Preparation method and application of cartilage extract

Abstract

The invention relates to the field of biological pharmacy, and particularly discloses a preparation method and application of a cartilage extract. The invention relates to a preparation method of a cartilage extract, which comprises the following steps: pretreatment, boiling, alkaline hydrolysis and enzymolysis: adjusting the pH value of an alkaline hydrolysis product to 7-11 at 50-65 DEG C, adding alkaline protease, carrying out enzymolysis for 1-2 hours, adjusting the pH value to 6-8, adding neutral protease, carrying out enzymolysis for 4-6 hours, adjusting the pH value to 5-7 after the enzymolysis is finished, heating to 60-70 DEG C, standing, filtering, washing, and drying to obtain the cartilage extract. Discharging the material liquid, performing primary filtration, performing nanofiltration, sterilizing, performing secondary filtration, and performing spray drying. The preparation method has the advantages that waste is turned into wealth, natural resources are reasonably and fully utilized, the cartilage extract is fully utilized, environmental pollution is reduced, the extraction rate of the cartilage extract is increased, and the cartilage extract can be used for a wound healing agent and has the advantages of being anti-inflammatory, antibacterial, analgesic and capable of effectively promoting wound healing.

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical technology, and more specifically, to a method for preparing and using a cartilage extract. Background Technology

[0002] Cartilage is a tough yet elastic connective tissue, primarily found in joints and areas supporting bones. Cartilage extract is prepared from high-quality animal cartilage using a special enzymatic hydrolysis process. It typically contains many bioactive components, such as collagen, chondroitin sulfate sodium, and other natural bioactive substances. Therefore, cartilage extract is widely used in the health food and functional food industries.

[0003] Cartilage extract is a complex collagen containing both type II collagen and glycosaminoglycans, prepared using a special enzymatic hydrolysis process. To improve the utilization value of cartilage extract, many studies have been conducted on it. For example, Chinese invention patent application CN2020114258457 discloses a method for preparing a high-purity, high-tasting cartilage extract, including the following steps: 1) cleaning the cartilage; 2) enzymatic hydrolysis using directional enzymatic digestion technology, with an enzyme dosage of 0.2-0.5% of the cartilage weight, using a single enzyme, one of alkaline protease, neutral protease, bromelain, or papain; 3) activated carbon adsorption; 4) solid-liquid separation; 5) desalting and impurity removal using advanced membrane separation technology; 6) spray drying to obtain off-white granular cartilage extract. This paper uses only a single enzyme, which improves product quality, simplifies the process, and enhances the color and taste of the cartilage extract. However, the extraction rate of the cartilage extract using this process needs further improvement. Summary of the Invention

[0004] To improve the extraction rate of cartilage extract, this application provides a method for preparing cartilage extract and its application.

[0005] In a first aspect, this application provides a method for preparing a cartilage extract, employing the following technical solution: A method for preparing a cartilage extract, S1, pretreatment: thawing and pulverizing animal cartilage to obtain a pulverized product; S2. Cooking: Add water to the pulverized material and cook at 85-95℃. Let it stand to remove the grease, and then cook at ≥99℃ for ≥30 minutes. S3. Alkaline hydrolysis: Cool the boiled product to 50-65℃, adjust the pH to 9-12, and perform alkaline hydrolysis for 2-5 hours. S4. Enzymatic hydrolysis: Adjust the pH of the alkaline hydrolysis product to 7-11 at 50-65℃, add alkaline protease, hydrolyze for 1-2 hours, adjust the pH to 6-8, add neutral protease, hydrolyze for 4-6 hours, adjust the pH to 5-7 after hydrolysis, heat to 60-70℃, let stand, and release the liquid. S5. Primary filtration: The liquid is filtered through a filter aid to obtain a primary filtrate; S6. Nanofiltration: The primary filtrate is nanofiltered at a temperature ≥60℃, concentrated to a concentration ≥18 Brix, and then concentrated again to a concentration of 45-70 Brix to obtain a concentrated solution. S7. Sterilization: Heat the concentrated liquid to 80-90℃ and keep it warm for 30-60 minutes; S8. Secondary filtration: The sterilized concentrate is filtered through a filter screen with a pore size of no more than 80 mesh to obtain secondary filtrate. S9. Spray drying: The secondary filtrate is spray-dried and then packaged to obtain cartilage extract.

[0006] By employing the above-mentioned technical solution, the effective components in cartilage are encapsulated in a dense three-dimensional network structure, tightly bound to minerals and proteoglycans. Therefore, through the synergistic action of physical, chemical, and biological methods, barriers are overcome layer by layer. Pulverization increases the specific surface area, providing more contact sites for subsequent chemical and enzymatic hydrolysis reactions. Boiling dissolves some proteins and fats and kills heat-resistant enzymes. Alkaline hydrolysis effectively decalcifies and breaks down the cross-links between collagen fibers, making the structure more porous and providing more sites for subsequent protease attack. Then, proteases specifically cleave the peptide bonds of collagen, efficiently converting it into soluble collagen peptides and amino acids, while simultaneously releasing the bound chondroitin sulfate. Compared to strong acid and strong alkali methods, enzymatic hydrolysis conditions are milder, better preserving the sulfate groups of chondroitin sulfate and the bioactivity of collagen peptides. Therefore, the hydrophobic structure after alkaline hydrolysis creates better conditions for enzymatic hydrolysis, significantly improving product yield.

[0007] Optionally, the amount of alkaline protease added is 0.04-0.08 wt% of the pulverized material; The amount of the central Y-type protease added is 0.01-0.03 wt% of the pulverized material.

[0008] By adopting the above technical solution, at the optimal amount of protease added, the protease can fully interact with the substrate, hydrolyzing most of the soluble proteins and bound polysaccharides, thereby obtaining a high extraction rate.

