JP6948152B2 - Composition for promoting cartilage formation - Google Patents

Description

The present invention relates to a chondrogenesis-promoting composition, a chondrogenesis-promoting supplement, and foods and drinks, which are useful for preventing joint function deterioration and the like.

Osteoarthritis (OA), along with osteoporosis, reduces activities of daily living (ADL) and quality of life (QOL) of the elderly, and therefore, establishment of a curative treatment method is desired. Articular cartilage tissue is composed of collagen, hyaluronic acid, and proteoglycan produced by chondrocytes, and the presence of proteoglycan and hyaluronic acid between collagen fibers having a network structure retains a large amount of water. That is, articular cartilage retains its cushioning property by collagen and proteoglycan.

When the blood circulation around the joint is deteriorated and the supply of oxygen is reduced, the production of proteoglycan and the like by chondrocytes is reduced, and the dead chondrocytes stimulate and inflame the synovium, causing pain in the joint. Furthermore, the release of cytokines during inflammation further induces chondrocyte death and exacerbates the symptoms of pain. Therefore, OA symptomatic treatments include administration of painkillers and anti-inflammatory preparations, injection of high molecular weight hyaluronic acid (sodium hyaluronate) into the joint cavity, and other methods other than symptomatic treatments. In recent years, approaches such as promotion of chondrocyte differentiation or suppression of chondrocyte hypertrophy, chondrocyte transcription of collagen and proteoglycan, and promotion of synthesis have begun to be taken.

It is known that the function of a transcription factor called Sox9 is important in cartilage formation, and analysis of genetically modified mice lacking the Sox9 gene shows that Sox9 is essential at all stages of cartilage formation. Therefore, it is considered that promotion of Sox9 activity is important for promotion of cartilage formation (Non-Patent Document 1 and Non-Patent Document 2).

According to the results of the 2009 ROAD project survey, the number of patients in Japan is reported to be 25 million for the knee and 38 million for the lumbar spine, and the breakdown of men and women is 8.6 million for men and 1670 for women. There are 10,000 men and 19 million women in the lumbar spine, but this number is increasing year by year.
Against this background, inventions relating to the prevention and treatment of OA have been disclosed. Patent Document 1 discloses a cartilage formation promoter containing a milk-derived basic protein. Patent Document 2 discloses the use of a PEDF-derived polypeptide for treating osteoarthritis. Patent Document 3 discloses cartilage regeneration using chondrocytes and TGF-β, but new foods and the like that promote chondrogenesis are still required.

International Publication No. WO2013 / 164992 Special Table 2015-530392 Japanese Patent Publication No. 2005-519698

Haruhiko Akiyama, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B: The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 16 (21), 2813 -28, 2002. Haruhiko Akiyama, H. Scott Stadler, James F. Martin, Takahiro M. Ishii, Philip A. Beachy, Takashi Nakamura, and Benoit de Crombrugghe. Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice. , 224-233, 2007

An object of the present invention is to provide an unprecedented composition for promoting cartilage formation. Another object of the present invention is to provide a food or drink for promoting cartilage formation containing such a substance.

The present invention includes the following aspects.
(1) A composition for promoting cartilage formation containing E-cadherin and / or a decomposition product of E-cadherin as an active ingredient.
(2) The composition for promoting cartilage formation according to (1), wherein the E-cadherin decomposition product is obtained by decomposing E-cadherin with a proteolytic enzyme.
(3) The cartilage according to (2), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Composition promoting composition. (4) The composition for promoting cartilage formation according to any one of (1) to (3), wherein the E-cadherin decomposition product has a molecular weight of 500 or more and 8000 or less.
(5) A food or drink for promoting cartilage formation containing the E-cadherin and / or the decomposition product of E-cadherin according to any one of (1) to (4).

