Bullfrog-source micromolecular collagen peptide repair material and preparation method thereof

Abstract

The invention discloses a bullfrog source micromolecular collagen peptide repair material and a preparation method thereof in the technical field of repair materials. The bullfrog source micromolecular collagen peptide repair material is prepared from the following components in parts by weight: bullfrog source collagen peptide, N-succinimido-S-acetylthioacetate (SATA), modified cellulose, chondroitin sulfate, geniposin and pinoresinol diglucoside. According to the preparation method, micromolecular collagen peptide is extracted from bullfrog skin, the collagen peptide is modified and grafted to a side chain of a cellulose molecular chain through sulfydryl to form a brush-shaped structure, cellulose molecules can provide rigid support, the side chain of the collagen peptide forms a fiber network with biological affinity, and meanwhile, the fiber network can provide rigid support for the bullfrog skin. And a plurality of small molecule auxiliary compounds with cartilage repair performance are loaded, so that the repair performance and the biological fusion performance of the collagen peptide repair material are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of repair materials, and specifically refers to a bullfrog-derived small-molecule collagen peptide repair material and a preparation method thereof. Background Art

[0002] Collagen is the most abundant structural protein in the human body, primarily found in the skin, bones, tendons, ligaments, cartilage, and blood vessels. Collagen not only has extremely high biocompatibility but also promotes tissue repair and regeneration. Collagen is a protein with a unique structure composed of three polypeptide chains wrapped in a triple helix. Its primary functions are to provide structural support, maintain mechanical strength of tissues, and promote wound healing. Collagen is highly biocompatible and biodegradable, leading to its widespread application in biomedical materials, wound dressings, and bone repair materials.

[0003] Bullfrog, as an amphibian, has high biological value, and the collagen derived from it has unique advantages. The molecular weight of bullfrog-derived collagen peptides is small, which enables them to be quickly absorbed by the human body and promote tissue repair. At the same time, bullfrog-derived collagen peptides have good solubility, plasticity and good biological activity, and can be widely used in skin repair, soft tissue repair, bone repair and other fields.

[0004] The source of bullfrog collagen peptides has superior biosafety compared to traditional collagen sources such as pig skin and cowhide. Since bullfrogs grow in a natural environment and antibiotics and hormones are rarely used in the breeding process, the collagen peptides derived from them have certain advantages in safety and biocompatibility.

[0005] Collagen peptides are small molecule polypeptides obtained from collagen through enzymatic hydrolysis. Their molecular weight is usually between 1000 and 5000 Daltons. Small molecule collagen peptides have a smaller molecular weight and stronger biological activity, and have shown outstanding effects in tissue repair, anti-aging, and anti-oxidation. Especially in the fields of wound healing, skin repair, and joint diseases, the application of small molecule collagen peptides has shown excellent effects. Its mechanism of action mainly includes the following aspects: Promoting cell proliferation and migration: Small molecule collagen peptides can promote the proliferation and migration of fibroblasts, keratinocytes, etc., thereby accelerating the wound healing process; Enhancing the antioxidant capacity of tissues: Collagen peptides can regulate the body's antioxidant enzyme system, reduce the damage of free radicals to tissues, thereby promoting tissue repair and regeneration; Improving blood circulation: Collagen peptides can promote the growth of microvessels and improve blood circulation, thereby providing better nutritional support for repairing tissues. Animal-derived small molecule collagen peptides have huge application potential in current technology, but still face some technical defects. The main problems include: limited raw material sources: there are challenges in the breeding environment and resource sustainability of bullfrogs, and may put certain pressure on the ecology; quality control issues of collagen peptides: inconsistent molecular weight and insufficient purity may affect the effect of the product, and the current enzymatic hydrolysis process is difficult to fully standardize; high production costs: complex production processes and equipment requirements lead to high production costs, affecting market competitiveness; individual differences: the repair effect of collagen peptides is affected by individual differences, and bioavailability and absorption effects vary from person to person; stability issues: collagen peptides are easily affected by the environment during storage and use, which may lead to a decrease in biological activity; side effects and safety: some individuals may experience allergic reactions, and the long-term use effects and safety have not yet been fully clarified; lack of unified standards: there is currently a lack of unified industry technical standards, resulting in differences in product quality and effects. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a bullfrog-derived small-molecule collagen peptide repair material and a preparation method thereof. The present invention extracts small-molecule collagen peptides from bullfrog skin, modifies the collagen peptides through thiol groups, and grafts them to the side chains of cellulose molecular chains to form a brush-like structure. The cellulose molecules can provide rigid support, while the collagen peptide side chains form a fiber network with biological affinity. At the same time, a variety of small-molecule auxiliary compounds with cartilage repair properties are loaded to improve the repair performance and biofusion performance of the collagen peptide repair material.

