Composition for promoting chondrocyte proliferation and cartilage repair as well as preparation method and application of composition

Abstract

The invention discloses a composition for promoting cartilage cell proliferation and cartilage repair. The composition comprises a combination of a small molecule cartilage extract and collagen peptide, the average molecular weight of the small molecular cartilage extract is less than or equal to 5000 Da, and the average molecular weight of the collagen peptide is 2000 Da to 4000 Da; the composition is prepared by the following method: (1) cleaning and crushing animal cartilage, performing primary depolymerization by using protease, performing microwave-assisted protease secondary depolymerization, and performing membrane filtration to obtain a small molecular cartilage extract of which the average molecular weight is less than or equal to 5000 Da; and (2) mixing the small molecular cartilage extract obtained in the step (1) with collagen peptide with the average molecular weight of 2000 Da to 4000 Da to obtain the composition. Under the same mass concentration, compared with single-component collagen peptide, cartilage extract or UC-II, the collagen peptide-cartilage extract combination has more outstanding inflammation inhibition effect and cartilage repair effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition for promoting cartilage repair, a preparation method and an application thereof. Background Art

[0002] Cartilage extract is a bioactive substance extracted from animal cartilage. Its main components include chondroitin sulfate, hyaluronic acid, collagen, and various growth factors. It is an important natural remedy for treating bone and joint diseases and is also widely used in food, health supplements, and cosmetics. Cartilage extract lubricates joints, enhances cartilage elasticity, and promotes cartilage repair. It also serves as an antioxidant and nutritional supplement, providing support for patients with diabetes and arthritis. Studies have shown that cartilage extract and other test substances increase the expression of type II collagen and promote cartilage repair by activating downstream signaling pathways including nuclear factor kappa-B (NF-κB), p38, and c-Jun N-terminal kinase (JNK). Due to its large molecular weight, cartilage extract has relatively low absorption efficiency in the human body and generally requires further degradation before it can be utilized. Due to its complex composition, it primarily works through the synergistic action of multiple components to provide the substances necessary for cartilage repair, while also maintaining and repairing cartilage health by protecting existing cartilage and improving the joint environment. However, the effect of cartilage extract is often closely related to its dosage. Too low a dosage cannot achieve the expected effect, while too high a dosage will increase the risk of side effects.

[0003] Cartilage extracts have complex extraction processes, unstable product quality, and potentially fluctuating active ingredient content. They lack comprehensive repair capabilities for the extracellular matrix (ECM) of cartilage cells, and their promotion of chondrocyte proliferation and differentiation is insufficient. Collagen peptides are small molecule peptides obtained by hydrolysis of collagen. They are rich in amino acids required by the human body, such as glycine, proline, and hydroxyproline, and provide essential nutrients for chondrocyte proliferation and differentiation. When used alone, they lack cartilage-specific repair effects and lack effective inhibition of inflammatory factors during the cartilage repair process.

[0004] Therefore, it is difficult for a single component of cartilage extract or collagen peptide to simultaneously achieve multiple effects such as protecting chondrocytes, promoting cartilage matrix synthesis, and inhibiting inflammatory responses.

[0005] Therefore, it is necessary to develop a new combination of substances to improve the protection of chondrocytes, promote cartilage matrix synthesis, and inhibit inflammatory responses, etc., to overcome the above-mentioned defects in the existing technology, which is a technical problem that needs to be solved urgently by technicians in this field. Summary of the Invention

[0006] The purpose of the present invention is to provide a composition that promotes chondrocyte proliferation and cartilage repair, so as to solve the defects in the prior art that low-dose cartilage extract cannot achieve the expected effect, high-dose cartilage extract increases the risk of side effects, and single components of cartilage extract or collagen peptide are difficult to simultaneously achieve multiple effects such as protecting chondrocytes, promoting cartilage matrix synthesis and inhibiting inflammatory response, and collagen peptide alone has little direct effect on inflammatory response.

[0007] The first object of the present invention is to provide a composition for promoting chondrocyte proliferation and cartilage repair, wherein the composition comprises a combination of a small molecule cartilage extract and a collagen peptide; the average molecular weight of the small molecule cartilage extract is ≤5000 Da, and the average molecular weight of the collagen peptide is 2000 Da~4000 Da.

[0008] The “average molecular weight” described in the present invention is obtained by liquid phase analysis, and therefore means “number average molecular weight”.

