Cartilage peptide and application thereof in preparation of cartilage injury repair product

Abstract

The application belongs to the technical field of biological medicine, functional food and the like, and particularly relates to a cartilage peptide and application of the cartilage peptide in preparation of a cartilage injury repair product. The cartilage peptide contains at least one of Phe-Gly-Qn, Val-Gly-Phe, Leu-Gly-Glu-Leu-Phe and the like. Research results prove that the cartilage peptide can significantly improve zebra fish cartilage structure injury induced by dexamethasone. The cartilage peptide can also increase glycosaminoglycan (GAG) content, promote expression of a key transcription factor Sox9 of cartilage, and inhibit activity of matrix metalloproteinase MMP-13, thereby effectively reducing three-dimensional cartilage cell injury caused by IL-1β, and achieving protection and repair of cartilage tissue. Based on the above-mentioned mechanism, the active peptide is suitable for development of related products for prevention and auxiliary treatment of osteoarthritis, including but not limited to ordinary food, health products, functional food, daily chemical products, drugs or biological products and the like. The component has clear structure, good stability, safety and reliability, and has good industrialization prospect and market application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chondroitin peptide and its application in the preparation of cartilage damage repair products. Background Technology

[0002] Osteoarthritis (OA) is a degenerative disease caused by various factors such as aging, obesity, and joint injury, resulting in degeneration and damage of articular cartilage, and reactive hyperplasia of the joint margins and subchondral bone. It is also known as osteoarthritis or degenerative arthritis. Clinical manifestations include slowly progressing joint pain, tenderness, stiffness, swelling, limited range of motion, and joint deformities. The prevalence of osteoarthritis increases with age, and it is more common in women than men. Currently, there are over 300 million OA patients worldwide, and the overall prevalence of OA in people over 40 years of age in my country has reached as high as 46.3%. Moreover, with the continuous aging of my country's population, the prevalence of OA is on the rise, increasingly affecting people's health and quality of life.

[0003] Matrix metalloproteinase-13 (MMP-13), also known as collagenase-3, is the most potent cartilage-degrading enzyme in the MMP family and is closely linked to the pathogenesis of osteoarthritis (OA). This enzyme can directly or indirectly initiate the degradation of cartilage matrix and collagen components through specific signaling pathways and related regulators, including endogenous inhibitors, transcription factors, and growth factors. MMP-13 expression is upregulated as the disease progresses. SRY-box transcription factor 9 (Sox9) is an important intracellular transcription factor that plays a crucial regulatory role in the extracellular matrix metabolism of articular cartilage cells. Sox9 promotes the transcription of genes for cartilage markers type II collagen, proteoglycans, and type IX collagen, and inhibits the expression of proteoglycans and matrix metalloproteinases, thereby maintaining the phenotype of articular cartilage cells. Sox9 expression is compensatorily upregulated in the early stages of osteoarthritis, and downregulated as the disease progresses. In other words, MMP-13 and Sox9 are important targets for the progressive development of osteoarthritis.

[0004] Therefore, it is of great significance to develop a chondroitin peptide that can inhibit MMP-13 expression, increase Sox9 expression, and increase glycosaminoglycan (GAG) content for use in cartilage injury repair products.

[0005] Patent CN101711591B, "A Method for Preparing Fish Cartilage Extract and the Obtained Product," discloses a method for preparing a fish cartilage extract from marine fish. The steps include alkaline protease hydrolysis of fish cartilage to obtain an enzymatic hydrolysate, followed by flavor protease hydrolysis, then enzyme inactivation and impurity removal. Molecular sieves are used to separate collagen and chondroitin sulfate, thus achieving product purification and separation, yielding two single-component products: low-cost, high-content collagen peptides and chondroitin sulfate. The fish collagen peptides are small-molecule collagen peptides with a molecular weight of 206–400 Daltons, containing more than 35% hydroxyproline, glycine, and proline. The chondroitin sulfate has glycosyl groups of glucuronic acid and acetylgalactosamine, with a molecular weight of 5000–15000 Da. However, the collagen peptide sequence in this patent is unclear.

