JP6143218B2 - Tendon and ligament function improving agent and pharmaceutical composition, food and feed containing the same - Google Patents

Description

  The present invention relates to a novel tendon and ligament function improving agent and pharmaceutical compositions, foods and feeds containing the same.

  Elastin is an insoluble protein that is a major constituent of elastic fibers widely distributed in various elastic tissues in the living body such as the aorta, ligaments of the skin, skin, and lungs. Elastin in the skin and blood vessels is known to cause a decrease and a decrease in function with aging. A substance that promotes the production of elastin in skin fibroblasts that produce elastin in the skin has potential as a skin improvement agent. For example, Patent Documents 1 to 3 propose an elastin production promoter containing a plant extract.

  In addition, reports that elastin derived from pigs and horses brought about an improvement effect and a whitening effect on the skin (refer to Patent Document 4), and reports that elastin derived from animals (bovine ligament) improved vascular conditions ( Patent Document 5). In Patent Document 6, 79 to 84% of amino acids constituting elastin are composed of proline, glycine, alanine and valine, 2-3% are composed of aspartic acid and glutamic acid, and 0.7 to 1.3% are composed of An arteriosclerosis inhibitor comprising lysine, histidine and arginine, 0.2 to 0.4% consisting of desmosine and isodesmosine, and having a molecular weight of about 1 to 30,000 and having a low molecular weight water-soluble elastin as an active ingredient is disclosed. .

  Patent Document 7 contains an elastin peptide extracted from an arterial sphere of fish as an active ingredient, and the total content of glycine, alanine, valine and proline per 1000 residues is 650 residues or more, and aspartic acid and asparagine content The total of glutamic acid and glutamine content is 20 to 50 residues, the total of lysine, histidine and arginine content is 20 to 50 residues, and the desmosine and isodesmosine content A skin improving agent and a blood vessel improving agent having a total of 0.3 residues or more and a hydroxyproline content of 10 residues or less are disclosed.

  Elastin is also present in many ligaments and tendons, including the ligaments of the knee and knee ligaments, and is expected to play an important role in mechanical and physiological functions, for example, in the knee ligaments. Similar to the skin and the like, regarding tendons and ligaments, it is considered that a decrease in function due to a decrease in the content of elastin due to aging or the like contributes to joint pain such as knees. Therefore, increasing the proliferative ability of tendon cells and ligament cells, and promoting the gene expression of proteins that constitute tendons and ligaments can prevent locomotive syndromes such as knee arthropathy, tendon and ligament damage caused by sports, etc., tendons and ligaments It is expected to lead to improvement of tendon and ligament functions such as strengthening of the skin.

As a composition intended to improve, promote or prevent healing of tendon or ligament damage, improve or strengthen tendon or ligament, for example, a degradation product obtained by degrading collagen or gelatin with collagenase, and having an amino acid sequence Is a peptide represented by (Gly-X—Y) _n (wherein Gly represents a glycine residue, X and Y represent any amino acid residue other than glycine, and n represents a positive integer). The thing which uses what is contained as an active ingredient (for example, refer patent document 8) is proposed. Patent Document 9 also discloses a platelet-derived growth factor and a pharmaceutically acceptable solid carrier at a concentration ranging from about 0.1 mg / mL to about 1.0 mg / mL in a pharmaceutically acceptable liquid carrier. Disclosed is a method for promoting bone, periodontal tissue, ligament or cartilage growth by applying a composition comprising it to bone, periodontal tissue, ligament or cartilage.

JP 2002-293747 A JP 2005-22993 A Japanese Patent Laid-Open No. 2005-60341 JP 2002-205913 A JP 2007-045722 A JP 2007-045722 A (Claim 6 etc.) JP 2010-155820 A JP 2005-281186 A JP 2009-195739 A

  To date, however, there is no known improvement in ligament function by promoting elastin production in tendon and ligament cells.

  The present invention has been made in view of such circumstances, and has a high safety, low risk of health damage due to long-term ingestion, and can be provided at low cost, and a pharmaceutical composition containing the same. It aims to provide food and feed.