[0009] As one embodiment of the present invention, the mass ratio of collagen peptides to small molecule cartilage extract is 50:1 to 1:50. Low doses of cartilage extract (mass ratio of collagen peptides to small molecule cartilage extract > 50:1) fail to achieve the desired effect, while high doses (mass ratio of collagen peptides to small molecule cartilage extract < 1:50) increase the risk of side effects such as gastrointestinal discomfort, allergic reactions, blood sugar fluctuations, and liver and kidney burden.

[0010] As a specific embodiment of the present invention, the mass ratio of collagen peptide to small molecule cartilage extract is 20:1~1:5, and the mass ratio of collagen peptide to small molecule cartilage extract can be further optionally 10:1~1:5, and more preferably the mass ratio of collagen peptide to small molecule cartilage extract is 1:1~1:5.

[0011] The second object of the present invention is to provide a method for preparing a composition for promoting chondrocyte proliferation and cartilage repair, comprising the following steps: (1) After washing and crushing the animal cartilage, it is first depolymerized using protease, then depolymerized using microwave-assisted protease for a second time, and then filtered through a membrane to obtain a small molecule cartilage extract with an average molecular weight of ≤5000 Da; (2) The small molecule cartilage extract obtained in step (1) is mixed with collagen peptides having an average molecular weight of 2000 Da to 4000 Da to obtain a composition.

[0012] As an embodiment of the present invention, the protease used in the primary depolymerization is any one or a combination of papain, bromelain, flavor protease, and keratinase.

[0013] As an embodiment of the present invention, the amount of protease added for the primary depolymerization is 1-5 g / L, the pH is 6.5-8.5, the reaction temperature is 25-60° C., and the enzymatic hydrolysis time is 4-24 h.

[0014] In one embodiment of the present invention, the protease used in the microwave-assisted protease secondary depolymerization is an acidic protease, the protease addition amount is 1-3 g / L, the microwave frequency is 400-700 W, the pH of the microwave-assisted depolymerization is 2-4, the temperature of the microwave-assisted depolymerization is 40-60°C, and the microwave-assisted depolymerization time is 1-3 hours. Glycosidic bonds in cartilage extract are more easily broken under acidic conditions and the action of microwaves.

[0015] As an embodiment of the present invention, the mass ratio of the collagen peptide and the small molecule cartilage extract is 50:1~1:50, and the optional mass ratio is 20:1~1:5.

[0016] The third object of the present invention is to provide the use of the composition in preparing a product for promoting chondrocyte proliferation and / or promoting cartilage repair.

[0017] The composition of the present invention is used in preparing a product for promoting cartilage repair by promoting the expression of type II collagen Col II, the expression of proteoglycan Aggrecan, and / or inhibiting the expression of matrix metalloproteinase MMP-1.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Using membrane filtration technology, the molecular weight of small molecule cartilage extract products can be precisely controlled to ≤5000 Da, which is simple to operate and has low industrial cost.

[0019] (2) The production cost of small molecule cartilage extract is higher than that of collagen peptide. When used in combination with collagen peptide, the cost of use can be greatly reduced.

[0020] (3) After the combination of small molecule cartilage extract and collagen peptide, it supports cartilage modification from different angles, provides structural support, supplements necessary nutrients, and has better efficacy in reducing inflammatory response. The direct anti-inflammatory mechanism of small molecule cartilage extract and the indirect anti-inflammatory mechanism of collagen peptide complement each other; and the preparation of small molecule cartilage extract and collagen peptide in different ratios can reduce the risk of low-dose cartilage extract not achieving the expected effect, while high-dose use increases the risk of side effects, and meet the needs of different customers for small molecule cartilage extract and collagen peptide combination products. Compared with large molecule cartilage extract and collagen, small molecule cartilage extract and collagen peptide are more easily absorbed by cells in the intestine and other parts after decomposition or processing, and have higher bioavailability. The idea of ​​compounding cartilage extract and collagen peptide is based on the structural characteristics of cartilage tissue to provide precursors of key components; at the same time, the relationship between molecular size and bioavailability is used to optimize the absorption effect. The multi-target regulation theory is adopted to achieve comprehensive cartilage repair, exert synergistic effects, and achieve the effect of "1+1>2" on the basis of reducing the cost per kilogram.

