Yak cartilage peptide with function of repairing cartilage injury and preparation method and application thereof

Abstract

The application provides yak cartilage peptide with a function of repairing cartilage injury, and a preparation method and application thereof, and belongs to the technical field of active peptides. The application selects crescent bones of yaks as raw materials, adopts papain and trypsin for complex enzymolysis, and combines with ultrasonic-assisted enzymolysis technology, so that the enzymolysis efficiency and peptide yield are greatly improved. The yak cartilage peptide prepared by the application is proved to have good antioxidant activity and the effect of promoting cartilage cell proliferation through in-vitro cell experiments, and the effect is significantly better than that of traditional enzymolysis methods and commercial pig bone peptide, fetal bovine bone peptide and other products. Therefore, the yak cartilage peptide prepared by the application can be used for repairing cartilage injury, treating cartilage degeneration and improving symptoms related to osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a yak cartilage peptide with cartilage repair function, its preparation method, and its application. Background Technology

[0002] Various types of cartilage injuries are common joint diseases in clinical practice, mainly manifested as degenerative changes, wear and tear, and defects in articular cartilage, leading to joint pain, swelling, and limited mobility, which seriously affects patients' quality of life. In addition, acute cartilage defects caused by sports injuries, trauma, and other factors are also common clinical problems. Currently, commonly used treatments include microfracture surgery, cartilage transplantation, and arthroscopic debridement, but these methods have problems such as large trauma, poor quality of tissue repair, and unstable long-term efficacy.

[0003] Yaks are a rare and precious livestock species unique to the Qinghai-Tibet Plateau, and their bone tissue is rich in collagen, proteoglycans, and various bioactive components. Currently, research on bioactive peptides from yak bone mainly focuses on long bones (such as the femur and tibia) or whole bone tissue; studies on differentiated screening and functional specificity of cartilage from different locations are still lacking. These different cartilage tissues exhibit varying peptide release kinetics during enzymatic hydrolysis due to differences in the degree of collagen cross-linking, the sulfation pattern of glycosaminoglycans, and the composition of non-collagenous proteins, thus affecting the bioactivity of the prepared peptides. However, there are currently no reports on the selection of peptide yields and optimal activity from specific locations for preparing functional peptides to repair cartilage damage. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing yak cartilage peptides with the function of repairing cartilage damage, which can significantly improve the enzymatic hydrolysis efficiency and peptide yield of yak cartilage peptides.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing yak cartilage peptides, comprising the following steps: using the crescent bone of Gannan yak as raw material, performing ultrasonic-assisted enzymatic hydrolysis under the action of papain and trypsin, and collecting peptide fragments with a density of 3000 Da or higher after enzyme inactivation of the hydrolysate to obtain yak cartilage peptides.

[0006] Preferably, the amount of papain or trypsin added is 2% to 4% of the dry weight of the raw material.

[0007] Preferably, the ultrasonic power of the ultrasonic machine-assisted enzymatic hydrolysis is 150~200 W, the heating power is 220~400 W, and the enzymatic hydrolysis time is 2~4 h.

[0008] This invention provides a yak cartilage peptide obtained by the preparation method described above.

[0009] The present invention provides a medicament for treating and / or preventing osteoarthritis and / or cartilage damage, comprising the yak cartilage peptide.

[0010] This invention provides the use of the yak cartilage peptide in the preparation of a medicament for treating and / or preventing osteoarthritis and / or cartilage damage.

[0011] Preferably, the osteoarthritis includes osteoarthritis caused by oxidative stress.

[0012] Preferably, the treatment or prevention of osteoarthritis includes enhancing the antioxidant function of chondrocytes; the antioxidant function includes the ability to scavenge DPPH free radicals and / or the ability to scavenge ABTS free radicals.

[0013] Preferably, the cartilage injury includes degenerative cartilage disease or cartilage defects caused by exercise.

[0014] Preferably, the treatment or prevention of cartilage damage includes promoting chondrocyte proliferation.

