Cartilage function improver

Abstract

To provide a cartilage function-improving agent, which can be taken on a daily basis for a long time, promotes the proliferation of chondrocytes, and / or has an effect of suppressing calcification of chondrocytes, and is useful for the prevention and treatment of various joint diseases, such as osteoarthritis, particularly knee osteoarthritis, and also provide a cartilage function-improving food or drink, cartilage function-improving nutritional composition, and cartilage function-improving feed, which contain the cartilage function-improving agent.SOLUTION: According to the present invention, a cartilage function-improving agent comprises, as an active ingredient, at least one selected from lactoperoxidase, cystatin, HMG-like protein, and / or degradation products thereof, which are proteins contained in milk and dairy products. The cartilage function-improving agent of the present invention has remarkable effects of correcting or regenerating deformed or disappeared cartilage, and is therefore useful for prevention and treatment of various joint diseases, such as osteoarthritis, particularly osteoarthritis of the knee.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cartilage function improving agent that has the effect of promoting the proliferation of chondrocytes and / or inhibiting the calcification of chondrocytes, has an excellent effect of correcting or regenerating deformed or lost cartilage, and is useful for the prevention and treatment of various joint diseases such as osteoarthritis, particularly knee osteoarthritis. The present invention also relates to a food or drink for improving cartilage function, a nutritional composition for improving cartilage function, and a feed for improving cartilage function that contain the cartilage function improving agent. [Background technology]

[0002] In recent years, the average life expectancy of Japanese people has exceeded 80 years, and about one in four people is over 65 years old, becoming an ultra-aging society. As a result, the incidence of musculoskeletal disorders has been steadily increasing, and in 2007, the Japanese Orthopaedic Association proposed a new term, "locomotor syndrome," to promote awareness among the public and doctors regarding the maintenance and promotion of musculoskeletal health and nursing care. Locomotive syndrome refers to a state in which the need for nursing care or the risk of needing nursing care is increased due to dysfunction of the musculoskeletal system. The musculoskeletal system generally includes organs that support and move the body, such as bones, joints, ligaments, spine, spinal cord, muscles, tendons, and peripheral nerves. Representative diseases and functional disorders of the musculoskeletal system include osteoporosis, sarcopenia, and osteoarthritis.

[0003] Osteoarthritis is said to affect 20-30% of people over 50 years old, making it the most common joint disease. In recent years, the number of patients has been increasing, and many osteoarthritis patients are forced to live a restricted life. The cause of this disease is thought to be the degeneration of joint components due to aging and genetic factors, or stress on the joints due to obesity, labor, and sports. The disease develops when the cartilage that exists on the surface of the epiphysis and is responsible for bone movement is reduced and deformed or lost, causing the joint to no longer move smoothly. In addition, cartilage does not have blood vessels or nerve cells, so once it is damaged, it is difficult to restore it naturally. Therefore, when the symptoms of osteoarthritis are mild, physical therapy such as heat therapy and traction, or palliative therapy using analgesics and anti-inflammatory drugs are used, but when the symptoms are severe, hyaluronic acid is injected into the joint or artificial joint replacement is performed by surgery. However, in order to cure osteoarthritis and improve the patient's QOL (Quality of Life), due to the nature of the disease, it is necessary to repair or regenerate the patient's own deformed or lost cartilage by safely consuming foods or active ingredients contained in foods on a daily basis for a long period of time. Cartilage is composed of chondrocytes differentiated from mesenchymal stem cells and cartilage matrix such as collagen, hyaluronic acid, and proteoglycan produced by the chondrocytes. In general, cartilage is calcified after chondrocytes proliferate, mature, and enlarge, and the cartilage matrix is ​​degenerated and replaced by bone tissue. However, in joints and intervertebral discs, chondrocytes maintain their properties throughout life, providing smooth mobility to the body without being replaced by bone. Therefore, in order to repair or regenerate deformed or lost cartilage and cure osteoarthritis, it is important to promote the proliferation of chondrocytes present in the cartilage and the production of cartilage matrix in chondrocytes, as well as to inhibit calcification of chondrocytes to prevent replacement by bone. Food ingredients that have the effect of promoting the proliferation of chondrocytes present in cartilage, the production of cartilage matrix in chondrocytes, or inhibiting the calcification of chondrocytes to prevent their replacement with bone, such as the promotion of matrix production in chondrocytes and the inhibition of calcification of chondrocytes by glucosamine hydrochloride (Non-Patent Document 1) and the promotion of matrix production in chondrocytes and the inhibition of calcification of chondrocytes by prolylhydroxyproline, a dipeptide derived from collagen (Non-Patent Document 2), have been disclosed. In addition, milk and dairy products are foods that have been eaten since ancient times and can be safely ingested on a daily basis for a long period of time, and it is known that the milk basic protein fraction and its decomposition products contained therein have the effect of promoting the production of cartilage matrix, and that lactoferrin has the effect of promoting the proliferation of chondrocytes and inhibiting the calcification of chondrocytes (Patent Documents 1, 2, Non-Patent Document 3). However, it is not known that lactoperoxidase, cystatin, HMG-like protein (High Mobility Group-like protein), and their decomposition products, which are proteins contained in milk and dairy products, have the effect of promoting the proliferation of chondrocytes and / or inhibiting the calcification of chondrocytes, and correct or regenerate deformed or lost cartilage. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2010 / 058679 [Patent Document 2] International Publication No. 2013 / 164992 [Non-patent literature]

