JP4813741B2 - Nutritional supplement for promoting chondrocyte proteoglycan production - Google Patents

Description

【００２１】
【発明の効果】
本発明により、コンドロシンを有効成分として含有する栄養補助剤が提供される。この栄養補助剤は、軟骨細胞プロテオグリカン生成促進作用を有する。
コンドロシンは、低分子化合物であるため、コンドロイチン硫酸などのムコ多糖よりも消化吸収性に優れている。しかも、プロテオグリカンを構成するコンドロイチン硫酸の基本骨格を有するので、吸収された後、コンドロイチン硫酸やプロテオグリカンへの生合成がグルコサミンやコンドロイチン硫酸よりも速く行われることが期待される。
さらに、コンドロシンは安定的に大量に入手することができる上に、このコンドロシンを含む栄養補助剤は副作用がなく、安全性が高いものである。
コンドロシンは、軟骨障害の予防又は治療に対しても有効であり、組織細胞培養に利用できる可能性がある。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chondrocyte proteoglycan production promoter, more particularly chondrocyte proteoglycan production promoting agent containing as an active ingredient a salt thereof chondrosine or physiologically acceptable. Further relates to the prophylactic or therapeutic agent for cartilage disorder containing the salts the chondrosine or physiologically acceptable active ingredients.
[0002]
Chondrocin is a known substance also referred to as β (1,3) -D-glucuronosyl-D-galactosamine. For example, chondroitin motif obtained by hydrolyzing chondroitin sulfate with an acid or a chondroitin motif produced by a specific microorganism It is a disaccharide obtained by hydrolyzing an acidic mucopolysaccharide containing (basic skeleton) with an acid. The present invention has developed a novel use of chondrosin that can be obtained industrially and stably by the latter method.
[0003]
[Prior art]
Cartilage is a connective tissue composed of chondrocytes and a cartilage matrix that surrounds them. The main components of the cartilage matrix are proteoglycan and collagen, which are involved in the regulation of important cellular activities such as cell adhesion, proliferation and differentiation through complex interactions with other functional molecules.
Most of the sugar chains of proteoglycans are acidic mucopolysaccharides such as chondroitin sulfate, which bind to proteins to form macromolecules. When proteoglycan release from the cartilage tissue is promoted due to various causes and the ability to produce proteoglycan in the tissue is reduced, the destruction of the cartilage tissue proceeds and diseases such as osteoarthritis occur.
There have been reports on proteoglycan expression promoting substances for the purpose of preventing or treating various cartilage diseases. For example, benzothiepine derivatives (Japanese Patent Laid-Open No. 2000-72678), HGF (Japanese Patent Laid-Open No. 8-59502), proteoglycan production Accelerators (JP 2000-281577 A), hyaluronic acid (JP 11-60609 A) and the like.
However, a nutritional supplement or medicine for promoting chondrocyte proteoglycan production that is excellent in safety, stability and effectiveness has not been developed yet.
[0004]
In recent years, the importance of amino sugars in vivo and various useful effects due to their intake have been confirmed. Amino sugars exist as structural units in various complex carbohydrates in the body, and typical ones are proteoglycans containing acidic mucopolysaccharides such as chondroitin sulfate and hyaluronic acid, and connective tissue, skin tissue, cartilage, Many are distributed in joint fluid.
These have been reported to maintain the function and morphology of cells and to have a function of alleviating osteoarthritis, moisturizing and softening skin tissue as a lubricant (Fortschr Medicine, 98 , 801-806, 1980 and New Food Industry, 41, 1-4, 1999).
[0005]
Acidic mucopolysaccharides such as chondroitin and chondroitin sulfate, which are composed of glucuronic acid and N-acetyl-galactosamine, are used for the prevention and treatment of connective tissue diseases in humans such as osteoarthritis. The demand has increased significantly.
However, the production of chondroitin and chondroitin sulfate is limited to those derived from animals such as bovine cartilage and shark cartilage, and recently bovine cartilage is difficult to use due to the influence of mad cow disease (BSE). In addition, the supply and quality of shark cartilage is unstable from the standpoint of resource conservation.
[0006]
In addition, conventional treatments are not intended to directly resolve the cause, but by administering an anti-inflammatory agent or the like, the disorder such as pain based on the disease is suppressed. It was only symptomatic such as a method and a method of injecting hyaluronic acid preparations into joints to lubricate joint movement. In addition, it is known that some drugs used cause adverse effects such as gastrointestinal tract disorders.