[0007] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is as follows: The present invention proposes a bullfrog-derived small molecule collagen peptide repair material, which comprises the following components in parts by weight:

[0008] Bullfrog collagen peptide 10 parts, N-succinimidyl-S-acetylthioacetate (SATA) 10-22 parts, modified cellulose 100-200 parts, chondroitin sulfate 20-30 parts, genipoic acid glycoside 10-20 parts, pinoresinol diglucoside 10-20 parts;

[0009] Preferably, the method for extracting bullfrog-derived collagen peptides specifically comprises the following steps:

[0010] ① Take fresh frozen bullfrog skin and bullfrog cartilage as raw materials, wash the impurities on the surface of the bullfrog skin and bullfrog cartilage with deionized water to obtain pre-treated extraction raw materials;

[0011] ② The pretreated extraction raw material prepared in step ① is placed in a NaCl aqueous solution, soaked for 10-20 minutes, washed with clean water to remove residual salt and impurities, and then soaked in an anhydrous ethanol solution for 20-30 minutes, washed with clean water to remove excess ethanol, to obtain a defatted extraction raw material;

[0012] Preferably, in step ②, the mass concentration of the NaCl aqueous solution is 0.5-1.5%;

[0013] ③ Place the fat-free extraction raw material prepared in step ② in a hydrochloric acid solution, soak for 5-10 minutes, and then wash with clean water until neutral to obtain a pre-enzymatic extraction raw material;

[0014] Preferably, in step ③, the concentration of the hydrochloric acid solution is 0.1-0.2 mol / L;

[0015] ④ Place the pre-enzymatic extraction raw material prepared in step ③ into the enzymatic solution, maintain the reaction temperature at 40-60°C, stir and react for 4-6 hours, centrifuge, collect the supernatant, and filter to obtain a crude collagen extract;

[0016] Preferably, in step ④, the enzymatic solution comprises the following components in mass concentrations: alkaline protease 0.05-0.07 g / mL, trypsin 0.08-0.1 g / mL, neutral protease 0.1-0.2 g / mL;

[0017] ⑤ Separate and purify the crude collagen extract prepared in step ④, filter it using an ultrafiltration membrane, rotary evaporate the filtrate, and freeze-dry it to obtain bullfrog-derived collagen peptides.

[0018] The present invention also provides a method for preparing a bullfrog-derived small molecule collagen peptide repair material, which specifically comprises the following steps:

[0019] S1. Dissolve bullfrog-derived collagen peptide in PBS buffer solution, slowly add N-succinimidyl-S-acetylthioacetate (SATA) solution, and stir to react under neutral conditions. After the reaction is completed, add Tris hydrochloride buffer solution, let it stand, and then centrifuge through an ultrafiltration tube to collect the product. Add hydroxylamine hydrochloride and EDTA, mix well, and stir to react to obtain thiol-modified collagen peptide;

[0020] Preferably, in step S1, the mass concentration of the bullfrog-derived collagen peptide in the PBS buffer solution is 5-10 mg / mL;

[0021] Preferably, in step S1, the mass ratio between the bullfrog-derived collagen peptide and SATA is 1:1-2.2;

[0022] S2. Dissolve cellulose in DMF, add p-toluenesulfonyl chloride and pyridine, raise the reaction temperature to 40-60°C, react for 6-8 hours, remove excess solvent, wash with deionized water, and freeze-dry to obtain activated cellulose;

[0023] Preferably, in step S2, the mass concentration of the cellulose in DMF is 0.1-0.2 g / mL;

[0024] Preferably, in step S2, the mass ratio between the cellulose and p-toluenesulfonyl chloride is 1:1.5-2;

[0025] Preferably, in step S2, the mass volume ratio between the cellulose and pyridine is 0.3-0.5 g / mL;

[0026] S3, dissolving the activated fiber prepared in step S2 in 1-butyl-3-methylimidazolium chloride ionic liquid, heating until the activated cellulose is completely dissolved, adding chloroacetyl chloride, maintaining the reaction temperature at 50-70° C., reacting for 20-30 hours, adding deionized water for precipitation, filtering, collecting the precipitate, washing, and vacuum drying to obtain modified cellulose;

[0027] Preferably, in step S3, the mass concentration of the activated cellulose in the 1-butyl-3-methylimidazolium chloride ionic liquid is 20-40 mg / mL;

[0028] Preferably, in step S3, the mass ratio between the activated cellulose and chloroacetyl chloride is 1:10-20;

[0029] S4. Dissolve the modified cellulose prepared in step S3 in DMSO, heat and stir until the modified cellulose is completely dissolved, dissolve the thiol-modified collagen peptide prepared in step S1 in DMSO and transfer it to the reaction system, add a catalyst, mix well, slowly add CS2, maintain the reaction temperature at 30-50°C, continue the reaction for 9-15 hours, add deionized water to wash the precipitate, filter, repeatedly wash with deionized water, and vacuum dry to obtain collagen-complexed cellulose;

[0030] Preferably, in step S4, the mass concentration of the modified cellulose in DMSO is 10-20 mg / mL;

[0031] S5. Dissolve the collagen composite cellulose prepared in step S4 in hexafluoroisopropanol and stir to form a homogeneous transparent solution. Add chondroitin sulfate, genipoic acid glycoside and pinoresinol diglucoside, perform ultrasonic treatment, vacuum degassing, adjust the electrospinning parameters for electrospinning, use aluminum foil to cover a rotating drum to collect the cellulose membrane, gently peel off the composite fiber membrane accumulated on the rotating drum, place it in a drying oven at room temperature and let it stand for 9-12 hours, wash to remove residual solvent, and vacuum dry to obtain a collagen peptide repair material;

[0032] Preferably, in step S5, the mass concentration of the collagen-cellulose composite in hexafluoroisopropanol is 10-20 mg / mL.

