A method for producing a hemostatic material and a hemostatic material

Abstract

This application provides a method for preparing a hemostatic material and the hemostatic material itself. The method includes: preparing an ink mixture using a washing buffer and ink sac; adding a cryoprotectant composed of trehalose, ascorbate palmitate, and alginate oligosaccharides to form a protective mixture; obtaining cuttlefish ink powder through rapid freezing and staged temperature-increasing drying; mixing the cuttlefish ink powder with a phosphate extraction buffer, followed by ultrasonic disruption and enzymatic hydrolysis with a mixed enzyme; obtaining the supernatant by centrifugation and freeze-drying to obtain an active ingredient powder; dissolving the active ingredient powder in an acetate-sodium acetate buffer solution, adding chitosan and ε-polylysine, and adjusting the pH to 7.0–8.5 to form a cuttlefish ink composite solution; adding CaCl2 solution to construct a gel-like substance, followed by freeze-drying to obtain the hemostatic material. This method improves the stability and physiological activity of the active ingredients in the hemostatic material, resulting in a hemostatic material with small particle size, strong antioxidant properties, and good hemostatic effect.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a method for preparing a hemostatic material and the hemostatic material itself. Background Technology

[0002] Hemostatic materials are widely used in clinical settings such as emergency care, surgery, and wound treatment to quickly control bleeding and promote coagulation. Currently, common clinical hemostatic materials include gelatin sponges, chitosan dressings, oxidized cellulose, and human or animal-derived coagulation factors. However, these materials generally suffer from high sourcing costs, poor antibacterial properties, limited adaptability, and the potential to induce immune responses. Therefore, developing novel natural hemostatic materials with widely available sources, well-defined hemostatic mechanisms, and multifunctional bioactivity has become a research hotspot.

[0003] In traditional medicine, there is an empirical therapy that believes "blood stops when it comes into contact with black," suggesting that black medicinal materials can quickly stop bleeding and astringe blood. This concept is reflected in various ancient prescriptions, such as palm charcoal, cuttlebone powder, and black ink, which are used to treat symptoms such as metrorrhagia, hematochezia, or traumatic bleeding. Modern research shows that cuttlebone ink is rich in natural melanin, polysaccharides, polypeptides, and various trace minerals, possessing good antioxidant, immunomodulatory, and antibacterial biological activities. The melanin and biopolysaccharide complexes are believed to induce a rapid hemostatic response at the wound site by regulating platelet aggregation and procoagulant factor activity. However, current research on cuttlebone ink-related products mainly focuses on antioxidant or immunomodulatory aspects. A mature system for the controllable extraction and precise release of active ingredients has not yet been established, hindering its widespread clinical application. Summary of the Invention

[0004] In view of this, this application provides a method for preparing a hemostatic material and a hemostatic material, which can be used to prepare a hemostatic material with small particle size, strong antioxidant properties and significant hemostatic effect using ink sacs.

[0005] Specifically, this application is implemented through the following technical solution:

[0006] The first aspect of this application provides a method for preparing a hemostatic material, the method comprising:

[0007] An ink mixture was prepared using a cleaning buffer and an ink sac, and a cryoprotectant was added to the ink mixture to obtain a protective mixture; the cryoprotectant included trehalose, ascorbate palmitate, and alginate oligosaccharide.

[0008] The protective mixture is cooled to a specified sub-zero temperature at a specified cooling rate and held at that specified sub-zero temperature for a first specified duration, followed by a staged heating and drying process to obtain cuttlefish ink powder.

[0009] The cuttlefish ink powder and phosphate extraction buffer are mixed, and after ultrasonic disruption, a mixed enzyme is added to carry out an enzymatic hydrolysis reaction to release the active ingredients in the cuttlefish ink powder, thereby obtaining a cuttlefish ink enzymatic hydrolysate; the mixed enzyme includes trypsin, chitinase and β-glucanase.

[0010] The cuttlefish ink enzymatic hydrolysate was centrifuged, the supernatant was collected, and the supernatant was freeze-dried to obtain cuttlefish ink active ingredient powder.

[0011] The active ingredient powder of cuttlefish ink was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added in sequence. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution.

[0012] Adding CaCl2 solution to the cuttlefish ink composite solution forms a gel-like substance with a three-dimensional network structure. Freeze-drying the gel-like substance yields a hemostatic material.

[0013] The second aspect of this application provides a hemostatic material, which is prepared using the preparation method described in any one of the first aspects of this application.

[0014] The hemostatic material preparation method and hemostatic material provided in this application achieve the synergistic release of multiple active ingredients in cuttlefish ink by integrating techniques such as cryoprotectant treatment, staged high-speed heating treatment, ultrasonic disruption, and mixed enzymatic hydrolysis reaction, while maintaining the stability of its structure and function. The hemostatic material prepared using this method is naturally derived, has a clearly defined composition, fully releases active ingredients, and exhibits good biocompatibility and hemostatic properties, making it suitable for various medical scenarios such as trauma emergency care, intraoperative hemostasis, and functional dressings. Attached Figure Description

[0015] Figure 1 This is a flowchart of Example 1 of the preparation method of the hemostatic material provided in this application. Detailed Implementation

[0016] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0017] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used herein are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0018] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0019] The following specific embodiments are given to illustrate the technical solution of this application in detail.

[0020] Figure 1 This is a flowchart of Example 1 of the preparation method of the hemostatic material provided in this application. Please refer to... Figure 1 The method provided in this embodiment may include:

[0021] S101. Prepare an ink mixture using a cleaning buffer and an ink cartridge, and add a cryoprotectant to the ink mixture to obtain a protective mixture; the cryoprotectant includes trehalose, ascorbate palmitate, and alginate oligosaccharide.

[0022] Specifically, the ink cartridges can be cleaned using a cleaning buffer solution, and concentrated ink can be prepared using the cleaned ink cartridges. The concentrated ink can then be mixed with the cleaning buffer solution to obtain an ink buffer mixture.

[0023] It should be noted that cryopreserved squid ink sacs and / or cuttlefish ink sacs are selected as raw materials. The ink sacs are gently washed 3-5 times with a washing buffer solution of 1-3 times their volume to thoroughly remove residual blood, impurities, and metabolites from the ink sac surface, ensuring the purity and stability of the subsequently extracted active ingredients. Furthermore, the aforementioned washing buffer solution is specifically a phosphate buffer solution with a pH of 7.4, containing 0.9-1.3% (w / v) NaCl. This washing buffer solution effectively simulates the physiological osmotic environment, preventing premature rupture or structural changes in cell components.

[0024] Furthermore, the cleaned ink sacs are cut into pieces and placed in a sterile slurry cup, and then subjected to high-speed homogenization at 4°C to obtain a dark brown concentrated ink. This concentrated ink is rich in natural melanin, polysaccharides, polypeptides, and a small amount of lipids and minerals.

[0025] Furthermore, the concentrated ink is mixed with the washing buffer at a volume ratio of 1:1 to form a homogeneous ink mixture. This ink mixture maintains the stability of the particle size distribution of melanin in the concentrated ink, which is beneficial to the full dissolution and uniform action of the subsequent cryoprotectant.

[0026] To further improve the structural stability and activity retention rate during the subsequent freeze-drying process, a cryoprotectant was added to the above ink mixture and stirred thoroughly to form a protective mixture.