[0015] This invention provides a method for preparing yak cartilage peptides with cartilage repair function. The method uses the crescent bone of Gannan yak, which is high in protein and low in fat, as raw material. A combination of papain and trypsin is used, along with ultrasound-assisted enzymatic hydrolysis, increasing the yield of yak cartilage peptides to 28.64%, significantly higher than the peptide yield of the traditional water bath method (23.71%). Furthermore, the DPPH scavenging rate, ABTS scavenging rate, and chondrocyte proliferation-promoting activity of the yak cartilage peptides are significantly superior to those of the traditional method. After enzymatic hydrolysis, the yak cartilage peptides are directionally retained through a 3000 Da ultrafiltration membrane, precisely collecting the molecular weight range of peptides most conducive to cartilage repair and anti-oxidation, while removing impurities of varying sizes to ensure concentrated activity and stable quality, making the product suitable for industrial production. Attached Figure Description

[0016] Figure 1 The graph shows the protein and fat content of different cartilage parts; the bar graph represents protein content, and the line graph represents fat content. Figure 2 The results show the peptide yield from different cartilage sites. Figure 3 The figure shows the detection results of ABTS clearance rate of yak cartilage peptides obtained by ultrasound-assisted and conventional water bath enzymatic hydrolysis. Figure 4 The figure shows the detection results of the DPPH free radical scavenging rate of yak cartilage peptides obtained by ultrasound-assisted and traditional water bath enzymatic hydrolysis. Detailed Implementation

[0017] This invention provides a method for preparing yak cartilage peptides, comprising the following steps: using the crescent bone of Gannan yak as raw material, performing ultrasonic-assisted enzymatic hydrolysis under the action of papain and trypsin, and collecting peptide fragments with a density of 3000 Da or higher after enzyme inactivation of the hydrolysate to obtain yak cartilage peptides.

[0018] In this invention, the type of raw material directly affects the yield of yak cartilage peptides. By comparing the protein and fat content of five cartilage parts from Gannan yaks—lunar bone, nasal bone, skull, trachea, and fossa—high protein content and low fat content were selected as the optimal raw material. The lunar bone has a protein content of 78.53%, which provides sufficient collagen and proteoglycan substrates for enzymatic hydrolysis, facilitating the release of more bioactive peptides during subsequent enzymatic hydrolysis and directly improving peptide yield and the production of the target product. The fat content of the lunar bone is only 3.2%, far lower than other parts, reducing the burden of defatting before enzymatic hydrolysis, simplifying the process, and avoiding the inhibitory effect of excessive fat on protease activity, thus improving the enzymatic hydrolysis efficiency of papain and trypsin.

[0019] In this invention, the residual fat in the crescent bone coats the protein surface or adsorbs into the interstitial spaces, inhibiting the activity of papain and trypsin, reducing the effective contact between the enzyme and the substrate, thereby reducing peptide yield and the release of active peptides. Simultaneously, the fat is easily oxidized in subsequent processes, producing an off-odor and affecting the quality of the final product. To improve peptide yield, the enzymatic hydrolysis process preferably includes pulping and defatting the crescent bone from Gannan yaks. The pulping process preferably involves crushing the solid crescent bone into a homogeneous slurry, filtering, collecting the sieve residue, and drying to obtain crescent bone powder. In this embodiment, the sieve used for filtering has a 40-mesh pore size. The crushing method is preferably using a crusher or a high-speed tissue homogenizer. During the crushing process, water is preferably added to assist in the crushing. In this embodiment, a crusher is used for pulping. The amount of water added is preferably sufficient to assist in crushing, facilitate material flow and collection, for example, the water mass percentage is 40%~65%. The pulping process breaks down the crescent bone's tissue structure, significantly reducing the particle size of cartilage particles and greatly increasing their specific surface area. This provides more contact sites for proteases during subsequent enzymatic hydrolysis, improving hydrolysis efficiency and peptide release. After pulping, the pulping product is preferably coarsely filtered to remove excess water. The coarse filtration is preferably performed using 20-40 mesh gauze to obtain the filtered product. In this specific embodiment, 40 mesh gauze is used for filtration. The filtered product is preferably degreased.

[0020] In this invention, the defatting treatment is preferably performed by boiling, lipase treatment, washing with alkaline aqueous solution, or soaking in organic solvent. In this embodiment, boiling is used to remove fat. The boiling temperature is preferably 90-100°C. The boiling time is preferably 10-40 min, but can be 20-30 min or 25 min. The amount of water added during boiling is 8-12 times the mass of the crescent bone powder. Defatting by boiling melts and floats the fat, facilitating subsequent removal. Simultaneously, moderate heat denaturation loosens the protein structure, exposing more enzyme cleavage sites, which improves enzymatic hydrolysis efficiency.