[0021] (4) Through a series of efficacy experiments, it was proved that the combination of collagen peptides and cartilage extracts in different proportions (a) significantly promoted the synthesis of type II collagen (Collagen II) and aggrecan, and improved the cartilage repair effect; (b) significantly inhibited the overexpression of matrix metalloproteinase 1 (MMP-1), and alleviated the inflammatory response; and as the proportion of cartilage extract in the composite sample increased, the repair effect gradually increased. At the same mass concentration, compared with single-component collagen peptides, large-molecule cartilage extracts, small-molecule cartilage extracts or UC-II, the combination of collagen peptides and cartilage extracts had more prominent anti-inflammatory and cartilage repair effects. The combination of collagen peptides and cartilage extracts had a significant synergistic effect in inhibiting inflammatory response, promoting cartilage matrix synthesis, and repairing cartilage damage, far exceeding the repair effect of a single component. This discovery provides new ideas and treatment strategies for the treatment of cartilage damage, and has important clinical application prospects and innovations. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This figure shows the effect of cartilage extracts with different molecular weights on the expression of type II collagen (Collagen II) content.

[0023] Figure 2 This figure shows the effect of cartilage extracts with different molecular weights on the expression of aggrecan content.

[0024] Figure 3 This figure shows the effect of cartilage extracts with different molecular weights on the expression of matrix metalloproteinase (MMP-1).

[0025] Figure 4 This is a graph showing cell viability of human chondrocytes after treatment with collagen peptide-cartilage extract (20:1).

[0026] Figure 5 This is a graph showing cell viability of mouse chondrocytes after treatment with collagen peptide-cartilage extract (20:1).

[0027] Figure 6 This is a diagram showing the effects of different test substances on the expression of type II collagen (Collagen II) content.

[0028] Figure 7 This figure shows the effects of different test substances on the expression of aggrecan content.

[0029] Figure 8 This figure shows the effects of different test substances on the expression of matrix metalloproteinase (MMP-1). DETAILED DESCRIPTION

[0030] The following examples are included to illustrate preferred embodiments of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. Based on this disclosure, those skilled in the art will recognize that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, that is, any equivalent results or equivalent process changes made using the contents of the present specification, or directly or indirectly applied in other related technical fields, should be included within the scope of protection of this patent.

[0031] The experimental data obtained in the present invention were analyzed according to the corresponding statistical methods. Unless otherwise specified, P < 0.05 was considered to be statistically significant. In the present invention, the statistical differences between the control group and the model group, and between the test substance group and the model group were mainly analyzed. It is specified that "#" represents the model group vs. the control group, "#" represents P < 0.05, "##" represents P < 0.01, "###" represents P < 0.001, and "####" represents P < 0.0001; it is specified that "*" represents each test substance group vs. the model group, "*" represents P < 0.05, "**" represents P < 0.01, "***" represents P < 0.001, and "****" represents P < 0.0001.

[0032] Example 1 After cleaning and crushing the animal cartilage, it was first depolymerized using bromelain at a concentration of 1 g / L, a pH of 7.0, a reaction temperature of 40°C, and a duration of 10 hours. A second microwave-assisted depolymerization with acidic protease was then performed. The acidic protease concentration was 3 g / L, the microwave frequency was 700 W, the pH of the microwave-assisted depolymerization was 2, the temperature was 50°C, and the duration was 1 hour. After the reaction, the pH was adjusted to 7.0, and the filtrate was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The filtrate was then desalted and concentrated using a 100 Da nanofiltration membrane. The extract was sterilized by 0.22 μm filtration and spray-dried to obtain a low-molecule cartilage extract with an average molecular weight of approximately 2000 Da.

[0033] Example 2 After cleaning and crushing the animal cartilage, it was first depolymerized using bromelain at a concentration of 3 g / L, a pH of 6.5, a reaction temperature of 60°C, and an enzymatic hydrolysis time of 8 hours. A second microwave-assisted depolymerization with acidic protease was then performed. The acidic protease was added at a concentration of 2 g / L, the microwave frequency was 700 W, the pH of the microwave-assisted depolymerization was 2, the temperature was 60°C, and the depolymerization time was 3 hours. After the reaction, the pH was adjusted to 7.0, and the filtrate was collected by ultrafiltration using a 2000 Da ultrafiltration membrane. The filtrate was then desalted and concentrated using a 100 Da nanofiltration membrane. The extract was sterilized by 0.22 μm filtration and spray-dried to obtain a low-molecule cartilage extract with an average molecular weight of approximately 1000 Da.

[0034] Example 3 After cleaning and crushing the animal cartilage, it was first depolymerized using bromelain at a concentration of 2 g / L, a pH of 7.5, a reaction temperature of 50°C, and a decomposition time of 6 hours. A second depolymerization step was then performed using microwave-assisted acidic protease at a concentration of 2 g / L, a microwave frequency of 500 W, a pH of 2, a temperature of 40°C, and a decomposition time of 2 hours. After the reaction, the pH was adjusted to 7.0, and the filtrate was collected by ultrafiltration using a 4000 Da ultrafiltration membrane. The filtrate was then desalted and concentrated using a 100 Da nanofiltration membrane. The extract was sterilized by 0.22 μm filtration and spray-dried to obtain a low-molecule cartilage extract with an average molecular weight of approximately 3000 Da.