[0005] [Non-Patent Document 1] Nakatani et al., Biological & Pharmaceutical Bulletin, 30:433-438 (2007) [Non-Patent Document 2] Nakatani et al., Osteoarthritis and Cartilage, 17:1620-1627 (2009) [Non-Patent Document 3] Takayama et al., Biometals, 23:477-484 (2010) Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of the present invention is to provide a cartilage function improving agent that can be ingested safely on a daily basis over a long period of time, has the effect of promoting the proliferation of chondrocytes and / or inhibiting the calcification of chondrocytes, is highly effective in correcting or regenerating deformed or lost cartilage, and is useful for the prevention and treatment of various joint diseases such as osteoarthritis, particularly knee osteoarthritis, as well as food and beverage products, nutritional compositions, and feed for improving cartilage function that contain the cartilage function improving agent.

[0007] The present inventors have conducted extensive research to solve the above problems and have discovered that proteins contained in milk and dairy products, such as lactoperoxidase, cystatin, HMG-like protein, and their degradation products, have the effect of promoting the proliferation of chondrocytes and / or inhibiting the calcification of chondrocytes, and thus correct or regenerate deformed or lost cartilage, thereby completing the present invention.

[0008] That is, the present invention has the following configuration. (1) A cartilage function improving agent comprising as an active ingredient one or more selected from lactoperoxidase, cystatin, HMG-like protein, and their decomposition products. (2) The cartilage function improving agent according to (1), wherein the lactoperoxidase, cystatin, and HMG-like protein are derived from mammalian milk. (3) The cartilage function improving agent according to (1) or (2), characterized in that the lactoperoxidase, cystatin, and HMG-like protein are derived from milk. (4) A cartilage function improving agent according to any one of (1) to (3), characterized in that the hydrolysis products of lactoperoxidase, cystatin, and HMG-like protein are obtained by hydrolyzing them with proteolytic enzymes. (5) The cartilage function improving agent according to (4), characterized in that the protease is at least one selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin and papain. (6) A food or drink, a nutritional composition, or a feed for improving cartilage function, which comprises the cartilage function improver according to any one of (1) to (5). (7) A method for improving cartilage function by ingesting 1 mg or more of lactoperoxidase, cystatin, HMG-like protein, and / or their degradation products according to any one of (1) to (5) per day. Effect of the Invention

[0009] The cartilage function improving agent of the present invention has the effect of promoting the proliferation of chondrocytes and / or inhibiting the calcification of chondrocytes, and is highly effective in correcting or regenerating deformed or lost cartilage, and is therefore useful in the prevention and treatment of various joint diseases such as osteoarthritis, particularly knee osteoarthritis. [Brief description of the drawings]