[0009] Optionally, in step S3, when the product obtained from cooking is cooled to 75-80°C, an auxiliary agent is added. The mass ratio of the auxiliary agent to the product obtained from cooking is 1:20-30. The mixture is subjected to ultrasonic reaction for 2-4 hours, followed by enzymatic hydrolysis. The auxiliary agent is prepared by mixing choline chloride and lactic acid in a molar ratio of 1:2-3, adding 20-30 wt% of water (based on their total weight), stirring to dissolve at 75-80°C, and adjusting the pH to 3.5-4.

[0010] By employing the above-mentioned technical solution, collagen (mainly type II) in cartilage and polysaccharides such as chondroitin sulfate form a dense and stable three-dimensional network structure through complex hydrogen bonds and electrostatic interactions. Choline and lactic acid have extremely strong hydrogen bond breaking effects, effectively penetrating into collagen fibers, breaking the intramolecular and intermolecular hydrogen bond network of collagen molecules, as well as the connection between collagen and polysaccharides. This makes the originally dense cartilage matrix loose and swollen, greatly increasing the contact area between proteases and substrates. Moreover, choline and lactic acid disrupt the extracellular matrix, allowing proteases to more easily diffuse into the tissue interior and come into contact with the originally encapsulated collagen and chondroitin sulfate. Furthermore, choline and lactic acid can improve enzyme stability and reduce protease inactivation during the reaction process, thereby maintaining high catalytic activity for a longer period of time and improving the extraction rate of cartilage extract.

[0011] Optionally, the inlet air temperature during spray drying is 200-300℃, the exhaust air temperature is ≥80℃, and the spray pressure is 15-30MPa.

[0012] Optionally, impurities are removed during nanofiltration. The specific method is to add water at a temperature ≥60℃ to the filtrate and repeat this process 1-5 times until nanofiltration is complete.

[0013] Optionally, the animal cartilage includes at least one of the following: chicken short bone, chicken breastbone, duck trachea, duck cartilage, chicken trachea, chicken bone, bovine trachea, and bovine cartilage.

[0014] Secondly, this application provides an application of a cartilage extract, employing the following technical solution: The application of a cartilage extract in wound healing preparations.

[0015] By employing the above-mentioned technical solutions, the cartilage extract, rich in collagen, guides the orderly growth of fibroblasts and epithelial cells, providing an extracellular matrix scaffold for cell migration, attachment, and growth. Furthermore, the chondroitin sulfate it contains can bind a large number of water molecules, creating a moist healing environment for the wound, accelerating cell migration, reducing scab formation, and promoting healing. Chondroitin sulfate can also bind to various growth factors. The cartilage extract is slowly released at the wound site, continuously stimulating cell proliferation and tissue repair. In addition, the cartilage extract also has a certain anti-inflammatory effect, regulating the inflammatory response in the early stages of wound healing and preventing excessive inflammation from damaging tissues.

[0016] Optionally, the wound healing agent comprises chitosan hydrogel in a mass ratio of 1:0.3-0.5 and microspheres loaded with human platelet lysis solution and lithium magnesium silicate, wherein the microspheres have a cartilage extract as the core and methacrylamide gelatin as the shell.

[0017] By employing the above-mentioned technical solution, hydrogel microspheres with cartilage extract as the core and methacrylamide gelatin as the shell are developed. Methacrylamide gelatin acts as a temporary scaffold for cell migration, proliferation, and differentiation, filling the gaps in the wound. The hydrogel properties absorb wound exudate, providing a moist healing environment and preventing tissue dehydration. The small-molecule collagen peptides and chondroitin sulfate rich in the cartilage extract attract surrounding fibroblasts and keratinocytes to migrate to the wound area, promoting proliferation and anti-inflammatory effects. Encapsulating the cartilage extract within the microspheres protects it from ultraviolet light and external environmental sensitivity, allowing for continuous and slow release throughout the wound healing process. Providing continuous nutrition and support during the inflammatory, proliferative, and remodeling phases enables long-term treatment. Human platelet lysate is rich in various growth factors that can promote granulation tissue formation, stimulate angiogenesis, and accelerate re-epithelialization. Lithium magnesium silicate contains silicate and lithium ions that can significantly promote the proliferation of vascular endothelial cells and promote angiogenesis. Furthermore, the alkaline environment released by lithium magnesium silicate has an inhibitory effect on bacteria. Therefore, through the physical support of methacryloyl gelatin in the microspheres, the tissue-specific guidance of cartilage extract, the strong driving force of growth factors in human platelet lysate, and the angiogenesis-promoting effect of lithium magnesium silicate, wound closure is significantly accelerated and tissue repair is promoted.

[0018] Chitosan hydrogel is formed by electrostatic cross-linking of rhein and chitosan. It has high water content, strong absorption capacity, excellent biocompatibility, good antibacterial properties, and special temperature sensitivity, which allows it to gel in situ at temperatures close to the skin surface. It is suitable for the treatment of various irregular wounds. By reducing bacterial load, reducing inflammation, promoting the proliferation and migration of keratinocytes, clearing excess reactive oxygen species, and providing a moist wound environment, it accelerates wound healing.

[0019] Optionally, the microspheres loaded with human platelet lysis buffer and lithium magnesium silicate are made using the following methods: Methacrylamide gelatin and cartilage extract were added to PBS solution and then mixed evenly to prepare a precursor solution. The precursor solution was pumped out as droplets at a flow rate of 3-10 mL / h and irradiated with ultraviolet light to form microspheres. Lithium magnesium silicate was added to human platelet lysis buffer and sonicated to prepare an enhanced solution. Mix the microspheres with the enhancement solution and incubate at 35-37°C for 2-4 hours. Wash with PBS buffer 1-2 times.