[0006] Patent CN109265531A, "Chicken Breast Cartilage Peptide, its Preparation Method and Application," discloses a chicken breast cartilage peptide, its preparation method, and its application. The specific steps for preparing the cartilage peptide are as follows: chicken breast cartilage is pulverized, the pH is adjusted to 7.0, and under 55°C, 0.2%–0.3% alkaline protease and 0.3%–0.5% flavor protease are added for enzymatic hydrolysis for 3 hours. After enzyme inactivation, the supernatant is centrifuged, filtered through silk cloth, and then concentrated and dried to obtain the chicken breast cartilage peptide. Its characteristics include a type II collagen content ≥50% and a chondroitin sulfate content ≥20%.

[0007] Patent CN113599397B, "A Cartilage Extract and Its Preparation Method and Application," discloses a cartilage extract and its preparation method and application. A two-step enzymatic hydrolysis method is used to prepare a cartilage extract composed of low molecular weight chondroitin sulfate and low molecular weight polypeptides. The specific steps for preparing the cartilage extract are as follows: chicken breast cartilage is mixed with 0.5-1% (w / w) of papain and enzymatically hydrolyzed at 50-55°C and pH 6.5-6.8 for 3-4 hours to obtain a first hydrolysate; the first hydrolysate is then mixed with chondroitin sulfate at 12-15 U / g and enzymatically hydrolyzed at 37°C and pH 4.0-7.2 for 4 hours to obtain a second enzymatic hydrolysis, yielding a cartilage extract solution. This solution is then purified and dried to obtain the final product. The glucosamine content in the cartilage extract is 12-17% (w / w); 97% of the II collagen peptides have a molecular weight below 1000 Da. The active peptide is characterized by having the sequences Ala-Pro-Asp-Asp-Val-His-Pro-Pro-Glu (APDDVHPPE) and Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu-Pro-Lys (KPAPPKPEPK).

[0008] Patent CN119366608A, "Method for Preparing Highly Active Small Molecule Cartilage Extract from Animal Cartilage Tissue, Cartilage Extract and Its Application," discloses a method for preparing highly active small molecule cartilage extract from bovine and porcine cartilage tissue. The method employs multi-enzyme, multi-level, directional enzymatic hydrolysis to obtain a cartilage extract with a type II collagen content of over 65%, a GAGs content of over 30%, and a hyaluronic acid content of over 17%. Selective enzyme selection can selectively obtain target peptides: DVGEKGPEGA(HYP)PGKDGAR, GESGSP(HYP)GENGSP(HYP)GPM, GPAGPSGPRGPP(HYP)GPVGPS, SGSP(HYP)GENGSP(HYP)GPM, GDRGP(HYP)PGPVGP(HYP)PGLT, GESGSP(HYP)GENGSP(HYP)GPM, and GAP(HYP)GDKGEAGPP. The patent lists the following peptides: (HYP)GPAGPT, GNSGKP(HYP)GEQGPEGEAGPT, GDQGPP(HYP)GNNGPAGPR, GARGQP(HYP)GVM, SGSP(HYP)GENGSPGP(HYP)M, GDRGPP(HYP)GPVGPP(HYP)GLT, AGPP(HYP)GADGQP(HYP)GAK, TGPIGPP(HYP)GPAGPN, GPSGPRGPP(HYP)GPVGPS, GPAGPP(HYP)GEKGEPGEDGPAGPDGPP(HYP)GPQ, and GDRGP(HYP)PGPVGPP(HYP)GLTGPA. While the patent mentions target peptides, it does not specify which combination of enzymes can be used to obtain these peptides, nor does it perform peptide identification on the obtained cartilage extract.

