JP2006327979A - Articular cartilage lesion treatment-accelerating agent, tendon lesion treatment-accelerating agent and food and drink containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an articular cartilage lesion treatment-accelerating agent and a tendon lesion treatment-accelerating agent containing collagen peptide derived from fishes and high in safety as an active ingredient, and to provide foods and drinks containing these agents. <P>SOLUTION: The collagen peptide derived from fishes is contained as an active ingredient for the articular cartilage lesion treatment-accelerating agent and the tendon lesion treatment-accelerating agent. The articular cartilage lesion treatment-accelerating agent and the tendon lesion treatment-accelerating agent may contain the collagen peptide derived from fishes and N-acetylglucosamine as active ingredients. The foods and drinks labeled that they are usable for accelerating articular cartilage lesion treatment or accelerating tendon lesion treatment are obtained by containing the collagen derived from fishes as an active ingredient. The foods and drinks may contain the collagen peptide derived from fishes and N-acetylglucosamine as active ingredients. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an articular cartilage injury healing promoter and a tendon injury healing promoter containing a fish-derived collagen peptide as an active ingredient, and a food or drink containing them.

  Joints, muscles, tendons, etc. play an important role in the movement of animals. For example, a joint, which is a joint between bones, can be bent at a free angle, and the tip of the muscle becomes a tendon and adheres to the bone, giving the skeleton and joints mobility and support.

  Articular cartilage rich in viscoelasticity is present on the surface of the joint where the bone is in contact, protecting the joint and smoothing the joint surface to smooth the movement of the joint.

  However, it is known that when articular cartilage is lost or damaged due to intense exercise or injury, the articular cartilage is poor in natural healing power and thus deteriorates and easily shifts to osteoarthritis. Conventional articular cartilage regeneration methods include: i) a method of transplanting an intact cartilage to a damaged place (mosaic plastic), ii) using an arthroscope, taking out the articular cartilage and taking it out A method (curtisel) for transplanting chondrocytes in articular cartilage into damaged cartilage is known.

  Similarly, tendon damage may occur due to intense exercise or injury, but as a treatment for tendon damage, the torn tendon is sutured, the affected area is casted, the tendon is fused, and rehabilitation is performed. The mainstream method is to rebuild the affected area to a normal state.

  However, the rate of fusion of the sutured tendon is very slow, and after completion of the fusion, the tendon strength is substantially reduced and tends to result in loss of mobility.

  Therefore, in recent years, not only surgical treatment as described above but also attempts to orally administer collagen and N-acetylglucosamine in order to promote healing of cartilage and tendon damage. No. 1 discloses a joint-strengthening drink characterized by containing a collagen peptide and a glucosamine salt and having a pH of 2-5.

  Patent Document 2 listed below contains an oral joint injury therapeutic agent characterized by containing at least one selected from collagen and collagen peptides, amino sugar, mucopolysaccharide and uronic acid, or Functional foods are disclosed.

  Patent Document 3 listed below discloses a composition containing, as essential components, hydrolyzed collagen protein having a molecular weight of 1 to 300 kilodaltons; and at least one of glucosamine or a pharmaceutically acceptable salt thereof.

Patent Document 4 listed below discloses an oral composition characterized by containing an extract of cat's claw, glucosamine, chondroitin sulfate, and collagen.
JP 2002-125638 A JP 2003-48850 A JP 2001-524080 A JP 2003-155250 A

  However, in the conventional products for promoting healing of cartilage and tendon damage, collagen and collagen peptides derived from livestock such as cattle and pigs are used, and recent mad cow disease, swine and foot-and-mouth disease, and bird flu. There was a problem in terms of safety due to the occurrence of livestock plague.

  On the other hand, although collagen and collagen peptides derived from fish have high safety, it has hardly been known whether they have the same physiological activity as the above-described collagen and collagen peptides derived from livestock.

  Accordingly, an object of the present invention is to provide an articular cartilage injury healing promoter and tendon injury healing promoter containing a highly safe collagen peptide derived from fish as an active ingredient, and a food or drink containing them.

  In order to achieve the above object, one of the present invention is to provide an articular cartilage injury healing promoter characterized by containing a fish-derived collagen peptide as an active ingredient.

  Another aspect of the present invention is to provide an articular cartilage damage healing promoter characterized by containing fish-derived collagen peptides and N-acetylglucosamine as active ingredients.

  The articular cartilage injury healing promoter of the present invention is safer because it contains a fish-derived collagen peptide as an active ingredient. In addition, by containing a fish-derived collagen peptide excellent in digestibility and N-acetylglucosamine as active ingredients, it is possible to expect more excellent healing of articular cartilage damage due to the synergistic effect of these ingredients.