The present inventors promoted the proliferation of tendon cells and ligament cells for peptides obtained by fragmenting fish-derived elastin or a polypeptide chain thereof (hereinafter collectively referred to as “elastin peptide” in the present invention). As a result, it has been found that it has an activity of improving the function of the ligament, and has completed the present invention.
That is, the first aspect of the present invention that meets the above-mentioned object is a hydrolyzate of an insoluble protein obtained by removing lipids, soluble proteins and collagen from fish arterial spheres, and the dipeptide proline-glycine (Pro-Gly). and wherein 1 or several selected from their salts or Ranaru group as an active ingredient, to promote the growth of tendon cells and ligament cells, in tendon cells and ligament cells, elastin mRNA, I type collagen mRNA, III type It has an activity of promoting the expression of collagen mRNA and tenomodulin mRNA, and has the activity of inhibiting alkaline phosphatase in tendon cells and ligament cells and / or suppressing the expression of alkaline phosphatase in tendon cells and ligament cells. By providing a tendon and ligament function improving agent It is intended to solve the problem.

  “Peptide” means a polymer of amino acids in which two or more amino acid residues are polycondensed and joined by peptide bonds.

  In the tendon and ligament function improving agent according to the first aspect of the present invention, the insoluble protein is preferably elastin.

In the tendon and ligament function improving agent according to the first aspect of the present invention, 70% by weight or more of the peptide preferably has a molecular weight of 1000 or less.
In the present invention, “%” means “% by weight” unless otherwise specified.

The second aspect of the present invention solves the above-mentioned problems by providing a pharmaceutical composition for improving tendon and ligament function comprising the tendon and tendon and ligament function improving agent according to the first aspect of the present invention. .

The third aspect of the present invention solves the above problems by providing a food for improving tendon and ligament function, which contains the tendon and ligament function improving agent according to the first aspect of the present invention.

The fourth aspect of the present invention solves the above problems by providing a tendon and a ligament function improving feed containing the tendon and ligament function improving agent according to the first aspect of the present invention.

  ADVANTAGE OF THE INVENTION According to this invention, the tendon and ligament function improving agent which uses the elastin peptide derived from the arterial sphere which is an elastic tissue of fish as an active ingredient, and a pharmaceutical composition, food, and feed containing this can be provided. The tendon and ligament function improving agent of the present invention does not use a protein derived from an elastic tissue of an animal at risk of infection of livestock plague as a raw material, and since it is composed of a peptide, it is highly safe, and risk of health damage due to long-term intake Can be provided at low cost. The tendon and ligament function-improving agent of the present invention promotes the proliferation of tendon cells and ligament cells, thereby preventing ligament functions such as prevention of ligament damage due to locomotive syndrome such as knee joint disease and sports, and strengthening of the ligament. It is expected to be useful for improvement.

It is a figure which shows the effect of the elastin peptide and Pro-Gly on the proliferation rate of the cultured cell derived from the anterior cruciate ligament. It is a figure which shows the effect of the elastin peptide and Pro-Gly on the expression level of the elastin mRNA in the cultured cell derived from an anterior cruciate ligament. It is a figure which shows the effect of the elastin peptide and Pro-Gly on the expression level of type I collagen mRNA in cultured cells derived from the anterior cruciate ligament. It is a figure which shows the effect of the elastin peptide and Pro-Gly on the expression level of type III collagen mRNA in the cultured cell derived from the anterior cruciate ligament. It is a figure which shows the effect of the elastin peptide and Pro-Gly on the expression level of tenomodulin mRNA in the cultured cell derived from the anterior cruciate ligament. It is a figure which shows the effect of the elastin peptide and Pro-Gly on the activity of the alkaline phosphatase in the cultured cell derived from the anterior cruciate ligament.

Subsequently, an embodiment in which the present invention is embodied will be described for the understanding of the present invention.
The tendon and ligament function improving agent according to one embodiment of the present invention is one or a plurality of peptides (insoluble protein hydrolysates obtained by removing lipids, soluble proteins and collagen from fish arterial spheres). Hereinafter, it may be abbreviated as “elastin peptide”.), Containing one or more compounds selected from the group consisting of the dipeptide proline-glycine (Pro-Gly), salts thereof and derivatives thereof as an active ingredient It is out.
Of the above active ingredients, the elastin peptide will be described first.