[0035] Example 4 After cleaning and crushing the animal cartilage, it was first depolymerized using bromelain at a concentration of 4 g / L, a pH of 8.0, a reaction temperature of 55°C, and a decomposition time of 4 hours. A second depolymerization step was then performed using microwave-assisted acidic protease at a concentration of 1 g / L, a microwave frequency of 600 W, a pH of 4, a temperature of 60°C, and a decomposition time of 2 hours. After the reaction, the pH was adjusted to 7.0, and the filtrate was collected by ultrafiltration using a 5000 Da ultrafiltration membrane. The filtrate was then desalted and concentrated using a 100 Da nanofiltration membrane. The extract was sterilized by 0.22 μm filtration and spray-dried to obtain a low-molecule cartilage extract with an average molecular weight of approximately 4000 Da.

[0036] Example 5 After cleaning and crushing the animal cartilage, it was first depolymerized using bromelain at a concentration of 5 g / L, a pH of 8.5, a reaction temperature of 25°C, and a duration of 24 hours. A second depolymerization step was then performed using microwave-assisted acidic protease at a concentration of 2 g / L, a microwave frequency of 400 W, a pH of 3, a temperature of 55°C, and a duration of 3 hours. After the reaction, the pH was adjusted to 7.0, and the filtrate was collected by ultrafiltration using a 6000 Da ultrafiltration membrane. The filtrate was then desalted and concentrated using a 100 Da nanofiltration membrane. The extract was sterilized by 0.22 μm filtration and spray-dried to obtain a low-molecule cartilage extract with an average molecular weight of approximately 5000 Da.

[0037] Example 6 The smaller the molecular weight of the small molecule cartilage extract, the more difficult it is to prepare and the lower the yield. Taking into account the sample yield and product cost, a collagen peptide with an average molecular weight of 3000 Da was selected and mixed with the small molecule cartilage extract with an average molecular weight of about 3000 Da prepared in Example 1 in mass ratios of 20:1, 1:1 and 1:5, respectively, to obtain collagen peptide-cartilage extract compositions with mass ratios of 20:1, 1:1 and 1:5.

[0038] Example 7. Cell experiment preparation A cellular inflammation model was established by stimulating C28 / I2 human chondroblasts with lipopolysaccharide (LPS).

[0039] A cellular inflammation model was established by stimulating ATDC5 mouse chondroblasts with interleukin-1β (IL-1β).

[0040] The efficacy of each test substance in cartilage repair was investigated under this cellular inflammatory model, simulating the mechanisms of inflammation at the cellular level. Cell viability was used to assess the effects of the test substances on cell proliferation and cytotoxicity. Enzyme-linked immunosorbent assays were also used to investigate the effects of each test substance on the expression of type II collagen (Collagen II), aggrecan, and matrix metalloproteinase-1 (MMP-1) in C28 / I2 cells.

[0041] (1) Cell culture: (1.1) C28 / I2 human chondrocyte culture Take well-growing C28 / I2 cells and sample them at a rate of 1×10 4 Cells were seeded into 96-well plates at a density of 1 × 10 cells / mL. 5 Cells were seeded into 6-well plates at a density of 100 cells / mL, and an appropriate amount of cell suspension was added to each well. The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24 h and then taken out.

[0042] The control group was added with complete cell culture medium and cultured in a cell culture incubator at 37°C and 5% CO2. After 24 h, the cells were removed and replaced with serum-free culture medium. After starvation for 17 h, the cells were removed again and added with complete cell culture medium to the control group. The other groups were added with 5 μg / mL LPS complete cell culture medium containing or not containing the test substances (test substances: collagen peptide, commercially available macromolecular cartilage extract, 3000 Da small molecule cartilage extract prepared in Example 3, collagen peptide-cartilage extract prepared in Example 6 (mass ratio 20:1), collagen peptide-cartilage extract prepared in Example 6 (mass ratio 1:1), collagen peptide-cartilage extract prepared in Example 6 (mass ratio 1:5), UC-II) and cultured in a cell culture incubator at 37°C and 5% CO2 for another 24 h.

[0043] (1.2) Culture of ATDC5 mouse chondrocytes ATDC5 cells with good growth were taken at a rate of 5×10 3 Cells were seeded into 96-well plates at a density of 2 × 10 5 Cells were seeded into 6-well plates at a density of 100 cells / mL, and an appropriate amount of cell suspension was added to each well. The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24 h and then taken out.