[0021] In this invention, the preferred amount of papain or trypsin added is 2% to 4% of the dry weight of the raw material. In the embodiments of this application, the amount of papain or trypsin added is 3% of the dry weight of the raw material. Too little enzyme will lead to insufficient enzymatic digestion and low peptide yield; too much enzyme may lead to over-hydrolysis, producing free amino acids or bitter peptides, which will reduce the content of the target active peptide. The amount of each enzyme added is within the range of 2% to 4%, which can ensure sufficient enzymatic digestion without causing over-hydrolysis. Using papain and trypsin to enzymatically hydrolyze the lunula is beneficial to achieve deep hydrolysis of the lunula and efficient release of peptides. Papain has a broad spectrum of action sites and can hydrolyze a variety of peptide bonds; trypsin specifically cleaves the carboxyl termini of lysine and arginine, and the two enzymatic sites are well complementary. The combined use of the two can more thoroughly hydrolyze collagen and proteoglycans in yak lunula, releasing more small molecule active peptides.

[0022] In this invention, the enzymatic hydrolysis step preferably includes adding papain and trypsin to the raw materials for enzymatic hydrolysis, followed by ultrasound-assisted enzymatic hydrolysis. The enzymatic hydrolysis is preferably performed using a water bath heating method. The water bath heating temperature is preferably 50-60°C, and can be 53-57°C. The water bath heating time is preferably 20-40 minutes. In this embodiment, enzymatic hydrolysis is performed using a 55°C water bath for 30 minutes. The optimal operating temperature range for papain is 50-65°C, and the optimal operating temperature range for trypsin is 50-55°C. The temperature range of 50-60°C is an overlapping region where both enzymes maintain high catalytic activity. Within this temperature range, the spatial conformation of the enzyme molecules is in the most favorable active state for substrate binding, ensuring efficient substrate recognition and cleavage.

[0023] In this invention, the ultrasonic power of the ultrasonic-assisted enzymatic hydrolysis is preferably 150-200 W, and can be 160 W, 170 W, or 190 W. The heating power of the ultrasonic-assisted enzymatic hydrolysis is preferably 220-400 W, and can be 240 W, 290 W, 350 W, or 380 W. The ultrasonic-assisted enzymatic hydrolysis time is preferably 2-4 h, and can be 2.5-3.5 h, or even 3 h. In this embodiment of the invention, an ultrasonic power of 180 W and a heating power of 300 W are used to assist enzymatic hydrolysis for 2.5 h. The cavitation effect and mechanical vibration generated by the ultrasonic machine can effectively destroy the dense cartilage tissue structure in the lunula of yak, increase the movement frequency of solvent molecules and enzyme molecules, promote effective contact between substrate and protease, thereby increasing the enzymatic hydrolysis rate and peptide release. The heating power affects the temperature of the enzymatic hydrolysis system; a heating power of 220-400 W can maintain the enzymatic hydrolysis system within the optimal temperature range of 50-60°C. The selection of the enzymatic hydrolysis time is directly related to the degree of adequacy of the enzymatic hydrolysis reaction. When the enzymatic hydrolysis time is too short, the interaction between the enzyme and the substrate is insufficient, and the collagen and proteoglycans in the lunula are not completely hydrolyzed. A large number of large protein fragments remain in the retentate, resulting in insufficient release of the target peptides, low peptide yield, and affected product activity. As the enzymatic hydrolysis time increases, the enzymatic reaction progresses, and the peptide yield gradually increases. Under ultrasound-assisted conditions, the peptide yield of the lunula is above 28.64%, and the peptide content is above 82.36%. In this embodiment of the invention, the yields of yak cartilage peptides obtained by ultrasound-assisted enzymatic hydrolysis and traditional water bath enzymatic hydrolysis were compared. The results showed that the yield of ultrasound-assisted enzymatic hydrolysis (28.64%) was significantly higher than that of traditional water bath enzymatic hydrolysis (23.71%), indicating that ultrasound assistance significantly improved the peptide yield.

[0024] In this invention, enzyme inactivation is preferably performed after enzymatic hydrolysis. The enzyme inactivation method is preferably heat inactivation. The preferred temperature for heat inactivation is 90–100°C, but can be 95°C; the preferred heat inactivation time is 10–20 min, but can be 12–18 min, or even 15 min. Complete inactivation of both proteases at a temperature of 90–100°C has advantages such as simple operation, low cost, and no introduction of exogenous chemicals. Enzyme inactivation causes irreversible thermal denaturation of papain and trypsin, resulting in the loss of their catalytic activity, thereby terminating the enzymatic hydrolysis reaction. This prevents residual proteases from continuing to exert catalytic activity in subsequent processing, further cleaving released active peptides, leading to further hydrolysis of peptides within the target molecular weight range into inactive small oligopeptides or free amino acids, resulting in product activity loss. The enzyme inactivation treatment can also be terminated by adjusting the pH to an extreme value, adding protease inhibitors, or TCA precipitation.