[0010] [Figure 1] Mice were divided into six groups and fed a standard diet containing a standard amount of phosphorus, a high-phosphorus diet, or a high-phosphorus diet plus lactoperoxidase, etc., and after 10 weeks of feeding, the thickness of the articular cartilage layer in the right hind leg was compared. [Diagram 2] Mice were divided into six groups and fed a standard diet containing a standard amount of phosphorus, a high-phosphorus diet, or a high-phosphorus diet plus lactoperoxidase, etc., and after 10 weeks of feeding, the number of chondrocytes present in the articular cartilage layer of the right hind leg was compared. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The feature of the present invention is that the lactoperoxidase, cystatin, HMG-like protein, and / or their decomposition products, which are proteins contained in milk and dairy products, are used as active ingredients.The lactoperoxidase, cystatin, and HMG-like protein used in the present invention can be prepared from mammalian milk such as cow's milk, human milk, goat's milk, and sheep's milk, or can be chemically synthesized, produced by genetic engineering, or purified from blood or organs, and can also use the reagents of purified and commercially available lactoperoxidase, cystatin, and HMG-like protein.In addition, the lactoperoxidase, cystatin, and HMG-like protein used in the present invention can be prepared from mammalian milk such as cow's milk, human milk, goat's milk, and sheep's milk, or can be chemically synthesized, produced by genetic engineering, or purified from blood or organs, and can also use the reagents of purified lactoperoxidase, cystatin, and HMG-like protein. Decomposition products of HMG-like proteins can be prepared by treating them with proteolytic enzymes.

[0012] The method of preparing the lactoperoxidase of the present invention, which is the active ingredient, is known to be, for example, the method of purifying it from milk using polysaccharide affinity carrier with sulfonic group (JP Patent Publication No. 109400 / 1991).In addition, the decomposition product of the lactoperoxidase of the present invention, which is the active ingredient, can be prepared by using proteolytic enzymes such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, V8 protease, etc., so that the molecular weight is 10,000 or less (preferably, the molecular weight is 500 or more).

[0013] As a method for preparing cystatin, which is an active ingredient of the present invention, for example, a method for purifying it from milk using column chromatography (JP Patent Publication No. 2000-281587) is known. In addition, a decomposition product of cystatin, which is an active ingredient of the present invention, may be prepared by, for example, preparing cystatin using a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease so that the molecular weight is 8,000 or less (preferably, 500 or more).

[0014] As a method for preparing the HMG-like protein, which is an active ingredient of the present invention, for example, a method for purifying it from milk using column chromatography (JP Patent Publication 2007-227403) is known. In addition, the decomposition product of the HMG-like protein, which is an active ingredient of the present invention, may be prepared by, for example, preparing the HMG-like protein using a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease so that the molecular weight is 6,000 or less (preferably, 500 or more).

[0015] In the present invention, any one or more of lactoperoxidase, cystatin, HMG-like protein, and their degradation products may be used as is in the cartilage function improving agent of the present invention, but if necessary, they can also be formulated into powder, granules, tablets, capsules, drinks, etc. according to conventional methods.

[0016] In the present invention, when the active ingredients lactoperoxidase, cystatin, HMG-like protein, and their decomposition products are added to formulations or foods, beverages, etc., there is no particular limitation on the method of addition, the method of blending, etc. For example, to add or blend in solution, any one or more of lactoperoxidase, cystatin, HMG-like protein, and their decomposition products can be suspended or dissolved in deionized water, stirred and mixed, and then formulated, or prepared in the form of foods, beverages, or feeds for use. The condition of stirring and mixing is that lactoperoxidase, cystatin, HMG-like protein, and their decomposition products can be mixed uniformly, and it is also possible to use an ultra-disperser, TK homomixer, etc. for stirring and mixing. In addition, the solution of the composition can be used after desalting or concentrating with an RO membrane, UF membrane, etc., or after lyophilization, as necessary, so that it can be easily used in formulations, foods, beverages, and feeds. In the present invention, sterilization treatment that is usually used in the manufacture of medicines, foods, beverages, and feeds is possible, and dry heat sterilization is also possible in powder form. As described above, the cartilage function improving agent of the present invention can be made into various forms such as liquid, gel, powder, granules, etc., and after being formulated, it can be incorporated into nutritional supplements, foods and beverages such as yogurt, milk drinks, wafers, and nutritional compositions.