  Another aspect of the present invention provides a tendon damage healing promoter characterized by containing a fish-derived collagen peptide as an active ingredient.

  Another aspect of the present invention is to provide a tendon injury healing promoter characterized by containing fish-derived collagen peptides and N-acetylglucosamine as active ingredients.

  Since the tendon injury healing promoter of this invention contains the collagen peptide derived from fish as an active ingredient, it is safer. Moreover, by containing the collagen peptide derived from fish excellent in digestibility and absorptive and N-acetylglucosamine as an active ingredient, the healing promotion of the superior tendon damage can be anticipated by the synergistic effect of these ingredients.

  Another aspect of the present invention is to provide a food or drink with a label indicating that it is used for promoting healing of articular cartilage damage, comprising a collagen peptide derived from fish as an active ingredient. .

  In addition, another aspect of the present invention is a food and drink with a label indicating that it is used for promoting healing of articular cartilage damage, comprising a collagen peptide derived from fish and N-acetylglucosamine as active ingredients. Goods.

  Another aspect of the present invention is to provide a food or drink with a label indicating that it is used for promoting healing of tendon injury, comprising a fish-derived collagen peptide as an active ingredient.

  Another aspect of the present invention is a food or drink with a label indicating that it is used for promoting healing of tendon damage, comprising a collagen peptide derived from fish and N-acetylglucosamine as active ingredients. Is to provide.

  The food / beverage product of the present invention contains fish-derived collagen peptides as active ingredients, so it is highly safe and can be expected to promote healing of articular cartilage damage and tendon damage by eating. In addition, by containing a fish-derived collagen peptide excellent in digestibility and N-acetylglucosamine as active ingredients, the synergistic effect of these ingredients can further promote healing of articular cartilage damage and tendon damage. I can expect. In the present invention, “with a label” means not only those directly displayed on packaging materials such as product containers, bags and boxes, but also printed materials and products enclosed in product bags and boxes. This means that it includes information displayed on pamphlets, sales promotion materials for agents, etc., as well as product information described on Internet homepages.

  According to the present invention, by containing a fish-derived collagen peptide as an active ingredient, a safer joint cartilage injury healing promoter and tendon injury healing promoter can be provided. Moreover, the food-drinks provided with physiological functions, such as joint cartilage damage healing promotion and tendon damage healing promotion, can be provided by mix | blending these with food-drinks.

  As the fish-derived collagen peptide used in the present invention, those commercially available as food materials can be used, but those produced by the following method described in JP-A-2003-238597 are preferably used.

(1) Process for preparing an extract containing collagen from fish raw materials There is no limitation on the fish species that are used as collagen raw materials, but for example, cod, flatfish, salmon, trout, sharks, rays, tilapia , Horse mackerel, mackerel, snapper, grouper, skipjack, tuna, swordfish, puffer fish, scorpion, Thailand, eel, sardines, herring, saury, rockfish, yellowtail, etc. Tuna such as bluefin tuna, southern bluefin tuna, yellowfin tuna, butterfly tuna, and bigeye tuna, cod, such as cod, komai, and grasshopper, and fish skin of flounder such as flounder, scorpion, halibut, flounder, etc. Thai fish scales are preferably used.

  As a method for extracting collagen from the fish raw material, a known method can be adopted, but a method of heating extraction or pressure heating extraction by adding water is preferably used. According to this extraction method, insoluble proteins such as elastin which are hardly soluble in water are not eluted, and collagen can be extracted efficiently. In order to increase the extraction efficiency, the fish raw material is preferably used after being cut, shredded, pulverized, or minced by appropriate means, and the fish bones and fish scales are used after being decalcified with hydrochloric acid or the like in advance. preferable.

  For example, 100 to 500 parts by mass of water may be added to 100 parts by mass of the fish raw material and heated and extracted at 60 to 100 ° C. for 0.5 to 3 hours. Moreover, what is necessary is just to extract for 0.5 to 3 hours at 110-120 degreeC, when extracting by pressurization heating.

(2) Process of enzymatically degrading an extract containing collagen The collagen-containing extract obtained in the above process is treated with a protein hydrolase to peptideize collagen.