[0027] In one possible implementation, the cryoprotectant comprises, by mass-volume ratio, 0.08-0.26% trehalose, 0.03-0.06% ascorbate palmitate and 0.04-0.08% alginate oligosaccharide.

[0028] It should be noted that trehalose, ascorbate palmitate, and alginate oligosaccharides work synergistically at low temperatures to enhance the low-temperature tolerance and antioxidant capacity of cell membranes and protein structures. Trehalose, by forming hydrogen bonds with protein amino groups, constructs an amorphous structure, acting as a "molecular glass" protective membrane. Ascorbate palmitate, a fat-soluble vitamin C derivative, inserts into the membrane structure to improve lipid stability and scavenge cryogenic free radicals. Alginate oligosaccharides stabilize the three-dimensional conformation of proteins and melanin granules through electrostatic adsorption and chelation effects, while also inhibiting oxidative degradation induced by metal ions.

[0029] S102. The protective mixture is cooled to a specified sub-zero temperature at a specified cooling rate and held at the specified sub-zero temperature for a first specified duration, followed by a staged heating and drying process to obtain cuttlefish ink powder.

[0030] In practice, the protective mixture can be rapidly cooled to -40°C at a rate of at least 5°C / min and maintained at this temperature for 4 hours. This rapid cooling causes the water in the system to quickly form a dense and uniform ice crystal structure, preventing damage to proteins and polysaccharides caused by irregular ice crystal growth. Trehalose and alginate oligosaccharides in the cryoprotectant compete with water molecules to form hydrogen bond networks during cooling, thus stabilizing cellular components and polypeptide structures. Ascorbate palmitate, with its antioxidant properties, synergistically reduces the risk of oxidative degradation of melanin and enzyme components during freezing. These three substances work together to provide cryoprotection, protecting the active ingredients in squid ink and preventing freezing damage and reduced activity.

[0031] Furthermore, in one possible implementation, the step of subjecting the cooled mixture to a staged heating and drying process to obtain cuttlefish ink powder includes:

[0032] The cooling mixture is heated to a first specified temperature according to a preset heating rate and maintained for a first preset duration; wherein the first specified temperature is a sub-zero temperature.

[0033] The temperature of the cooling mixture is raised to a second specified temperature and maintained for a second preset duration; wherein the second specified temperature is a sub-zero temperature and the second preset duration is less than the first preset duration;

[0034] The temperature of the cooling mixture is raised to 0°C and maintained for a third preset duration.

[0035] In practice, for example, the temperature is first raised to -20°C and held for 2-3 hours to allow some free water to sublimate; then the temperature is raised to -10°C and held for 1-2 hours to further remove bound water; finally, the temperature is slowly raised to 0°C and held for 1-2 hours. This process ensures uniform thawing of the sample while completing the final drying process, resulting in powdered, structurally intact cuttlefish ink powder. This staged heating and drying process avoids prolonged low-temperature freezing, preserving the active ingredients and facilitating later applications.

[0036] It should be noted that, as described above, the cuttlefish ink powder is squid ink powder, cuttlefish ink powder, or a mixture of squid ink powder and cuttlefish ink powder. Although the raw materials differ, squid ink powder and cuttlefish ink powder have strong similarities in the structure of melanin, mucopolysaccharides, and protein components. Therefore, they exhibit good consistency during freeze-drying and can both produce cuttlefish ink powder with good flowability and uniform color.

[0037] S103. The cuttlefish ink powder and phosphate extraction buffer are mixed, and after ultrasonic crushing, a mixed enzyme is added to carry out an enzymatic hydrolysis reaction to release the active ingredients in the cuttlefish ink powder, thereby obtaining a cuttlefish ink enzymatic hydrolysate; the mixed enzyme includes trypsin, chitinase and β-glucanase.

[0038] Furthermore, cuttlefish ink powder was mixed with a pH 6.0 phosphate extraction buffer to form a preliminary extract. The preliminary extract was then subjected to ultrasonic disruption using a probe-type ultrasonic device to break down larger particles, releasing cuttlefish ink pigments and small-molecule active ingredients. In addition, ultrasonic disruption helps to break down the physical barrier between ink particles and mucopolysaccharides, promoting the deconstruction of cell membrane structures, thereby improving the permeability and efficiency of subsequent enzymatic reactions.

[0039] It should be noted that, in one possible implementation, the parameters for the ultrasonic fragmentation treatment are set as follows: frequency of 20~25kHz, power density of 300~500 W / cm2, treatment time of 10~20 min, and temperature of 0~4℃. The combination of ultrasound and enzymatic hydrolysis can promote the release of a large amount of active ingredients from the ink sac, resulting in a synergistic effect.

[0040] Furthermore, in one possible implementation, the addition of mixed enzymes for enzymatic hydrolysis includes: adding 2000-2800 U / g trypsin, 900-1000 U / g chitinase, and 800-1300 U / g β-glucanase, maintaining the reaction temperature at 35-40°C, and the hydrolysis time at 2-5 hours.

[0041] It should be noted that trypsin can effectively cleave structural proteins and polypeptide bonds in cuttlefish ink, releasing low-molecular-weight peptides with procoagulant activity; chitinase mainly acts on chitosan or chitin residues that may be present in cuttlefish ink, assisting in the degradation of cell wall or shell residues; in addition, β-glucanase can break down heteropolysaccharide chains in cuttlefish ink, making active polysaccharides easier to dissolve and homogenize, improving the uniformity and biostability of subsequent gelation and compounding. The synergistic effect of the above mixed enzymes not only enhances the release efficiency of the target active factors, but also significantly improves the physicochemical homogeneity of the raw material system.

[0042] S104. Centrifuge the cuttlefish ink enzymatic hydrolysate, collect the supernatant, and freeze-dry the supernatant to obtain cuttlefish ink active ingredient powder.

[0043] Specifically, in order to further remove residual undegraded particles and enzyme reaction residues from the cuttlefish ink enzymatic hydrolysate, in one possible implementation, the cuttlefish ink enzymatic hydrolysate is placed in a centrifuge tube and centrifuged at 9000~12000 g for 10~20 min, which can effectively settle residual tissue fragments, unreacted macromolecules and excess enzymes in the reaction system, and obtain a clear supernatant.

[0044] It should be noted that the supernatant is rich in various functional active components, including natural melanin, polysaccharides, bioadhesive peptides, and small molecule metabolites from cuttlefish ink. Melanin mainly exists as aggregates with a particle size of tens of nanometers, exhibiting good dispersibility and biocompatibility, and possessing potential hemostatic, antioxidant, and antibacterial effects. The polysaccharide component has strong liquid absorption and swelling capacity and gel-forming ability, which can form a protective barrier on the wound surface. The supernatant obtained at this stage is the crude extract of the functional active substances from cuttlefish ink, a core intermediate for constructing hemostatic materials.

[0045] Furthermore, to facilitate long-term preservation and subsequent formulation operations, the supernatant needs to be freeze-dried to produce squid ink active ingredient powder. First, the supernatant is rapidly frozen at -40°C, converting the water in the system into ice crystals while preserving the natural configuration of the functional components to the greatest extent possible. Subsequently, sublimation drying is performed under vacuum, directly converting the water from solid ice to gas and removing it, avoiding molecular rearrangement or structural denaturation that might be caused by liquid water. This achieves gentle dehydration without high-temperature interference. This process preserves the original bioactivity of sensitive components such as melanin and polysaccharides in squid ink. The resulting dry powder is dark gray to black, lightweight, has good flowability, and possesses excellent solubility and resolubility.