[0009] The current technology for cartilage extract has the following disadvantages: (1) The cartilage extract is mostly prepared from marine fish, cattle, and pig cartilage. Marine fish sources are prone to causing allergies, cattle sources have the potential risk of mad cow disease, and pig sources are subject to religious restrictions; (2) The prepared products are mostly large molecular weight collagen and chondroitin sulfate, not the traditional low molecular weight cartilage peptides; (3) The few small molecular weight peptides prepared have not been identified, and their peptide composition is unclear; (4) Although the current technology can prepare cartilage extract through different routes, it is lacking in the preservation of activity. In terms of application, a series of activity experiments have not been conducted, and systematic and comprehensive activity and mechanism verification has not been carried out. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a chondroitin peptide and its application in the preparation of cartilage damage repair products.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a chondroitin peptide with cartilage damage repair function, wherein the preparation method of the chondroitin peptide includes the following steps: S1. After cleaning the chicken breast cartilage, mince it and then heat it. S2. Chicken breast cartilage was hydrolyzed with protease to obtain a protease hydrolysate; S3. Filter the protease hydrolysate and adsorb it with activated carbon; S4. The adsorbent solution is decarbonized to obtain a clear solution; the clear solution is concentrated and spray-dried to obtain chondroitin.

[0012] More preferably, in step S1, the pH of the alkaline cleaning is 8.0-12.0.

[0013] More preferably, in step S1, the alkali is at least one of sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution.

[0014] More preferably, the concentration of the sodium hydroxide solution is 0.2-2 mol / L.

[0015] More preferably, in step S1, the heat treatment temperature is 80℃-120℃.

[0016] More preferably, in step S1, the heat treatment time is 0.5h-2h.

[0017] More preferably, in step S2, the solid-liquid ratio of the chicken breast cartilage and water is 1g:(1.5-3)mL.

[0018] More preferably, in step S2, the enzymatic hydrolysis temperature is 50°C. 65℃.

[0019] More preferably, in step S2, the protease is at least one of neutral protease, alkaline protease, papain, trypsin, aminopeptidase, and flavor protease.

[0020] More preferably, in step S2, the pH of the enzymatic hydrolysis is 6.0-10.0.

[0021] More preferably, in step S2, the enzymatic hydrolysis time is 4 hours. 8h.

[0022] More preferably, in step S2, the pH adjuster for enzymatic hydrolysis is at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, hydrochloric acid, sulfuric acid, acetic acid, citric acid, and phosphoric acid.

[0023] Alternatively, in the above-mentioned chondroitin peptide, the amino acid sequence of the chondroitin peptide is at least one of Phe-Gly-Gln (FGQ), Val-Gly-Phe (VGF), or Leu-Gly-Glu-Leu-Phe (LGELF).

[0024] Alternatively, the chondroitin peptides mentioned above may contain, by mass ratio, Phe-Gly-Gln, Val-Gly-Phe, and Leu-Gly-Glu-Leu-Phe of a relative content ratio of (0.7-1.4):(0.1-0.4):(0.2-0.5).

[0025] Alternatively, in the above-mentioned chondroitin peptides, the molecular weight of each component is ≤3000 Da, and the average molecular weight is <1000 Da.

[0026] The chondroitin peptide of the present invention improves dexamethasone-induced cartilage damage in zebrafish; at the same time, the chondroitin peptide of the present invention improves IL-1β-induced 3D chondrocyte damage by increasing GAG content, inhibiting MMP-13 protein expression, and increasing Sox9 protein expression, thereby achieving the purpose of cartilage damage repair.

[0027] The chondroitin peptide of this invention is an active amino acid sequence released by enzymatic decomposition of chicken breast cartilage, which has the effect of repairing cartilage damage. Compared with large protein molecules, the chondroitin peptide is a short-chain amino acid, which is not only structurally stable, but also has a small molecular weight and is easily absorbed. In addition, compared with large protein molecules, the chondroitin peptide is a small molecule and has extremely low immunogenicity. Most importantly, the amino acid structure of the chondroitin peptide has good compatibility with cartilage tissue, so it is safe and non-toxic and can be used for a long time.