  The arterial sphere used as a raw material for elastin peptide is an organ peculiar to fish, is connected to the ventricle through a valve, and is involved in blood flow regulation of blood sent from the ventricle to the aorta. There is no particular limitation on the origin of the arterial sphere used as a raw material, and arterial spheres derived from any fish species can be used, but it is necessary to have a certain size in order to collect arterial spheres from the heart. Therefore, the arterial sphere used as a raw material is preferably derived from large fish such as bonito, tuna, swordfish, cod, yellowtail, yellowtail, salmon, trout, etc. More preferably, it is derived from tuna, cod, hamachi, salmon.

The elastin peptide is prepared, for example, by the following method.
First, in order to remove blood from the arterial sphere used as a raw material, it is washed with running water and then pulverized. The pulverization can be performed by any known means such as a homogenizer or a food cutter. Next, by removing lipids, soluble proteins, and collagen from the pulverized arterial spheres, insoluble protein containing elastin as a main component can be obtained. As a pretreatment for facilitating further washing of the raw material and subsequent processing. It is preferable to perform an immersion treatment using an alkaline solution.

  Since the treatment conditions with the alkaline solution vary depending on the fish species, it is preferable to determine in advance, but when using bonito-derived arterial spheres, the alkaline solution used is sodium hydroxide or calcium hydroxide, preferably Is a solution of sodium hydroxide. The concentration of the alkaline solution is 0.01 to 0.1 mol / L, preferably 0.02 mol / L. The immersion temperature is 20 ° C. or less, and the immersion period is several days to 2 weeks, preferably 1 week. Moreover, during immersion, it is preferable to change an alkaline solution twice or more per day. After soaking, excess alkali is removed by washing with running water, and then neutralized as necessary. Any acid used in the art can be used for neutralization.

  Removal of lipid and collagen from the alkali-treated raw material can be performed, for example, by adding distilled water and heating at a high temperature (for example, 95 ° C.). By heating at a high temperature, the helical structure of collagen collapses, the trimer dissociates, and water-soluble tropocollagen (gelatin) is released. When the supernatant is separated and removed by any known method such as filtration, centrifugation, or decantation, an insoluble protein is obtained.

  The elastin peptide can be obtained by subjecting the insoluble protein thus obtained to fragmentation of the polypeptide chain and solubilization treatment for improving water solubility. At this time, the insoluble protein may be further fragmented prior to the solubilization treatment.

  The solubilization treatment can be performed using any known method, and specific examples include enzymatic degradation using a proteolytic enzyme. For enzymatic degradation, any proteolytic enzyme used in food, pharmaceutical and cosmetic production can be used, but those with high titers such as Alcalase 2.4L FG (Novoenzyme), Protin AC-10F (Yamato) Kasei), protease N “Amano” G, and pepsin (Amano Enzyme) are preferred. Degradation conditions are appropriately determined according to the enzyme used, arterial sphere, desired molecular weight distribution, and the like. The amount of enzyme added (enzyme / substrate weight ratio) is the usual amount used for proteolysis in the art, for example, 1:50 to 1: 10000. Moreover, although these enzymes can also be used independently, it is preferable to use combining 2 or more types. The enzymatic degradation reaction is performed at a temperature at which the proteolytic enzyme is not deactivated (for example, room temperature to 37 ° C.) over a predetermined time (30 minutes to 96 hours). After the reaction, the enzymatic decomposition reaction is terminated by heat deactivation of the enzyme.

  The solubilization treatment can also be performed by an acid decomposition method in which an insoluble protein is heated in an inorganic acid solution. Examples of the acid to be used include any inorganic acid, but oxalic acid is preferable, and the concentration and heating temperature are preferably 0.25 N and 90 ° C. After the solubilization treatment, neutralization is performed with an alkali, and sodium hydroxide and calcium hydroxide are preferred as the alkali used at this time. In particular, when oxalic acid is used, neutralization with calcium hydroxide is essential in order to completely remove it.

  Alternatively, the solubilization treatment can also be performed by an alkali-ethanol method in which an insoluble protein is treated with an alkaline aqueous ethanol solution. The solution used at this time is preferably a 1N sodium hydroxide 80% ethanol solution, and the treatment temperature is preferably room temperature.