[0046] S105. Dissolve the active ingredient powder of cuttlefish ink in an acetate-sodium acetate buffer solution, add chitosan and ε-polylysine in sequence, and adjust the pH to 7.0-8.5 to obtain a cuttlefish ink composite solution.

[0047] Furthermore, in this step, the active ingredient powder of cuttlefish ink is dissolved in an acetate-sodium acetate buffer solution with a pH of 4.5–5.0 to promote the full dissolution of the subsequent functional polymers and the smooth occurrence of intermolecular interactions. The acetate-sodium acetate system not only has good pH buffering capacity but also effectively dissolves natural polysaccharides and alkaloids, providing a stable reaction environment for constructing functional composite solutions.

[0048] Further, chitosan and ε-polylysine are added sequentially; wherein the mass of the added chitosan is equal to 60-85% of the mass of the cuttlefish ink active ingredient powder; and the mass of the added ε-polylysine is equal to 25-40% of the mass of the cuttlefish ink active ingredient powder.

[0049] It should be noted that chitosan is a cationic polysaccharide, naturally derived, and contains a large number of amino and hydroxyl groups in its structure. Chitosan not only possesses good biodegradability and biocompatibility, but it can also form stable network structures through hydrogen bonding and electrostatic interactions with various anionic molecules. Furthermore, chitosan exhibits excellent hemostatic and antibacterial properties. ε-Polylysine is a natural linear cationic peptide with antibacterial activity. Under neutral to weakly alkaline conditions, it exhibits strong charge density and molecular flexibility. When combined with chitosan, it can synergistically form a denser gel network, and its hydrophobic side chains enhance the mechanical properties and interfacial adhesion of the composite material.

[0050] Furthermore, the above solution is stirred thoroughly, and the pH is gradually adjusted to a neutral or slightly alkaline environment of 7.0–8.5. This triggers an electrostatically driven self-assembly process between cationic polymers such as chitosan and ε-polylysine and proteins, melanin aggregates, and polysaccharide molecules in the cuttlefish ink active ingredient powder. After this self-assembly process, a cuttlefish ink composite solution is obtained. This composite solution is a nanoscale composite polymer with uniform size and stable structure. This cuttlefish ink composite solution not only effectively integrates the cuttlefish ink active ingredient powder with functional biopolymers but also significantly improves the overall stability, antibacterial properties, and biocompatibility of the hemostatic material.

[0051] S106. Add CaCl2 solution to the cuttlefish ink composite solution to form a gel-like substance with a three-dimensional network structure. Freeze-dry the gel-like substance to obtain a hemostatic material.

[0052] Specifically, 0.1–0.3 mol / L CaCl2 solution is slowly added to the cuttlefish ink composite solution to induce ionic cross-linking, and the reaction is maintained for 30–45 min. During this process, chitosan, ε-polylysine, and the active ingredient powder of cuttlefish ink form a complex through hydrogen bonding or electrostatic interactions in the cuttlefish ink composite solution; Ca... 2+ It undergoes ionic cross-linking with the hydroxyl and amino groups of chitosan to form a gel-like substance.

[0053] It should be noted that the gel-like substance exhibits both ionic cross-linking and molecular self-assembly characteristics. Specifically, the amino (-NH2) and hydroxyl (-OH) groups on the chitosan molecular backbone serve as typical Lewis base sites, capable of reacting with Ca... 2+ Ions bond together to form a stable three-dimensional network structure. ε-Polylysine, a cationic linear peptide chain, also provides electrostatic interaction sites with its side-chain amine groups, facilitating cross-linking with other components in the system. Furthermore, the polysaccharides, peptides, and melanin macromolecules in the cuttlefish ink active ingredient powder contain abundant carboxyl groups, phenolic hydroxyl groups, and other polar functional groups, which can also form hydrogen bonds or electrostatic adsorption with chitosan and ε-polylysine, thus forming a denser and biofunctionally superior composite network system. This step not only significantly improves the mechanical strength and morphological stability of the hemostatic material but also enhances its liquid adsorption capacity, hemostatic efficiency, and bioadhesion.

[0054] Furthermore, the gel-like material is freeze-dried in a vacuum environment to achieve gentle dehydration and avoid structural damage and loss of activity under high temperature conditions. This process preserves the three-dimensional porous structure of the gel-like material and the biological functions of the squid ink active ingredients, ultimately yielding a dense, uniformly pore-sized nanocomposite hemostatic material with excellent hemostatic and resolubility properties.

[0055] The following specific embodiments are provided to illustrate the technical solutions of this application in detail:

[0056] Example 1:

[0057] Specifically, Table 1.1 shows the preparation conditions of the hemostatic material shown in Example 1 of this application:

[0058] Table 1.1 Preparation conditions of the hemostatic material shown in Example 1

[0059]

[0060] Please refer to Table 1.1. In Example 1, frozen squid or cuttlefish ink sacs were selected as raw materials. The ink sacs were gently washed 3-5 times with a washing buffer (pH 7.4 phosphate buffer containing 0.9-1.3% (w / v) NaCl) at 1-3 times their volume. The washed ink sacs were then cut into small pieces and placed in a sterile slurry cup for high-speed homogenization at 4°C to obtain a dark brown concentrated ink. The concentrated ink was then mixed with the washing buffer at a 1:1 volume ratio to form a homogeneous ink mixture. A cryoprotectant (including 0.20% trehalose, 0.05% ascorbate palmitate, and 0.06% alginate oligosaccharide) was added to the ink mixture and thoroughly stirred to form a protective mixture, completing the first stage of processing. The protective mixture was cooled to -40°C at a rate of 5°C / min or higher and held at this temperature for 4 hours. Following this, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 3 hours, then raised to -10°C and held for 2 hours, and finally raised to 0°C and held for 2 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with a phosphate extraction buffer and dried at 4°C using a frequency of 20 kHz and a power density of 350 W / cm². 2 The mixture was subjected to ultrasonic disruption for 15 minutes to promote structural depolymerization. Subsequently, 2000 U / g trypsin, 1000 U / g chitinase, and 1000 U / g β-glucanase were added for enzymatic hydrolysis at 35℃ for 4 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The cuttlefish ink active ingredient powder was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The mass of chitosan added was 60-85% of the mass of the cuttlefish ink active ingredient powder, and the mass of ε-polylysine added was 25-40% of the mass of the cuttlefish ink active ingredient powder. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained.

[0061] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under these experimental conditions are shown in Table 1.2.

[0062] Table 1.2 Performance parameters of the hemostatic material prepared in Example 1

[0063]

[0064] Referring to both Tables 1.1 and 1.2, it can be seen that Example 1 used a cryoprotectant composed of 0.20% trehalose, 0.05% ascorbate palmitate, and 0.06% alginate oligosaccharides, combined with a phased temperature-increasing drying process (-20℃ / 3h → -10℃ / 2h → 0℃ / 2h, vacuum degree ≤8 Pa). This preparation condition helps stabilize cell structure and protect the activity of functional factors during freeze-drying. Trehalose stabilizes the membrane structure by replacing cell membrane water, ascorbate palmitate provides lipid-soluble antioxidant protection, and alginate oligosaccharides form a gel network to prevent aggregation and inactivation. During the temperature-increasing drying process, the gradual transition of the temperature gradient effectively avoids structural collapse, helps maintain the pore structure of the composite system, and improves the rehydration and biological properties of the material.