[0033] The beneficial effects achieved by the present invention are as follows:

[0034] The present invention provides a bullfrog-derived small-molecule collagen peptide repair material and a preparation method thereof. The present invention extracts small-molecule collagen peptides from bullfrog skin, modifies the collagen peptides through thiol groups, and grafts them onto the side chains of cellulose molecular chains to form a brush-like structure. The cellulose molecules can provide rigid support, while the collagen peptide side chains form a fiber network with biological affinity. At the same time, a variety of small-molecule auxiliary compounds with cartilage repair properties are loaded to improve the repair performance and biofusion performance of the collagen peptide repair material. In the present invention, acid hydrolysis + multi-enzyme combined enzymatic hydrolysis combined with 3kDa ultrafiltration purification are used to obtain bullfrog-derived collagen peptides with small molecular weight and easy absorption. Small molecule peptide chains are more easily taken up by body cells, can quickly promote fibroblast adhesion, migration and proliferation, and accelerate the formation of new tissue. Controllable -SH groups are introduced through SATA to form stable chemical bonds between collagen peptides and modified cellulose, thereby enhancing the cross-linking density and structural stability of the material and avoiding excessive swelling or degradation of nanofibers after electrospinning during use. The cellulose is sequentially modified by toluenesulfonylation and chloroacetylation to make it soluble in DMF / DMSO and covalently bonded to thiol collagen peptides, providing excellent mechanical strength and flexibility. After electrospinning, the three-dimensional porous structure can be maintained to support cell infiltration and tissue growth. By electrospinning through hexafluoroisopropanol solution, the fiber diameter is controllable, the surface is smooth, and the pores are interconnected. The porous fiber network can simulate the natural extracellular matrix, promote the exchange of nutrients and oxygen, and at the same time block pathogenic microorganisms. In the present invention, chondroitin sulfate provides moisturizing, anti-inflammatory and chondrocyte matrix synthesis-promoting effects, and is suitable for soft tissue and joint surface repair. Genipoic acid glycoside has significant antioxidant and anti-inflammatory activities. Pinoresinol diglucoside has both antibacterial and antibacterial film-forming activities, promotes the osteogenic differentiation of osteoblasts cultured in vitro, increases the formation area of ​​calcified nodules and improves the expression level of osteogenesis-related gene proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the elution peak results of the bullfrog-derived collagen peptide in Example 1 of the present invention;

[0036] Figure 2 This is an infrared spectrum image of the collagen peptide repair material prepared in Example 1 of the present invention;

[0037] Figure 3 This is a diagram showing the effect of the collagen peptide repair material prepared in Example 1 on promoting the growth of hBMSCs.

[0038] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0041] The experimental methods in the following examples, unless otherwise specified, are conventional methods; the test materials and test strains used in the following examples, unless otherwise specified, are purchased from commercial channels.

[0042] Example 1

[0043] This embodiment provides a bullfrog-derived small molecule collagen peptide repair material, which comprises the following components in parts by weight: 10 parts of bullfrog-derived collagen peptide, 11 parts of SATA, 100 parts of modified cellulose, 20 parts of chondroitin sulfate, 10 parts of geniposide, and 10 parts of pinoresinol diglucoside;

[0044] The method for extracting bullfrog-derived collagen peptides specifically comprises the following steps:

[0045] ① Take fresh frozen bullfrog skin and bullfrog cartilage, wash the bullfrog skin and cartilage thoroughly with deionized water to remove surface impurities and blood stains, and obtain pre-treated extraction raw materials;

[0046] ② Place the pretreated raw material in a 0.8% NaCl aqueous solution for 15 minutes, thoroughly wash with clean water to remove residual salt and impurities, soak the raw material in an anhydrous ethanol solution for 25 minutes, and wash with clean water to remove excess ethanol to obtain a defatted extraction raw material;

[0047] ③ Soak the defatted extraction raw material in 0.12 mol / L hydrochloric acid solution for 7 minutes, rinse thoroughly with clean water to ensure that the pH value is close to neutral, and obtain the pre-enzymatic extraction raw material;

[0048] ④ Place the pre-enzymatically extracted raw materials into the enzymatic solution containing 0.05 g / mL alkaline protease, 0.1 g / mL trypsin, and 0.2 g / mL neutral protease. Maintain the reaction temperature at 55°C, stir for 5 hours, and centrifuge (9000 rpm for 20 minutes). Collect the supernatant and filter to obtain a crude collagen extract.

[0049] ⑤ Separate and purify the crude collagen extract prepared in step ④ using a 3 kDa ultrafiltration membrane to remove macromolecular impurities, concentrate the filtrate by rotary evaporation to remove part of the solvent, and freeze-dry the concentrate (-50°C, 20 Pa) to obtain bullfrog-derived collagen peptides;

[0050] The bullfrog collagen peptide was separated by gel chromatography to obtain a single compound. In the gel chromatography, the gel was selected as Sephadex G-25, which was swollen and evenly filled into the chromatography column and equilibrated overnight. The bullfrog collagen was dissolved in distilled water at a concentration of 200 μg / mL, and the sample was loaded into the gel column at a sample volume of 5 mL. Distilled water was used as the mobile phase, and the flow rate was controlled to be 50 mL / h. The absorption peak was roughly measured at 220 nm, and the sample was received according to the absorption peak. Figure 1 This is the result diagram of the elution peak of bullfrog-derived collagen peptide. The sample liquid with different absorption peaks was freeze-dried to obtain bullfrog-derived collagen peptide-I, bullfrog-derived collagen peptide-II, bullfrog-derived collagen peptide-III, bullfrog-derived collagen peptide-IV, and bullfrog-derived collagen peptide-V.