When the elastin peptide obtained as described above is used as a solution, the solution is adjusted to a pH suitable for a desired application, and desalting is performed if necessary. Desalting can be performed by any method such as an ultrafiltration method or an ion exchange method.
Moreover, when an insoluble matter exists, it can remove using arbitrary methods, such as filtration, centrifugation, and a decantation. In the case of removal by filtration, adsorbents and filter aids such as activated carbon, bentonite, and celite are used to remove impurities (coloring components other than elastin peptides) other than insoluble substances contained in the solution, if necessary. May be added. In particular, when used as a solution, it is preferable to perform sterilization filtration with a membrane filter or the like.
The elastin peptide thus obtained may be used as a solution as it is, or may be used in the form of a powder obtained by further performing spray drying or freeze drying after concentration.

  Among the elastin peptides obtained as described above, the proportion occupied by those having a molecular weight of 1000 or less is 70% or more, preferably 75% or more, more preferably 80% or more. The tendon and ligament function improving agent having the above-mentioned conditions each show high activity and are excellent in absorption characteristics at the time of administration. The molecular weight of the elastin peptide and its abundance ratio (weight ratio) can be determined using any known method such as size exclusion chromatography (gel filtration chromatography), mass spectrometry and the like. If the proportion of the elastin peptide having a molecular weight of 1000 or less is less than 70%, problems such as a decrease in absorption efficiency or expression of mutagenicity may occur.

  The elastin peptide thus obtained has an action of promoting proliferation of tendon cells and ligament cells. Thereby, it is considered effective for promoting recovery of ligament damage, preventing locomotive syndrome such as knee arthropathy, ligament damage caused by sports, etc., and strengthening functions of ligaments such as elderly people, athletes and racehorses.

Elastin peptide has the activity of promoting the expression of elastin mRNA, type I collagen mRNA, type III collagen mRNA and tenomodulin mRNA in tendon cells and ligament cells. Elastin, type I collagen, and type III collagen are the main constituents of the ligament, and their production is promoted, whereby effects such as enhancement of ligament function and promotion of recovery of damage to the ligament can be obtained. Tenomodulin is a 317 amino acid glycoprotein expressed in ligaments, tendons and the like, and contains a cysteine-rich functional domain having homology with chondromodulin-I. The 16 kD C-terminal domain of tenomodulin cleaved by proteolysis is known to exhibit tendon cell proliferation activity, and tenomodulin is thought to play some role in strengthening the functions of ligaments and tendons. Therefore, it seems that promoting the production of tenomodulin also plays an advantageous role in enhancing the function of the ligament.

  The elastin peptide has an activity of inhibiting alkaline phosphatase in tendon cells and ligament cells and / or suppressing expression of alkaline phosphatase in tendon cells and ligament cells. Alkaline phosphatase is an enzyme required for bone formation, but it has been reported that expression of alkaline phosphatase from ligament cells increases with aging and the like, and tendon cells and ligament cells differentiate like osteoblasts. Since such an increase in alkaline phosphatase activity is thought to lead to ligament stiffening with aging, inhibition of alkaline phosphatase activity and suppression of alkaline phosphatase expression in tendon cells and ligament cells are associated with reduced ligament function. Useful for prevention.

  The elastin peptide is fractionated using a known method such as column chromatography, and each activity is assayed for each fraction, whereby a fraction having one or more of the above-mentioned activities is singly or arbitrarily selected. Two or more fractions may be used in combination, or the mixture may be used as it is without separation and purification of the elastin peptide.

The elastin peptide can be used as a pharmaceutical composition for improving tendon and ligament function having an action of promoting the proliferation of tendon cells and ligament cells by mixing with any carrier or the like that is usually used for pharmaceutical use. Examples of the dosage form of the pharmaceutical composition for humans or animals include oral, rectal, parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.), and the dosage is the formulation of the pharmaceutical composition. However, in the case of humans, the active ingredient generally contained in the formulation is difficult to determine and to be determined uniquely depending on the administration method, purpose of use, and age, weight, and symptoms of the subject to be applied. The amount is about 0.1 to 1000 mg, preferably about 0.5 to 500 mg, more preferably about 1 mg to 100 mg per day per adult, and this is administered in 1 to several divided doses. Of course, since the dosage varies depending on various conditions, an amount smaller than the above dosage may be sufficient or may be necessary beyond the range.