[0028] In a second aspect, the present invention provides the use of the chondroitin peptide described in the first aspect above in the preparation of products for cartilage damage repair and / or increasing GAG content and / or inhibiting MMP-13 protein expression and / or increasing Sox9 protein expression.

[0029] Alternatively, in the above applications, the chondroitin peptide is used to prevent or treat osteoarthritis.

[0030] Alternatively, in the above applications, the product may be at least one of the following: ordinary food, health products, functional food, daily chemical products, pharmaceuticals, or biological products.

[0031] In this invention, the form or carrier of the food includes, but is not limited to, compressed candy, solid beverage, beverage, oral liquid, gummy candy, soft capsule, tablet, granule, etc.

[0032] Alternatively, in the above applications, the dosage form of the product is at least one of liquid dosage form, solid dosage form, semi-solid dosage form, and gaseous dosage form.

[0033] In this invention, when the chondroitin peptide is applied to cartilage damage repair in the product, the chondroitin peptide can also be developed into a composition with other ingredients that also have cartilage damage repair effects; the ingredients that have cartilage damage repair effects include, but are not limited to, at least one of: glucosamine, turmeric, curcumin, non-denatured type II collagen, and Drynaria fortunei.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: The chondroitin peptide of the present invention improves dexamethasone-induced cartilage damage in zebrafish; at the same time, the chondroitin peptide of the present invention improves IL-1β-induced 3D chondrocyte damage by increasing GAG content, inhibiting MMP-13 protein expression, and increasing Sox9 protein expression, thereby achieving the purpose of cartilage damage repair.

[0035] The chondroitin peptide of this invention is an active amino acid sequence released by enzymatic decomposition of chicken breast cartilage, which has the effect of repairing cartilage damage. Compared with large protein molecules, the chondroitin peptide is a short-chain amino acid, which is not only structurally stable, but also has a small molecular weight and is easily absorbed. In addition, compared with large protein molecules, the chondroitin peptide is a small molecule and has extremely low immunogenicity. Most importantly, the amino acid structure of the chondroitin peptide has good compatibility with cartilage tissue, so it is safe and non-toxic and can be used for a long time. Attached Figure Description

[0036] Figure 1 The TIC diagrams are for Examples 1-5, Comparative Example 1, and Comparative Example 5.

[0037] Figure 2 MS / MS map of the amino acid sequence FGQ of chondroitin.

[0038] Figure 3 The image shows the amino acid sequence of chondroitin and the MS / MS map of VGF.

[0039] Figure 4 The amino acid sequence of chondroitin is shown in the MS / MS map of LGELF.

[0040] Figure 5 The effects of chondroitin, FGQ, LGELF, and VGF on GAGs that induce IL-1β-induced chondrocyte damage.

[0041] Figure 6 The mechanism of action of chondroitin peptide, FGQ, LGELF, and VGF in protecting chondrocytes from damage. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0044] Adult bone marrow mesenchymal stem cells were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd., catalog number HUXMA-90011.

[0045] Example 1: Preparation of chondroitin peptides This embodiment provides a method for preparing chondroitin peptides, including the following steps: S1. Take 1000 g of chicken breast cartilage, add 2000 mL of pH 11.0 NaOH solution, and wash for 0.5 h; take out the chicken breast cartilage, mince it with a meat grinder with a pore size of 2-5 mm.