[0065] In addition, during the active ingredient release stage, Example 1 employed ultrasonic disruption at 4°C, 20 Hz, and 350 W / cm² for 15 min. This effectively depolymerized the microstructure of the cuttlefish ink powder, disrupted the cell walls, and released the encapsulated components, significantly improving the efficiency of the enzymatic hydrolysis reaction. Subsequently, 2000 U / g trypsin, 1000 U / g chitinase, and 1000 U / g β-glucanase from the raw material were added, and enzymatic hydrolysis was carried out at 35°C for 4 hours. This effectively synergistically cleaved proteins, chitin, and polysaccharides, promoting the release of hemostatic-related active factors.

[0066] Example 2

[0067] Specifically, Table 2.1 shows the preparation conditions of the hemostatic material shown in Example 2 of this application:

[0068] Table 2.1 Preparation conditions of the hemostatic material shown in Example 2

[0069]

[0070] Referring to Table 2.1, in Example 2, frozen squid or cuttlefish ink sacs were selected as raw materials. The ink sacs were gently washed 3-5 times with a washing buffer (pH 7.4 phosphate buffer containing 0.9-1.3% (w / v) NaCl) at 1-3 times their volume. The washed ink sacs were then cut into small pieces and placed in a sterile slurry cup for high-speed homogenization at 4°C to obtain a dark brown concentrated ink. The concentrated ink was then mixed with the washing buffer at a 1:1 volume ratio to form a homogeneous ink mixture. A cryoprotectant (including 0.08% trehalose, 0.06% ascorbate palmitate, and 0.08% alginate oligosaccharide) was added to the ink mixture and thoroughly stirred to form a protective mixture, completing the first stage of processing. The protective mixture was cooled to -40°C at a rate greater than or equal to 5°C / min and held at this temperature for 4 hours. Subsequently, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 2.5 hours, then raised to -10°C and held for 1.5 hours, and finally raised to 0°C and held for 1.5 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with a phosphate extraction buffer and dried at 2°C using a frequency of 25 kHz and a power density of 300 W / cm². 2 The sample was subjected to ultrasonic disruption for 20 minutes to promote structural depolymerization. Subsequently, 2300 U / g trypsin, 900 U / g chitinase, and 800 U / g β-glucanase were added for enzymatic hydrolysis at 40℃ for 5 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The cuttlefish ink active ingredient powder was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The mass of chitosan added was 60-85% of the mass of the cuttlefish ink active ingredient powder, and the mass of ε-polylysine added was 25-40% of the mass of the cuttlefish ink active ingredient powder. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained.

[0071] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured to determine the yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under these experimental conditions are shown in Table 2.2.

[0072] Table 2.2 Performance parameters of the hemostatic material prepared in Example 2

[0073]

[0074] Please refer to Tables 2.1 and 2.2 simultaneously. It can be seen that Example 2 adjusted the composition of the cryoprotectant, the ultrasonic treatment, and the enzymatic hydrolysis conditions. Specifically, Example 2 reduced the trehalose content from 0.20% in Example 1 to 0.08%, while appropriately increasing the proportion of alginate oligosaccharides to 0.08%, and slightly increasing ascorbate palmitate to 0.06%. This reduced the interference of carbohydrate components on the cell membrane structure to a certain extent, which is beneficial for maintaining membrane permeability stability under short-term heating and drying conditions. Furthermore, Example 2 adopted a short-path heating program of -20℃ / 2.5h → -10℃ / 1.5h → 0℃ / 1.5h, which shortened the processing time compared to Example 1, improving preparation efficiency while achieving good protective effects.

[0075] Furthermore, in Example 2, the ultrasound frequency was increased to 25 Hz and the treatment time was extended to 20 min. Although the power density decreased slightly (300 W / cm², compared to 350 W / cm² in Example 1), the longer treatment time and higher frequency resulted in more complete cell lysis. In the enzymatic digestion step, the total enzyme dosage remained at 4000 U / g, but the proportion of trypsin in Example 2 increased to 2300 U / g, indicating that it focused more on the degradation of protein structures, which helped to improve the release efficiency of hemostatic factors.

[0076] Experimental results showed that the yeast cell survival rate of Example 2 was 77.8%, the total antioxidant capacity was 27.42 U / g, and the hemostatic effect reached 59.1%, which was slightly lower than that of Example 1 (80.3%, 29.65 U / g, and 62.5%, respectively). However, while maintaining high performance, it achieved formulation optimization and process time compression, and has good practical value and process flexibility.

[0077] Example 3

[0078] Specifically, Table 3.1 shows the preparation conditions of the hemostatic material shown in Example 3 of this application:

[0079] Table 3.1 Preparation conditions of the hemostatic material shown in Example 3

[0080]

[0081] Referring to Table 3.1, in Example 3, frozen squid or cuttlefish ink sacs were selected as raw materials. The ink sacs were gently washed 3-5 times with a washing buffer (pH 7.4 phosphate buffer containing 0.9-1.3% (w / v) NaCl) at 1-3 times their volume. The washed ink sacs were then cut into small pieces and placed in a sterile slurry cup for high-speed homogenization at 4°C to obtain a dark brown concentrated ink. The concentrated ink was then mixed with the washing buffer at a 1:1 volume ratio to form a homogeneous ink mixture. A cryoprotectant (including 0.26% trehalose, 0.03% ascorbate palmitate, and 0.04% alginate oligosaccharide) was added to the ink mixture and thoroughly stirred to form a protective mixture, completing the first stage of processing. The protective mixture was cooled to -40°C at a rate of 5°C / min or higher and held at this temperature for 4 hours. Following this, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 3 hours, then raised to -10°C and held for 2 hours, and finally raised to 0°C and held for 2 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with a phosphate extraction buffer and dried at 0°C using a frequency of 20 kHz and a power density of 500 W / cm². 2 The mixture was subjected to ultrasonic disruption for 10 minutes to promote structural depolymerization. Subsequently, 2000 U / g trypsin, 900 U / g chitinase, and 1100 U / g β-glucanase were added for enzymatic hydrolysis at 40℃ for 2 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The cuttlefish ink active ingredient powder was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The mass of chitosan added was 60-85% of the mass of the cuttlefish ink active ingredient powder, and the mass of ε-polylysine added was 25-40% of the mass of the cuttlefish ink active ingredient powder. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained.

[0082] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured to determine the yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under these experimental conditions are shown in Table 3.2.

[0083] Table 3.2 Performance parameters of the hemostatic material prepared in Example 3

[0084]

[0085] Please refer to Tables 3.1 and 3.2 simultaneously. It can be seen that Example 3 used a combination of 0.26% trehalose, 0.03% ascorbate palmitate, and 0.04% alginate oligosaccharides, increasing the proportion of trehalose in the cryoprotectant. This helps to more effectively stabilize the cell membrane structure during freeze-drying and enhances the overall freeze resistance. The heating and drying time was also further shortened to 4 hours compared to Example 1 (7 hours) and Example 2 (5.5 hours), further improving drying efficiency while ensuring structural integrity.