  When prepared as an orally administered preparation, it can be prepared in the form of tablets, granules, powders, capsules, coatings, liquids, suspensions, etc. It can be prepared in the form of drops, suppositories and the like. Any known method can be used for formulation. For example, an elastin peptide and a pharmaceutically acceptable carrier or diluent, a stabilizer, and other desired additives can be blended to obtain the desired dosage form described above.

Tendon and ligament function-improving food containing tendon and ligament function-improving foods: Elastin peptide prepared as it is, added to other foods, or capsules, tablets, etc. Can take any form.

  When ingested or administered in foods, mix with excipients, extenders, binders, thickeners, emulsifiers, colorants, flavors, food additives, seasonings, etc. Accordingly, it can be formed into a powder, granule, tablet or the like. Moreover, it can mix suitably in a food raw material, can prepare a foodstuff, and can use it as a functional food which has the activity which promotes the proliferation of a tendon cell and a ligament cell.

The feed for improving tendon and ligament function including the tendon and ligament function improving agent can take various forms such as a prepared elastin peptide as it is, or a blend prepared in the feed. When mixed in feed and administered to animals such as livestock, it can be mixed in advance with feed ingredients to prepare a feed with functionality. It can also be added to the feed for administration. That is, the tendon and ligament function improving agent containing an elastin peptide as an active ingredient is added to livestock such as pigs, chickens, cows, horses, sheep, and feed such as fish, pets (dogs, cats, birds), It can be used as a functional feed that is safe and has the activity of promoting the proliferation of tendon cells and ligament cells.

  The elastin peptide may be contained in the tendon and ligament function improving agent in the form of a salt, derivative or solvate. As the salt of elastin peptide, any salt in which the amino group and carboxyl group in each amino acid residue in the elastin peptide form a salt with an acid and base that are harmless to the living body can be used. Specific examples of amino group salts include (1) hydrochlorides, sulfates, phosphates, diphosphates, hydrobromides, nitrates and other inorganic acid salts, (2) acetates, propionates , Maleate, fumarate, tartrate, malonate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, salicylate, oxalate, stearate, Examples include organic acid salts such as ascorbate, malate, adipate, and gluconate. Specific examples of the salt of the carboxyl group include (1) metal salts such as sodium salt, potassium salt, magnesium salt and calcium salt, and (2) ammonium salt, pyridinium salt and organic base such as salt with basic amino acid. Salt. Alternatively, a so-called inner salt (Zwitter ion) may be formed.

  Derivatives of elastin peptides are reaction products of amino and carboxyl groups in each amino acid residue in elastin peptides and other compounds that are harmless to the living body. Any derivative that can produce Gly can be used. Specific examples of the derivative include N-acylamide, O-alkyl ester and the like.

  In the present embodiment, elastin peptide is used as an active ingredient, but other peptides known to have the activity of promoting the proliferation of tendon cells and ligament cells may be used. Among such peptides, one having a known amino acid sequence includes Pro-Gly (L-prolylglycine). Among the elastin peptide metabolites, a peptide whose blood concentration increased after oral intake of elastin peptide was confirmed (Proceedings of the 63rd Annual Meeting of the Japan Society of Nutrition and Food Science, page 131, 2G-18P, Yasushi Shigemura et al., "Detection of diet-derived elastin peptide in human peripheral blood"), Pro-Gly is one of the dipeptides whose amino acid sequence is known and confirmed to have the activity of promoting the proliferation of tendon cells and ligament cells.

  Pro-Gly is a peptide whose blood concentration increases by oral ingestion of elastin peptide, so it is strongly suggested that oral ingestion of elastin peptide is effective in promoting proliferation of tendon cells and ligament cells, etc. A peptide containing Pro-Gly and Pro-Gly as a partial amino acid sequence and producing Pro-Gly in the digestion and absorption process is considered to be one of active ingredients having high activity in tendon and ligament function improving agents.

  As Pro-Gly, only Pro-Gly can be isolated from the degradation product of elastin peptide or digestive enzyme, and one synthesized by any known method may be used. Similarly, other peptides that are included in the elastin peptide, have the activity of promoting the proliferation of tendon cells and ligament cells, heal or prevent ligament damage, improve ligament function, and strengthen the ligament As for, the one isolated from the elastin peptide can be used, or the one synthesized by chemical or biological techniques after clarifying the amino acid sequence may be used. In addition, Pro-Gly, a salt of Pro-Gly and derivatives thereof (see above for specific examples of salts and derivatives) are used in combination of one or more compounds selected from the group Also good.