INDUSTRIAL APPLICABILITY The present invention provides a cartilage-forming production promoter containing E-cadherin and / or an E-cadherin decomposition product as an active ingredient, and foods and drinks for promoting cartilage-forming growth. The cartilage shape production promoter and the food and drink for promoting cartilage shape growth of the present invention have an action of promoting chondrogenesis and are useful for prevention and treatment of arthritis and joint dysfunction.

FIG. 1 shows the effect of changes in the concentration of E-cadherin on the expression of mRNA of Sox9, which is a chondrocyte differentiation regulator. FIG. 2 shows the effect of the time-dependent change of E-cadherin on the expression of mRNA of Sox9, which is a chondrocyte differentiation regulator.

In order to solve the above-mentioned problems, the present inventors diligently searched for a substance exhibiting a chondrogenesis-promoting action, which is widely contained in food materials, and found that a 120 kDa transmembrane cell adhesion molecule was used. We have found that a certain E-cadherin or an E-cadherin degradation product obtained by degrading the E-cadherin promotes chondrogenesis by promoting the expression of mRNA of Sox9, which is a chondrocyte differentiation regulator, and the present invention. Has been completed.

A feature of the composition for promoting cartilage formation of the present invention is that E-cadherin and / or an E-cadherin decomposition product is used as an active ingredient. The E-cadherin of the present invention can be of any origin. For example, human and bovine-derived E-cadherin has already been clarified in its gene sequence and can be produced by gene recombination. However, in the present invention, E-cadherin produced by a genetic engineering method is also used. It is possible, and those derived from cells recovered from the culture medium of cell culture can also be used. In addition, E-cadherin is contained in bovine primary milk and may be recovered from milk. From raw milk, milk powder, defatted milk, reduced milk, etc., heat treatment, salting treatment, ethanol treatment, ion exchange It can also be obtained by various chromatographic treatments such as chromatography and gel filtration chromatography, and ultrafiltration treatment. Further, the E-cadherin degradation product has an amino acid composition similar to that of E-cadherin, and can be obtained by treating E-cadherin with a proteolytic enzyme to have a molecular weight of 500 or more and 8000 or less. Examples of proteolytic enzymes include trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Moreover, you may use one or more of these proteolytic enzymes.

The cartilage formation promoting composition of the present invention exerts a cartilage formation promoting effect by oral administration or application. When the composition for promoting chondrogenesis of the present invention is orally administered, the active ingredient E-cadherin can be used as it is, but according to a conventional method, powders, granules, tablets, capsules, drinks It can also be formulated into a drug or the like and used.

In the present invention, oral preparations such as powders, granules, tablets and capsules are prepared by a conventional method using, for example, excipients such as starch, lactose, sucrose, mannit, carboxymethyl cellulose, cornstarch and inorganic salts. It is possible to make it. In addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, colorants, fragrances and the like may be appropriately used in this type of preparation.

Examples of the binder include starch, dextrin, arabic gum, gelatin, hydroxypropyl starch, sodium carboxymethyl cellulose, methyl cellulose, crystalline cellulose, ethyl cellulose and polyvinylpyrrolidone, and examples of the disintegrant include starch and hydroxypropyl starch. , Carboxymethyl cellulose, sodium carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, crystalline cellulose and the like. Further, as a surfactant, soybean lecithin, a sucrose fatty acid ester, etc., as a lubricant, talc, wax, a sucrose fatty acid ester, hydrogenated vegetable oil, etc., as a fluidity accelerator, silicic anhydride, dry aluminum hydroxide, etc. Examples thereof include magnesium silicate.

Furthermore, it is also possible to add these E-cadherins as they are or after formulating them to supplements, nutritional supplements, foods and drinks, and the like. Since E-cadherin is relatively stable to heat, it is possible to sterilize the raw material containing E-cadherin by heating under the conditions normally used.