[0086] Specifically, in the release phase of the active factor, Example 3 used ultrasonic disruption treatment at 0°C with a frequency of 20 Hz, a power density of 500 W / cm², and a duration of 10 min. The power density was significantly higher than that of Examples 1 and 2. Although the ultrasonic time and treatment duration were reduced at the same time, the cell wall disruption and aggregate deagglomeration effects were still effectively enhanced.

[0087] In terms of performance, the yeast cell survival rate (78.9%), total antioxidant capacity (30.10 U / g), total polysaccharide content (17.58 mg / g), and hemostatic effect (63.4%) of Example 3 were all better than those of Example 2, and some indicators were close to or slightly higher than those of Example 1, indicating that this example has excellent performance in enhancing the functional activity of cuttlefish ink hemostatic material.

[0088] The following comparative examples illustrate the technical solutions of this application. It should be noted that, in terms of the four experimental conditions—low-temperature protectant, heating and drying parameters, ultrasonic crushing parameters, and enzymatic hydrolysis conditions—at least one of the experimental conditions in the comparative examples is outside the set range.

[0089] Comparative Example 1:

[0090] Specifically, Table 4.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 1 of this application:

[0091] Table 4.1 Preparation conditions of the hemostatic material shown in Comparative Example 1

[0092]

[0093] Please refer to Table 4.1. In Comparative Example 1, squid or cuttlefish ink sacs that have been frozen were used as raw materials. The ink sacs were gently washed 3-5 times with a washing buffer (pH 7.4 phosphate buffer containing 0.9-1.3% (w / v) NaCl) at 1-3 times their volume. The washed ink sacs were then cut into small pieces and placed in a sterile slurry cup for high-speed homogenization at 4°C to obtain a dark brown concentrated ink. The concentrated ink was then mixed with the washing buffer at a 1:1 volume ratio to form a homogeneous ink mixture. A cryoprotectant (including 0.20% trehalose, 0.05% ascorbate palmitate, and 0.06% alginate oligosaccharide) was added to the ink mixture and thoroughly stirred to form a protective mixture, completing the first stage of treatment. The protective mixture was cooled to -40°C at a rate of 5°C / min or higher and held at this temperature for 4 hours. Following this, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 3 hours, then raised to -10°C and held for 2 hours, and finally raised to 0°C and held for 2 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with a phosphate extraction buffer and dried at 4°C using a frequency of 20 kHz and a power density of 350 W / cm². 2 Ultrasonic disruption was performed for 15 minutes to promote structural depolymerization. Subsequently, 2000 U / g trypsin and 2000 U / g chitinase were added for enzymatic hydrolysis at 35℃ for 4 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The cuttlefish ink active ingredient powder was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The mass of chitosan added was 60-85% of the mass of the cuttlefish ink active ingredient powder, and the mass of ε-polylysine added was 25-40% of the mass of the cuttlefish ink active ingredient powder. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained.

[0094] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 4.2.

[0095] Table 4.2 Performance parameters of the hemostatic material prepared in Comparative Example 1

[0096]

[0097] Please refer to Tables 4.1 and 4.2 simultaneously. It can be seen that although the total enzyme dosage in both Comparative Example 1 and Example 1 is 4000 U / g of raw material, Comparative Example 1 did not add β-glucanase, but instead increased the chitinase dosage to 2000 U / g of raw material. Although this enhanced the hydrolysis of chitosan to some extent, the absence of β-glucan significantly affected the release efficiency of functional components because β-glucan participates in the structural stability and activity release of polysaccharides in cuttlefish ink. This resulted in significantly lower total antioxidant capacity (16.25 U / g), average particle size of melanin aggregates (138.40 nm), and total polysaccharide content (11.20 mg / g) compared to Example 1, and the hemostatic effect also decreased to 35.1%. This demonstrates that a complete three-enzyme synergistic system plays an important role in improving the functionality and material uniformity of cuttlefish ink.

[0098] Comparative Example 2:

[0099] Specifically, Table 5.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 2 of this application:

[0100] Table 5.1 Preparation conditions of the hemostatic material shown in Comparative Example 2

[0101]

[0102] Referring to Table 5.1, in Comparative Example 2, squid or cuttlefish ink sacs that had been frozen were used as raw materials. The ink sacs were gently washed 3-5 times with a washing buffer (pH 7.4 phosphate buffer containing 0.9-1.3% (w / v) NaCl) at 1-3 times their volume. The washed ink sacs were then cut into small pieces and placed in a sterile slurry cup for high-speed homogenization at 4°C to obtain a dark brown concentrated ink. The concentrated ink was then mixed with the washing buffer at a 1:1 volume ratio to form a homogeneous ink mixture. A cryoprotectant (including 0.20% trehalose, 0.05% ascorbate palmitate, and 0.06% alginate oligosaccharide) was added to the ink mixture and thoroughly stirred to form a protective mixture, completing the first stage of treatment. The protective mixture was cooled to -40°C at a rate of 5°C / min or higher and held at this temperature for 4 hours. Following this, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 3 hours, then raised to -10°C and held for 2 hours, and finally raised to 0°C and held for 2 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with phosphate extraction buffer, and 2000 U / g trypsin, 1000 U / g chitinase, and 1000 U / g β-glucanase were added for enzymatic hydrolysis. The hydrolysis temperature was controlled at 35°C, and the reaction was carried out for 4 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The active ingredient powder of cuttlefish ink was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The mass of chitosan added was equal to 60-85% of the mass of the active ingredient powder of cuttlefish ink, and the mass of ε-polylysine added was equal to 25-40% of the mass of the active ingredient powder of cuttlefish ink. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained.

[0103] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were evaluated for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 5.2.

[0104] Table 5.2 Performance parameters of the hemostatic material prepared in Comparative Example 2

[0105]

[0106] Referring to Tables 5.1 and 5.2, it can be seen that, compared with Example 1, Comparative Example 2 omitted the ultrasonic disruption step with a frequency of 20 Hz, a power density of 350 W / cm², a treatment time of 15 min, and a temperature of 4°C. This resulted in insufficient deagglomeration of the cuttlefish ink powder structure and a reduced efficiency in releasing intracellular active factors, thus affecting subsequent enzymatic hydrolysis reactions. Specifically, the yeast cell survival rate decreased from 80.3% to 75.6%, the total antioxidant capacity decreased from 29.65 U / g to 23.92 U / g, and the melanin aggregate particle size significantly increased to 105.18 nm, indicating a decrease in structural uniformity. Simultaneously, the total polysaccharide content and hemostatic effect of Comparative Example 2 also showed a certain degree of decrease, demonstrating that ultrasonic disruption plays an important role in promoting substance release, improving particle distribution, and enhancing biological functions.

[0107] Comparative Example 3

[0108] Specifically, Table 6.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 3 of this application:

[0109] Table 6.1 Preparation conditions of the hemostatic material shown in Comparative Example 3

[0110]

[0111] Please refer to Table 6.1. The process steps of Comparative Example 3 and Example 1 are basically the same, with the only difference being the composition of the cryoprotectant. No alginate oligosaccharide was added in Comparative Example 3, while 0.06% alginate oligosaccharide was added in Example 1 to investigate the role of this component in the cryoprotection system. The remaining steps include ink sac cleaning, homogenization, cryoprotectant treatment, freeze drying, staged heating (-20℃ / 3h → -10℃ / 2h → 0℃ / 2h), ultrasonic disruption (20kHz, 350 W / cm², 15min), mixed enzyme hydrolysis (trypsin, chitinase, β-glucanase), and composite molding to finally obtain the hemostatic material.