[0020] By employing the above technical solution, methacrylamide gelatin is dissolved and mixed with a photoinitiator and cartilage extract to form a precursor solution. This solution is then used to form uniformly sized and structurally stable core-shell droplets at a specific flow rate. These droplets are then cross-linked and cured under ultraviolet light to form stable hydrogel microspheres. These microspheres are then mixed with a strengthening solution and incubated. A large number of growth factors and proteins from human platelet lysate are adsorbed onto the surface and porous structure of the microspheres through physical action. Lithium magnesium silicate particles adhere to the surface of the microspheres and continuously release bioactive ions such as lithium ions and silicate ions. These ions can penetrate the gel network, inducing the formation of a beneficial bioactive interface layer. After washing with PBS solution, unbound particles and proteins are removed, resulting in microspheres loaded with human platelet lysate and lithium magnesium silicate.

[0021] Optionally, the wound healing agent is prepared as follows: microspheres loaded with human platelet lysis buffer and magnesium silicate are immersed in PBS buffer containing (2,3-epoxypropyl)dodecyltrimethylammonium chloride, shaken at 35-37°C for 4-8 hours, centrifuged, washed, and mixed evenly with chitosan hydrogel.

[0022] By adopting the above technical solution, (2,3-epoxypropyl)dodecyltrimethylammonium chloride contains epoxy groups, which have high reactivity and can undergo ring-opening reactions with the primary amino and hydroxyl groups contained in the methacrylamide gelatin molecule, thereby introducing quaternary ammonium cationic groups into the structure of methacrylamide gelatin. Through covalent bonds, the quaternary ammonium salt antibacterial layer is firmly bound, achieving long-lasting and safe contact antibacterial effects. Thus, the wound healing agent has the functions of powerful healing promotion, angiogenesis promotion, and long-lasting antibacterial effects.

[0023] In summary, this application has the following beneficial effects: 1. Since this application uses animal cartilage as the extraction raw material, it reduces costs, turns waste into treasure, and reduces environmental pollution. At the same time, it uses processes such as crushing, boiling, alkaline hydrolysis, enzymatic hydrolysis, and filtration for extraction. Multiple technologies work in an orderly and synergistic manner to stably achieve a high extraction rate and maximize the content of effective ingredients. Moreover, it has good reproducibility and can balance efficiency, cost and product quality.

[0024] 2. In this application, cartilage extract is preferably used to prepare the wound healing agent. The cartilage extract is used as the core of the microspheres, and methacrylamide gelatin is used as the shell. Human platelet lysis solution and lithium magnesium silicate are loaded on the surface of the microspheres. This can release and support nutrients throughout the wound healing cycle, thereby significantly accelerating the wound closure speed, improving the quality and thickness of the new granulation tissue, promoting more orderly collagen deposition and epithelialization, and reducing scar formation.

[0025] 3. In this application, it is preferred to use quaternary ammonium salts with epoxy groups to impregnate microspheres loaded with human platelet lysate and lithium magnesium silicate. The epoxy groups can undergo ring-opening reactions with the primary amino groups in methacrylamide gelatin, thereby introducing quaternary ammonium cations and further improving the antibacterial properties of the wound healing agent. Attached Figure Description

[0026] Figure 1 This is a flowchart of the preparation method of the cartilage extract in Example 1 of this application. Detailed Implementation

[0027] The following embodiments provide a further detailed description of this application.

[0028] Examples of preparation of auxiliary agents 1-4 In the following preparation examples, choline chloride was analytical grade and selected from Langfang Qianyao Technology, lactic acid was selected from Tianrun Lactic Acid and was pharmaceutical grade lactic acid, betaine was selected from Shanghai Yuanye, catalog number S48731, and glucose was selected from Hebei Zhangshan Biotechnology.

[0029] Preparation Example 1: 14g of choline chloride and lactic acid were mixed in a molar ratio of 1:2, and 20wt% of water (total weight of both) was added. The mixture was stirred and dissolved at 80°C, and the pH was adjusted to 4 to obtain the auxiliary agent.

[0030] Preparation Example 2: 14g of choline chloride and lactic acid were mixed in a molar ratio of 1:3, and 30wt% of water (total weight of both) was added. The mixture was stirred and dissolved at 75°C, and the pH was adjusted to 3.5 to obtain the auxiliary agent.

[0031] Preparation Example 3: 14g of betaine and lactic acid were mixed in a molar ratio of 1:2, and 20wt% of water (total weight of both) was added. The mixture was stirred and dissolved at 80°C, and the pH was adjusted to 4 to obtain the auxiliary agent.

[0032] Preparation Example 4: 14g of choline chloride and glucose were mixed in a molar ratio of 1:2, and 20wt% of water was added. The mixture was stirred and dissolved at 80°C, and the pH was adjusted to 4 to obtain the auxiliary agent. Example

[0033] In the following examples, the alkaline protease was selected from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., model number FDY-2241, and the neutral protease was selected from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., model number FDG-2230.