[0017] The cartilage function improving agent of the present invention comprises lactoperoxidase, cystatin, HMG-like protein, and any one or more of their decomposition products as active ingredients, and can also use diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants, etc. that are commonly used in formulation.As excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium aluminometasilicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, calcium carboxymethylcellulose, etc. can be used alone or in combination of two or more. The cartilage function improving agent of the present invention can also be used in combination with stabilizers, sugars, lipids, flavors, vitamins, minerals, flavonoids, polyphenols, etc., and can be appropriately mixed and used when preparing foods, beverages, and feeds. It can also be used together with other components that have the effect of improving cartilage function, such as glucosamine hydrochloride and lactoferrin.

[0018] In the cartilage function improving agent of the present invention, as shown in the test example described below, by orally ingesting 1mg or more of any one of lactoperoxidase, cystatin, HMG-like protein, and their decomposition products per kg of body weight to mice, it is possible to promote the proliferation of chondrocytes and / or inhibit the calcification of chondrocytes.Since the amount of intake in experimental animals corresponds to the amount of intake per adult in terms of blood drug concentration (Nakajima Mitsuyoshi (1993) "Vol. 8 Drug Efficacy Evaluation" Hirokawa Shoten, pp. 2-18), generally, by ingesting 1mg or more of any one of lactoperoxidase, cystatin, HMG-like protein, and their decomposition products per adult per day, it is expected to improve the function of cartilage, particularly to prevent and treat osteoarthritis.Therefore, any one of lactoperoxidase, cystatin, HMG-like protein, and their decomposition products can be formulated or mixed into food, drink, etc. so as to ensure the required amount. For example, in order to allow an adult to take in 1mg or more of lactoperoxidase, cystatin, HMG-like protein, and any one or more of their decomposition products per day, depending on the form of medicine, food, drink, or feed, the final product should contain 0.0005%-5% (weight / weight), preferably 0.05-2.5% (weight / weight), of lactoperoxidase, cystatin, HMG-like protein, and any one or more of their decomposition products relative to the total mass.

[0019] The present invention will be described in detail below with reference to examples and test examples. However, these are merely illustrative and the present invention is not limited to these in any way. EXAMPLES

[0020] A column (diameter 5 cm x height 30 cm) packed with 400 g of sulfonated chitopearl (manufactured by Fujiboseki Co., Ltd.) which is a cation exchange resin was thoroughly washed with deionized water, and 40 L of non-sterilized skim milk (pH 6.7) was passed through the column at a flow rate of 25 ml / min. After passing the liquid through, the column was thoroughly washed with deionized water and eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. The eluted fraction containing lactoperoxidase was then adsorbed onto an S-Sepharose FF column (manufactured by GE Healthcare Co., Ltd.), thoroughly washed with deionized water, and equilibrated with 10 mM phosphate buffer (pH 7.0), after which the adsorbed fraction was eluted with a linear gradient of 0 to 2.0 M sodium chloride, and the fraction containing lactoperoxidase was collected. Then, this fraction is treated by gel filtration chromatography using HiLoad 16 / 60 Superdex 75 pg (GE Healthcare Co., Ltd.), to obtain 3.0g of lactoperoxidase.The purity of the lactoperoxidase thus obtained is 94%, and it can be used as cartilage function improving agent (Example product 1) as it is. EXAMPLES

[0021] 1g of lactoperoxidase obtained in Example 1 is dissolved in 200ml of water, and the protease pancreatin (Sigma-Aldrich) is added so that the final concentration is 0.01% by weight, and the enzyme is treated at 37°C for 5 hours.Then, the enzyme is inactivated by heating at 90°C for 5 minutes, and then lyophilized to obtain 0.8g of lactoperoxidase decomposition product.Then, the molecular weight of the lactoperoxidase decomposition product obtained in this way is less than 10,000, and it can be used as cartilage function improving agent (Example product 2) as it is. EXAMPLES