[0112] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were evaluated for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 6.2.

[0113] Table 6.2 Performance parameters of the hemostatic material prepared in Comparative Example 3

[0114]

[0115] Referring to both Tables 6.1 and 6.2, it can be seen that the difference between Comparative Example 3 and Example 1 lies in the absence of alginate oligosaccharides in the cryoprotectant composition. Experimental results show that the yeast cell survival rate of Comparative Example 3 was 72.1%, lower than the 80.3% of Example 1, indicating a weakened cell protection effect during freeze-drying. Furthermore, the melanin aggregate particle size increased to 90.5 nm, indicating decreased particle stability, possibly related to the loose structure of the composite system. The hemostatic effect decreased to 54.2%, and the polysaccharide content also decreased to 14.75 mg / g, both significantly lower than the 62.5% and 17.02 mg / g of Example 1. This indicates that alginate oligosaccharides play a role in structural stability and functional synergy in the three-component cryoprotectant, and their absence directly affects the physical stability and biological activity of the final product.

[0116] Comparative Example 4

[0117] Specifically, Table 7.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 4 of this application:

[0118] Table 7.1 Preparation conditions of the hemostatic material shown in Comparative Example 4

[0119]

[0120] Please refer to Table 7.1. The process steps of Comparative Example 4 are basically the same as those of Example 1. The only difference is that Comparative Example 4 did not use staged heating and drying treatment, but instead used conventional one-stage vacuum freeze drying. The remaining steps include ink sac cleaning, homogenization, cryoprotectant treatment, freeze drying, ultrasonic disruption (20kHz, 350 W / cm², 15 min), mixed enzyme hydrolysis (trypsin, chitinase, β-glucanase), and composite molding, finally obtaining the hemostatic material.

[0121] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 7.2.

[0122] Table 7.2 Performance parameters of the hemostatic material prepared in Comparative Example 4

[0123]

[0124] Referring to Tables 7.1 and 7.2, it can be seen that the difference between Comparative Example 4 and Example 1 is that Comparative Example 4 did not employ a phased temperature-increasing drying process, but instead used a conventional one-stage vacuum freeze-drying method. Due to the lack of a gradual temperature gradient during the drying process, the cell membrane is prone to sudden rupture during the temperature rise, affecting the integrity of the cell structure. Experimental results show that the cryopreservation survival rate of yeast cells in Comparative Example 4 decreased to approximately 75.5%, lower than the 80.3% in Example 1; simultaneously, the total antioxidant capacity and polysaccharide content also decreased to approximately 26.20 U / g and 15.02 mg / g, respectively, the melanin granule size increased to approximately 84.20 nm, and the hemostatic effect weakened (bleeding decreased to 52.8%).

[0125] Comparative Example 5:

[0126] Specifically, Table 8.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 5 of this application:

[0127] Table 8.1 Preparation conditions of the hemostatic material shown in Comparative Example 5

[0128]

[0129] Please refer to Table 8.1. The process steps of Comparative Example 5 are basically the same as those of Example 1, the difference being the composition of the cryoprotectant and the staged heating and drying treatment. Comparative Example 3 did not add ascorbate palmitate, while Example 1 added 0.05% ascorbate palmitate; furthermore, Comparative Example 3 did not use staged heating and drying treatment, but instead used conventional one-stage vacuum freeze-drying. The remaining steps all include ink sac cleaning, homogenization, cryoprotectant treatment, freeze-drying, ultrasonic disruption (20kHz, 350 W / cm², 15 min), mixed enzyme hydrolysis (trypsin, chitinase, β-glucanase), and composite molding, ultimately yielding the hemostatic material.

[0130] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were measured for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 8.2.

[0131] Table 8.2 Performance parameters of the hemostatic material prepared in Comparative Example 5

[0132]

[0133] Referring to Tables 8.1 and 8.2, it can be seen that, compared with Example 1, Comparative Example 5 did not add ascorbate palmitate during the preparation process and omitted the staged temperature-drying process. The results show that the yeast cell survival rate of Comparative Example 5 was only 50.8%, far lower than the 80.3% of Example 1; the total antioxidant capacity decreased to 19.65 U / g, the melanin particle size increased to 122.80 nm, and the hemostatic effect was only 44.2%, significantly lower than the 62.5% of Example 1.

[0134] Furthermore, compared to Comparative Example 4, which only omitted the stage of temperature-drying treatment, Comparative Example 5 showed slightly lower cell viability, antioxidant capacity, and hemostatic effect, indicating that the absence of ascorbate palmitate further weakened the protective effect. For example, the cell viability of Comparative Example 4 was 75.5%, while that of Comparative Example 5 decreased to 50.8%, demonstrating that the combined absence of both processes had a more significant impact on product performance.

[0135] Comparative Example 6:

[0136] Specifically, Table 9.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 6 of this application:

[0137] Table 9.1 Preparation conditions of the hemostatic material shown in Comparative Example 6

[0138]

[0139] Please refer to Table 9.1. The process steps of Comparative Example 6 are basically the same as those of Example 1, the difference being the composition of the mixed enzymes and whether ultrasonic disruption is used. The mixed enzymes in Comparative Example 6 include 2000 U / g raw trypsin and 2000 U / g raw chitinase (the mixed enzymes in Example 1 include 2000 U / g raw trypsin, 1000 U / g raw chitinase, and 1000 U / g raw β-glucanase); in addition, ultrasonic disruption is not used in Example 6. The remaining steps include ink sac cleaning, homogenization, cryoprotectant treatment, freeze drying, staged heating (-20℃ / 3h → -10℃ / 2h → 0℃ / 2h), mixed enzyme hydrolysis (trypsin, chitinase, β-glucanase), and composite molding, finally obtaining the hemostatic material.

[0140] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were evaluated for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 9.2.

[0141] Table 9.2 Performance parameters of the hemostatic material prepared in Comparative Example 6

[0142]

[0143] Please refer to Tables 9.1 and 9.2 simultaneously. It can be seen that the difference between Comparative Example 6 and Example 1 is that ultrasonic treatment was not used and β-glucanase was not added, which resulted in a significant decrease in all performance indicators, especially in total antioxidant capacity (from 29.65 U / g to 15.10 U / g), melanin particle size (from 72.25 nm to 145.2 nm), and hemostatic effect (from 62.5% to 32.5%).

[0144] Furthermore, the difference between Comparative Example 6 and Comparative Example 1 lies in the absence of ultrasonic disruption, and Comparative Example 6 also had a higher chitinase content. The results showed that Comparative Example 6 had slightly worse overall performance, lower total antioxidant capacity, and larger average particle size of melanin aggregates, indicating that the lack of ultrasonic treatment significantly affected the material's microstructure and antioxidant properties. Although Comparative Examples 6 and 2 used higher chitinase dosages, due to the absence of β-glucanase, Comparative Example 2 exhibited better cell viability and antioxidant capacity, as well as a higher total polysaccharide content, further validating the importance of enzyme synergy.