[0044] The control group was added with complete cell culture medium and cultured in a cell culture incubator at 37°C and 5% CO2. After 24 hours, the cells were removed and replaced with serum-free medium. After starvation for 24 hours, the cells were removed again and the control group was added with serum-free medium. The other groups were added with 10 ng / mL IL-1β serum-free medium with or without the test substance (the test substance was collagen peptide-cartilage extract 20:1) and cultured in a cell culture incubator at 37°C and 5% CO2 for another 24 hours.

[0045] (2) Experimental group design: Group 1: Control group (Control); Group 2: Model group (Model); Group 3: collagen peptides, from Gelita, Germany; Group 4: macromolecular cartilage extract, purchased from Hunan Wuxing Biotechnology Co., Ltd.; Group 5: small molecule cartilage extract, derived from the 3000 Da small molecule cartilage extract prepared by the process prepared in Example 3; Group 6: collagen peptide-cartilage extract 20:1, collagen peptide-cartilage extract 1:1, collagen peptide-cartilage extract 1:5, prepared by Example 6.

[0046] Group 7: Non-denatured type II collagen (UC-II), sourced from Lonza (China) Investments Co., Ltd.

[0047] (3) Collection and testing Supernatant collection: After the culture was completed, the six-well plate was removed and the cell supernatant was collected into a 2 mL centrifuge tube. The tube was centrifuged at 2500 rpm and 4°C for 5 min. After centrifugation, the cell supernatant was transferred and stored at -80°C for later use. The supernatant was used for enzyme-linked immunosorbent assay to detect the content of matrix metalloproteinase 1 (MMP-1), aggrecan, and type II collagen (Collagen II) in the cell supernatant.

[0048] Cell collection: After the culture was completed, the six-well plate was removed, the cell supernatant was removed, and 1 mL of PBS was added to each well to wash twice. The supernatant was discarded, and 1 mL of PBS was added to blow the cells to completely detach them. The cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 2500 rpm and 4°C for 5 min. The PBS was discarded, and according to the amount of collected cells, an appropriate amount of protein lysis buffer (1 mL of lysis buffer + 10 μL of phosphatase inhibitor + 1 μL of protease inhibitor + 10 μL of PMSF) was added to fully lyse the cells. The fully lysed cells were centrifuged at 12000 rpm and 4°C for 15 min, the supernatant was collected, and the protein concentration was determined using the BCA method.

[0049] Cell viability assay: CCK8 was equilibrated to room temperature before use; 10 μL of CCK-8 solution was added to each well; the cells were incubated at 37°C, 5% CO2, for 1 hour; and the absorbance was measured at 450 nm using a microplate reader.

[0050] Example 8. Study on the efficacy of small molecule cartilage extract A lipopolysaccharide (LPS)-induced human chondrogenic (C28 / I2) cell inflammation model was constructed to study the effects of small molecule cartilage extracts with different molecular weights (1000-5000 Da) prepared in Examples 1-5 on cartilage repair compared with commercially available large molecule cartilage extracts under inflammatory conditions.

[0051] (1) Effect on the expression of type II collagen (Collagen II) Type II collagen (Collagen II) is the main structural protein of cartilage tissue, which constitutes the elastic scaffold and protective layer of cartilage tissue.

[0052] The results are as follows Figure 1 As shown, the relative content of Collagen II in the model group was significantly lower than that in the control group, with significant differences, indicating that the model was successfully constructed. At the same mass concentration (1 mg / mL, 5 mg / mL, 10 mg / mL), the small molecule cartilage extracts (1000-5000 Da) prepared by the processes of Examples 1-5 showed better cartilage repair effects than the purchased large molecule cartilage extracts and were more conducive to the synthesis of type II collagen (Collagen II), indicating that the small molecule cartilage extracts have better bioavailability and can achieve the effect of increasing the content of Collagen II at lower mass concentrations. In addition, as the average molecular weight of the small molecule cartilage extracts decreases, the corresponding bioavailability also increases.

[0053] (2) Effect on Aggrecan expression Aggrecan is a proteoglycan macromolecule composed of glycosaminoglycan (GAG) and a core protein. Aggrecan non-covalently binds to hyaluronic acid to form a proteoglycan aggregate, which is a major component of the extracellular matrix (ECM) of cartilage.