[0022] 1g of lactoperoxidase obtained in Example 1 is dissolved in 200ml of water, and trypsin (Sigma-Aldrich Co., Ltd.), which is a protease, is added to the mixture so that the final concentration is 0.01% by weight, and the mixture is treated with enzyme at 37 ℃ for 5 hours.Then, the mixture is heated at 90 ℃ for 5 minutes to inactivate enzyme, and then freeze-dried to obtain 0.9g of lactoperoxidase decomposition product.The molecular weight of the lactoperoxidase decomposition product thus obtained is 10,000 or less, and it can be used as cartilage function improving agent (Example product 3) as it is. EXAMPLES

[0023] A column packed with 3,000 g of S-Sepharose was thoroughly washed with deionized water, 10,000 L of skim milk was passed through, and the column was thoroughly washed with deionized water, after which it was eluted with a linear concentration gradient of 0.1 to 1.0 M sodium chloride. The obtained fraction was heated at 90°C for 10 minutes and centrifuged to remove precipitates. The eluted fraction containing cystatin was then fractionated again by MonoS ion exchange chromatography. Furthermore, this fraction was subjected to MonoQ ion exchange chromatography and Superose12 gel filtration chromatography using an FPLC system, and further to hydroxyapatite chromatography and C4 reverse phase chromatography using an HPLC system in sequence to obtain 58 mg of cystatin. The cystatin thus obtained can be used as a cartilage function improving agent (Example Product 4) as it is. EXAMPLES

[0024] 10,000 L of 5% whey protein solution was heated at 90°C for 10 minutes and centrifuged to remove precipitates. Furthermore, a carrier in which carboxymethylated papain was bound to Tresyl-Toyopearl (manufactured by Tosoh Corporation) was packed into a column, which was then equilibrated with 0.5M sodium chloride solution, and the above whey protein solution was passed through. After passing through, the column was washed sequentially with 0.5M sodium chloride solution and 0.5M sodium chloride solution containing 0.1% Tween20. Next, cysteine ​​protease was eluted with 20 mM acetic acid-0.5M sodium chloride solution. The eluted fraction was immediately neutralized with 1M sodium hydroxide solution, and fractionated by MonoS anion exchange chromatography, hydroxyapatite chromatography and C4 reverse phase chromatography in an HPLC system, to obtain 48 mg of cystatin. The cystatin thus obtained can be used as a cartilage function improving agent (Example Product 5) as it is. EXAMPLES

[0025] 25 mg of the cystatin obtained in Example 4 was dissolved in 100 ml of water, and pancreatin (Sigma-Aldrich) was added to a final concentration of 1%, followed by enzymatic treatment at 37°C for 5 hours. The enzyme was then inactivated by heating at 90°C for 5 minutes, and the mixture was lyophilized to obtain 23 mg of a cystatin degradation product (Example Product 6A). Similarly, 25 mg of the cystatin obtained in Example 5 was treated to obtain 24 mg of a cystatin degradation product (Example Product 6B). The molecular weight of the cystatin degradation product thus obtained was 8,000 or less, and it can be used as a cartilage function improving agent (Example Products 6A and 6B) as it is. EXAMPLES

[0026] A column packed with 3,000 g of sulfonated chitopearl (manufactured by Fujiboseki Co., Ltd.) was thoroughly washed with deionized water, and then 300 L of skim milk was passed through. After thorough washing with deionized water, the basic proteins adsorbed on the resin were eluted with a linear concentration gradient of 0.1 M to 1.0 M NaCl in 0.02 M carbonate buffer (pH 6.7) and collected. Next, the eluted fraction containing HMG-like protein was fractionated by eluting with a linear concentration gradient of 0.1 M to 1.0 M NaCl using S-Sepharose cation exchange chromatography equilibrated with 0.1 M phosphate buffer (pH 6.5), and the obtained fraction was heated at 90°C for 10 minutes and then centrifuged to remove precipitates. This fraction was then fractionated by elution with a linear gradient of 0.1M to 1.0M NaCl using Mono Q ion exchange chromatography equilibrated with 0.1M phosphate buffer (pH 6.5), and further fractionated using Sepharose 12 gel filtration chromatography equilibrated with 0.1M phosphate buffer (pH 6.5). Finally, the fraction was treated with high performance liquid chromatography using C4 reverse phase chromatography to obtain 135mg of HMG-like protein. The HMG-like protein thus obtained can be used as a cartilage function improving agent (Example Product 7) as it is. EXAMPLES