[0046] S2. Add 2.5 times the amount of water to the minced chicken breast cartilage, adjust the pH to 8.5, add 0.2% of complex protease 1 (composed of alkaline protease, neutral protease, and papain) in a 55℃ water bath, and hydrolyze for 0.5 h; then add 0.03% of complex protease 2 (aminopeptidase), and continue hydrolysis for 3.5 h; after hydrolysis, heat in a 85℃ water bath for 30 min to inactivate the enzyme, cool to room temperature, centrifuge at 5000 rpm for 3 min, and collect the supernatant to obtain the protease hydrolysate; S3. Add 0.35% activated carbon to the enzymatic hydrolysate and adsorb at 55℃ for 0.5h; use diatomaceous earth as the filter medium for decarbonization treatment to obtain a clear liquid; concentrate the clear liquid under reduced pressure, and spray dry the concentrated liquid through an LPG-5 centrifugal spray dryer at an inlet air temperature of 130-160℃ and an outlet air temperature of 90-110℃ to obtain chondroitin peptides.

[0047] Example 2 The difference between this example and Example 1 is that in step S2, the pH is adjusted to 6.5.

[0048] Example 3 The difference between this example and Example 1 is that in step S2, the pH is adjusted to 7.5.

[0049] Example 4 The difference between this example and Example 1 is that in step S2, the pH is adjusted to 9.0.

[0050] Example 5 The difference between this example and Example 1 is that in step S2, the pH is adjusted to 10.0.

[0051] Comparative Example 1 The difference between this example and Example 1 is that in step S2, 0.5% alkaline protease and 0.15% complex protease 2 (aminopeptidase) are added.

[0052] Comparative Example 2 The difference between this example and Example 1 is that in step S2, 0.5% of complex protease 1 (composed of alkaline protease, neutral protease and papain) and 0.15% of complex protease 2 (aminopeptidase) are added.

[0053] Comparative Example 3 The difference between this example and Example 1 is that in step S2, 0.5% of complex protease 1 (composed of alkaline protease, neutral protease and papain) and 0.075% of complex protease 2 (aminopeptidase) are added.

[0054] Comparative Example 4 The difference between this example and Example 1 is that in step S2, 0.3% bromelain and 0.075% complex protease 2 (aminopeptidase) are added.

[0055] Comparative Example 5 Commercially available chondroitin peptide sample, batch number CJ23082705.

[0056] Test case 1. Determination of protein content in chondroitin peptides The protein content of chondroitin was determined according to GB 5009.5-2016, "National Food Safety Standard - Determination of Protein in Food". The results are shown in Table 1.

[0057] As shown in Table 1, the protein content of Examples 1-5 is all above 70%, significantly higher than that of Comparative Examples 1-4, indicating that a protein content of over 70% is only achievable with a specific enzyme combination. The protein content of Examples 1-5 is also significantly higher than that of Comparative Example 5, demonstrating that the chondroitin peptide obtained by this invention is superior to similar products on the market.

[0058] Table 1. Protein content of chondroitin peptides 2. Amino acid sequence identification of chondroitin peptides The amino acid sequence of the prepared chondroitin peptides was identified using HPLC / MS / MS. The specific detection method is as follows: A Dionex UltiMate 3000 DGLC system (ThermoFisher Scientific) and a Q-Exactive orbital ion trap mass spectrometer were used. A C18 column (HSS T3 1.8 μm, 100 × 2.1 mm, Waters Acquity) was employed at a flow rate of 0.15 mL / min. Solvent A (0.1% formic acid in water) and solvent B (acetonitrile) were used as elution solvents. The gradient elution operation was as follows: 0–4 min, 5% B; 6 min, 10% B; 15 min, 40% B; 18–21 min, 95% B. Mass spectrometry analysis was performed in positive ion mode, with a scan range of 100–1,500 m / z, a resolution of 70,000, a maximum injection time of 100 ms, and an automatic gain control (AGC) target set to 1e6. The instrument settings included a capillary spray voltage of 3.0 kV, a capillary temperature of 350°C, an auxiliary gas heating temperature of 320°C, and an s-lens setting of 65. Peptides were identified using PepOS 3.5.0 software. The number of identified peptides is shown in Table 2. Figure 1 As shown.