[0051] This embodiment provides a method for preparing a bullfrog-derived small molecule collagen peptide repair material, which specifically includes the following steps:

[0052] S1. Accurately weigh 10 mg of bullfrog collagen peptide III and dissolve it in 5 mL of PBS buffer solution, maintaining the pH of the reaction system at 7.4. Dissolve 10.8 mg of SATA in 0.5 mL of DMF solvent to obtain a SATA solution. Add the SATA solution to the reaction system at a rate of 50 μL / min. After the addition is complete, stir the reaction at 200 rpm for 60 minutes. After the reaction is complete, add 5 mL of Tris hydrochloride buffer solution (concentration of 50 mM). After standing at room temperature for 5 minutes, transfer the reaction system to a 3 kDa ultrafiltration tube, centrifuge at 4000 g and 4 ° C for 10 minutes, collect the product, add it to 2 mL of PBS buffer (pH 7.4), mix well, and add 1 mL of 100 mM hydroxylamine hydrochloride / PBS buffer solution to the reaction system. 50 mM EDTA / PBS buffer solution was added to the reaction system, and the reaction was stirred at 50 rpm for 120 min. After the reaction was completed, the mixture was transferred to a 3 kDa ultrafiltration tube and centrifuged at 4000 g and 4°C for 10 min. The product was collected and freeze-dried to obtain thiol-modified collagen peptide.

[0053] S2. Accurately weigh 2 g of cellulose and place it in a flask. Add 20 mL of DMF to fully dissolve the cellulose. Then add 3.0 g of p-toluenesulfonyl chloride and mix well. Add 5 mL of pyridine and raise the reaction temperature to 40°C. After reacting for 8 h, cool the reaction system to room temperature and remove excess solvent by distillation under reduced pressure. Repeatedly wash with deionized water until neutral, and freeze-dry to obtain activated cellulose.

[0054] S3. 1 g of the activated cellulose prepared in step S2 was placed in a flask, 50 mL of DMSO solvent was added, and the mixture was heated until the activated cellulose was completely dissolved. 3.4 g of chloroacetyl chloride was added, and the reaction temperature was maintained at 50° C. The reaction was continued for 24 h. Deionized water was added for sedimentation, and the precipitate was collected by suction filtration, washed repeatedly with deionized water, and dried under vacuum at 60° C. for 12 h to obtain modified cellulose.

[0055] S4. Accurately weigh 100 mg of the modified cellulose prepared in step S3 and place it in a flask. Add 10 mL of DMSO solvent and raise the temperature until the modified cellulose is completely dissolved. Then, transfer 20 mg of the thiol-modified collagen peptide prepared in step S1 to the reaction system and mix well. Add 0.5 mL of triethanolamine and stir at room temperature for 20 min. Then, add 0.2 mL of CS2 dropwise and continue mixing for 20 min. Raise the reaction temperature to 40°C and react for 12 h. After the reaction is cooled to room temperature, add deionized water for precipitation. Filter and collect the precipitate. Wash the precipitate with deionized water and dry it in a vacuum at 40°C for 12 h to obtain collagen-complexed cellulose.

[0056] S5. Take 100 mg of the collagen composite cellulose prepared in step S4 and place it in a centrifuge tube. Add 5 mL of hexafluoroisopropanol and stir at 200 rpm at 37 ° C for 2 hours to form a uniform transparent solution. Add 20 mg of chondroitin sulfate, 10 mg of geniposide and 10 mg of pinoresinol diglucoside. After ultrasonic treatment at 500 W for 4 hours, degassing treatment under vacuum conditions for 30 minutes, adjust the electrospinning parameters, adjust the flow rate to 0.5 mL / h, adjust the voltage to 15 kV, adjust the collection distance to 15 cm, adjust the ambient temperature to 25 ° C, adjust the relative humidity to 40%, and use aluminum foil to cover the rotating drum to collect the cellulose membrane. After spinning for 4 hours, gently peel off the composite fiber membrane accumulated on the rotating drum, place it in a room temperature drying oven for 12 hours, wash to remove the residual solvent, and place it in a vacuum drying oven at 40 ° C for 12 hours to ensure that the moisture and residual solvent are completely removed to obtain a collagen peptide repair material.

[0057] The collagen peptide repair material prepared in Example 1 was mixed with potassium bromide and ground evenly, pressed into thin slices, and detected by Fourier transform infrared spectrometer with a scanning range of 4000-500 cm-1 , with a resolution of 4cm -1 ; Figure 2 The infrared spectrum image of the collagen peptide repair material prepared in Example 1 of the present invention is shown in the figure, where A is the collagen composite cellulose prepared in Example 1, and B is the bullfrog-derived collagen peptide prepared in Example 1, 3400-3200 cm -1 The characteristic peak at (OH / NH broad band) indicates that an enhanced hydrogen bond network is formed between the cellulose hydroxyl groups and the collagen peptide chain amino groups. The peak position is slightly shifted to the low frequency and the peak shape is more symmetrical. -1 The characteristic peak at (-CH2- asymmetric / symmetric stretching) indicates that the methylene structure of the cellulose and collagen skeletons is completely retained, 1670-1630cm -1 (Amide I: C=O stretching) and 1550-1520 cm -1 The characteristic peak at (amide II: NH bending + CN stretching) represents the "fingerprint" absorption of collagen peptides. After compounding, it is slightly broadened or slightly shifted, reflecting the interaction between molecules, 1050-1000cm -1 The characteristic peaks at 700-600 cm-1 (C=S stretching) and 700-600 cm-1 (CS bending) correspond to the dithiocarbonate / dithiocarbamate structure generated by CS2 / triethanolamine cross-linking, and the peaks at 1200-1000 cm-1 correspond to the dithiocarbonate / dithiocarbamate structure generated by CS2 / triethanolamine cross-linking. -1 (COC stretching β-1,4 glycosidic bond), 900-880cm -1 (β-glucose ring vibration) The characteristic peaks in the cellulose fingerprint region are clear, indicating that the skeleton is not damaged, and the peak shape is sharper after compounding.