[0145] Comparative Example 7:

[0146] Specifically, Table 10.1 shows the preparation conditions of the hemostatic material shown in Comparative Example 7 of this application:

[0147] Table 10.1 Preparation conditions of the hemostatic material shown in Comparative Example 7

[0148]

[0149] Please refer to Table 10.1. Comparative Example 7 maintains the same basic process flow as Example 1, including ink sac cleaning, homogenization, cryoprotection, freeze-drying, enzymatic hydrolysis, centrifugation, freeze-drying, and composite molding. However, it differs from Example 1 in several key aspects: First, trehalose is not added to the cryoprotectant; it only contains 0.05% ascorbate palmitate and 0.06% alginate oligosaccharide. Second, a staged heating vacuum drying method is not used; instead, a one-stage vacuum freeze-drying method is employed. Third, ultrasonic disruption is not performed. Fourth, 2000 U / g trypsin and 2000 U / g chitinase are used during enzymatic hydrolysis, but β-glucanase is not added. This combination aims to investigate the impact of the lack of multiple synergistic treatments on the performance of hemostatic materials.

[0150] Furthermore, the hemostatic material was tested after being frozen at -18℃ for 4 weeks, and the results were evaluated for yeast cell survival rate (%), total antioxidant capacity (U / g), average particle size of melanin aggregates (nm), total polysaccharide content (mg / g), and hemostatic effect compared to the untreated mouse tail-cutting model. The performance parameters of the hemostatic material prepared under the control group conditions were obtained, as shown in Table 10.2.

[0151] Table 10.2 Performance parameters of the hemostatic material prepared in Comparative Example 7

[0152]

[0153] Referring to Tables 10.1 and 10.2, it can be seen that the difference between Comparative Example 7 and Example 1 lies in the lack of trehalose, staged heating, ultrasound, and β-glucanase, resulting in a significant decrease in all performance indicators. The difference between Comparative Example 7 and Comparative Example 5 is that ultrasound treatment was not used and trehalose was not added. Although more chitinase was used, cell protection was insufficient, and the total antioxidant capacity and hemostatic effect were lower than those of Comparative Example 5. The difference between Comparative Example 7 and Comparative Example 6 is that staged heating drying and a complete cryoprotectant were not used, resulting in Comparative Example 7 performing worse than Comparative Example 6 in yeast cell survival rate, polysaccharide retention, and hemostatic effect.

[0154] In summary, Table 11.1 shows the preparation conditions of the hemostatic materials for each experimental group and the control group, and Table 11.2 shows the performance parameters of the hemostatic materials prepared for each experimental group and the control group.

[0155] Table 11.1 Preparation conditions of hemostatic materials for each experimental group and control group

[0156]

[0157] Table 11.2 Performance parameters of hemostatic materials prepared in each experimental group and control group

[0158]

[0159] Please refer to Tables 11.1 to 11.2. It can be seen that the hemostatic material preparation method provided in this application achieves overall improvement in multiple aspects, such as cell activity protection, polysaccharide and bioactive component release, particle size control and hemostatic performance optimization, through the combined design of four key process conditions: low temperature protectant combination, staged heating and drying, ultrasonic crushing treatment and mixed enzyme hydrolysis.

[0160] The synergistic effect of the experimental conditions specified in this application is explained below:

[0161] First, the trehalose, ascorbate palmitate, and alginate oligosaccharides in the cryoprotectant combination each play different stabilizing and protective functions. Trehalose can replace water molecules to form hydrogen bonds with the phospholipid bilayer on the cell membrane during freezing, effectively inhibiting mechanical damage caused by ice crystal formation; ascorbate palmitate can embed itself in the lipid region of the cell membrane, slowing down lipid peroxidation; and alginate oligosaccharides have certain water-retention capacity and immune activity, stabilizing the extracellular microenvironment. The synergistic use of these three enzymes can significantly improve the integrity of cell structure and physiological activity during freeze-drying and thawing, thereby enhancing the retention of effective components and biosafety of hemostatic materials.

[0162] Secondly, the stabilizing effect of cryoprotectants needs to be coordinated with a phased heating and drying process to achieve optimal results. Rapid rewarming can cause a temperature difference between the inside and outside of cells, leading to drastic changes in osmotic pressure, which can induce cell rupture or denaturation of active ingredients. By setting a multi-stage heating path (e.g., -20℃ → -10℃ → 0℃), slow cell recovery and gradual structural restoration can be achieved, reducing stress load and allowing the cryoprotectant to continue its function during the heating process, thereby further improving material stability and functional retention.

[0163] Third, the mixed-enzyme hydrolysis technology can efficiently release functional polysaccharides and other bioactive substances from cuttlefish ink. Trypsin is responsible for cleaving protein structures, providing reaction channels for other enzymes; chitinase acts on the chitin backbone, disrupting the cell wall structure; β-glucanase hydrolyzes polysaccharide branches or assists in breaking the polysaccharide backbone, improving the polysaccharide dissolution rate. These three enzymes have a clear substrate division of labor and synergistic reaction mechanism, and can synergistically degrade multi-component structures in complex natural matrices, significantly improving the extraction efficiency of soluble functional components, especially suitable for cuttlefish ink raw materials with high adhesiveness and high structural complexity.

[0164] Fourth, ultrasonic disruption generates instantaneous high pressure and microjets through cavitation, which can rapidly lyse cell structures, expose enzymatic targets, and increase the contact area between enzymes and substrates, thereby improving enzymatic efficiency and reaction rate. Simultaneously, ultrasound also helps reduce particle size, improves material dispersibility and dissolution rate, and positively promotes hemostatic properties.

[0165] Fifth, the combined use of enzymatic hydrolysis and ultrasound can achieve complementary advantages. Ultrasound pre-disruption of the cuttlefish ink cell wall allows for more complete exposure of the endogenous components, which is conducive to the efficient hydrolysis of the mixed enzymes. Meanwhile, the reaction products of the mixed enzymes (such as low-molecular-weight polysaccharides and small-molecule peptides) are also more easily released and more evenly distributed due to the dispersion and promotion effect of ultrasound, thereby exerting coagulation activity or adsorption hemostasis effect more quickly on the tissue contact surface, forming an effective synergistic release mechanism.

[0166] Sixth, when the four conditions of cryoprotectant, temperature drying, ultrasonic treatment, and enzyme-assisted enzymatic hydrolysis are not applied within the optimal parameter range, the overall performance of the hemostatic material will be significantly inhibited. At this time, not only is the cell structure not adequately protected, leading to severe degradation of active components, but the release of key components such as functional polysaccharides is also hindered, and the particle size control is unbalanced, ultimately resulting in multiple performance degradations such as poor hemostatic effect, low polysaccharide extraction rate, and weak antioxidant capacity.

[0167] In summary, the hemostatic material preparation method provided in this application, through the synergistic effect of multiple factors such as cryoprotectant (0.08~0.26% trehalose, 0.03~0.06% ascorbate palmitate and 0.04~0.08% alginate oligosaccharide), staged temperature rise drying (-20℃ / 2~3h → -10℃ / 1~2h → 0℃ / 1~2h, vacuum degree ≤8 Pa), ultrasonic disruption treatment (frequency of 20~25kHz, power density of 300~500 W / cm2, treatment time of 10~20 min, temperature of 0~4℃), and mixed enzyme hydrolysis (2000-2800 U / g trypsin, 900-1000 U / g chitinase, 800~1300 U / g β-glucanase), can significantly improve the bioactivity and hemostatic performance of the hemostatic material, which helps to prepare hemostatic materials with stable performance, outstanding function, and meeting diverse clinical needs.