[0034] Example 1: A method for preparing a cartilage extract, comprising the following steps: S1. Pretreatment: Take frozen animal cartilage, thaw it first, and then crush it. During crushing, prevent foreign objects from being mixed in to obtain crushed material. The animal cartilage includes chicken short bone, chicken breastbone, duck trachea, duck cartilage, chicken trachea, chicken bone, cow trachea, and cow cartilage in a mass ratio of 1:1:1:1:1:1:1:1. S2. Cooking: The crushed material is transported to the cooking tank, with 9 tons of material fed into each tank. Water is added to the cooking tank to the required level. The steam valve is turned on and the mixture is stirred at 150 rpm. The mixture is heated to 85°C and stirred continuously until the oil is absorbed. After standing, the oil is skimmed off. Then the mixture is cooked at 105°C for 60 minutes. During the process, the water volume will decrease due to evaporation. Water is added to the required level. S3, Alkaline hydrolysis: Cool the boiled product to 50°C, add a 0.5wt% sodium hydroxide solution, adjust the pH to 12, maintain the temperature and pH, and perform alkaline hydrolysis for 2 hours; S4. Enzymatic hydrolysis: The alkaline hydrolysis product was adjusted to pH 7 with hydrochloric acid at 50℃. 0.067wt% of alkaline protease with an enzyme activity of 200,000 u / g was added to the total weight of the pulverized material. After 1 hour of enzymatic hydrolysis, the pH was adjusted to 6. 0.022wt% of neutral protease with an enzyme activity of 110,000 u / g was added to the total weight of the pulverized material. After 4 hours of enzymatic hydrolysis, hydrochloric acid was added to adjust the pH to 5. The temperature was raised to 60℃. After standing, the liquid was released. S5. First filtration: After coarse filtration, the liquid is transferred to a filter tank. 0.33 wt% of diatomaceous earth (by weight of the crushed material) is added to the filter tank as a filter aid. The mixture is pre-coated for 20 minutes and then discharged after the liquid has clarified to obtain the first filtrate. S6. Nanofiltration: Transfer the primary filtrate into a nanofiltration tank, control the temperature of the nanofiltration tank at 60℃, and concentrate it to a concentration of 20 Brix. During nanofiltration, further impurity removal is performed: add water at 60℃ to the primary filtrate to dilute it and then nanofilter it. The amount of water added is 1 / 3 of the volume of the primary filtrate. Repeat the impurity removal 3 times, and then concentrate it to a concentration of 60 Brix to obtain the concentrated solution. S7. Sterilization: Heat the concentrated liquid to 90℃ and keep it warm for 60 minutes; S8. Secondary filtration: The sterilized concentrate is filtered a second time using a filter screen with an 80-mesh pore size to obtain secondary filtrate. S9. Spray drying: Spray dry the secondary filtrate. The inlet air temperature is 200℃. When the outlet air temperature rises to 120℃, turn on the spray pump. The working pressure of the spray pump is 20MPa. Keep the pressure stable during the spraying process. Control the outlet air temperature at 100℃. After spraying, adjust the size of the dehumidifier outlet so that the temperature of the dry powder coming down from the total receiving rotary valve is not higher than 45℃. Then package it.

[0035] Example 2: A method for preparing a cartilage extract, comprising the following steps: S1. Pretreatment: Take frozen animal cartilage, thaw it first, and then crush it. During crushing, prevent foreign objects from being mixed in to obtain crushed material. The animal cartilage includes chicken short bone, chicken breastbone, duck trachea, duck cartilage, chicken trachea, chicken bone, cow trachea, and cow cartilage in a mass ratio of 1:1:1:1:1:1:1:1. S2. Cooking: The crushed material is transported to the cooking tank, with 9 tons of material added to each tank. Water is added to the cooking tank to the required level. The steam valve is turned on and the mixture is stirred at 200 rpm. The mixture is heated to 95°C and stirred continuously until the oil is absorbed. After standing, the oil is skimmed off. Then the mixture is cooked at 120°C for 50 minutes. During the process, the water volume will decrease due to evaporation. Water is added to the required level. S3, Alkaline hydrolysis: Cool the boiled product to 65°C, add a 0.5wt% sodium hydroxide solution, adjust the pH to 9, maintain the temperature and pH, and perform alkaline hydrolysis for 5 hours; S4. Enzymatic hydrolysis: Adjust the pH of the alkaline hydrolysis product to 11 at 65℃, add 0.067wt% alkaline protease of the total weight of the pulverized material with an enzyme activity of 200,000 u / g, and hydrolyze for 2 hours. Then adjust the pH to 8, add 0.022wt% neutral protease of the total weight of the pulverized material with an enzyme activity of 110,000 u / g, and hydrolyze for 6 hours. After the hydrolysis is completed, add hydrochloric acid to adjust the pH to 7, raise the temperature to 70℃, and release the liquid after standing. S5. First filtration: After coarse filtration, the liquid is transferred to a filter tank. 0.44 wt% of diatomaceous earth (by weight of the crushed material) is added to the filter tank as a filter aid. The mixture is pre-coated for 30 minutes and then discharged after the liquid has clarified to obtain the first filtrate. S6. Nanofiltration: Transfer the primary filtrate into a nanofiltration tank, control the temperature of the nanofiltration tank at 70℃, and concentrate it to a concentration of 18 Brix. During nanofiltration, further impurity removal is performed: add water at 60℃ to the primary filtrate to dilute it and then nanofilter it. The amount of water added is 1 / 3 of the volume of the primary filtrate. Repeat the impurity removal process 5 times, and then concentrate it to a concentration of 70 Brix to obtain the concentrated solution. S7. Sterilization: Heat the concentrated liquid to 80℃ and keep it warm for 60 minutes; S8. Secondary filtration: The sterilized concentrate is filtered a second time using a filter screen with an 80-mesh pore size to obtain secondary filtrate. S9. Spray Drying: Spray dry the secondary filtrate. The inlet air temperature is 250℃. When the outlet air temperature rises to 100℃, turn on the spray pump. The working pressure of the spray pump is 30MPa. Keep the pressure stable during the spraying process. Control the outlet air temperature at 100℃. After spraying, adjust the size of the dehumidifier outlet so that the temperature of the dry powder coming down from the total receiving rotary valve is kept no higher than 45℃. Then package it.