[0058] Example 2

[0059] This embodiment provides a bullfrog-derived small molecule collagen peptide repair material, which comprises the following components in parts by weight: 10 parts of bullfrog-derived collagen peptide, 16 parts of SATA, 150 parts of modified cellulose, 30 parts of chondroitin sulfate, 20 parts of geniposide, and 20 parts of pinoresinol diglucoside;

[0060] The method for extracting bullfrog-derived collagen peptides specifically comprises the following steps:

[0061] ① Take fresh frozen bullfrog skin and bullfrog cartilage, wash the bullfrog skin and cartilage thoroughly with deionized water to remove surface impurities and blood stains, and obtain pre-treated extraction raw materials;

[0062] ② Place the pretreated raw material in a 1.0% NaCl aqueous solution for 20 minutes, thoroughly wash with clean water to remove residual salt and impurities, soak the raw material in anhydrous ethanol solution for 30 minutes, and wash with clean water to remove excess ethanol to obtain a defatted extraction raw material;

[0063] ③ Soak the defatted extraction raw material in 0.15 mol / L hydrochloric acid solution for 8 minutes, rinse thoroughly with clean water to ensure that the pH value is close to neutral, and obtain the pre-enzymatic extraction raw material;

[0064] ④ Place the pre-enzymatically extracted raw materials into the enzymatic hydrolysis solution. The enzymatic hydrolysis solution composition is: alkaline protease: 0.06g / mL, trypsin: 0.09g / mL, neutral protease: 0.15g / mL. Maintain the reaction temperature at 50°C, stir the reaction for 6 hours, centrifuge (8000 rpm, 15 minutes), collect the supernatant and filter to obtain the crude collagen extract;

[0065] ⑤ Separate and purify the crude collagen extract prepared in step ④ using a 3 kDa ultrafiltration membrane to remove macromolecular impurities, concentrate the filtrate by rotary evaporation to remove part of the solvent, and freeze-dry the concentrate (-50°C, 20 Pa) to obtain bullfrog-derived collagen peptides;

[0066] This embodiment provides a method for preparing a bullfrog-derived small molecule collagen peptide repair material, which specifically includes the following steps:

[0067] S1. Accurately weigh 10 mg of bullfrog collagen peptide III and dissolve it in 10 mL of PBS buffer solution, maintaining the pH of the reaction system at 7.4. Dissolve 16.2 mg of SATA in 1 mL of DMF solvent to obtain a SATA solution. Add the SATA solution to the reaction system at a rate of 50 μL / min. After the addition is complete, stir the reaction at 200 rpm for 60 minutes. After the reaction is complete, add 5 mL of Tris hydrochloride buffer solution (concentration of 50 mM). After standing at room temperature for 5 minutes, transfer the reaction system to a 3 kDa ultrafiltration tube, centrifuge at 4000 g and 4°C for 10 minutes, collect the product, add it to 2 mL of PBS buffer (pH 7.4), mix well, and then add 1 mL of 100 mM hydroxylamine hydrochloride / PBS buffer solution to the reaction system. 50 mM EDTA / PBS buffer solution was added to the reaction system, and the reaction was stirred at 50 rpm for 120 min. After the reaction was completed, the mixture was transferred to a 3 kDa ultrafiltration tube and centrifuged at 4000 g and 4°C for 10 min. The product was collected and freeze-dried to obtain thiol-modified collagen peptide.

[0068] S2. Accurately weigh 2 g of cellulose and place it in a flask. Add 15 mL of DMF to fully dissolve the cellulose. Then add 3.5 g of p-toluenesulfonyl chloride and mix well. Add 4 mL of pyridine and raise the reaction temperature to 50°C. After reacting for 7 h, cool the reaction system to room temperature and remove excess solvent by distillation under reduced pressure. Repeatedly wash with deionized water until neutral, and freeze-dry to obtain activated cellulose.

[0069] S3. 1 g of the activated cellulose prepared in step S2 was placed in a flask, 40 mL of DMSO solvent was added, and the mixture was heated until the activated cellulose was completely dissolved. Then, 5.0 g of chloroacetyl chloride was added, and the reaction temperature was maintained at 60° C. The reaction was continued for 20 h. Deionized water was added for sedimentation, and the precipitate was collected by suction filtration, washed repeatedly with deionized water, and dried under vacuum at 60° C. for 12 h to obtain modified cellulose.

[0070] S4. Accurately weigh 150 mg of the modified cellulose prepared in step S3 and place it in a flask. Add 10 mL of DMSO solvent and raise the temperature until the modified cellulose is completely dissolved. Then, transfer 20 mg of the thiol-modified collagen peptide prepared in step S1 to the reaction system and mix well. Add 0.5 mL of triethanolamine and stir at room temperature for 20 min. Then, add 0.45 mL of CS2 dropwise and continue mixing for 20 min. Raise the reaction temperature to 30°C and react for 15 h. After the reaction is cooled to room temperature, add deionized water for precipitation. Filter and collect the precipitate. Wash the precipitate with deionized water and dry it in a vacuum at 40°C for 12 h to obtain collagen-complexed cellulose.