[0007]
[Problems to be solved by the invention]
Thus, a radical treatment method for cartilage damage and cartilage disorder has not yet been found, and osteoarthritis has a large number of patients, and an effective treatment method is eagerly desired.
An object of the present invention is to provide a nutritional supplement that is effective in the prevention and treatment of cartilage damage and cartilage damage and that is highly safe.
The present inventor has intensively studied to solve the above problems, and has focused on chondrosin produced by the above-mentioned specific microorganisms and examined its action. As a result, the inventor has an action of promoting proteoglycan production of chondrocytes. The present invention has been completed based on this finding. Furthermore, chondrosin is effective for the prevention or treatment of cartilage disorders, and it is considered that it may be used for tissue cell culture.
[0008]
[Means for Solving the Problems]
The present invention according to claim 1 is a chondrocyte proteoglycan production promoter comprising chondrosin or a physiologically acceptable salt thereof as an active ingredient.
The present invention according to claim 2 is a prophylactic or therapeutic agent for cartilage disorders, comprising chondrosin or a physiologically acceptable salt thereof as an active ingredient .
[0009]
DETAILED DESCRIPTION OF THE INVENTION
As described above, chondrosin used in the present invention is produced by microorganisms. That is, it can be obtained by hydrolyzing acidic mucopolysaccharides produced and accumulated in the culture of Pseudomonas sp. Strain PAK WAK-1 which is a marine microorganism (Japanese Patent Application No. 2001-64228). Chondrosin obtained by other methods such as chemical and enzymatic methods, such as chondroitin sulfate obtained by hydrolyzing chondroitin sulfate or a ground product of animal tissue containing the substance with an acid, can also be used in the present invention. .
In addition, as a physiologically acceptable salt, for example, an alkali metal salt such as sodium salt or potassium salt, an alkaline earth metal salt such as magnesium salt or calcium salt, or an inorganic base such as ammonium salt is formed. Examples thereof include salts, and hydrochlorides, and can be used in combination with chondrosin.
[0010]
The present invention according to claim 1 is a chondrocyte proteoglycan production promoter containing chondrosin or a physiologically acceptable salt thereof as an active ingredient. If desired, excipients (for example, cellulose, dextrin, lactose, tricalcium phosphate, etc.), stabilizers, coloring agents, fragrances, vitamins and the like can be appropriately added to the chondrocyte proteoglycan production promoter .
The chondrocyte proteoglycan production promoter according to the present invention can be in various forms such as tablets, powders, granules, capsules, and liquids. In addition, nutritional supplements can be added to foods.
The amount of the chondrocyte proteoglycan production promoter according to the present invention is not particularly limited, but usually 0.001 to 10 g, preferably about 0.01 to 1 g as chondrocin is taken per day. In addition, it may be used once or several times a day.
[0011]
【Example】
EXAMPLES Next, although an Example etc. demonstrate this invention in detail, this invention is not restrict | limited by these.
Production Example 1
In 10 mL of medium (basic medium) obtained by sterilizing seawater containing 0.5% peptone and 0.1% yeast extract, Pseudomonas sp. WAK-1 strain (Kagawa University Faculty of Agriculture, Department of Life Sciences, Okazaki Laboratory) 1 platinum ear ) was inoculated and cultured at 28 ° C. for 72 hours with shaking.
[0012]
The preculture solution thus obtained was inoculated into 250 mL of a medium supplemented with 3% sucrose and agar in a basic medium in a sterilized container (18 × 26 cm) and cultured at 28 ° C. for 72 hours. After culturing, the mucilage containing the bacterial cells was suspended in a 1% phenol solution, centrifuged, and twice the amount of ethanol was added to the supernatant after removing the bacterial cells to obtain a white precipitate.
The precipitate was collected and dissolved in water, and then twice the amount of ethanol was added again to precipitate the polysaccharide. The precipitate was dissolved in water, dialyzed against water, and freeze-dried to obtain a polysaccharide.
[0013]
The obtained polysaccharide was dissolved in 0.01M phosphate buffer (pH 70.) and equilibrated with 0.01M phosphate buffer (pH 70.), DEAE-cellulose column (2.3 × 22.5 cm). The fraction eluted with 0.2 M sodium chloride in 0.01 M phosphate buffer (pH 70.) was removed, and the fraction eluted with 0.4 M sodium chloride was collected and dialyzed.