[0059] The structures of three peptide fragments in the chondroitin peptides of Examples 1-5—FGQ (Phe-Gly-Gln), VGF (Val-Gly-Phe), and LGELF (Leu-Gly-Glu-Leu-Phe)—were identified by MS / MS. Their corresponding characteristic fragments are shown below. Figure 2-4 After identifying FGQ, VGF, and LGELF by mass spectrometry, a biotechnology company was commissioned to synthesize these three peptides for subsequent activity evaluation experiments. Furthermore, the content of FGQ, VGF, and LGELF in the chondroitin peptides was analyzed, and the results are shown in Table 2.

[0060] The relative contents of FGQ, VGF, and LGELF in chondroitin are in the range of 0.7%-1.4%, 0.1%-0.4%, and 0.2%-0.5%, respectively.

[0061] Table 2 Content of FGQ, VGF, and LGELF in chondroitin Note: In the table above, the relative content of FGQ = peak area of ​​FGQ / sum of peak areas of all identified peptides × 100%, the relative content of VGF = peak area of ​​VGF / sum of peak areas of all identified peptides × 100%, and the relative content of LGELF = peak area of ​​LGELF / sum of peak areas of all identified peptides × 100%.

[0062] Example of effect 1. Experiment on improving cartilage damage Zebrafish have become an important model organism for studying skeletal development and disease in basic and preclinical research. The potential of these bony fish lies in their small size, ease of care, genetic compliance, and high regenerative capacity. Furthermore, due to the transparency of the embryonic and larval stages, transgenic and mutant strains affecting specific cells or tissues allow for more detailed monitoring of bone formation and osteoblast activity.

[0063] Experimental steps: 1) Five AB-type zebrafish with dpf3 were used in each well, with three replicates and 3 mL per well. The experiment was divided into a control group, a model group, and a sample group. The control group was given embryo culture medium, while the model group and the sample group were given DEX (dexamethasone) 25µM. The sample group was also given samples of different concentrations. 2) After the experimental drug was administered to dpf5, the fish were anesthetized and killed with 0.2% MS-222 (tricaine); 3) 1 mL of 4% paraformaldehyde was added to each well for 2 hours for fixation; 4) 0.4% alicin blue stock solution was prepared with 50% ethanol and dissolved at 37℃. After fixation, a staining solution was prepared using 0.4% alicin blue, 95% ethanol, and 160mM MgCl2 in a ratio of 0.05:0.7:0.25; 5) The staining time ended when the cartilage was stained, which was expected to be about 3-5 hours; 6) The fish were bleached with a mixed solution of 1.5% H2O2 and 1% KOH (using a 1:1 mixture of 3% H2O2 and 2% KOH, diluted with PBS). The fish were considered healthy when no melanin was observed under a stereomicroscope; 7) 1 mL of... Wash once with PBS to remove bleach; 8) Store in 50% glycerol and take a picture, calculate the length of the palatal quadriceps cartilage, and calculate the cartilage regeneration rate according to the formula [Cartilage regeneration rate (%) = (length of palatal quadriceps cartilage in the treatment group - length of palatal quadriceps cartilage in the model group) / length of palatal quadriceps cartilage in the model group × 100%, the treatment group is the different concentration group of Example 1 or the comparative example 5 group].

[0064] The experimental results are shown in Table 3 below: Table 3. Protective effect of chondroitin peptides against cartilage damage Note: In the table above, compared with the control group, Compared with the model group, # P <0.05, #### P <0.0001. Compared with Comparative Example 5, △ P <0.05.

[0065] As shown in Table 3, compared with the model group, the concentrations of 5, 10, 25, and 50 μg / mL in Example 1 and the 50 μg / mL concentration in the comparative example group significantly increased the length of the palatal quadrate cartilage in zebrafish, demonstrating a significant protective effect against cartilage damage. The protective effects of the chondroitin peptides of the present invention at concentrations of 10 and 50 μg / mL are significantly better than those in the 50 μg / mL concentration of the comparative example group.