[0027] 25 mg of the HMG-like protein obtained in Example 7 was dissolved in 100 ml of water, and trypsin (Sigma-Aldrich), a protease, was added to a final concentration of 0.01% by weight, followed by enzyme treatment at 37°C for 5 hours. The enzyme was then inactivated by heating at 90°C for 5 minutes, and lyophilized to obtain 24 mg of HMG-like protein degradation product. The molecular weight of the HMG-like protein degradation product thus obtained was 6,000 or less, and it can be used as it is as a cartilage function improving agent (Example Product 8). (Test Example 1)

[0028] (Cell experiments) The effect of promoting the proliferation of chondrocytes was evaluated using lactoperoxidase (Example Product 1), lactoperoxidase decomposition product (Example Product 2), cystatin (Example Product 4), cystatin decomposition product (Example Product 6B), HMG-like protein (Example Product 7), HMG-like protein decomposition product (Example Product 8), a mixture of Examples Products 1 and 4, and a mixture of Examples Products 5 and 7. Mouse-derived chondrocyte precursor cell line (ATDC5) was cultured in a 96-well cell culture plate at 5 × 10 per well. 3 The cells were seeded at 1000 x 1000 cells / well and cultured in a Dulbecco's modified Eagle medium / Ham's F-12 mixed medium containing 5% fetal bovine serum at 37°C in an incubator under a 5% CO2 atmosphere for 24 hours. After that, the entire medium was replaced, and Example Products 1, 2, 4, 6B, 7, 8, a mixture of Example Products 1 and 4, and a mixture of Example Products 5 and 7 dissolved in PBS(-) were added to the medium to a final concentration of 1μg / mL, and cultured for 48 hours. For the negative control, PBS(-) was used as the solvent. The entire medium was removed, and cell proliferation reagent WST-1 (Roche Diagnostics) was added to the medium so that it was contained in 1 / 10 of the amount, followed by culture for 6 hours and measuring the absorbance at 440 nm using a plate reader. WST-1 is reduced to formazan dye by the metabolic activity of living cells, so the amount of formazan dye is proportional to the number of cells with metabolic activity. Therefore, the absorbance value reflecting the amount of formazan dye was used as an index of the chondrocyte proliferation promoting effect. The results are shown in Table 1.

[0029] [Table 1]

[0030] As a result, when the lactoperoxidase of Example 1, the lactoperoxidase decomposition product of Example 2, the cystatin of Example 4, the cystatin decomposition product of Example 6B, the HMG-like protein of Example 7, the HMG-like protein decomposition product of Example 8, and the mixture of Example 1 and 4, and the mixture of Example 5 and 7 are added to medium, the number of cartilage precursor cells increases significantly compared with the case where PBS (-) is added to medium.Therefore, it is found that the lactoperoxidase, cystatin, HMG-like protein and their decomposition products of the present invention promote the proliferation of chondrocytes. (Test Example 2)