[0025] In this invention, after obtaining the enzyme-inactivating product, it is preferably dried to obtain chondroitin peptide powder. The drying process refers to completely removing water from the concentrated active peptide solution collected after ultrafiltration, transforming the liquid peptide concentrate into a solid dry powder. The drying process preferably employs freeze-drying technology. The freeze-drying preferably involves pre-freezing the peptide concentrate at low temperatures, causing the water in the system to form ice crystals, and then, under vacuum, directly transforming the ice crystals from a solid to a gaseous state and removing them from the system through sublimation, thus completing the dehydration process without undergoing a liquid phase transition. Compared to traditional thermal drying, the entire freeze-drying process is carried out at low temperatures, effectively avoiding the damage to the spatial structure and amino acid sequence of the active peptides in the chondroitin peptides caused by high temperatures, and maximizing the preservation of the product's antioxidant activity in scavenging DPPH and ABTS free radicals, as well as its bioactivity in promoting chondrocyte proliferation.

[0026] This invention provides yak cartilage peptides obtained by the aforementioned preparation method.

[0027] The present invention provides a medicament for treating and / or preventing osteoarthritis and / or cartilage damage, characterized in that it comprises the yak cartilage peptide.

[0028] In this invention, the yak cartilage peptide is rich in collagen peptides and proteoglycan peptide fragments within a specific molecular weight range, with a peptide content as high as 82.36%. Functional verification has shown that this yak cartilage peptide possesses excellent antioxidant activity and cartilage damage repair effects. The antioxidant activity refers to its excellent DPPH and ABTS free radical scavenging abilities. The cartilage damage repair effect is preferably the promotion of chondrocyte proliferation. In an embodiment of this invention, mouse chondrocyte cell lines were used as experimental subjects, and the yak cartilage peptide was used for culture, resulting in a relative cell proliferation rate of 142.8%. It is evident that the yak cartilage peptide possesses both antioxidant and cartilage repair effects, laying a material foundation for its application in the field of bone and joint health.

[0029] This invention provides the use of the yak cartilage peptide in the preparation of medicaments for the treatment and / or prevention of osteoarthritis and / or cartilage damage.

[0030] In this invention, the osteoarthritis preferably includes osteoarthritis caused by oxidative stress. During the development of osteoarthritis, oxidative stress can directly attack chondrocyte membranes, mitochondrial DNA, and proteins, leading to lipid peroxidation, mitochondrial dysfunction, and apoptosis. Simultaneously, oxidative stress can activate inflammatory signaling pathways within chondrocytes, induce upregulation of matrix metalloproteinase expression, accelerate the degradation of type II collagen and proteoglycans, and further disrupt cartilage matrix homeostasis. Embodiments of this invention demonstrate that the yak cartilage peptide has significant DPPH and ABTS free radical scavenging capabilities, effectively neutralizing oxidative stress in the joint microenvironment, reducing oxidative stress damage to chondrocytes, thereby delaying or inhibiting the progression of oxidative stress-induced osteoarthritis.

[0031] In this invention, the treatment or prevention of osteoarthritis preferably includes enhancing the antioxidant function of chondrocytes. The antioxidant function of chondrocytes preferably includes the ability of cells to maintain redox balance through exogenous antioxidants. Enhancing antioxidant function means strengthening the ability of chondrocytes to resist oxidative stress damage, thereby protecting the structural and functional integrity of cells.

[0032] The antioxidant function preferably includes the ability to scavenge DPPH free radicals and / or ABTS free radicals. DPPH is a stable nitrogen-centered free radical; its ethanol solution is purple and has a characteristic absorption peak at 517 nm. When scavenged by antioxidants, the solution color lightens and absorbance decreases, making it a classic indicator for evaluating electron-transfer type antioxidant activity. ABTS, after oxidation, forms a stable blue-green cationic free radical with a characteristic absorption peak at 734 nm. When scavenged by antioxidants, the solution fades and absorbance decreases, often used to evaluate hydrogen atom-transfer type antioxidant activity. The yak cartilage peptide of this invention achieves a DPPH free radical scavenging rate of 66.09% and an ABTS free radical scavenging rate of 74.70%, indicating that it possesses both electron-transfer and hydrogen-transfer antioxidant mechanisms. It can efficiently scavenge free radicals through multiple pathways, enhancing the antioxidant defense capacity of chondrocytes, thereby exerting therapeutic and preventative effects on osteoarthritis.