  The protein hydrolase is not particularly limited, and neutral protease, alkaline protease, acidic protease, enzyme preparation containing them, or the like can be used. As a commercially available protein hydrolase preparation, a protein hydrolase generally used in food production can be used. For example, the trade name “Protease N” (manufactured by Amano Enzyme, neutral protease), the trade name “Protease P- 3 ”(manufactured by Amano Enzyme, alkaline protease), trade name“ Sumiteam AP ”(manufactured by Shin Nippon Chemical Industry Co., Ltd., acidic protease) and the like can be used.

  The amount of the enzyme added, the reaction time, and the treatment temperature can be appropriately set. Usually, the amount of the enzyme added is preferably 0.2 to 5% by mass and more preferably 1 to 2% by mass with respect to the solid content of the treatment liquid. The reaction time is preferably 0.5 to 5 hours, more preferably 1 to 3 hours. The reaction temperature is preferably 30 to 70 ° C, more preferably 45 to 65 ° C. If the enzyme reaction conditions are outside the above range, collagen may not be sufficiently hydrolyzed, or conversely, hydrolysis may proceed excessively, resulting in a decrease in yield. In addition, after completion | finish of an enzyme reaction, it is preferable to inactivate an enzyme by heating.

(3) A step of concentrating and purifying the enzyme degradation product of the extract using a reverse osmosis membrane. The enzyme degradation product obtained in the above step is subjected to a reverse osmosis membrane treatment to recover a concentrate. Concentrate and purify using reverse osmosis membranes to provide fish-specific taste and odor components (for example, amino acids, oligopeptides, nucleic acids, organic acids, minerals, volatile sulfur compounds, fatty acids, nitrogen compounds, carbonyl compounds, etc. ) And arsenic can be easily removed in one step. Further, collagen peptides having a small molecular weight can be recovered without loss. The collected concentrated liquid can be used as it is or after being appropriately dried and powdered.

  As the reverse osmosis membrane used here, one having a salt rejection of 10 to 50% is preferably used. Examples of such reverse osmosis membranes include trade name “NTR-7410”, trade name “NTR-7430”, trade name “NTR-7450” (all manufactured by Nitto Denko).

  When the salt rejection of the reverse osmosis membrane is outside the above range, it is not preferable because impurities such as a taste component, an odor component, and arsenic are not sufficiently removed, and loss of the collagen peptide increases.

  The conditions for the reverse osmosis membrane treatment can be set as appropriate. Usually, the treatment liquid is adjusted to a solid content concentration of 15% by mass or less, and the solid content concentration is circulated at a pH of 4 to 7 and a liquid temperature of 60 ° C. or less. It is preferable to concentrate until it becomes 25 mass% or more. At this time, it is preferable to add 1 to 10 times, preferably 3 to 5 times the amount of water of the stock solution while appropriately adding water to allow the solution to permeate. Impurities can be efficiently removed by repeating the hydration operation.

  In addition, it is preferable to perform decoloring and deodorizing treatment in any one of the above steps (1) to (3), and in particular, in the step before (3), the extract containing the collagen or its enzyme It is preferable to decolorize and deodorize the decomposed product. By performing decolorization and deodorization treatment in the step before (3) above, it is easy to remove coloring and odor caused by extraction and enzymatic decomposition, and it is possible to prevent clogging of the reverse osmosis membrane, so the efficiency of membrane treatment is improved. Can be improved.

  As the decolorization and deodorization methods, it is preferable to use a solid adsorbent from the viewpoint of workability. As the solid adsorbent, activated carbon, alumina, silica gel, activated clay and the like can be used in appropriate combination, and activated carbon is particularly preferably used among them.

  For example, if 0.5-30 mass% activated carbon is added to the extract containing collagen or its enzymatic decomposition solution, and stirred at 50-90 ° C. for 15 minutes-3 hours, then filtered to recover the liquid part Good. It can also be carried out by passing through a column filled with activated carbon or the like.

  The fish-derived collagen peptide obtained by the above production method preferably has a free amino acid content of 1.0% by mass or less and an arsenic content of 2 ppm or less in the solid content. Since such a collagen peptide has a low amino acid content, even if it is added to food and drink at a high concentration, discoloration during heating and adverse effects on taste do not occur. In addition, since the arsenic content is low, the safety is very high even when blended in food or drink at a high concentration. As such a fish-derived collagen peptide, for example, trade name “Marine Matrix” (manufactured by Yaizu Suisan Chemical Co., Ltd.) can be used.

  In the present invention, a fish-derived collagen peptide having an average molecular weight (number average molecular weight) of 1,000 to 10,000 is more preferably used. Collagen peptides having an average molecular weight within the above range have a low viscosity and are suitable for addition to food and drink. In addition, fish-derived collagen peptides in the above molecular weight range are excellent in digestibility, and higher synergistic effects with N-acetylglucosamine can be expected in terms of physiological functions.