[0036] Example 3: A method for preparing a cartilage extract, comprising the following steps: S1. Pretreatment: Take frozen animal cartilage, thaw it first, and then crush it. During crushing, prevent foreign objects from being mixed in to obtain crushed material. The animal cartilage includes chicken short bone, chicken breastbone, duck trachea, duck cartilage, chicken trachea, chicken bone, cow trachea, and cow cartilage in a mass ratio of 1:1:1:1:1:1:1:1. S2. Cooking: The crushed material is transported to the cooking tank, with 9 tons of material added to each tank. Water is added to the cooking tank to the required level. The steam valve is turned on and the mixture is stirred at 200 rpm. The mixture is heated to 90°C and stirred continuously until the oil is absorbed. After standing, the oil is skimmed off. Then the mixture is cooked at 110°C for 60 minutes. During the main process, the water volume will decrease due to evaporation. Water is added to the required level. S3, Alkaline hydrolysis: Cool the boiled product to 60°C, add a 0.5wt% sodium hydroxide solution, adjust the pH to 10, maintain the temperature and pH, and perform alkaline hydrolysis for 4 hours; S4. Enzymatic hydrolysis: Adjust the pH of the alkaline hydrolysis product to 9 at 60℃, add 0.067wt% alkaline protease of the total weight of the pulverized material with an enzyme activity of 200,000 u / g, and hydrolyze for 2 hours. Then adjust the pH to 7, add 0.022wt% neutral protease of the total weight of the pulverized material with an enzyme activity of 110,000 u / g, and hydrolyze for 5 hours. After the hydrolysis is completed, add hydrochloric acid to adjust the pH to 6, raise the temperature to 65℃, and release the liquid after standing. S5. First filtration: After coarse filtration, the liquid is transferred to a filter tank. 0.44 wt% of diatomaceous earth (by weight of the crushed material) is added to the filter tank as a filter aid. The mixture is pre-coated for 30 minutes and then discharged after the liquid has clarified to obtain the first filtrate. S6. Nanofiltration: Transfer the primary filtrate into a nanofiltration tank, control the temperature of the nanofiltration tank at 65℃, and concentrate it to a concentration of 22 Brix. During nanofiltration, further impurity removal is performed: add water at 65℃ to the primary filtrate to dilute it and then nanofilter it. The amount of water added is 1 / 3 of the volume of the primary filtrate. Repeat the impurity removal process 5 times, and then concentrate it to a concentration of 45 Brix to obtain the concentrated solution. S7. Sterilization: Heat the concentrated liquid to 85℃ and keep it warm for 40 minutes; S8. Secondary filtration: The sterilized concentrate is filtered a second time using a filter screen with an 80-mesh pore size to obtain secondary filtrate. S9. Spray Drying: Spray dry the secondary filtrate. The inlet air temperature is 300℃. When the outlet air temperature rises to 150℃, turn on the spray pump. The working pressure of the spray pump is 30MPa. Keep the pressure stable during the spraying process. Control the outlet air temperature at 140℃. After spraying, adjust the size of the dehumidifier outlet so that the temperature of the dry powder coming down from the total receiving rotary valve is not higher than 45℃. Then package it.

[0037] Example 4: A method for preparing a cartilage extract, which differs from Example 1 in that step S3, alkaline hydrolysis: the boiled product is cooled to 75°C, and an auxiliary agent prepared in Preparation Example 1 is added. The mass ratio of the auxiliary agent to the boiled product is 1:20. The product is ultrasonically reacted at 250W for 4 hours, then cooled to 50°C, and a 0.5wt% sodium hydroxide solution is added. The pH is adjusted to 12, and the temperature and pH are maintained for alkaline hydrolysis for 2 hours.

[0038] Example 5: A method for preparing a cartilage extract, which differs from Example 1 in that step S3, alkaline hydrolysis: the boiled product is cooled to 80°C, and an auxiliary agent prepared in Preparation Example 2 is added. The mass ratio of the auxiliary agent to the boiled product is 1:30. The product is ultrasonically reacted at 250W for 2 hours, then cooled to 50°C, and a 0.5wt% sodium hydroxide solution is added. The pH is adjusted to 12, and the temperature and pH are maintained for alkaline hydrolysis for 2 hours.

[0039] Example 6: A method for preparing a cartilage extract, which differs from Example 1 in that step S3, alkaline hydrolysis: the boiled product is cooled to 75°C, and an auxiliary agent prepared in Preparation Example 3 is added. The mass ratio of the auxiliary agent to the boiled product is 1:20. The product is ultrasonically reacted at 250W for 4 hours, then cooled to 50°C, and a 0.5wt% sodium hydroxide solution is added. The pH is adjusted to 12, and the temperature and pH are maintained for alkaline hydrolysis for 2 hours.

[0040] Example 7: A method for preparing a cartilage extract, which differs from Example 1 in that step S3, alkaline hydrolysis: the boiled product is cooled to 75°C, and an auxiliary agent prepared in Preparation Example 4 is added. The mass ratio of the auxiliary agent to the boiled product is 1:20. The product is ultrasonically reacted at 250W for 4 hours, then cooled to 50°C, and a 0.5wt% sodium hydroxide solution is added. The pH is adjusted to 12, and the temperature and pH are maintained for alkaline hydrolysis for 2 hours.

[0041] Comparative Example Comparative Example 1: A method for preparing a cartilage extract, which differs from Example 1 in that step S4, enzymatic hydrolysis, is as follows: the alkaline hydrolysis product is adjusted to pH 7 with hydrochloric acid at 50°C, 0.067 wt% of alkaline protease with an enzyme activity of 200,000 u / g is added, and enzymatic hydrolysis is carried out for 5 hours. After enzymatic hydrolysis, hydrochloric acid is added to adjust the pH to 5, the temperature is raised to 60°C, and the liquid is released after standing.

[0042] Comparative Example 2: A method for preparing a cartilage extract, which differs from Example 1 in that step S4, enzymatic hydrolysis, is as follows: the alkaline hydrolysis product is adjusted to pH 6 at 50°C, and 0.022 wt% of neutral protease with an enzyme activity of 110,000 u / g is added. Enzymatic hydrolysis is carried out for 5 hours. After enzymatic hydrolysis, hydrochloric acid is added to adjust the pH to 5, the temperature is raised to 60°C, and the liquid is released after standing.

[0043] Application examples In the following application examples, lithium magnesium silicate is selected from Qianfu Mineral Products in Lingshou County, with the product number 0321; methacrylamide gelatin is selected from Nanjing Xianfeng Nano, with the product number 105596 and the product code XFBM02, wherein 1g contains 0.05g of initiator LAP; and human platelet lysis buffer is selected from Ribo (Shanghai) Biochemical Technology, with the product number LBX111.