[0071] S5. Take 200 mg of the collagen composite cellulose prepared in step S4 and place it in a centrifuge tube. Add 15 mL of hexafluoroisopropanol and stir at 200 rpm at 37 ° C for 2 hours to form a uniform transparent solution. Add 30 mg of chondroitin sulfate, 20 mg of genipoic acid glycoside and 20 mg of pinoresinol diglucoside. After ultrasonic treatment at 500 W for 4 hours, degassing treatment under vacuum conditions for 30 minutes, adjust the electrospinning parameters, adjust the flow rate to 0.5 mL / h, adjust the voltage to 15 kV, adjust the collection distance to 15 cm, adjust the ambient temperature to 25 ° C, adjust the relative humidity to 40%, and use aluminum foil to cover the rotating drum to collect the cellulose membrane. After spinning for 4 hours, gently peel off the composite fiber membrane accumulated on the rotating drum, place it in a room temperature drying oven and let it stand for 12 hours, wash to remove the residual solvent, and place it in a vacuum drying oven at 40 ° C for 12 hours to ensure that the moisture and residual solvent are completely removed to obtain the collagen peptide repair material.

[0072] Example 3

[0073] This embodiment provides a bullfrog-derived small molecule collagen peptide repair material, which comprises the following components in parts by weight: 10 parts of bullfrog-derived collagen peptide, 22 parts of SATA, 200 parts of modified cellulose, 25 parts of chondroitin sulfate, 15 parts of geniposide, and 15 parts of pinoresinol diglucoside;

[0074] The method for extracting bullfrog-derived collagen peptides specifically comprises the following steps:

[0075] ① Take fresh frozen bullfrog skin and bullfrog cartilage, wash the bullfrog skin and cartilage thoroughly with deionized water to remove surface impurities and blood stains, and obtain pre-treated extraction raw materials;

[0076] ② Place the pretreated raw material in a 1.2% NaCl aqueous solution for 10 minutes, wash thoroughly with clean water to remove residual salt and impurities, soak the raw material in an anhydrous ethanol solution for 20 minutes, and wash with clean water to remove excess ethanol to obtain a defatted extraction raw material;

[0077] ③ Soak the defatted extraction raw material in 0.18 mol / L hydrochloric acid solution for 6 minutes, rinse thoroughly with clean water to ensure that the pH value is close to neutral, and obtain the pre-enzymatic extraction raw material;

[0078] ④ Place the pre-enzymatically extracted raw materials into the enzymatic hydrolysis solution. The enzymatic hydrolysis solution composition is: alkaline protease: 0.07g / mL, trypsin: 0.1g / mL, neutral protease: 0.18g / mL. Maintain the reaction temperature at 60°C, stir the reaction for 4 hours, centrifuge (10,000 r / min, 15 minutes), collect the supernatant and filter to obtain the crude collagen extract;

[0079] ⑤ Separate and purify the crude collagen extract prepared in step ④ using a 3 kDa ultrafiltration membrane to remove macromolecular impurities, concentrate the filtrate by rotary evaporation to remove part of the solvent, and freeze-dry the concentrate (-50°C, 20 Pa) to obtain bullfrog-derived collagen peptides;

[0080] This embodiment provides a method for preparing a bullfrog-derived small molecule collagen peptide repair material, which specifically includes the following steps:

[0081] S1. Accurately weigh 10 mg of bullfrog collagen peptide III and dissolve it in 7.5 mL of PBS buffer solution, maintaining the pH of the reaction system at 7.4. Dissolve 21.6 mg of SATA in 1 mL of DMF solvent to obtain a SATA solution. Add the SATA solution to the reaction system at a rate of 50 μL / min. After the addition is complete, stir the reaction at 200 rpm for 60 minutes. After the reaction is complete, add 5 mL of Tris hydrochloride buffer solution (concentration of 50 mM). After standing at room temperature for 5 minutes, transfer the reaction system to a 3 kDa ultrafiltration tube, centrifuge at 4000 g and 4°C for 10 minutes, collect the product, add it to 2 mL of PBS buffer (pH 7.4), mix well, and then add 1 mL of 100 mM hydroxylamine hydrochloride / PBS buffer solution to the reaction system. 50 mM EDTA / PBS buffer solution was added to the reaction system, and the reaction was stirred at 50 rpm for 120 min. After the reaction was completed, the mixture was transferred to a 3 kDa ultrafiltration tube and centrifuged at 4000 g and 4°C for 10 min. The product was collected and freeze-dried to obtain thiol-modified collagen peptide.

[0082] S2. Accurately weigh 2 g of cellulose and place it in a flask. Add 10 mL of DMF to fully dissolve the cellulose. Then add 5.0 g of p-toluenesulfonyl chloride and mix well. Add 6 mL of pyridine and raise the reaction temperature to 60°C. After reacting for 6 h, cool the reaction system to room temperature and remove excess solvent by distillation under reduced pressure. Wash the mixture repeatedly with deionized water until neutral. After freeze-drying, obtain activated cellulose.

[0083] S3. 1 g of the activated cellulose prepared in step S2 was placed in a flask, 25 mL of DMSO solvent was added, and the mixture was heated until the activated cellulose was completely dissolved. 1.7 g of chloroacetyl chloride was added, and the reaction temperature was maintained at 70° C. The reaction was continued for 30 h. Deionized water was added for sedimentation, and the precipitate was collected by suction filtration, and the precipitate was repeatedly washed with deionized water, and then vacuum-dried at 60° C. for 12 h to obtain modified cellulose.