  In the present invention, a collagen peptide solution before pulverization may be used, or a powder obtained by pulverization by spray drying or the like may be used.

  When the articular cartilage injury healing promoter and tendon injury healing promoter of the present invention contain the above-described fish-derived collagen peptide alone as an active ingredient, the effective intake is 0 in terms of collagen peptide derived from fish per day for adults. 0.1 to 10 g is preferable, and 0.5 to 5 g is more preferable.

  The articular cartilage injury healing promoter and tendon injury healing promoter of the present invention can also contain the above-mentioned collagen peptide derived from fish and N-acetylglucosamine as active ingredients.

  The origin of N-acetylglucosamine used in the present invention is not particularly limited, and for example, a decomposition method using chitin as a raw material, a decomposition method using an acid or an enzyme, or a fermentation method using a saccharide such as glucose (Japanese Patent Laid-Open No. 2003-1993) However, it is preferably obtained by partially hydrolyzing chitin with an acid from the viewpoint of production efficiency, and chitin is partially hydrolyzed with hydrochloric acid. After dehydration by ion exchange membrane electrodialysis, the coexisting glucosamine hydrochloride is adsorbed and removed with an ion exchange resin, and N-acetylglucosamine is liberated by enzymatic decomposition. preferable.

  Chitin used as a raw material is prepared by removing the calcium content by treating the crustacean crust, such as shrimp, crab, krill, etc., with hydrochloric acid, and then removing the protein by sodium hydroxide treatment. Chitin obtained by an acquisition route, a preparation means, etc. can also be used.

  Partial hydrolysis of chitin with hydrochloric acid is carried out by adding 2 to 20 times the amount of chitin mass and reacting at 30 to 60 ° C. for 2 to 8 hours with stirring. More preferably, 3 to 10 times the amount of chitin mass of concentrated hydrochloric acid is added, and the mixture is reacted at 40 to 50 ° C. for 3 to 5 hours with stirring. It is not preferred that the amount of hydrochloric acid added is outside the above range because the decomposition efficiency deteriorates or the amount of neutralized salt increases and it takes time for desalting. Moreover, it is not preferable that the reaction temperature and the reaction time are out of the above ranges because the decomposition efficiency deteriorates and the amount of glucosamine hydrochloride produced increases.

  Next, in order to complete the hydrolysis reaction, it is diluted with about the same volume of water as the partially hydrolyzed solution, and an alkaline agent such as 25-50% sodium hydroxide solution is used so that the temperature does not rise. Neutralize to pH 3-7.

  The neutralized partially hydrolyzed solution contains N-acetylglucosamine, chitin oligosaccharides, glucosamine hydrochloride and the like, as well as undegraded insoluble chitin, which is somewhat brownish due to the decomposition of sugar. Decomposed insoluble chitin and coloring can be removed by filtration using a small amount of activated carbon and a filter.

  Then, the neutralized partially hydrolyzed solution is desalted by the method (ion exchange membrane electrodialysis method) described in Japanese Patent No. 2134244 (Japanese Patent Publication No. 5-86399). The ion exchange membrane used for the ion exchange membrane electrodialysis is not particularly limited. For example, Neocepta CL-25T, CM-1 to 2, AM-1 to 3 (all trade names, manufactured by Tokuyama Soda Co., Ltd.), Selemion CMV / AMV (trade name, manufactured by Asahi Glass Co., Ltd.) and the like.

  Next, the desalted partial hydrolysis solution is treated with an ion exchange resin to adsorb and remove glucosamine hydrochloride in the solution. The ion exchange resin treatment is performed by a combination of a strongly acidic ion exchange resin and a weakly basic ion exchange resin. Thereby, compared with N-acetylglucosamine and chitin oligosaccharide, the taste is bad and the glucosamine hydrochloride which causes the browning of the powder in the spray drying process can be removed very efficiently.

  Specifically, the desalted partially hydrolyzed solution is treated with a strongly acidic ion exchange resin and subsequently treated with a weakly basic ion exchange resin. The processing method may be a column type or a batch type. As the strong acid ion exchange resin, for example, trade name “Diaion SK1-B” (manufactured by Mitsubishi Chemical) can be used, and as the weakly basic ion exchange resin, trade name “Diaion WA-30” (Mitsubishi). Chemical) etc. can be used.