  Next, examples carried out for confirming the effects of the present invention will be described.

Example 1: Manufacture of bonito-derived elastin peptide powder Arterial spheres (100 g) were collected from fresh bonito, washed with running water and ground. As a pretreatment of the raw material, it was immersed in a 0.02N aqueous sodium hydroxide solution for 1 week in a refrigerator. After immersion, excess alkali was removed by running water washing, and running water washing was performed until the discharged liquid became neutral. To this was added 3 volumes of distilled water, heated to 95 ° C., and the supernatant was removed to remove lipids and collagen. The residue was cut into pieces with a food cutter, and Protin AC-10F (manufactured by Yamato Kasei, 0.5%) and protease N “Amano” G (manufactured by Amano Enzyme, 0.1%) were added at 1% of the base mass, Decomposition was performed for 10 hours. Heat deactivation was performed at a temperature of 85 ° C. or higher, and the residue was separated by filtration and centrifugation. Thereafter, the extract clarified by microfiltration was spray-dried to obtain water-soluble elastin peptide powder (8 g).

Example 2: Production of Hamachi-derived elastin peptide powder An elastin peptide powder was obtained in the same manner as in Example 1 using arterial spheres collected from fresh Hamachi as a raw material.

Example 3 Production of Tuna-Derived Elastin Peptide Powder Elastin peptide powder was obtained in the same manner as in Example 1 using arterial spheres collected from fresh tuna as a raw material.

  The amino acid analysis results of the elastin peptides obtained according to Examples 1 to 3 are shown in Table 1 below.

  For any elastin peptide, the total of glycine, alanine, valine and proline content per 1000 residues is 650 residues or more, the sum of aspartic acid and asparagine content is 10 to 35 residues, glutamic acid and glutamine content The total content of lysine, histidine and arginine is 20 to 50 residues, the total content of desmosine and isodesmosine is 0.3 residues or more, and the hydroxyproline content is It turns out that it is 10 residues or less.

  The results of protein, lipid, moisture, and ash content analysis of the elastin peptides obtained in Examples 1 to 3 are shown in Table 2 below.

  Table 3 below shows the molecular weight measurement results of the elastin peptides obtained in Examples 1 to 3 (measured by size exclusion chromatography using the Japan Food Analysis Center: TSKgel G2500PWXL column). In Table 3, “%” means wt%.

  As for any elastin peptide, it can be seen that the proportion occupied by those having a molecular weight of 1000 or less is 70% or more.

Example 4: Human-derived anterior cruciate ligament cell proliferation test In each of the tests in this example and the following examples, the anterior cruciate ligament removed from a rheumatoid arthritis patient or a knee osteoarthritis patient was isolated after tissue culture. Cells were used. Human-derived anterior cruciate ligament-derived cells cultured in a culture flask are placed in a 96-well plate at 5,000.
Cells / cm ² were seeded (medium: 10% FBS DMEM). After culturing at 37 ° C. under 5% CO ₂ conditions for 24 hours, the culture medium in the wells was removed, and DMEM containing 0.5% FBS was added, followed by further culturing under the same conditions for 24 hours. The culture solution in the well was removed, washed with DPBS, and then DPBS was removed. Prepare bonito-derived elastin peptide and Pro-Gly (isolated from bonito-derived elastin peptide) to a concentration of 0, 5, 50, 500, 5000, 50000 ng / mL in DMEM containing 0.5% FBS. Added to wells. The cells were cultured for 2 days under conditions of 37 ° C. and 5% CO ₂ , and the number of cells was confirmed using cell counting kit-8.

  The obtained results are shown in FIG. From comparison with the control, it was confirmed that both bonito-derived elastin peptide and Pro-Gly significantly increased the proliferation of human anterior cruciate ligament-derived cells at a concentration of 5 ng / mL or more. Similar results were obtained for elastin peptides derived from hamachi and tuna.