[0033] In this invention, the cartilage damage preferably includes degenerative cartilage lesions or exercise-induced cartilage defects. The degenerative cartilage lesions are preferably caused by factors such as aging, prolonged joint weight-bearing, or metabolic abnormalities, leading to an imbalance between the synthesis and degradation of the extracellular matrix of chondrocytes, gradual loss of type II collagen and proteoglycans, resulting in thinning of the cartilage tissue, roughness of the surface, and decreased elasticity, ultimately developing into structural damage to the cartilage. The exercise-induced cartilage defects are preferably focal cartilage injuries caused by acute trauma or chronic strain, commonly found in weight-bearing joints such as the knee and hip joints. The defect area has clear boundaries, and the surrounding cartilage tissue is usually relatively healthy, allowing for effective repair through promoting cell proliferation. The yak cartilage peptide of this invention can repair both types of cartilage damage by promoting chondrocyte proliferation.

[0034] In this invention, the treatment or prevention of cartilage damage preferably includes promoting chondrocyte proliferation. Chondrocytes are the only resident cell type in articular cartilage, responsible for synthesizing and maintaining the extracellular matrix (mainly type II collagen and proteoglycans). Normal chondrocyte proliferation and apoptosis are crucial for maintaining cartilage tissue homeostasis. When cartilage damage occurs, the number of chondrocytes in the damaged area decreases, their synthetic function declines, extracellular matrix degradation accelerates, and cartilage repair capacity is limited. Promoting chondrocyte proliferation can increase the number of functional chondrocytes in the damaged area, thereby enhancing their ability to secrete type II collagen and proteoglycans, promoting the resynthesis and deposition of the cartilage matrix, and filling the defect area. In an embodiment of this invention, CCK-8 assay showed that the yak chondrocyte peptide at a concentration of 3.0 mg / mL had a relative proliferation rate of 142.8% on chondrocyte cell lines, indicating that the chondrocyte peptide can effectively activate the proliferative activity of chondrocytes, thereby accelerating the repair process of cartilage damage.

[0035] This invention provides a method for preparing yak cartilage peptides. Using crescent bone as raw material, the method employs a combination of papain and trypsin for enzymatic hydrolysis, combined with ultrasound assistance. The cavitation effect is utilized to disrupt the dense structure of the cartilage, increasing the yield of crescent bone peptides from 23.71% in the traditional water bath enzymatic hydrolysis method to 28.64%, an improvement of 20.79%. The hydrolysate is then directionally filtered through a 3000 Da ultrafiltration membrane to obtain the yak cartilage peptides. The reaction process is mild, environmentally friendly, and simple to operate, demonstrating good feasibility for industrial production.

[0036] This invention provides the application of the aforementioned yak cartilage peptide in the preparation of medicaments for treating or preventing osteoarthritis and / or cartilage damage. Regarding antioxidant properties, the yak cartilage peptide exhibits a concentration-dependent scavenging rate of 66.09% against DPPH free radicals and 74.70% against ABTS free radicals, demonstrating that it possesses both electron transfer and hydrogen atom transfer antioxidant mechanisms. This effectively neutralizes excess reactive oxygen species in the joint microenvironment, reduces oxidative stress levels, and protects chondrocytes from free radical attack. In promoting chondrocyte proliferation, the yak chondrocyte peptide at a concentration of 3.0 mg / mL achieved a relative proliferation rate of 142.8% against small chondrocyte cell lines, which is significantly better than the traditional water bath enzymatic hydrolysis method. It is also superior to commercially available porcine limb bone peptides, fetal bovine bone peptides, and bone peptides derived from other parts of the yak. ​​This indicates that the yak chondrocyte peptide described in this invention, on the one hand, scavenge free radicals to block the damage of oxidative stress to cartilage, and on the other hand, promotes chondrocyte proliferation to increase the number of functional cells and accelerate matrix resynthesis. It is suitable for the treatment and prevention of various joint diseases such as osteoarthritis, degenerative cartilage lesions, and exercise-induced cartilage defects.

[0037] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0038] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1 Screening of yak cartilage peptide substrates Five parts were collected from 3-4 year old Gannan yaks (sampling within 2 hours of slaughter): crescent bone, nasal bone, skull, trachea, and fossa bone. Fascia and fat were removed, and the samples were washed and frozen at -20℃. The cartilage from each part was crushed, passed through a 40-mesh sieve, and the crude protein content was determined by the Kjeldahl method (GB 5009.5-2016), while the crude fat content was determined by Soxhlet extraction (GB 5009.6-2016).