  Next, an enzyme having hydrolytic ability is allowed to act on the chitin oligosaccharide to decompose the chitin oligosaccharide contained in the partially hydrolyzed solution treated with the ion exchange resin to further release N-acetyl-glucosamine. . As such an enzyme, any enzyme capable of degrading chitin oligosaccharide to monosaccharide N-acetylglucosamine may be used. For example, lysozyme, chitinase, chitobiase (β-N-acetyl) Hexosaminidase) and the like. In addition, some lysozyme and chitinase have low hydrolytic ability for disaccharides and trisaccharides of chitin oligosaccharides, so use an enzyme with high hydrolytic ability for low molecular weight oligosaccharides such as chitobiase. Is preferred.

  Commercially available enzymes can be used as the enzyme. For example, lysozyme is generally chicken egg white lysozyme. Examples of the chitinase include enzymes derived from microorganisms such as Streptomyces griseus, Serratia martzens (Sigma), Aeromonas hydrophila (joint sake spirit), Streptomyces antibiotecus (Calbiochem). It is also possible to use a crude enzyme obtained by culturing a microorganism producing chitin or chitin oligosaccharide-degrading enzyme such as chitinase or chitobiase and extracting the enzyme from this culture. Moreover, many commercially available enzyme preparations (cellulase preparations, pectinase preparations, amylase preparations, protease preparations, etc.) contain chitinase, chitobiase, etc., and these commercially available enzyme preparations can also be used.

  Conditions for the enzymatic degradation reaction of chitin oligosaccharide contained in the partially hydrolyzed solution treated with the ion exchange resin can be appropriately set according to the type of enzyme and the amount of the enzyme, but the sugar composition finally obtained However, it is preferable to set the conditions for the enzymatic decomposition reaction so that N-acetylglucosamine is contained in an amount of 80 to 99% by mass and chitin oligosaccharide 1 to 20% by mass. If the ratio of the chitin oligosaccharide is higher than the above range, the solubility of the saccharide composition in water is reduced, which is unfavorable for use in foods and drinks. In addition, after completion | finish of reaction, what is necessary is just to inactivate an enzyme by heating a reaction liquid etc. Furthermore, you may perform a decoloring process suitably using adsorption agents, such as activated carbon.

  In the present invention, N-acetylglucosamine is selected by a separation membrane by the method described in JP-A-2000-281696 as it is, or after the enzyme reaction and / or after the enzyme reaction. The N-acetylglucosamine can be used with increased purity.

  When the articular cartilage injury healing promoter and tendon injury healing promoter of the present invention contain the above-mentioned fish-derived collagen peptide and N-acetylglucosamine as active ingredients, the effective intake is the fish-derived collagen peptide per adult day 0.1 to 10 g in terms of conversion, 0.1 to 15 g in terms of N-acetylglucosamine is preferred, more preferably 0.5 to 5 g in terms of fish-derived collagen peptide, and 0.5 to 1 g in terms of N-acetylglucosamine is there.

  The articular cartilage injury healing promoter and tendon injury healing promoter of the present invention include, in addition to the above active ingredients, shark cartilage extract, vitamin B group, MSM (methyl sulfonyl methane), ginger extract, excipient, Stabilizers, preservatives, preservatives, brighteners, thickeners, colorants, minerals, vitamins, sugars, fragrances, fats and oils, amino acids and the like can be included as appropriate. For example, by including a shark cartilage extract, biosynthesis of mucopolysaccharide in vivo is promoted, and a synergistic effect with fish-derived collagen peptides and N-acetylglucosamine can be expected.

  The product form of the articular cartilage injury healing promoter and tendon injury healing promoter of the present invention is not particularly limited as long as it is in a form suitable for oral administration, and examples thereof include tablets, powders, granules, solutions, capsules and the like. .