When the composition for promoting cartilage formation of the present invention is applied, it is prepared into various dosage forms such as a liquid agent, a solid agent, and a semi-solid agent by blending it with a known component usually used according to the purpose of use. Possible and preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the composition for promoting cartilage formation of the present invention includes hydrocarbons such as vaseline, stearyl alcohol, lower alkyl esters of higher fatty acids such as isopropyl myristate, animal fats and oils such as lanolin, polyhydric alcohols such as glycerin, and glycerin fatty acid esters. Cosmetics for promoting cartilage formation by mixing with surfactants such as monostearic acid and polyethylene glycol, inorganic salts, waxes, resins, water, and preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate, if necessary. And can manufacture pharmaceuticals.

The effective amount of the composition for promoting cartilage formation of the present invention by administration is 10 μg or more per adult per day. It may be added to foods and drinks so that this required amount can be secured, or it may be administered as a medicine. The administration can be divided into several times a day as needed.

The effective amount of the composition for promoting cartilage formation of the present invention by application varies depending on the dosage form, but E-cadherin is preferably added in an amount of 0.001 to 2% by weight based on the total amount of the composition to be applied. It should be mixed. However, the amount of bath salts that are diluted at the time of use can be further increased.

(Evaluation method)
Using CDH1 (E-cadherin) Recombinant Human Protein purchased from Thermo Fisher Scientific Co., Ltd. as sample A, ATDC5, which is a mouse EC-derived mesenchymal cell, was used for the effect of Sox9, a chondrocyte differentiation regulator, on mRNA expression. Confirmed using cell experiments and real-time PCR.

(Example product 1)
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 the column, and after sufficient washing with deionized water, a straight line of 0.1 to 1.0 M sodium chloride was obtained. It eluted with a concentration gradient. Then, the eluted fraction containing E-cadherin was again fractionated by phenyl S sepharose hydrophobic column chromatography. Further, this fraction was sequentially treated by C4 and C8 reverse phase chromatography and gel filtration chromatography in an HPLC system to obtain 2500 mg of E-cadherin. The E-cadherin thus obtained can be used as it is as a cartilage formation promoter.

[Test Example 1]
Using CDH1 (E-cadherin) Recombinant Human Protein purchased from Thermo Fisher Scientific Co., Ltd. as sample A, the effect of changes in the concentration of CDH1 on the mRNA expression of Sox9, a chondrocyte differentiation regulator, was examined in mouse EC-derived mesenchymal cells. It was confirmed by a cell experiment using a certain ATDC5 and a real-time PCR method.
Specifically, seeded ATDC5 cells which become 0.5 × 10 ⁵ cells / well in 24-well plates, 37 ° C. in DMEM / F12 medium (manufactured by Sigma), under 5% CO ² environment It was cultured for 7 days. For the final 4 hours of the 7-day culture period, CDH1 was dissolved in DMEM / F12 medium so as to be 10 nM, 50 nM, and 100 nM, respectively, and then added to the cells, and then total RNA was collected and cDNA was synthesized.
After adding 0.5 ml of ISOGEN (manufactured by Nippon Gene Co., Ltd.), which is an RNA extractant, to the cultured cells and allowing the cells to stand for 5 minutes, the extracellular fluid solubilized by pipetting was collected in a 1.5 ml tube. After adding 0.1 ml of chloroform to the cell fluid and stirring thoroughly, the upper layer (aqueous layer) separated into two layers was collected in a new 1.5 ml tube. 0.25 ml of 2-propyl alcohol was added to the recovered solution, and the mixture was allowed to stand for 10 minutes and then centrifuged at 15,000 rpm at 4 ° C. for 15 minutes to obtain a total RNA precipitate. The obtained precipitate was washed with 70% ethanol and then dissolved in DEPC water to prepare an RNA solution. CDNA was synthesized from 1 μg of RNA using the trade name “Takara PrimeScript ^TM RT reagent Kit”. Using the obtained cDNA as a template, real-time PCR using SYBR Green (Takara SYBR Prime Ex Taq II) was performed. The reaction conditions were 95 ° C., 30 seconds of initial denaturation, 95 ° C., 5 seconds of denaturation, 57 ° C., 15 seconds of annealing, 72 ° C., 20 seconds of extension, for a total of 40 cycles of reaction. As the primer, the Sox9 gene expression confirmation primer shown in Table 1 was used. The results are shown in FIG.