[0031] (Cell experiments) The inhibitory effect on calcification of chondrocytes was evaluated using lactoperoxidase (Example Product 1), lactoperoxidase decomposition product (Example Product 3), cystatin (Example Product 5), cystatin decomposition product (Example Product 6A), HMG-like protein (Example Product 7), HMG-like protein decomposition product (Example Product 8), a mixture of Examples Products 1 and 7, and a mixture of Examples Products 4 and 8. Mouse-derived chondrocyte precursor cell line (ATDC5) was cultured in a 96-well cell culture plate at 3 x 10 per well. 3The cells were seeded one by one and cultured in a Dulbecco's modified Eagle medium / Ham's F-12 mixed medium containing 5% fetal bovine serum at 37°C in an incubator under a 5% CO2 atmosphere for 24 hours. After that, the entire medium was replaced, and Example products 1, 3, 5, 6A, 7, 8, a mixture of Example products 1 and 7, and a mixture of Example products 4 and 8 dissolved in PBS(-) were added to the medium to a final concentration of 1 μg / mL, and the cells were cultured for 7 days while changing the medium every day. For the negative control, PBS(-) used as the solvent was used. After the culture was completed, the cells were fixed in 20% formalin solution for 20 minutes, washed with water, and left to stand for 15 minutes in water. Then, they were stained for 20 minutes at 37°C with alkaline phosphatase (ALP) staining solution prepared by dissolving Naphthol AS-BI phosphate and Fast Red Violet LB Salt in 0.05M 2-Amino-2-methyl-1,3-propanediol (AMP) solution. After staining, the cells were washed five times with water and air-dried. The stained area was calculated using image processing software from the stained image of the culture plate captured with a scanner. The stained area was shown as a relative value with the negative control value set as 1. In addition, since ALP activity is considered to be an early marker of calcification in chondrocytes, the smaller the area of ​​the stained area, the more calcification is suppressed. The results are shown in Table 2.

[0032] [Table 2]

[0033] As a result, when the lactoperoxidase of Example 1, the lactoperoxidase decomposition product of Example 3, the cystatin of Example 5, the cystatin decomposition product of Example 6A, the HMG-like protein of Example 7, the HMG-like protein decomposition product of Example 8, and the mixture of Example 1 and 7, and the mixture of Example 4 and 8 are added to the medium, the stained area of ​​ALP staining is significantly reduced compared with the case where PBS (-) is added to the medium.Therefore, it is found that the lactoperoxidase, cystatin, HMG-like protein and their decomposition products of the present invention suppress the calcification of cartilage cell. (Test Example 3)

[0034] (Animal experiments) Using the lactoperoxidase of Example Product 1, the lactoperoxidase decomposition product of Example Product 3, the cystatin of Example Product 4, and the HMG-like protein of Example Product 7, the effect on the early degeneration of articular cartilage layer was verified. 10-week-old male C57BL / 6J mice were divided into 6 groups, each with 10 mice, so that there was no variation in body weight. The group that did not take Example Product but took a standard amount of phosphorus (0.2g / 100g-feed) (standard diet) was group A, and the group that did not take Example Product but took a high amount of phosphorus (1.2g / 100g-feed) (high phosphorus diet) was group B. In addition, the group that took Example Product 1 was group C, the group that took Example Product 4 was group D, the group that took Example Product 7 was group E, and the group that took Example Product 3 was group F, and the daily intake of Example Product was mixed into the high phosphorus diet so that it was 1mg per kg of mouse body weight. After 10 weeks of feeding, the right hind limbs were removed and fixed in 10% neutral buffered formalin, and paraffin-embedded sections of the knee joints were prepared and stained with hematoxylin and eosin. The thickness of the articular cartilage layer and the number of chondrocytes present in the articular cartilage layer were measured to evaluate the effect of treatment on the early degeneration of the articular cartilage layer. The results are shown in Figures 1 and 2.

[0035] As a result, in group B, which does not take example product but takes high phosphorus diet, compared with group A, which does not take example product but takes standard diet, the thickness of articular cartilage layer and the number of chondrocytes in articular cartilage layer are significantly decreased by excessive phosphorus intake.On the other hand, in group C to group F, which takes example products 1, 3, 4, and 7, the decrease in the thickness of articular cartilage layer and the number of chondrocytes are significantly suppressed compared with group B.Therefore, it is found that the lactoperoxidase, cystatin, HMG-like protein and their decomposition products of the present invention suppress the early degeneration of articular cartilage. EXAMPLES

[0036] (Preparation of tablets for improving cartilage function) After mixing the raw materials according to the composition shown in Table 3, the mixture was molded into 1g tablets by a conventional method and compressed to produce tablets for improving cartilage function. Each 1g tablet contained 1mg of lactoperoxidase of Example Product 1.