  Furthermore, the articular cartilage injury healing promoter and tendon injury healing promoter of the present invention can be incorporated into various foods and drinks, for example, (1) soft drinks, carbonated drinks, fruit drinks, vegetable juices, lactic acid bacteria drinks, Beverages such as milk drinks, soy milk, mineral water, tea drinks, coffee drinks, sports drinks, alcoholic drinks, jelly drinks, (2) processed vegetables such as tomato puree, canned mushrooms, dried vegetables, pickles, (3) Processed fruit products such as dried fruit, jam, fruit puree, canned fruit, (4) spices such as curry powder, wasabi, ginger, spice blend, seasoning powder, (5) noodles such as pasta, udon, buckwheat noodles, macaroni (Including raw noodles and dried noodles), (6) breads such as bread, confectionery bread, cooked bread, donuts, (7) alpha rice, oatmeal, rice cake, batter flour, 8) Baked confectionery, biscuits, rice confectionery, candy, chocolate, chewing gum, snack confectionery, frozen confectionery, candied confectionery, Japanese confectionery, Western confectionery, half confectionery, pudding, ice cream confectionery, (9) red beans, tofu, natto , Bean products such as kinako, yuba, boiled beans, peanuts, (10) processed products of honey, royal jelly, (11) meat products such as ham, sausage, bacon, (12) yogurt, pudding, condensed milk, cheese, fermented milk, butter Dairy products such as ice cream, (13) processed egg products, (14) processed fish such as dried fish, salmon, chikuwa, fish sausage, processed seaweed such as dried seaweed, kelp, boiled fish, tarako, number child, salmon roe (15) Dashi-no-moto, soy sauce, vinegar, mirin, consomme base, Chinese base, concentrated broth, dressing, mayo Seasonings such as sesame, ketchup, miso, edible oils and fats such as salad oil, sesame oil, linole oil, diacylglycerol, safflower oil, (16) cooking soup (including powder and liquid), semi-cooked foods , Side dishes, retort foods, chilled foods, semi-cooked foods (for example, cooked rice, crab balls).

  What is necessary is just to set suitably the compounding quantity of the articular cartilage injury healing promoter or tendon injury healing promoter in the said food / beverage products based on the kind of food / beverage products, and the effective intake mentioned above. For example, when mix | blending with drinks, 0.01-30 mass% is preferable normally and 0.1-10 mass% is more preferable. If the amount of articular cartilage injury healing promoter or tendon injury healing promoter is too small, the intake per fish will be greatly increased in order to take the collagen peptide derived from fish and N-acetylglucosamine in an amount that can be expected to have physiological activity. Therefore, if it is too much, it may cause precipitation due to crystallization in the product or loose stool due to excessive intake.

  The healing promotion effect on cartilage damage by oral administration of fish-derived collagen peptide and N-acetylglucosamine was tested by the following method.

1) Experimental material Rabbit: Japanese white species, female, 12 weeks old, 9 animals weighing 2.0-3.0 kg were used, and a 1-week acclimatization period was set for the experiment.
Test substances: fish-derived collagen peptide (hereinafter abbreviated as “FCP”) having an amino acid composition shown in Table 1 and an average molecular weight of 3,000, and chitin-derived N-acetylglucosamine (hereinafter abbreviated as “GlcNAc”) Was used.

2) Experimental method Production of cartilage damage model: After cartilage damage was surgically completely exposed to the patellofemoral joint, the inner longitudinal ridge of the distal part of the femur, and above and below the thigh pulley groove, a total of three places A hole with a diameter of 2 mm and a depth of 4 mm was made with an orthopedic electric drill. These rabbits were divided into three groups (three per group): a control group, an FCP administration group (Test Group 1), and an FCP + GlcNAc administration group (Test Group 2).

  And during the test period (14 days), tap water for the control group, 1.2 g / head / day FCP for test group 1, 1.2 g / head · day FCP and 1.0 g / head for test group 2. / day GlcNAc was mixed with tap water and administered.

  After completion of the test period, the degree of repair of each defect hole (upper and lower sides of the medial longitudinal ridge and thigh pulley groove) created at the distal part of the femur at the time of necropsy was visually observed, and the degree of repair was scored and evaluated. (Criteria: less than 50%: 0 point, less than 60%: 1 point, less than 80%: 2 points, 80% or more: 3 points).

  The left femur was sampled and fixed with a 10% neutral buffered formalin aqueous solution (formaldehyde solution), and then decalcified with a 5% formic acid solution under shaking. The decalcified tissue piece was cut out so that the repaired site had a longitudinal cross section, and then embedded in paraffin according to a conventional method, and sliced into 5 μm with a microtome.

  Using the obtained tissue piece, hematoxylin and eosin (HE) heavy staining, safranin O staining for the purpose of proteoglycan staining, and alcian blue staining (pH 2.5) for the purpose of staining of glucosaminoglycan, It was observed with a microscope.

  Further, the depth of the damaged hole was measured using a micrometer under a microscope, and the degree of repair with respect to the initial depth of 4 mm was determined in percentage (%).

3) Results Macroscopic findings of the damaged site are shown in FIG. From FIG. 1, it can be seen that repair of the damaged site has progressed further in the order of test group 2> test group 1> control group.

  Moreover, the result of having scored the degree of repair is shown in FIGS. 2 shows the evaluation of the pulley groove (upper), FIG. 3 shows the evaluation of the pulley groove (lower), FIG. 4 shows the evaluation of the inner longitudinal ridge, and FIG. 2-5, it turns out that the test group 2 is a significantly high point with respect to a control group in each damage location and a comprehensive point. Moreover, it turns out that the test group 1 is also a significantly higher point in the overall score than the control group.