As shown in FIG. 1, when CDH1 was added to ATDC5 cells, the Sox9 expression level of the gene was significantly increased depending on the concentration of CDH1. In addition, Sox9 mRNA expression enhancing effect is higher than that of CDH2 (N-cadherin), which is one of the cadherin families and has 47% amino acid homology with CDH1, lactoferrin (LF), and lactoperoxidase (LPO). It became clear that it has.

The effect of sample A on the expression of cartilage differentiation regulator on mRNA was examined using a real-time PCR method. The method was based on the method described in Test Example 1. That is, after sample A was added to ATDC5 cells from 2 hours to 48 hours, total RNA was collected, cDNA was synthesized, and real-time PCR was performed. In the control group, total RNA was recovered from ATDC5 cells cultured for 4 to 24 hours without administration of CDH1, cDNA was synthesized, and real-time PCR was performed. The results are shown in FIG.

As shown in FIG. 2, the mRNA expression of Sox9 by CDH1 was significantly increased 4 hours after the action of CDH1 on ATDC5 cells, and was almost constant and significantly increased up to 24 hours.

(Example 1)
Beverages for promoting cartilage formation having the formulations shown in Table 2 were produced by a conventional method. The flavor of the produced beverage was good and there were no problems such as precipitation.

(Example 2)
Doughs having the formulations shown in Table 3 were prepared by a conventional method, molded, and then roasted to produce biscuits for promoting cartilage formation.

(Example 3)
The composition for promoting cartilage formation having the formulation shown in Table 4 was produced by a conventional method.

(Example 4)
The lotion containing the formulations shown in Table 5 was produced by a conventional method.

（実施例６）
Ｅ−カドヘリン３００ｍｇを精製水３０ｍＬに溶かした後、１Ｎ塩酸にてｐＨ２〜３に調整し、３７℃に保持してペプシンを３ｍｇ添加し、一晩反応させた。１Ｎ水酸化ナトリウムにてｐＨ７．３に調整し、凍結乾燥してＥ−カドヘリン分解物３００ｍｇを得た。 (Example 5)
The creams having the formulations shown in Table 6 were produced by a conventional method.

(Example 6)
After dissolving 300 mg of E-cadherin in 30 mL of purified water, the pH was adjusted to 2-3 with 1N hydrochloric acid, kept at 37 ° C., 3 mg of pepsin was added, and the reaction was carried out overnight. The pH was adjusted to 7.3 with 1N sodium hydroxide and lyophilized to obtain 300 mg of E-cadherin decomposition product.

[Test Example 2]
A one-year clinical trial was conducted in 20 patients with mild pain due to osteoarthritis, who drank 100 g of the beverage of Example 1 once a day. Evaluation of joint pain and function is performed using the visual analog scale (VAS) for pain and the Western Antonio and McMaster Universities (WOMAC) index for pain, function, and rigidity in arthritic joints. rice field. The results are shown in Table 7.

The present invention relates to a composition for promoting chondrogenesis, a supplement for promoting chondrogenesis, food and drink, and a cosmetic for promoting chondrogenesis, which are useful for preventing deterioration of joint function and the like.

Claims (5)

A composition for promoting cartilage formation containing E-cadherin and / or a decomposition product of E-cadherin as an active ingredient. A supplement for promoting cartilage formation containing the E-cadherin and / or the decomposition product of E-cadherin according to claim 1. A food or drink for promoting cartilage formation containing the E-cadherin and / or the decomposition product of E-cadherin according to claim 1. A cosmetic for promoting cartilage formation containing the E-cadherin and / or the decomposition product of E-cadherin according to claim 1. A composition for improving joint pain containing E-cadherin and / or E-cadherin degradation products for oral intake of 10.0 μg or more of E-cadherin and / or E-cadherin degradation products per day. The composition.

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