[0044] Application Example 1: A wound healing agent comprising 10g chitosan hydrogel and 5g microspheres loaded with human platelet lysis solution and lithium magnesium silicate. The microspheres have a cartilage extract prepared in Example 1 as the core and methacrylamide gelatin as the shell. The mass ratio of human platelet lysis solution, lithium magnesium silicate, and microspheres is 0.1:0.1:1. The microspheres loaded with human platelet lysis solution and lithium magnesium silicate are prepared by the following method: Methacrylated gelatin was added to PBS solution (pH 7.4) to prepare a 15 wt% solution. Cartilage extract was added to PBS solution (pH 7.4) to a concentration of 5 mg / ml. The methacrylated gelatin solution and cartilage extract solution were then mixed evenly at a volume ratio of 1:3 to prepare the precursor solution. Add Span 80 to paraffin wax at a concentration of 2% (v / v) and stir for 30 minutes to obtain the oil phase. The precursor solution was loaded into a syringe and pumped dropwise into the oil phase at a rate of 3 ml / h. After the addition was completed, the mixture was stirred for 30 min. Then, it was irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 9 W / cm2 for 3 min. The microspheres were then centrifuged, washed, and collected. Lithium magnesium silicate was added to human platelet lysis buffer and sonicated for 10 min to prepare an enhanced solution. Mix the microspheres and the enhancement solution, incubate at 37°C for 2 hours, wash twice with PBS buffer, centrifuge, and store at 4°C.

[0045] The preparation method of the above-mentioned wound healing agent is as follows: 14 mg of rhein was dissolved in 5 mL of 0.1 M sodium bicarbonate solution and sonicated at 100 W for 15 min. 100 mg of chitosan was added to 5 mL of 1% (V / V) acetic acid solution and stirred for 10 min. 5 mL of chitosan solution and 5 mL of rhein solution were placed on ice (temperature below 4℃) and stirred for 15 min. The rhein solution was then added to the chitosan solution until completely mixed to obtain chitosan hydrogel. Microspheres loaded with human platelet lysis buffer and magnesium silicate were immersed in 2% (2,3-epoxypropyl)dodecyltrimethylammonium chloride PBS buffer, shaken at 35°C for 8 h, centrifuged, washed, and mixed evenly with chitosan hydrogel.

[0046] Application Example 2: A wound healing agent comprising 10g chitosan hydrogel and 3g microspheres loaded with human platelet lysis solution and lithium magnesium silicate. The microspheres have a cartilage extract prepared in Example 1 as the core and methacrylamide gelatin as the shell. The mass ratio of human platelet lysis solution, lithium magnesium silicate, and microspheres is 0.1:0.1:1. The microspheres loaded with human platelet lysis solution and lithium magnesium silicate are prepared by the following method: Methacrylated gelatin was added to PBS solution (pH 7.4) to prepare a 15 wt% solution. Cartilage extract was added to PBS solution (pH 7.4) to a concentration of 5 mg / ml. The methacrylated gelatin solution and cartilage extract solution were then mixed evenly at a volume ratio of 1:3 to prepare the precursor solution. Add Span 80 to paraffin wax at a concentration of 2% (v / v) and stir for 30 minutes to obtain the oil phase. The precursor solution was loaded into a syringe and pumped dropwise into the oil phase at a rate of 5 ml / h. After the addition was completed, the mixture was stirred for 30 min, and then irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 9 W / cm2 for 3 min. The microspheres were then centrifuged, washed, and collected. Lithium magnesium silicate was added to human platelet lysis buffer and sonicated for 10 min to prepare an enhanced solution. Mix the microspheres and the enhancement solution, incubate at 35°C for 4 hours, wash twice with PBS buffer, centrifuge, and store at 4°C.

[0047] The preparation method of the above-mentioned wound healing agent includes the following steps: 14 mg of rhein was dissolved in 5 mL of 0.1 M sodium bicarbonate solution and sonicated at 100 W for 15 min. 100 mg of chitosan was added to 5 mL of 1% (V / V) acetic acid solution and stirred for 10 min. 5 mL of chitosan solution and 5 mL of rhein solution were placed on ice (temperature below 4℃) and stirred for 15 min. The rhein solution was then added to the chitosan solution until completely mixed to obtain chitosan hydrogel. Microspheres loaded with human platelet lysis buffer and magnesium silicate were immersed in 2% (2,3-epoxypropyl)dodecyltrimethylammonium chloride PBS buffer, shaken at 37°C for 4 h, centrifuged, washed, and mixed evenly with chitosan hydrogel.

[0048] Application Example 3: A wound healing agent, which differs from Application Example 1 in that the microspheres are not loaded with human platelet lysis solution, but only with lithium magnesium silicate. The mass ratio of lithium magnesium silicate to microspheres is 0.1:1. The reinforcing solution is made of lithium magnesium silicate and an equal mass of deionized water. The remaining methods and parameters are the same as in Application Example 1.

[0049] Application Example 4: A wound healing agent, which differs from Application Example 1 in that the microspheres are not loaded with lithium magnesium silicate and human platelet lysis fluid. The wound healing agent contains 10g of chitosan hydrogel and 5g of microspheres. The microspheres are prepared by adding methacrylamide gelatin to PBS solution (pH 7.4) to prepare a solution with a concentration of 15wt%. Cartilage extract is added to PBS solution (pH 7.4) to a concentration of 5mg / ml. Then, the methacrylamide gelatin solution and the cartilage extract solution are mixed evenly at a volume ratio of 1:3 to obtain the precursor solution. Add Span 80 to paraffin wax at a concentration of 2% (v / v) and stir for 30 minutes to obtain the oil phase. The precursor solution was loaded into a syringe and pumped dropwise into the oil phase at a rate of 3 ml / h. After the addition was complete, the mixture was stirred for 30 min, and then irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 9 W / cm2 for 3 min. The microspheres were then centrifuged, washed, and collected.