[0168] The method for preparing hemostatic materials provided in this embodiment integrates techniques such as cryoprotectant treatment, staged high-speed heating treatment, ultrasonic disruption, and mixed enzymatic hydrolysis to achieve the synergistic release of multiple active ingredients in cuttlefish ink while maintaining its structural and functional stability. Specifically, the ink sac of cuttlefish or squid is first treated with a pH 7.4 phosphate buffer to prepare concentrated ink, which is then mixed with a washing buffer to form an ink-buffered mixture. A cryoprotectant, comprising 0.08–0.26% trehalose, 0.03–0.06% ascorbate palmitate, and 0.04–0.08% alginate oligosaccharides, is then added to the mixture. The protective mixture was cooled to -40°C at a rate of 5°C / min or higher and held at this temperature for 4 hours. Following this, a staged heating and drying process was performed: under a vacuum of ≤8 Pa, the temperature was raised to -20°C and held for 2-3 hours, then raised to -10°C and held for 1-2 hours, and finally raised to 0°C and held for 1-2 hours to obtain cuttlefish ink powder. The cuttlefish ink powder was mixed with phosphate extraction buffer and subjected to ultrasonic disruption at 0-4°C using a frequency of 20-25 kHz and a power density of 300-500 W / cm² for 10-20 minutes to promote structural depolymerization. Subsequently, 2000-2800 U / g trypsin, 900-1000 U / g chitinase, and 800-1300 U / g β-glucanase were added for enzymatic hydrolysis at 35-40°C for 2-5 hours to enhance the release efficiency of melanin, polysaccharides, peptides, and other biological functional factors. The supernatant was collected by centrifugation and freeze-dried to obtain cuttlefish ink active ingredient powder. The cuttlefish ink active ingredient powder was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added sequentially. The added chitosan was 60-85% of the mass of the cuttlefish ink active ingredient powder, and the added ε-polylysine was 25-40% of the mass of the cuttlefish ink active ingredient powder. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. CaCl2 solution was added dropwise to the composite solution to form a gel-like substance with a three-dimensional network structure. After freeze-drying, the final hemostatic material was obtained. The hemostatic material prepared by this method is naturally derived, has a clearly defined composition, fully releases its active ingredients, and exhibits good biocompatibility and hemostatic properties. It is suitable for various medical scenarios such as trauma emergency care, intraoperative hemostasis, and functional dressings.

[0169] The second aspect of this application also provides a hemostatic material, which is prepared based on the preparation method described in any one of the first aspects of this application.

[0170] It should be noted that the average particle size of the melanin aggregates in the hemostatic material provided in this application is 72.25±3.35nm, the polysaccharide content is 17.02±0.29 mg / g, and the antioxidant capacity is 29.65±0.36 U / g.

[0171] The hemostatic material provided in this application is characterized by its small particle size, stable structure, and enrichment of functional factors. Its particle size is much smaller than that of melanin aggregates in ordinary hemostatic materials, which enhances its density and adsorption capacity in contact with the wound surface. Its high polysaccharide content provides hydrogel properties and moisturizing effects, and also helps accelerate the activation of coagulation factors. Furthermore, the significantly enhanced antioxidant capacity of this hemostatic material not only stops bleeding but also alleviates local oxidative stress and promotes wound healing. Animal experimental results show that this hemostatic material reduced bleeding by up to 62.5% in a mouse tail amputation model, significantly outperforming existing conventional treatment methods, demonstrating excellent hemostatic performance and bioactivity.

[0172] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing a hemostatic material, characterized in that, The preparation method includes: An ink mixture was prepared using a cleaning buffer and an ink sac, and a cryoprotectant was added to the ink mixture to obtain a protective mixture; the cryoprotectant included trehalose, ascorbate palmitate, and alginate oligosaccharide. The protective mixture is cooled to a specified sub-zero temperature at a specified cooling rate and held at the specified sub-zero temperature for a first specified duration, followed by a staged heating and drying process to obtain cuttlefish ink powder. The cuttlefish ink powder and phosphate extraction buffer are mixed, and after ultrasonic disruption, a mixed enzyme is added to carry out an enzymatic hydrolysis reaction to release the active ingredients in the cuttlefish ink powder, thereby obtaining a cuttlefish ink enzymatic hydrolysate; the mixed enzyme includes trypsin, chitinase and β-glucanase. The cuttlefish ink enzymatic hydrolysate was centrifuged, the supernatant was collected, and the supernatant was freeze-dried to obtain cuttlefish ink active ingredient powder. The active ingredient powder of cuttlefish ink was dissolved in an acetate-sodium acetate buffer solution, and chitosan and ε-polylysine were added in sequence. The pH was adjusted to 7.0-8.5 to obtain a cuttlefish ink composite solution. Adding CaCl2 solution to the cuttlefish ink composite solution forms a gel-like substance with a three-dimensional network structure. Freeze-drying the gel-like substance yields a hemostatic material.

2. The preparation method according to claim 1, characterized in that, According to the mass-volume ratio, the cryoprotectant includes 0.08~0.26% trehalose, 0.03~0.06% ascorbate palmitate and 0.04~0.08% alginate oligosaccharide.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction by adding mixed enzymes includes: Add 2000-2800 U / g trypsin, 900-1000 U / g chitinase, and 800-1300 U / g β-glucanase. Maintain the reaction temperature at 35-40℃ and the enzymatic hydrolysis time at 2-5 hours.

4. The preparation method according to claim 1, characterized in that, The step of subjecting the cooled mixture to a phased heating and drying process to obtain cuttlefish ink powder includes: The cooling mixture is heated to a first specified temperature according to a preset heating rate and maintained for a first preset duration; wherein, the first specified temperature is a sub-zero temperature; The temperature of the cooling mixture is raised to a second specified temperature and maintained for a second preset duration; wherein the second specified temperature is a sub-zero temperature and the second preset duration is less than the first preset duration; The temperature of the cooling mixture is raised to 0°C and maintained for a third preset duration.

5. The preparation method according to claim 1, characterized in that, The parameters for the ultrasonic fragmentation process are set as follows: frequency 20~25kHz, power density 300~500 W / cm². 2 The processing time is 10~20 min, and the temperature is 0~4℃.

6. The preparation method according to claim 1, characterized in that, The specified cooling rate is greater than or equal to 5℃ / min, the specified sub-zero temperature is -40℃, and the first specified duration is 4 h.

7. The preparation method according to claim 1, characterized in that, The sequential addition of chitosan and ε-polylysine includes: Chitosan and ε-polylysine are added sequentially; wherein the mass of the added chitosan is equal to 60-85% of the mass of the cuttlefish ink active ingredient powder; and the mass of the added ε-polylysine is equal to 25-40% of the mass of the cuttlefish ink active ingredient powder.

8. The preparation method according to claim 1, characterized in that, The cuttlefish ink powder is squid ink powder, cuttlefish ink powder, or a mixture of squid ink powder and cuttlefish ink powder.

9. A hemostatic material, characterized in that, The hemostatic material is prepared according to the preparation method described in any one of claims 1-8.

10. The hemostatic material according to claim 9, characterized in that, The hemostatic material has an average melanin aggregate particle size of 72.25±3.35nm, a polysaccharide content of 17.02±0.29 mg / g, and an antioxidant capacity of 29.65±0.36 U / g.

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