  Moreover, the tissue image which observed the dye | stained tissue piece under the microscope is shown to FIGS. Histologically, regeneration of cartilage was not observed in the control group (see FIG. 6), but clear cartilage regeneration was seen in test group 1 (see FIG. 7) and test group 2 (see FIG. 8). . In particular, in Test Group 2, it was found that regeneration of subchondral bone, proteoglycan and glucosaminoglucan were significantly increased.

  Moreover, the depth of the damaged hole was actually measured using a micrometer, and the degree of repair (%) with respect to the initial depth of 4 mm was obtained. The results are shown in FIG.

  From FIG. 9 and Table 2, it can be seen that the degree of repair of test groups 1 and 2 is higher than that of the control group, and in particular, test group 2 is significantly higher.

  From the above results, it has been clarified that FCP has a cartilage repairing effect equivalent to or higher than that of avian-derived collagen peptide, and shows a synergistic effect when GlcNAc is administered simultaneously.

  The following method was used to test the healing promotion effect on tendon damage caused by oral administration of fish-derived collagen peptides.

1) Experimental material Rabbit: Japanese white species, body weight of about 2.0 to 3.0 kg, female, 8 weeks old 8 animals were used, and the experiment was allowed to acclimatize for 1 week.
Test substance: The same FCP as in Example 1 was used.

2) Experimental method Creation of a tendon injury model: After the Achilles tendon was surgically exposed, the tendon injury was identified 1 cm from the tendon rib attachment site, and 10 23G needles were randomly inserted through the site. It was produced by letting. These rabbits were divided into 2 groups (1 group, 3 birds) of control group 1 and FCP administration group (test group 1). Further, the remaining two rabbits were randomly penetrated with 20 23G injection needles at the same site as above, and divided into a control group 2 and an FCP administration group (test group 2), respectively.

  During the test period (14 days), tap water was administered to the control groups 1 and 2, and FCP 2.4 g / head / day (2.4 g as the amount of collagen) was administered to the test groups 1 and 2.

  After completion of the test period, the degree of repair of the damaged site created in the Achilles tendon at the time of necropsy was visually observed. In addition, paratendon tissue at the damaged site and tissue pieces of Achilles tendon were prepared, stained with HE, and observed with a microscope.

3) Results Figures 10 and 11 show the macroscopic findings of the damaged site. Moreover, the microscope observation image (x100) of the dyed tendon tissue is shown in FIG. 10 to 12, it can be seen that the control group 1 exhibits marked angiogenesis and paratendon tissue adhesion / thickening as compared to the test group 1. Further, it can be seen that the control group 2 is more markedly vascularized than the control group 1 and the test group 2. Furthermore, in the FCP administration group (test groups 1 and 2), it can be seen that the same healing process can be observed even if the degree of damage is high. The difference in healing between the control group and the test group was clearer in the control group 2 and the test group 2 punctured 20 times than in the control group 1 and the test group 1 punctured 10 times.

  Further, FIGS. 13 and 14 show tissue images (× 200) of the Achilles tendon and the surface layer portion of the Achilles tendon. 13 and 14, a large number of large scars were formed in the control group (see FIGS. 13 (a) and 14 (a)), whereas in test group 1, a small number of small scars were observed at the damaged site. (See FIGS. 13 (b) and 14 (b)).

  In addition, the ratio of cell nuclei in the entire visual field (12000 pixels) was calculated using image analysis software (NIH Image / IPLab / Openlab2 / Media Juicer). The result is shown in FIG. It can be seen from FIG. 15 that there is a difference between the control group and the test group. Here, collagen of the tendon is formed from fibroblasts at the time of repair, but if damage repair is delayed, the fibroblasts are indefinitely activated, and the activated fibroblasts have a large nucleus size and histology In some cases, the area of the nucleus occupying the damaged tissue is large, but in the tendon tissue that is well repaired, more fibroblasts are mature and the nucleus area relative to the cytoplasmic area is small. Therefore, the result that the ratio of the nuclear area (the number of pixels of the nucleus when the nucleus is blue) is smaller in the test groups 1 and 2, the result is that the test groups 1 and 2 are compared with the control groups 1 and 2. This means that tendon repair has been completed, and the FCP administration has proved the effect of promoting tendon repair.

  The healing promotion effect for tendon damage by oral administration of fish-derived collagen peptide and N-acetylglucosamine was tested by the following method.