[0066] 2. 3D Chondroblastic Cell Injury Protection Experiment The effects of chondroitin peptides, FGQ, LGELF, and VGF on GAG in 3D chondrocyte injury were evaluated according to the following experimental procedure. The specific test methods are as follows: Human BMSCs (OriCell, HUXMA-90011) passage 7 cells were collected by centrifugation at 300×g for 5 minutes. The cells were then incubated in chondrocyte differentiation medium (OriCell, HUXMA-90041, 100 mL) for 30 minutes, and then seeded at a rate of 80,000 cells / well (200 µL) in U-shaped plates (CON-7007, 96 wells) for 3 days to form chondrocyte spheres. The culture medium was changed every 3 days. Three days later, the cells were divided into the following groups and cultured for another 12 days with the following medication: 14 groups were established, namely: normal group (PBS), model group (IL-1β 10 ng / mL), low-dose chondroitin group (IL-1β 10 ng / mL + chondroitin 0.5 mg / mL), medium-dose chondroitin group (IL-1β 10 ng / mL + chondroitin 1 mg / mL), high-dose chondroitin group (IL-1β 10 ng / mL + chondroitin 2 mg / mL), low-dose FGQ group (IL-1β 10 ng / mL + FGQ 5 µM), medium-dose FGQ group (IL-1β 10 ng / mL + FGQ 10 µM), high-dose FGQ group (IL-1β 10 ng / mL + FGQ 20 µM), and low-dose LGELF group (IL-1β 10 ng / mL + LGELF). 5µM), medium-dose LGELF group (IL-1β 10 ng / mL + LGELF 10µM), high-dose LGELF group (IL-1β 10 ng / mL + LGELF 20µM), low-dose VGF group (IL-1β 10 ng / mL + VGF 5µM), medium-dose VGF group (IL-1β 10 ng / mL + VGF 10µM), high-dose VGF group (IL-1β 10 ng / mL + VGF 20µM).

[0067] Twelve days later, cartilage spheres were collected in 100 μL PBS and cells were sonicated (300 W, 3 seconds sonication, 3 seconds pause, 3 min total). The total content of glycosaminoglycans (GAGs) was determined using a dimethylmethylene blue (DMMB) colorimetric assay kit (HPBIO-JM9047). The results are as follows. Figure 5 As shown: Depend on Figure 5 It was found that, compared with the model group, the GAG ​​content in the groups containing 1 mg / mL chondroitin peptide, 20µM FGQ, 5µM VGF, 10µM VGF, 20µM VGF, 5µM LGELF, 10µM LGELF, and 20µM LGELF was significantly increased, indicating that the chondroitin peptide, FGQ, LGELF, and VGF of the present invention have a protective effect against cartilage damage. Moreover, the effects of FGQ, LGELF, and VGF are superior to those of chondroitin peptide, which clearly shows that chondroitin peptide mainly exerts its cartilage protective effect through the amino acid sequences FGQ, LGELF, and VGF.

[0068] 3. Mechanism of 3D chondrocyte damage protection To further clarify the chondrogenic mechanism of the amino acid sequences FGQ, LGELF, and VGF in chondroitin, the inventors used IL-1β to prepare an inflammatory stimulation chondrocyte injury model. A control group, a model group, and a sample group were set up and cultured together for 4 days. The expression levels of chondrocyte-related proteins Collagen-II, Aggrecan, Sox9, MMP13, and ADAMTS4 were detected by Western blotting.