[0050] Application Example 5: A wound healing agent, which differs from Application Example 1 in that the microspheres loaded with human platelet lysis solution and magnesium lithium silicate were not impregnated in PBS buffer of 2% (2,3-epoxypropyl)dodecyltrimethylammonium chloride. The wound healing agent was prepared by impregnating the microspheres loaded with human platelet lysis solution and magnesium silicate with chitosan hydrogel and mixing them evenly.

[0051] Application Example 6: A wound healing agent, which differs from Application Example 1 in that it does not use methacrylamide gelatin, human platelets and lithium magnesium silicate, and is not soaked in PBS buffer of 2% (2,3-epoxypropyl)dodecyltrimethylammonium chloride. The wound healing agent is prepared by uniformly mixing 10g of chitosan hydrogel and 5g of cartilage extract prepared in Example 1.

[0052] Application Example 7: A wound healing agent, which differs from Application Example 1 in that it contains only chitosan hydrogel.

[0053] Performance testing I. Testing of cartilage extract: Cartilage extract was prepared according to the methods in the examples and comparative examples. Chondroitin sulfate, which is unique to cartilage extract, was used as an indicator. The extraction rate and content of chondroitin sulfate were determined by the carbazole sulfate method, with chondroitin sulfate standard as a reference. The test results are recorded in Table 1.

[0054] Table 1. Extraction rate detection of cartilage extract In Examples 1-3, different amounts of enzymes and process parameters were used to enzymatically hydrolyze animal cartilage, and it was observed that the extraction rate and content of chondroitin sulfate were high.

[0055] Compared to Example 1, Examples 4 and 5 further involved treating the cooked products with an auxiliary agent before alkaline hydrolysis. As shown in Table 1, the extraction rate of chondroitin sulfate in the cartilage extracts obtained in Examples 4 and 5 was increased.

[0056] In Example 6, the excipient prepared in Preparation Example 3 was used. The excipient contained betaine and lactic acid. In Example 7, the excipient prepared in Preparation Example 4 was used. The excipient contained choline chloride and glucose. It can be seen that the extraction rate of chondroitin sulfate in the cartilage extracts prepared in Examples 6 and 7 was higher than that in Example 1, but still lower than that in Examples 5 and 6. This indicates that using appropriate choline chloride and lactic acid as excipients can improve the extraction rate of chondroitin sulfate.

[0057] Compared with Example 1, Comparative Examples 1 and 2 used only one protease for enzymatic hydrolysis. As can be seen from the data in Table 1, the extraction rate of chondroitin sulfate in the cartilage extracts obtained by Comparative Examples 1 and 2 decreased, and the content also decreased.

[0058] II. Detection of wound healing agent: (1) Wound healing rate: The wound healing agent prepared in the application examples was used as the experimental group, and physiological saline was used as the control group; 80 SPF mice, half male and half female, weighing 20g, were selected and divided into 8 groups of 10 mice each; the mice were anesthetized with 1% sodium pentobarbital intraperitoneally, their hair was clipped, and they were disinfected with povidone-iodine. A longitudinal full-thickness skin incision of 1cm was made in the center of the neck and back of the mice. The mice were housed in separate cages to establish the injury model. After the model was established, the experimental group used the wound healing agent prepared in application examples 1-7, and applied it to the wound of the corresponding mice at a dosage of 0.1ml per mouse (extending to 2-3mm, with a thickness of about 0.5mm). The control group used physiological saline. The observation was continued, and the wound healing rate was calculated according to the following formula: Wound healing rate = (A0-At) / A0×100%, where A0 is the initial wound area and At is the wound area at each time point. The test results were recorded in Table 2.

[0059] (2) Antibacterial activity was tested using the plate count method. 50 μL of frozen *Escherichia coli* and *Staphylococcus aureus* were added to 10 mL of LB broth and incubated overnight at 37°C in a bacterial incubator (160 rpm). 200 μL of the reactivated plastisol was then transferred to a sterile 96-well plate. The bacterial solution was serially diluted with physiological saline, and the OD value was measured at 600 nm using a multi-functional microplate reader. The bacterial concentration was adjusted to 1 × 10⁻⁶. 8 CFU / mL, further dilution yielded a bacterial concentration of 2×10⁻⁶. 5CFU / mL was prepared for use. After washing the 24-well plate with physiological saline, 500 μL of the prepared bacterial culture was added to each well. A control group was set up with bacterial culture plus PBS. The experimental group was treated with sterilized wound healing agent. Each group was divided into 3 replicates. After incubation at 37°C for 24 h, the liquid in each well was diluted 500 times with physiological saline, and 50 μL was added to agar medium and plated. The plate was incubated at 37°C for another 24 h, and the inhibition rate was calculated.

[0060] Table 2 Performance testing of wound healing agents In Application Examples 1 and 2, a wound healing agent prepared by mixing microspheres loaded with human platelet lysis fluid and lithium magnesium silicate with chitosan hydrogel was continuously applied to the wounds of mice. It was observed that more than 87% of the wounds healed within 15 days, and 100% of the wounds were healed within 20 days. This indicates that the prepared wound healing agent has anti-inflammatory and antibacterial effects, can reduce infection and inflammation at the wound site, and exhibits good wound healing effect.

[0061] Compared with Application Example 1, Application Example 3 did not load human platelet lysate on the surface of the microspheres, but only loaded lithium magnesium silicate on the surface of the microspheres. As can be seen from the data in Table 2, the wound healing effect of the wound healing agent prepared in this way was reduced, but the antibacterial rate against Escherichia coli and Staphylococcus aureus did not change significantly, indicating that the growth factors in human platelet lysate can promote granulation tissue formation, thereby promoting rapid wound healing.