[0037] [Table 3] EXAMPLES

[0038] (Preparation of nutritional composition for improving cartilage function) 0.5g of the lactoperoxidase decomposition product of Example 3 is dissolved in 4,999.5g of deionized water, heated to 45℃, and then stirred and mixed at 6,000rpm for 30 minutes with TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.), to obtain a solution containing 0.5g / 5.0kg of the lactoperoxidase decomposition product of Example 3. 5.0kg of this solution is added with the raw materials of Table 4, stirred and mixed for another 10 minutes, then filled into a 200ml retort pouch, and sterilized at 121℃ for 20 minutes with a retort sterilizer (first class pressure vessel, TYPE: RCS-4CRTGN, manufactured by HISAKA MFG. CO., LTD.) to produce 50kg of the liquid nutritional composition for improving cartilage function of the present invention. Note that this liquid nutritional composition for improving cartilage function contains 1mg of the lactoperoxidase decomposition product of Example 3 per 100g.

[0039] [Table 4] EXAMPLES

[0040] (Preparation of beverage for improving cartilage function) 140g of skim milk powder was dissolved in 569.95g of deionized water, and then 0.05g of cystatin (Example Product 4) was dissolved. After heating to 45°C, the mixture was stirred and mixed for 30 minutes at 9,500 rpm using an Ultra Disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan Co., Ltd.). The raw materials in Table 5 were added, and the mixture was stirred and mixed for another 15 minutes. The mixture was then filled into a 100ml glass bottle, sterilized at 95°C for 15 seconds, and then sealed to prepare 10 bottles (100ml) of the beverage for improving cartilage function of the present invention. This beverage for improving cartilage function contained 5mg of cystatin (Example Product 4) per 100ml.

[0041] [Table 5] EXAMPLES

[0042] (Preparation of food for improving cartilage function in dogs) 0.1g of the HMG-like protein of Example Product 7 was dissolved in 99.9g of deionized water, heated to 45°C, and then stirred and mixed for 40 minutes at 9,500 rpm with an Ultra Disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan Co., Ltd.) to obtain a solution containing 0.1g / 100g of the HMG-like protein of Example Product 7. The raw materials in Table 6 were added to 100g of this solution, stirred and mixed for another 40 minutes, and then sterilized at 120°C for 4 minutes to produce 1kg of the canine cartilage function improving feed of the present invention. This canine cartilage function improving feed contained 10mg of the HMG-like protein of Example Product 7 per 100g.

[0043] [Table 6]

Claims

1. A chondrocyte proliferation promoter comprising as an active ingredient at least one selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and degradation products thereof.

2. An agent for promoting the proliferation of chondrocytes and inhibiting calcification of chondrocytes, comprising as an active ingredient at least one selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and degradation products thereof.

3. The agent for promoting the proliferation of chondrocytes according to claim 1, characterized in that the lactoperoxidase, cystatin, and HMG-like protein are derived from mammalian milk, or the agent for promoting the proliferation of chondrocytes and inhibiting calcification of chondrocytes according to claim 2.

4. The agent for promoting the proliferation of chondrocytes or the agent for promoting the proliferation of chondrocytes and inhibiting the calcification of chondrocytes according to any one of claims 1 to 3, characterized in that the lactoperoxidase, cystatin, and HMG-like protein are derived from milk.

5. The agent for promoting the proliferation of chondrocytes or the agent for promoting the proliferation of chondrocytes and inhibiting the calcification of chondrocytes according to any one of claims 1 to 4, characterized in that the hydrolysis products of lactoperoxidase, cystatin, and HMG-like protein are obtained by hydrolyzing them with proteolytic enzymes.

6. The agent for promoting chondrocyte proliferation or an agent for promoting chondrocyte proliferation and inhibiting chondrocyte calcification as described in claim 5, characterized in that the protease is at least one selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin and papain.

7. A food, beverage, nutritional composition or feed for promoting the proliferation of chondrocytes, or for promoting the proliferation of chondrocytes and inhibiting the calcification of chondrocytes, characterized by containing as an active ingredient one or more selected from lactoperoxidase, cystatin, HMG-like protein, and their degradation products.

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