[0069] The mechanism of action of chondroitin peptides, FGQ, LGELF, and VGF in protecting against 3D chondrocyte damage in Example 1 was evaluated according to the following experimental procedures. The specific test methods are as follows: Human BMSCs (OriCell, HUXMA-90011) passage 7 cells were collected by centrifugation at 400×g for 5 minutes. The cells were then incubated in 100 mL of chondrocyte differentiation medium (OriCell, HUXMA-90041) for 30 minutes, and then seeded into centrifuge tubes at a rate of 300,000 cells / 15 mL (1000 µL) for 3 days to form chondrocyte spheres. The culture medium was changed every 3 days. After 3 days, the cells were divided into the following groups and cultured for another 4 days with the following drugs: 6 groups were established, namely normal group (PBS), model group (IL-1β 10 ng / mL), chondroitin group (IL-1β 10 ng / mL + chondroitin 1 mg / mL), FGQ group (IL-1β 10 ng / mL + FGQ 10µM), LGELF group (IL-1β 10 ng / mL + LGELF 10µM), and VGF group (IL-1β 10 ng / mL + VGF 10µM).

[0070] Four days later, cells were disrupted by sonication using 150 μL RIPA / chondroid cells (300 W, 3 seconds sonication, 3 seconds pause, 3 min total). Western blotting was then used to detect the expression of cartilage-related proteins Collagen-II, Aggrecan, Sox9, MMP13, and ADAMTS4. The results are as follows: Figure 6 As shown.

[0071] Depend on Figure 6 It can be seen that, compared with the model group, the expression of Aggrecan and Sox9 in chondroitin peptides, FGQ, VGF, and LGELF was significantly increased, while MMP13 was significantly decreased. This indicates that the chondroitin peptides, FGQ, LGELF, and VGF of the present invention mainly exert their cartilage protective effects by increasing the protein expression of Sox9, decreasing the protein expression of MMP13, and increasing Aggrecan.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A chondroitin peptide with cartilage damage repair function, characterized in that, The preparation method of the chondroitin peptide includes the following steps: S1. After cleaning the chicken breast cartilage, mince it and then heat it. S2. Chicken breast cartilage was hydrolyzed with protease to obtain a protease hydrolysate; S3. Filter the protease hydrolysate and adsorb it with activated carbon; S4. The adsorbent solution is decarbonized to obtain a clear solution; the clear solution is concentrated and spray-dried to obtain chondroitin.

2. The chondroitin peptide as described in claim 1, characterized in that, In step S2, the protease includes at least one of neutral protease, alkaline protease, papain, trypsin, aminopeptidase, or flavor protease.

3. The chondroitin peptide as described in claim 1, characterized in that, The method for preparing the chondroitin peptide includes at least one of the following (1)-(3): (1) In step S1, the pH of the cleaning solution is 8.0-12.0; (2) In step S1, the heat treatment temperature is 80℃-120℃; (3) In step S2, the pH of the enzymatic hydrolysis is 6.0-10.

0.

4. The chondroitin peptide according to any one of claims 1 to 3, characterized in that, The characteristic amino acid sequence of the chondroitin is at least one of Phe-Gly-Gln, Val-Gly-Phe, or Leu-Gly-Glu-Leu-Phe.

5. The chondroitin peptide as described in claim 4, characterized in that, The chondroitin contains, by mass ratio, Phe-Gly-Gln, Val-Gly-Phe, and Leu-Gly-Glu-Leu-Phe, with a relative content ratio of (0.7-1.4):(0.1-0.4):(0.2-0.5).

6. The chondroitin peptide according to any one of claims 1 to 5, characterized in that, The molecular weight of each component in the chondroitin peptide is ≤3000 Da, and the average molecular weight is <1000 Da.

7. The use of the chondroitin peptide according to any one of claims 1 to 6 in the preparation of products for cartilage damage repair and / or increasing GAG content and / or inhibiting MMP-13 protein expression and / or increasing Sox9 protein expression.

8. The application as described in claim 7, characterized in that, The chondroitin peptide is used to prevent or treat osteoarthritis.

9. The application as described in claim 7, characterized in that, The product is at least one of the following: ordinary food, health products, functional food, daily chemical products, pharmaceuticals, or biological products.

10. The application as described in claim 7, characterized in that, The dosage form of the product is at least one of liquid dosage form, solid dosage form, semi-solid dosage form, and gas dosage form.

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