[0054] The results are as follows Figure 2As shown, the relative content of Aggrecan in the model group was significantly lower than that in the control group, with a significant difference, indicating that the model was successfully constructed. At equivalent mass concentrations (1 mg / mL, 5 mg / mL, and 10 mg / mL), the small molecule cartilage extracts (1000-5000 Da) prepared by the processes of Examples 1-5 demonstrated superior cartilage repair efficacy compared to purchased large molecule cartilage extracts and were more conducive to the synthesis of Aggrecan. This indicates that small molecule cartilage extracts have better bioavailability and can achieve the effect of increasing Aggrecan content at lower mass concentrations. Furthermore, as the average molecular weight of the small molecule cartilage extracts decreases, their bioavailability also increases.

[0055] (3) Effect on the expression of matrix metalloproteinase (MMP-1) Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases that degrade the extracellular matrix. MMP-1 is a key player in cartilage matrix degradation, contributing to inflammation. The more severe the inflammatory response, the higher the MMP-1 level.

[0056] The results are as follows Figure 3 As shown, the relative content of MMP-1 in the model group was significantly increased compared to the control group, with a significant difference, indicating that the model was successfully established. At equivalent mass concentrations (1 mg / mL, 5 mg / mL, and 10 mg / mL), the small-molecule cartilage extracts (1000-5000 Da) prepared using the processes of Examples 1-5 demonstrated superior anti-inflammatory effects compared to purchased large-molecule cartilage extracts, and were more effective in inhibiting the overexpression of matrix metalloproteinase 1 (MMP-1). This indicates that the small-molecule cartilage extracts have better bioavailability and can inhibit MMP-1 overexpression at lower mass concentrations. Furthermore, as the average molecular weight of the small-molecule cartilage extracts decreases, their bioavailability also increases.

[0057] Example 9. Study on the efficacy of collagen peptide-cartilage extract composition (1) Effect on chondrocyte proliferation The collagen peptide-cartilage extract composition prepared in Example 6 (mass ratio of 20:1) was prepared into different mass concentrations and treated with human / mouse chondroblasts. The viability of human / mouse chondroblasts was detected (absorbance was measured at 450 nm using a microplate reader) to evaluate the effect of the collagen peptide-cartilage extract composition on cell proliferation and to preliminarily evaluate the cytotoxicity of the collagen peptide-cartilage extract composition, as shown in Figure 5. Figure 4 (Human chondroblast cell viability chart) and Figure 5 (Image of mouse chondroblast cell viability).

[0058] The results show that if Figure 4 As shown in the figure, after treating human chondrocytes with the collagen peptide-cartilage extract composition at a concentration of 1-10 mg / mL, the relative cell viability increased to varying degrees compared with the control group. Therefore, the collagen peptide-cartilage extract composition has the effect of promoting the proliferation of human chondrocytes within the concentration range of 1-10 mg / mL, and no inhibitory phenomenon occurs, indicating that it has no cytotoxicity. Figure 5 As shown, after treating mouse chondroblasts with the collagen peptide-cartilage extract composition at a concentration of 1-100 mg / mL, the relative cell viability increased to varying degrees compared with the control group. Therefore, the collagen peptide-cartilage extract composition has the effect of promoting the proliferation of mouse chondroblasts within the concentration range of 1-100 mg / mL, and the absence of inhibition indicates that it has no cytotoxicity.

[0059] (2) Impact on inflammatory indicators A lipopolysaccharide (LPS)-induced inflammation model of human chondrogenic (C28 / I2) cells was constructed to investigate the effects of different mass ratios of collagen peptide-cartilage extract on cartilage repair compared with single component collagen peptide, cartilage extract or non-denatured type II collagen (UC-II) under inflammatory conditions.

[0060] (2.1) Effects on the expression of type II collagen Type II collagen (Collagen II) is the main structural protein of cartilage tissue, which constitutes the elastic scaffold and protective layer of cartilage tissue.

[0061] The results are as follows Figure 6As shown, the relative content of Collagen II in the model group was significantly lower than that in the control group, and there was a significant difference, indicating that the model was successfully constructed. At the same mass concentration, the 3000 Da small molecule cartilage extract prepared by the process of Example 3 showed better cartilage repair effect than single-component commercially available large molecule cartilage extract, collagen peptide, or UC-II, and was more conducive to the synthesis of type II collagen (Collagen II). After using collagen peptides to compound with the 3000 Da small molecule cartilage extract prepared in Example 3, at the same mass concentration, the collagen peptide-cartilage extract compositions in different ratios (20:1, 1:1, and 1:5) showed the advantages of the combination, significantly promoting the synthesis of type II collagen. As the proportion of cartilage extract in the composition increased, the relative content of type II collagen also increased, and the cartilage repair effect gradually enhanced. Compared with single-ingredient collagen peptides, commercially available macromolecular cartilage extracts, the small-molecule cartilage extracts prepared in Example 3, or UC-II, the collagen peptide-cartilage extract compositions (20:1, 1:1, and 1:5) have better efficacy in promoting the expression of Collagen II and thus achieving cartilage repair.