[0040] Protein content determination: Accurately weigh 2 g of each cartilage powder, place the sample in a digestion tube, add 0.2 g of anhydrous copper sulfate and 3 g of potassium sulfate, and digest in a graphite digester for 5 h. Prepare 500 mL of 0.1 mol / L hydrochloric acid standard solution, 1000 mL of 40% sodium hydroxide solution, 1000 mL of 2% boric acid solution, and 10 mL of methyl red indicator. Measure the protein content of the sample using an automatic Kjeldahl nitrogen analyzer.

[0041] Fat content determination: First, fold quantitative filter paper into a filter paper tube and dry it to constant weight. Accurately weigh 5g of each cartilage sample powder, put it into the tube, and cover it with filter paper to prevent floating. Then, add 60mL of petroleum ether and zeolite to the round-bottom flask of the Soxhlet extractor. After assembling the sealing device, place it in a 70℃ water bath for reflux extraction for 6h (reflux 6-8 times per hour until the extract is colorless and transparent). After extraction and cooling, recover the petroleum ether in an 80℃ water bath. Dry the flask with residual fat at 105℃ for 2h, cool it, and weigh it accurately. Each sample is measured in parallel 3 times. Finally, calculate the dry basis fat content according to Formula I, ensuring that the RSD of the parallel experiments is ≤0.2%.

[0042] Fat content , Formula I.

[0043] Where M1 represents the mass of the receiving bottle and the sample (g); M2 represents the mass of the receiving bottle (g); and M3 represents the initial mass of the sample (g).

[0044] The results of protein and fat content tests are shown below. Figure 1 Among them, the crescent bone is the best, with a protein content of 78.53% and a fat content of 3.21%.

[0045] Example 2 Determination of chondroitin yield from different sites The crescent bone, nasal bone, skull, trachea, and fossa of Gannan yak were ground into bone powder with a particle size of 1-3 mm. Deionized water was added at a dry weight to water ratio of 1:10, and the mixture was steamed in a 100℃ water bath for 25 minutes to remove fat. After cooling to room temperature, papain and trypsin (3% each by dry weight of the bone powder) were added, and the mixture was stirred in a 55℃ water bath for 0.5 hours. Then, it was subjected to ultrasonic-assisted enzymatic hydrolysis at the same temperature for 2.5 hours. After enzymatic hydrolysis, the enzymes were inactivated at 95℃ for 15 minutes. After cooling, the mixture was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected by vacuum filtration through a 15-20 μm pore size membrane. The supernatant was then retained through a 3000 Da ultrafiltration membrane. Finally, the mixture was freeze-dried and ground into powder to obtain yak cartilage peptides.

[0046] The enzyme hydrolysate after enzyme inactivation was quantified, and 20% TCA solution was added at a 1:1 volume ratio. After vortexing and mixing, the mixture was allowed to stand for 30 min and then centrifuged (8000 r / min, 4℃, 10 min). The total nitrogen content of the enzyme hydrolysate, the total nitrogen content of the supernatant after TCA acid precipitation and centrifugation, and the free ammonia nitrogen content were determined respectively.

[0047] Meanwhile, a control experiment was conducted on cartilage from five sites using the traditional water bath method. The difference between this method and the enzymatic hydrolysis method mentioned above is that only the ultrasound assistance was not performed; the other steps were completely identical.

[0048] The peptide content can be calculated using Formula II: , Formula II.

[0049] The peptide yield is calculated using Formula III: , Formula III.

[0050] The improvement rate of ultrasound-assisted hydrolysis compared to the traditional enzymatic hydrolysis method is calculated using Formula IV: , Formula IV.

[0051] Table 1 shows the peptide yield, improvement rate, and peptide content results of the traditional enzymatic hydrolysis method and the ultrasound-assisted enzymatic hydrolysis method. The experimental results indicate that, compared with the traditional water bath method, the ultrasound-assisted enzymatic hydrolysis technology significantly improved the peptide yield of all five yak cartilage parts tested, with an average improvement rate of 20.72%. Specifically, the peptide yield of the crescent bone was 23.71% under traditional water bath enzymatic hydrolysis, which increased to 28.64% after ultrasound assistance, representing an improvement rate of 20.79%. Furthermore, the peptide content of the product obtained from the crescent bone using the ultrasound-assisted enzymatic hydrolysis method was as high as 82.36%, the highest among the five parts.

[0052] Table 1. Peptide yield and content of different parts by traditional enzymatic hydrolysis and ultrasound-assisted enzymatic hydrolysis.