[0062] In Application Example 4, no human platelet lysis solution and lithium magnesium silicate were loaded onto the surface of the microspheres. Compared with Application Example 1, the wound healing agent prepared in Application Example 4, when applied to the wounds of mice, resulted in a slower wound healing rate and a decrease in the antibacterial rate against Escherichia coli and Staphylococcus aureus. This indicates that human platelet lysis solution and lithium magnesium silicate can work together to promote granulation tissue formation, stimulate angiogenesis, and inhibit bacteria, thereby accelerating wound healing.

[0063] Compared with Application Example 1, Application Example 5 was not soaked in (2,3-epoxypropyl)dodecyltrimethylammonium chloride solution, that is, it was not grafted with quaternary ammonium salt. As shown in Table 2, the antibacterial rate of the wound healing agent prepared in Application Example 5 was significantly reduced and the wound healing speed was slowed down.

[0064] In Application Example 6, only chitosan hydrogel and cartilage extract were used to prepare a wound healing agent. Compared with Example 1, the wound healing rate was slightly increased at 5 days, but the healing rate did not improve significantly with the extension of time, and the antibacterial rate also decreased significantly.

[0065] In Application Example 7, only chitosan hydrogel was used as a wound healing agent. The data comparison in Table 2 shows that this wound healing agent slowed down the healing effect of the wound and had a poorer effect on promoting healing.

[0066] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a cartilage extract, characterized in that, Includes the following steps: S1. Pretreatment: The animal cartilage is thawed and crushed to obtain a powder; S2. Cooking: Add water to the pulverized material and cook at 85-95℃. Let it stand to remove the grease, and then cook at ≥99℃ for ≥30 minutes. S3. Alkaline hydrolysis: Cool the boiled product to 50-65℃, adjust the pH to 9-12, and perform alkaline hydrolysis for 2-5 hours. S4. Enzymatic hydrolysis: Adjust the pH of the alkaline hydrolysis product to 7-11 at 50-65℃, add alkaline protease, hydrolyze for 1-2 hours, adjust the pH to 6-8, add neutral protease, hydrolyze for 4-6 hours, adjust the pH to 5-7 after hydrolysis, heat to 60-70℃, let stand, and release the liquid. S5. Primary filtration: The liquid is filtered through a filter aid to obtain a primary filtrate; S6. Nanofiltration: The primary filtrate is nanofiltered at a temperature ≥60℃, concentrated to a concentration ≥18 Brix, and then concentrated again to a concentration of 45-70 Brix to obtain a concentrated solution. S7. Sterilization: Heat the concentrated liquid to 80-90℃ and keep it warm for 30-60 minutes; S8. Secondary filtration: The sterilized concentrate is filtered through a filter screen with a pore size of no more than 80 mesh to obtain secondary filtrate. S9. Spray drying: The secondary filtrate is spray-dried and then packaged to obtain cartilage extract.

2. The method for preparing cartilage extract according to claim 1, characterized in that: The amount of alkaline protease added is 0.04-0.08 wt% of the pulverized material. The amount of the central Y-type protease added is 0.01-0.03 wt% of the pulverized material.

3. The method for preparing cartilage extract according to claim 1, characterized in that: In step S3, when the product obtained from boiling is cooled to 75-80℃, an auxiliary agent is added. The mass ratio of the auxiliary agent to the product obtained from boiling is 1:20-30. The mixture is ultrasonically reacted for 2-4 hours, and then enzymatic hydrolysis is performed. The auxiliary agent is prepared by mixing choline chloride and lactic acid in a molar ratio of 1:2-3, adding 20-30 wt% of water, stirring and dissolving at 75-80℃, and adjusting the pH to 3.5-4.

4. The method for preparing cartilage extract according to claim 1, characterized in that: The spray drying process involves an inlet air temperature of 200-300℃, an exhaust air temperature of ≥80℃, and a spray pressure of 15-30MPa.

5. The method for preparing cartilage extract according to claim 1, characterized in that: The nanofiltration process removes impurities by adding water at a temperature ≥60℃ to the filtrate and repeating this process 1-5 times until the nanofiltration is complete.

6. The method for preparing cartilage extract according to claim 1, characterized in that: The animal cartilage includes at least one of the following: chicken short bone, chicken breastbone, duck trachea, duck cartilage, chicken trachea, chicken bone, bovine trachea, and bovine cartilage.

7. The application of the cartilage extract prepared by the method according to any one of claims 1-6, characterized in that: Used in wound healing preparations.

8. The application of the cartilage extract according to claim 7, characterized in that: The wound healing preparation comprises chitosan hydrogel in a mass ratio of 1:0.3-0.5 and microspheres loaded with human platelet lysis solution and lithium magnesium silicate. The microspheres have a cartilage extract as the core and methacrylamide gelatin as the shell.

9. The application of the cartilage extract according to claim 8, characterized in that: The microspheres loaded with human platelet lysate and lithium magnesium silicate are prepared using the following methods: Methacrylamide gelatin and cartilage extract were added to PBS solution respectively and then mixed evenly to prepare the precursor solution. The precursor solution was pumped out as droplets at a flow rate of 3-10 mL / h and irradiated with ultraviolet light to form microspheres; Lithium magnesium silicate was added to human platelet lysis buffer and sonicated to prepare an enhanced solution. Mix the microspheres with the enhancement solution and incubate at 35-37°C for 2-4 hours. Wash with PBS buffer 1-2 times.

10. The application of the cartilage extract according to claim 8, characterized in that: The wound healing agent is prepared as follows: microspheres loaded with human platelet lysis buffer and magnesium silicate are immersed in PBS buffer containing (2,3-epoxypropyl)dodecyltrimethylammonium chloride, shaken at 35-37°C for 4-8 hours, centrifuged, washed, and mixed evenly with chitosan hydrogel.

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