[0062] By comparing the relative content data of Collagen II in the collagen peptide group (concentrations of 1 mg / mL, 10 mg / mL and 20 mg / mL) and the collagen peptide-cartilage extract combination group (concentrations of 1 mg / mL, 5 mg / mL and 10 mg / mL), it was found that the collagen peptide-cartilage extract combination group was able to achieve the effect of increasing the content of Collagen II at a lower concentration.

[0063] (2.2) Effects on Aggrecan Expression Aggrecan is a proteoglycan macromolecule composed of glycosaminoglycan (GAG) and a core protein. Aggrecan non-covalently binds to hyaluronic acid to form a proteoglycan aggregate, which is a major component of the extracellular matrix (ECM) of cartilage.

[0064] The results are as follows Figure 7As shown, the relative content of Aggrecan in the model group was significantly lower than that in the control group, and the difference was significant, indicating that the model was successfully constructed. At the same mass concentration, the 3000 Da small molecule cartilage extract prepared by the process of Example 3 showed better cartilage repair effect than the single component commercially available large molecule cartilage extract or collagen peptide, and was more conducive to the synthesis of aggrecan. After using collagen peptide and the 3000 Da small molecule cartilage extract prepared in Example 3, at the same mass concentration, the relative content of Aggrecan in the collagen peptide-cartilage extract composition at a ratio of 20:1 was significantly higher than that of the collagen peptide under the same concentration conditions, but lower than the effect of the small molecule cartilage extract under the same concentration conditions. This may be because the amount of small molecule cartilage extract added was too small, and the synergistic effect of the composition in promoting Aggrecan expression was not obvious. However, as the proportion of low-molecule cartilage extract in the composition increased, the 1:1 and 1:5 ratios of collagen peptide-cartilage extract demonstrated clear advantages, significantly promoting Aggrecan synthesis. Furthermore, as the proportion of cartilage extract in the composition increased, the content of aggrecan also increased, gradually enhancing the cartilage repair effect. Compared with single-ingredient collagen peptides, commercially available large-molecule cartilage extracts, and low-molecule cartilage extracts, the collagen peptide-cartilage extract combinations (1:1 and 1:5) demonstrated superior cartilage repair efficacy by promoting Aggrecan expression.

[0065] By comparing the relative content data of Aggrecan in the collagen peptide group (concentrations of 1 mg / mL, 10 mg / mL and 20 mg / mL) and the collagen peptide-cartilage extract combination group (concentrations of 1 mg / mL, 5 mg / mL and 10 mg / mL), it was found that the collagen peptide-cartilage extract combination group could achieve the effect of increasing the Aggrecan content at a lower concentration.

[0066] (2.3) Effects on the expression of matrix metalloproteinases (MMP-1) Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases that degrade the extracellular matrix. MMP-1 is a key player in cartilage matrix degradation, contributing to inflammation. The more severe the inflammatory response, the higher the MMP-1 level.

[0067] The results are as follows Figure 8As shown, the relative content of MMP-1 in the model group was significantly increased compared to the control group, with a significant difference, indicating successful model construction. At equivalent mass concentrations, the 3000 Da low-molecule cartilage extract prepared by the process of Example 3 exhibited superior anti-inflammatory effects compared to single-component commercially available macromolecular cartilage extracts, collagen peptides, or UC-II. When collagen peptides were combined with the 3000 Da low-molecule cartilage extract prepared in Example 3, the 20:1 ratio of collagen peptide-cartilage extract combination exhibited significantly better anti-inflammatory effects than the collagen peptide alone at the same mass concentration, but lower than the effect of the low-molecule cartilage extract at the same concentration. This may be due to the low amount of low-molecule cartilage extract added, resulting in a less pronounced anti-inflammatory effect or a synergistic effect of the combination. However, as the ratio of low-molecule cartilage extract in the combination increased, the 1:1 and 1:5 ratios of collagen peptide-cartilage extract demonstrated significant advantages, significantly inhibiting the overexpression of matrix metalloproteinase-1 (MMP-1) and alleviating the inflammatory response, surpassing the low-molecule cartilage extract at the same concentration. Compared with single-ingredient collagen peptides, commercially available large-molecule cartilage extracts, small-molecule cartilage extracts or UC-II, the combination (1:1 and 1:5) has a better effect of reducing inflammatory response by inhibiting the overexpression of MMP-1.