[0053] Note: Data in the table represent the results of three parallel experiments, expressed as mean ± standard deviation. Improvement rate = (ultrasound-assisted peptide yield - conventional method peptide yield) / conventional method peptide yield × 100%. Different lowercase letters in the same column indicate significant differences between different sites (p < 0.05), while identical letters indicate no significant differences.

[0054] Example 3 Detection of antioxidant capacity of yak cartilage peptides The crescent bone peptide powder prepared in Example 2 was used to prepare a series of concentration gradient solutions with deionized water: 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.0 mg / mL and 2.0 mg / mL. DPPH powder was accurately weighed and dissolved in anhydrous ethanol to prepare a 0.1 mmol / L DPPH ethanol solution, which was then stored in the dark for later use.

[0055] Take 2.0 mL of sample solutions of different concentrations into 10 mL centrifuge tubes, add 2.0 mL of DPPH ethanol solution, and mix well. React at room temperature in the dark for 30 min. Zero the sample with deionized water and measure the absorbance at 517 nm (standard). Simultaneously measure the absorbance of the mixture of 2.0 mL sample solution and 2.0 mL anhydrous ethanol (sample), and the absorbance of the mixture of 2.0 mL deionized water and 2.0 mL DPPH ethanol solution (blank). Use Vc solution (with the same concentration gradient) as a positive control. The calculation of DPPH free radical scavenging rate is given by formula V: , Formula V.

[0056] 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution were prepared separately, mixed in equal volumes, and reacted at room temperature in the dark for 14 h to obtain ABTS free radical cation stock solution. Before use, it was diluted with deionized water to achieve an absorbance of 0.61 at 734 nm, serving as the ABTS working solution. For measurement, 2.0 mL of sample solutions of different concentrations were added to 2.0 mL of ABTS working solution, mixed well, and reacted at room temperature in the dark for 10 min. The absorbance was measured at 734 nm. Simultaneously, the absorbance of the sample solution mixed with deionized water was measured as the sample background. Deionized water was used as a blank control, and vitamin C solution was used as a positive control. The ABTS free radical scavenging rate was calculated using formula VI. , Formula VI.

[0057] Where A1 is the absorbance of the sample solution after mixing with ABTS working solution; A2 is the absorbance of the sample solution after mixing with deionized water; and A0 is the absorbance of the deionized water after mixing with ABTS working solution.

[0058] The traditional enzymatic hydrolysis method was performed without any ultrasonic assistance, otherwise identical to Example 2 above. Yak crescent bone peptides prepared using the traditional enzymatic hydrolysis method were used as a process control.

[0059] The DPPH free radical scavenging results are shown in Table 2, and the ABTS free radical scavenging results are shown in Table 3. Based on the experimental results of Example 3, it can be seen that the yak crescent bone peptide prepared by ultrasound-assisted enzymatic hydrolysis has significantly better antioxidant activity than the traditional water bath enzymatic hydrolysis method, and this effect is clearly concentration-dependent. Regarding DPPH free radical scavenging, when the concentration increased from 0.1 mg / mL to 2.0 mg / mL, the scavenging rate of the ultrasound-assisted group increased from 18.62% to 66.09%, while that of the traditional enzymatic hydrolysis group increased from 14.89% to 60.90%. At all tested concentrations, the ultrasound-assisted group was superior to the traditional group. Regarding ABTS free radical scavenging, the advantage of the ultrasound-assisted group was even more pronounced: at a concentration of 10.0 mg / mL, the scavenging rate of the ultrasound-assisted group reached 74.70%, while that of the traditional enzymatic hydrolysis group was only 56.72%; and throughout the entire concentration range from 0.5 mg / mL to 10.0 mg / mL, the ultrasound-assisted group consistently outperformed the traditional group. This indicates that the crescent bone peptide obtained in this invention has efficient and stable antioxidant activity, providing strong experimental support for its ability to resist oxidative stress and protect chondrocytes in bone and joint health products.

[0060] Table 2 Results of antioxidant activity assay of DPPH at different concentrations of lunula bone peptide

[0061] Table 3. Results of antioxidant activity assay for different concentrations of ABTS (lunula bone peptide).

[0062] Example 4 Effects of yak chondroitin peptides on the proliferation of mouse chondrocyte line ATDC5 cells The ATDC5 mouse chondrocyte cell line in its growth phase was used at a concentration of 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 hours, after which the supernatant was discarded. Solutions containing different concentrations (0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL) of yak crescent bone peptide powder prepared in Example 2 of this invention were added, with 6 replicates per group, and cultured for another 48 hours. 10 μL of CCK-8 solution was added to each well, and after incubation for 2–4 hours, the absorbance was measured at 450 nm to calculate the relative cell proliferation rate. The calculation method is given in Formula VII. , Formula VII.