[0084] S4. Accurately weigh 200 mg of the modified cellulose prepared in step S3 and place it in a flask. Add 10 mL of DMSO solvent and raise the temperature until the modified cellulose is completely dissolved. Then, transfer 20 mg of the thiol-modified collagen peptide prepared in step S1 to the reaction system and mix well. Add 0.5 mL of triethanolamine and stir at room temperature for 20 min. Then, add 0.30 mL of CS2 dropwise and continue mixing for 20 min. Raise the reaction temperature to 50°C and react for 9 h. After the reaction is cooled to room temperature, add deionized water for precipitation. Filter and collect the precipitate. Wash the precipitate with deionized water and dry it in a vacuum at 40°C for 12 h to obtain collagen-complexed cellulose.

[0085] S5. Take 200 mg of the collagen composite cellulose prepared in step S4 and place it in a centrifuge tube. Add 20 mL of hexafluoroisopropanol and stir at 200 rpm at 37 ° C for 2 hours to form a uniform transparent solution. Add 25 mg of chondroitin sulfate, 15 mg of genipoic acid glycoside and 15 mg of pinoresinol diglucoside. After ultrasonic treatment at 500 W for 4 hours, degassing treatment under vacuum conditions for 30 minutes, adjust the electrospinning parameters, adjust the flow rate to 0.5 mL / h, adjust the voltage to 15 kV, adjust the collection distance to 15 cm, adjust the ambient temperature to 25 ° C, adjust the relative humidity to 40%, and use aluminum foil to cover the rotating drum to collect the cellulose membrane. After spinning for 4 hours, gently peel off the composite fiber membrane accumulated on the rotating drum, place it in a room temperature drying oven and let it stand for 12 hours, wash to remove the residual solvent, and place it in a vacuum drying oven at 40 ° C for 12 hours to ensure that the moisture and residual solvent are completely removed to obtain a collagen peptide repair material.

[0086] Comparative Example 1

[0087] This comparative example provides a collagen peptide repair material and a preparation method thereof, which differs from Example 1 only in that SATA is not included in all components and the modified cellulose is replaced by cellulose in equal parts by weight, and the remaining components and component contents are the same as those in Example 1.

[0088] Comparative Example 2

[0089] This comparative example provides a collagen peptide repair material and a preparation method thereof, which differs from Example 1 only in that the modified cellulose is replaced by the same weight portion of cellulose in all components, and the remaining components and component contents are the same as those in Example 1.

[0090] Comparative Example 3

[0091] This comparative example provides a collagen peptide repair material and a preparation method thereof, which differs from Example 1 only in that modified cellulose is not included in all components, and the remaining components and component contents are the same as those in Example 1.

[0092] Experimental Example 1

[0093] This experimental example tests the cell compatibility and retrograde properties of the collagen peptide repair materials prepared in Examples 1-3 and Comparative Examples 1-3. The electrospun collagen peptide repair materials were cut into discs with a diameter of 10 mm and a thickness of about 1 mm. The discs were placed in a 24-well cell culture plate, with one disc placed in each well. The 24-well plate containing the material discs was placed under a UV biosafety cabinet and irradiated with UV254 nm for 30 min on both the front and back sides. After rinsing twice with sterile PBS, 500 μL of α-MEM medium containing 10% fetal bovine serum (FBS) was added and pre-incubated at 37°C and 5% CO2 for 1 h. 3-5 bone marrow mesenchymal stem cells (mBMSCs) were taken, routinely revived and passaged to the logarithmic growth phase, digested with 0.25% trypsin, and the final concentration was adjusted to 1×10 3 cells / mL, add 200 μL of cell suspension (i.e. 2×10 4 cells / well), and placed in a 37°C, 5% CO2 incubator for 30 min to allow the cells to initially attach. 800 μL of fresh complete medium was gently added, and the culture was continued. Samples were taken at the sampling point, the supernatant was removed to 100 μL per well, 10 μL of CCK-8 reagent was added, and the cells were incubated at 37°C, 5% CO2 for 2 h. The absorbance (OD) was measured at 450 nm using a microplate reader. 450 ), blank control wells (no cells, only culture medium + CCK-8) were used to subtract background.

[0094] Figure 3 This is a diagram showing the effect of the collagen peptide repair material prepared in Example 1 on promoting the growth of hBMSCs. As shown in the figure, the collagen peptide repair materials prepared in Examples 1-3 have a significant promoting effect on the differentiation of bone marrow cells. The present invention promotes the repair and regeneration of cartilage damage by regulating the proliferation, differentiation, migration and matrix synthesis of chondrocytes.

[0095] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

[0096] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A bullfrog-derived small molecule collagen peptide repair material, characterized by: The collagen peptide repair material includes the following components in parts by weight: 10 parts of bullfrog-derived collagen peptide, 10-22 parts of N-succinimidyl-S-acetylthioacetate, 100-200 parts of modified cellulose, 20-30 parts of chondroitin sulfate, 10-20 parts of genipoic acid glycoside, and 10-20 parts of pinoresinol diglucoside.

