to claim 4, characterized in that: The step of adjusting the pH value to be neutral is also included after the oxidation reaction.

11. The preparation method according to claim 4, characterized in that: The wavelength of the ultraviolet light is 250-260 nm.

12. The preparation method according to claim 4, characterized in that: The cross-linking time is 3.5-4.5 h.

13. The preparation method according to claim 4, characterized in that: The strength of the porous sponge after cross-linking is 2-10 J / cm 2 .

14. The preparation method according to claim 4, characterized in that: The deodorization temperature is 30-50°C and the deodorization time is 24-72 hours.

15. The preparation method according to claim 4, characterized in that: The deodorization is carried out by alternately filling with nitrogen and evacuating the air.

16. The preparation method according to claim 4, characterized in that: The particle size of the crushed hemostatic particles is 50-300 meshes.

17. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: (1) dissolving gelatin in water at a material-liquid ratio of 1:(10-15) to obtain a gelatin aqueous solution, mixing the gelatin aqueous solution, an auxiliary hemostatic material and xylo-oligosaccharide, dissolving the solution, cooling the solution to 15-30° C., adding silk fibroin, and mixing well to obtain a mixed solution; (2) adding any one of hydrogen peroxide, sodium percarbonate or peracetic acid to the mixed solution and performing an oxidation reaction at 2-6° C. for 10-30 min. After the reaction is completed, adjusting the pH value to neutral and freeze-drying for 36-48 h to obtain a porous sponge; (3) The porous sponge is cross-linked under ultraviolet light with a wavelength of 250-260 nm for 3.5-4.5 h, and the strength after cross-linking is 2-10 J / cm 2 Then, the mixture is deodorized at 30-50° C. by alternately filling with nitrogen and evacuating with vacuum for 24-72 h, and then crushed to 50-300 meshes and sterilized to obtain the hemostatic granules.

18. Use of the hemostatic granules for diffuse bleeding according to any one of claims 1 to 3 in the preparation of a medicine and / or hemostatic material for treating diffuse visceral bleeding.

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