[0068] By comparing the relative content data of MMP-1 in the collagen peptide group (concentrations of 1 mg / mL, 10 mg / mL and 20 mg / mL) and the collagen peptide-cartilage extract combination group (concentrations of 1 mg / mL, 5 mg / mL and 10 mg / mL), it was found that the collagen peptide-cartilage extract combination group could achieve the effect of downregulating MMP-1 content at a lower concentration.

[0069] The results of the above examples are summarized as follows: (1) The patented small molecule cartilage extract has a lower molecular weight and higher bioavailability, and has better cartilage repair and anti-inflammatory effects than the large molecule cartilage extract; (2) The collagen peptide-cartilage extract composition significantly promotes the synthesis of type II collagen (Collagen II) and aggrecan (Aggrecan), thereby improving the cartilage repair effect; (3) The collagen peptide-cartilage extract composition significantly inhibits the overexpression of matrix metalloproteinase 1 (MMP-1) and reduces the inflammatory response; and as the proportion of cartilage extract in the composite sample increases, the repair effect gradually increases.

[0070] In summary, small-molecule cartilage extracts have better bioavailability and are more beneficial for promoting cartilage repair and inhibiting inflammatory responses. The combination of collagen peptides and cartilage extracts exhibits significant synergistic effects. At equivalent concentrations, they are superior in inhibiting inflammation, promoting cartilage matrix synthesis, and repairing cartilage damage, far exceeding the repair effects of a single component. This discovery provides new insights and therapeutic strategies for the treatment of cartilage damage, with significant clinical application prospects and innovative value.

[0071] The above descriptions are merely embodiments of the present invention and are not intended to limit the scope of the present invention. Any equivalent results or equivalent process transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, should be included in the scope of protection of this patent.

Claims

1. A composition for promoting chondrocyte proliferation and cartilage repair, characterized in that: The composition comprises a combination of a small molecule cartilage extract and a collagen peptide; the average molecular weight of the small molecule cartilage extract is ≤5000 Da, and the average molecular weight of the collagen peptide is 2000 Da~4000 Da.

2. A composition for promoting chondrocyte proliferation and cartilage repair according to claim 1, characterized in that: The mass ratio of the collagen peptide to the small molecule cartilage extract is 50:1 to 1:

50.

3. A composition for promoting chondrocyte proliferation and cartilage repair according to claim 2, characterized in that: The mass ratio of the collagen peptide to the small molecule cartilage extract is 20:1 to 1:

5.

4. A method for preparing the composition for promoting chondrocyte proliferation and cartilage repair according to claim 1, characterized in that: (1) After washing and crushing the animal cartilage, it is first depolymerized using protease, then depolymerized using microwave-assisted protease for a second time, and then filtered through a membrane to obtain a small molecule cartilage extract with an average molecular weight of ≤5000 Da; (2) The small molecule cartilage extract obtained in step (1) is mixed with collagen peptides having an average molecular weight of 2000 Da to 4000 Da to obtain a composition.

5. The method for preparing a composition for promoting chondrocyte proliferation and cartilage repair according to claim 4, characterized in that: The protease used in the primary depolymerization is any one of papain, bromelain, flavor protease, and keratinase, or a combination of multiple proteases.

6. The method for preparing a composition for promoting chondrocyte proliferation and cartilage repair according to claim 4, characterized in that: The amount of protease added for the primary depolymerization is 1-5 g / L, the pH is 6.5-8.5, the reaction temperature is 25-60° C., and the enzymatic hydrolysis time is 4-24 h.

7. The method for preparing a composition for promoting chondrocyte proliferation and cartilage repair according to claim 4, characterized in that: The protease used in the microwave-assisted protease secondary depolymerization is acidic protease, the protease addition amount is 1-3 g / L, the microwave frequency is 400-700 W, the pH of the microwave-assisted depolymerization is 2-4, the temperature of the microwave-assisted depolymerization is 40-60° C., and the time of the microwave-assisted depolymerization is 1-3 h.

8. The method for preparing a composition for promoting chondrocyte proliferation and cartilage repair according to claim 4, characterized in that: The mass ratio of the collagen peptide and the small molecule cartilage extract is 50:1 to 1:50, and the optional mass ratio is 20:1 to 1:

5.

9. Use of the composition according to claim 1 in preparing a product for promoting chondrocyte proliferation and / or cartilage repair.

10. The use according to claim 9, characterized in that: The composition is used in preparing a product that promotes cartilage repair by promoting the expression of type II collagen Col II, the expression of proteoglycan Aggrecan, and / or inhibiting the expression of matrix metalloproteinase MMP-1.

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