2. The bullfrog-derived small molecule collagen peptide repair material according to claim 1, characterized in that: The method for extracting bullfrog-derived collagen peptides specifically comprises the following steps: ① Take fresh frozen bullfrog skin and bullfrog cartilage as raw materials, wash the impurities on the surface of the bullfrog skin and bullfrog cartilage with deionized water to obtain pre-treated extraction raw materials; ② The pretreated extraction raw material prepared in step ① is placed in a NaCl aqueous solution, soaked for 10-20 minutes, washed with clean water to remove residual salt and impurities, and then soaked in an anhydrous ethanol solution for 20-30 minutes, washed with clean water to remove excess ethanol, to obtain a defatted extraction raw material; ③ Place the fat-free extraction raw material prepared in step ② in a hydrochloric acid solution, soak for 5-10 minutes, and then wash with clean water until neutral to obtain a pre-enzymatic extraction raw material; ④ Place the pre-enzymatic extraction raw material prepared in step ③ into the enzymatic solution, maintain the reaction temperature at 40-60°C, stir and react for 4-6 hours, centrifuge, collect the supernatant, and filter to obtain a crude collagen extract; ⑤ Separate and purify the crude collagen extract prepared in step ④, filter it using an ultrafiltration membrane, rotary evaporate the filtrate, and freeze-dry it to obtain bullfrog-derived collagen peptides.

3. The bullfrog-derived small molecule collagen peptide repair material according to claim 2, characterized in that: In step ②, the mass concentration of the NaCl aqueous solution is 0.5-1.5%.

4. The bullfrog-derived small molecule collagen peptide repair material according to claim 3, characterized in that: In step ③, the concentration of the hydrochloric acid solution is 0.1-0.2 mol / L.

5. The bullfrog-derived small molecule collagen peptide repair material according to claim 4, characterized in that: In step ④, the enzymatic hydrolysis solution includes the following components in mass concentrations: alkaline protease 0.05-0.07 g / mL, trypsin 0.08-0.1 g / mL, and neutral protease 0.1-0.2 g / mL.

6. The method for preparing a bullfrog-derived small molecule collagen peptide repair material according to any one of claims 1 to 5, characterized in that: The specific steps include: S1. Dissolve bullfrog-derived collagen peptide in PBS buffer solution, slowly add N-succinimidyl-S-acetylthioacetate solution, and stir to react under neutral conditions. After the reaction is completed, add Tris hydrochloride buffer solution, let it stand, and centrifuge through an ultrafiltration tube to collect the product. Add hydroxylamine hydrochloride and EDTA, mix well, and stir to react to obtain thiol-modified collagen peptide; S2. Dissolve cellulose in DMF, add p-toluenesulfonyl chloride and pyridine, raise the reaction temperature to 40-60°C, react for 6-8 hours, remove excess solvent, wash with deionized water, and freeze-dry to obtain activated cellulose; S3, dissolving the activated fiber prepared in step S2 in 1-butyl-3-methylimidazolium chloride ionic liquid, heating until the activated cellulose is completely dissolved, adding chloroacetyl chloride, maintaining the reaction temperature at 50-70° C., reacting for 20-30 hours, adding deionized water for precipitation, filtering, collecting the precipitate, washing, and vacuum drying to obtain modified cellulose; S4. Dissolve the modified cellulose prepared in step S3 in DMSO, heat and stir until the modified cellulose is completely dissolved, dissolve the thiol-modified collagen peptide prepared in step S1 in DMSO and transfer it to the reaction system, add a catalyst, mix well, slowly add CS2, maintain the reaction temperature at 30-50°C, continue the reaction for 9-15 hours, add deionized water to wash the precipitate, filter, repeatedly wash with deionized water, and vacuum dry to obtain collagen-complexed cellulose; S5. Dissolve the collagen composite cellulose prepared in step S4 in hexafluoroisopropanol, stir to form a uniform transparent solution, add chondroitin sulfate, genipoic acid glycoside and pinoresinol diglucoside, ultrasonically treat, vacuum degas, adjust the electrospinning parameters for electrospinning, use aluminum foil to cover the rotating drum to collect the cellulose membrane, gently peel off the composite fiber membrane accumulated on the rotating drum, place it in a drying oven at room temperature and let it stand for 9-12 hours, wash to remove the residual solvent, and vacuum dry to obtain the collagen peptide repair material.

7. The method for preparing a bullfrog-derived small molecule collagen peptide repair material according to claim 6, characterized in that: In step S1, the mass concentration of the bullfrog-derived collagen peptide in the PBS buffer solution is 5-10 mg / mL; the mass ratio between the bullfrog-derived collagen peptide and SATA is 1:1-2.

2.

8. The method for preparing a bullfrog-derived small molecule collagen peptide repair material according to claim 7, characterized in that: In step S2, the mass concentration of the cellulose in DMF is 0.1-0.2 g / mL; the mass ratio of the cellulose to p-toluenesulfonyl chloride is 1:1.5-2; and the mass-to-volume ratio of the cellulose to pyridine is 0.3-0.5 g / mL.

9. The method for preparing a bullfrog-derived small molecule collagen peptide repair material according to claim 8, characterized in that: In step S3, the mass concentration of the activated cellulose in the 1-butyl-3-methylimidazolium chloride ionic liquid is 20-40 mg / mL; and the mass ratio of the activated cellulose to chloroacetyl chloride is 1:10-20.

10. The method for preparing a bullfrog-derived small molecule collagen peptide repair material according to claim 8, characterized in that: In step S4, the mass concentration of the modified cellulose in DMSO is 10-20 mg / mL; in step S5, the mass concentration of the collagen-complexed cellulose in hexafluoroisopropanol is 10-20 mg / mL.

Images