Example 5: Gene Expression Quantitative Test Cells from the anterior cruciate ligament cultured in a culture flask were seeded in a 6-well plate so as to be 5,000 cells / cm ² (medium: 10% FBS DMEM). After culturing at 37 ° C. under 5% CO ₂ until pre-confluence for 3 days, the culture medium in the well was removed, washed with DPBS, and DPBS was removed. 0, 50, 500 in DMEM containing 1.0% FBS
Skipjack tuna-derived elastin peptide and Pro-Gly were prepared to a concentration of ng / mL and added to the wells. Culture was performed for 5 days under conditions of 37 ° C. and 5% CO ₂ , and each gene expression was quantified by RT-PCR. At that time, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as an internal standard. The verified genes are elastin mRNA, type I collagen mRNA, type III collagen mRNA and tenomodulin mRNA.

  The results obtained for elastin mRNA, type I collagen mRNA, type III collagen mRNA and tenomodulin mRNA are shown in FIGS. From comparison with the control, it was confirmed that both bonito-derived elastin peptide and Pro-Gly significantly increased the expression of each of the above genes in human anterior cruciate ligament-derived cells at a concentration of 50 ng / mL or more. Similar results were obtained for elastin peptides derived from hamachi and tuna.

Example 6: Examination of bone differentiation inhibition of ligament cells by measuring alkaline phosphatase activity Human anterior cruciate ligament-derived cells (P8) cultured in a culture flask were seeded in a 48-well plate at 5,000 cells / cm ² , 0, 50, 500 in DMEM containing 10% FBS
Prepare bonito-derived elastin peptide and Pro-Gly to a concentration of ng / mL and add to wells. The cells were cultured for 10 days under conditions of 37 ° C. and 5% CO ₂ . The culture supernatant of each well was removed, the plate was washed with PBS, and then 0.05 μL of 0.05% Triton X was added to each well. The well was transferred to an Eppendorf tube and centrifuged (4C, 15,000 rpm, 15 minutes). The supernatant was collected and used as a measurement sample. The alkaline phosphatase activity of each sample was confirmed by measuring the activity with a kit.

  The obtained result is shown in FIG. From comparison with the control, it was confirmed that both bonito-derived elastin peptide and Pro-Gly significantly reduce alkaline phosphatase activity in human anterior cruciate ligament-derived cells at a concentration of 50 ng / mL or more. Similar results were obtained for elastin peptides derived from hamachi and tuna.

  Based on the above results, bonito-derived elastin peptide and Pro-Gly have the effect of promoting the proliferation of human anterior cruciate ligament-derived cells, the expression of each gene, and the activity inhibition or suppression of alkaline phosphatase activity. This was confirmed, suggesting that it may be effective for strengthening the knee ligament and preventing injury.

  The tendon and ligament function improving agent provided by the present invention can be used to promote the recovery of tendon and ligament damage, the prevention of tendon and ligament damage due to locomotive syndrome such as knee arthropathy and sports, the elderly, athletes, racehorses, etc. It can be used for pharmaceuticals, foods, feeds, etc. having effects such as strengthening the functions of tendons and ligaments.

Claims (6)

Fishes arterial spheres, lipid hydrolysis of insoluble protein obtained by removing soluble proteins and collagen, proline dipeptide - glycine (Pro-Gly) and one or multiple selected from their salts or Ranaru group Including the number as an active ingredient, promoting the proliferation of tendon cells and ligament cells, and having the activity of promoting the expression of elastin mRNA, type I collagen mRNA, type III collagen mRNA and tenomodulin mRNA in tendon cells and ligament cells, A tendon and ligament function improving agent characterized by having an activity of inhibiting alkaline phosphatase in tendon cells and ligament cells and / or suppressing expression of alkaline phosphatase in tendon cells and ligament cells.   The tendon and ligament function improving agent according to claim 1, wherein the insoluble protein is elastin.   The tendon and ligament function improving agent according to claim 1 or 2, wherein 70% by weight or more of the peptide has a molecular weight of 1000 or less.   A pharmaceutical composition for improving tendon and ligament function, comprising the tendon and ligament function improving agent according to any one of claims 1 to 3.   A food for improving tendon and ligament function, comprising the tendon and ligament function improving agent according to any one of claims 1 to 3.   A tendon and ligament function improving feed comprising the tendon and ligament function improving agent according to any one of claims 1 to 3.

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