[0063] The traditional enzymatic hydrolysis method was performed without any ultrasound assistance, otherwise identical to Example 2 above. The crescent-shaped bone peptide prepared using the traditional enzymatic hydrolysis method served as a process control. Simultaneously, commercially available porcine limb bone peptides (Guyue Bone Peptide Tablets purchased from Nanjing Xinbai Pharmaceutical Co., Ltd.), commercially available bovine bone peptide powder (purchased from Beijing Kaitai New Century Biotechnology Co., Ltd.), and bone peptide powders from different parts of Gannan yak prepared in Example 2 were used as control samples. All control samples were added to the ATDC5 cell culture system at a concentration of 3.0 mg / mL, and the detection method was the same as in this example.

[0064] The cell proliferation assay results are shown in Tables 4-6. The yak crescent bone peptide prepared in this invention exhibits significant concentration-dependent and superior effects in promoting the proliferation of the mouse chondrocyte cell line ATDC5. As shown in Table 4, as the concentration of crescent bone peptide increased from 0.5 mg / mL to 3.0 mg / mL, the relative cell proliferation rate continuously increased from 106.2% to 142.8%, reaching a peak. However, when the concentration increased to 4.0 mg / mL, the proliferation rate slightly decreased to 133.5%, indicating that the optimal concentration was 3.0 mg / mL. Table 5 further shows that, under the same conditions, the crescent bone peptide prepared by ultrasound-assisted enzymatic hydrolysis induced a relative cell proliferation rate of 142.8% at 3.0 mg / mL, which is higher than the 135.6% induced by the traditional water bath enzymatic hydrolysis method. Table 5 compares the proliferation-promoting effects of various commercially available and control samples: at a concentration of 3.0 mg / mL, the proliferation rates of commercially available porcine limb bone peptides and fetal bovine bone peptides were 126.7% and 130.2%, respectively, while the proliferation rates of bone peptides from other parts of yak (skull, fossa, trachea, and nasal bone) ranged from 119.8% to 136.4%. In contrast, the proliferation rate of the yak crescent bone peptide of this invention reached as high as 142.8%, superior to all controls. This result fully demonstrates that this invention, through the synergistic effect of raw material screening, process optimization, and molecular weight-directed retention, has obtained a crescent bone peptide with stronger chondrocyte proliferation-promoting activity, providing crucial functional validation data for its repair of cartilage damage.

[0065] Table 4. Relative cell proliferation rates of crescent bone peptide at different concentrations

[0066] Table 5 Comparison of traditional enzymatic hydrolysis method and ultrasound-assisted enzymatic hydrolysis method

[0067] Table 6. Effects of different sources of bone peptides / cartilage-related bioactive substances on the relative proliferation rate of ATDC5 cells.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing yak cartilage peptides, characterized in that, Includes the following steps: Using yak crescent bone as raw material, ultrasonic-assisted enzymatic hydrolysis was performed under the action of papain and trypsin. After enzyme inactivation, peptides with a value of more than 3000 Da were collected to obtain yak cartilage peptides.

2. The preparation method according to claim 1, characterized in that, The amount of papain or trypsin added is 2% to 4% of the dry weight of the raw material.

3. The preparation method according to claim 1, characterized in that, The ultrasonic power of the ultrasonic machine-assisted enzymatic hydrolysis is 150~200 W, the heating power is 220~400 W, and the enzymatic hydrolysis time is 2~4 h.

4. Yak cartilage peptides obtained by the preparation method according to any one of claims 1 to 3.

5. A medicament for treating and / or preventing osteoarthritis and / or cartilage damage, characterized in that, It contains the yak cartilage peptide as described in claim 4.

6. The use of the yak cartilage peptide according to claim 4 in the preparation of a medicament for treating and / or preventing osteoarthritis and / or cartilage damage.

7. The application according to claim 6, characterized in that, The osteoarthritis mentioned includes osteoarthritis caused by oxidative stress.

8. The application according to claim 6 or 7, characterized in that, The treatment and / or prevention of osteoarthritis includes enhancing the antioxidant function of chondrocytes; The antioxidant function includes the ability to scavenge DPPH free radicals and / or the ability to scavenge ABTS free radicals.

9. The application according to claim 6, characterized in that, The cartilage injury includes degenerative cartilage disease or cartilage defects caused by exercise.

10. The application according to claim 6 or 9, characterized in that, The treatment and / or prevention of cartilage damage includes promoting chondrocyte proliferation.

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