1) Experimental materials Rabbits: Japanese white species, body weight of about 2.0 to 3.0 kg, females, females of 12 weeks of age, 20 birds were used, and an acclimatization period of 1 week was allowed for the experiment.
Test substance: The same FCP and GlcNAc as in Example 1 were used.

2) Experimental method Creation of a tendon injury model: After surgically exposing the Achilles tendon to the injury of the tendon, a site 1 cm away from the rib attachment site of the tendon was specified, and 20 23G needles were randomly penetrated through the site. It was produced by letting. These rabbits were divided into four groups (four per group): a control group, an FCP administration group (test group 1), a GlcNAc administration group (comparison group), and a GlcNAc + FCP administration group (test group 2). The remaining 4 rabbits were not subjected to surgical treatment (normal group).

  During the test period (14 days), tap water was used for the control group, FCP 2.4 g / head / day (2.4 g as the amount of collagen) for the test group 1, and GlcNAc 1 g / head / day for the comparison group. Group 2 was administered FCP 2.4 g / head / day (2.4 g as the amount of collagen) and GlcNAc 1 g / head / day.

  At the end of the test period, the degree of repair of the damaged site created in the Achilles tendon at the time of necropsy was observed histologically, and in 400-fold images of each group, 10 fields (randomly extracted) i) fiber state, ii) The state of the nucleus was evaluated in five stages according to the criteria shown in Tables 3 and 4.

3) Results Table 5 shows the histological observation results of the damaged site. From Table 5, it can be seen that the test group 2 has the closest results to the normal group (normal cells) in both the collagen fiber state and the nucleus state as compared with the test group 1 and the comparison group. Histologically, many large traces were formed in the control group, but a small number of small traces were observed in the test group 1. In addition, the neutrophils were slightly wet in the comparative group as compared with the test group 1.

  From the above results, it was clarified that FCP has a tendon repair effect equivalent to or higher than that of avian-derived collagen peptides, and shows a synergistic effect when coadministered with GlcNAc.

  The articular cartilage injury healing promoter and tendon injury healing promoter of the present invention is suitable as a functional food material that assists in repairing and promoting faster healing when cartilage or tendon is damaged due to sports or accidents. .

It is a figure which shows the macroscopic finding of each damage site | part in a control group and the test groups 1 and 2. FIG. It is a figure which shows the result of having scored and evaluated about the restoration degree of a pulley groove | channel (upper). It is a figure which shows the result evaluated by scoring about the restoration degree of a pulley groove (lower). It is a figure which shows the result evaluated by scoring about the repair degree of an inside vertical ridge. It is a figure which shows the result of having scored and evaluated about the comprehensive repair degree. It is a figure which shows the structure | tissue image of a control group. 2 is a diagram showing a tissue image of test group 1. FIG. FIG. 4 is a diagram showing a tissue image of test group 2. It is a figure which shows a repair rate. It is a figure which shows the macroscopic findings of the damage site | part in the control group 1 and the test group 1. FIG. It is a figure which shows the macroscopic finding of the damage site | part in the control group 2 and the test group 2. FIG. It is a figure which shows the tissue image of the paratendon tissue in the control group 1 and the test group 1. FIG. It is a figure which shows the tissue image of the Achilles tendon in the control group 1 and the test group 1. FIG. It is a figure which shows the tissue image of the Achilles tendon surface layer part in the control group 1 and the test group 1. FIG. It is a figure which shows the result of having measured the ratio of the cell nucleus which occupies for the whole visual field.

Claims (8)

  An articular cartilage injury healing promoter comprising a fish-derived collagen peptide as an active ingredient.   An articular cartilage injury healing promoter comprising a fish-derived collagen peptide and N-acetylglucosamine as active ingredients.   A tendon injury healing promoter comprising a collagen peptide derived from fish as an active ingredient.   A tendon injury healing promoter comprising a fish-derived collagen peptide and N-acetylglucosamine as active ingredients.   A food or drink with a label indicating that it is used for promoting healing of articular cartilage damage, comprising a collagen peptide derived from fish as an active ingredient.   A food and drink with a label indicating that it is used for promoting healing of articular cartilage damage, comprising a collagen peptide derived from fish and N-acetylglucosamine as active ingredients.   A food or drink with a label indicating that it is used for promoting healing of tendon injury, comprising a collagen peptide derived from fish as an active ingredient.   A food or drink with a label indicating that it is used for promoting healing of tendon injury, comprising a collagen peptide derived from fish and N-acetylglucosamine as active ingredients.