Next, lyophilization was performed to obtain acidic mucopolysaccharide. This acidic mucopolysaccharide was confirmed for homogeneity using cellulose acetate membrane electrophoresis, and analyzed by saccharide composition analysis, pyruvic acid content analysis, nuclear magnetic resonance analysis, etc. (M. Matsuda et al., Fisheries Science, 62 , 983-986, 1997) and confirmed that it is the same acidic mucopolysaccharide.
[0014]
The obtained acidic mucopolysaccharide was dissolved in water to make a 1.0 wt% solution, and hydrolyzed by heating at 100 ° C. for 6 hours at 1.0 N hydrochloric acid concentration. The reaction solution was cooled and neutralized with sodium hydroxide.
Using this neutralized solution, chondrocin was separated from monosaccharides and inorganic salts and collected by gel filtration Bio-Gel P2 column (2.5 × 100 cm). Bio-Gel P2 column chromatography was developed using a buffer solution (water: pyridine: acetic acid = 500: 5: 2) as an eluent at a flow rate of 4.0 mL / 30 min, and each 4.0 mL was fractionated.
[0015]
Subsequently, fractions containing only chondrosin were collected and lyophilized. For the detection of chondrosin and galactosamine in each fraction, a certain amount of sample was taken from each fraction, (i) reduced after N-acetylation, wakopak WBT-130E column (7.8 × 300 mm, manufactured by Wako Pure Chemical Industries) Applied. Water was used for the mobile phase, and it was developed at a flow rate of 0.5 mL / min at 60 ° C. and measured with a differential refractometer. (Ii) Not applied as a derivative, but applied to a Mightysil RP-18GP column (4.6 × 250 mm, manufactured by Kanto Chemical). The mobile phase used was 0.001N HCl or 2.4 mM phthalic acid-containing 2.5 mM trisaminomethane (pH 4.0), developed at 35 ° C. with a flow rate of 0.5 mL / min, and measured with a differential refractometer. .
[0016]
Activated carbon was added to the neutralized solution and stirred well, then filtered through a glass fiber filter paper (Whatman GFF) on a Buchner funnel, washed well with water, and further washed with 2.5% ethanol. Next, the fraction eluted with 5% ethanol was collected, evaporated to dryness with a rotary evaporator, dissolved in 300 mL of water, and an electrodialysis system equipped with an AC-220-550 cartridge (Asahi Kasei Co., Ltd., S3). Was used to purify and freeze-dried.
[0017]
Production Example 2
Commercially available chondroitin sulfate (manufactured by San-Ei Gen FFI, shark cartilage extract) was dissolved in 1.0N hydrochloric acid to make a 1.0 wt% solution, and heated at 100 ° C. for 6 hours for hydrolysis treatment. . The reaction solution was cooled and neutralized with sodium hydroxide.
This neutralized solution is added as it is or after adding activated carbon to the neutralized solution and stirred well, filtered through a glass fiber filter paper (Whatman GFF) on a Buchner funnel, washed well with water, and further washed with 2.5% ethanol. Washed. Next, the fraction eluted with 5% ethanol was collected, evaporated to dryness with a rotary evaporator, dissolved in 300 mL of water, and an electrodialysis system equipped with an AC-220-550 cartridge (Asahi Kasei Co., Ltd., S3). Was used to purify and freeze-dried.
[0018]
Example 1
The ATDC5 (RIKEN BANK, RBC0565) cell lines that differentiate into cartilage cells like derived from mouse embryonic cells were seeded at a density of 5 × 104 cells / well in 48-well plates, containing 5% FCS (fetal calf serum) The cells were cultured at 37 ° C. using 5% CO 2 in Eagle MEM medium.
After culturing for 2 days, the wells were washed twice with serum-free Eagle MEM medium, 450 μL of Eagle's MEM medium containing 5% FCS was added to each well, and then 50 μL of test solution was added for 4 days at 37 ° C. In culture. The test solution was prepared by dissolving chondrosin prepared in Production Example 1 or 2 in an Eagle's MEM medium containing no serum so as to have a concentration of 5 mg / mL, and then sterilizing with a 0.45 nm filter.
[0019]
After completion of the culture, the cell line obtained by removing the culture solution was washed twice with PBS-A (phosphate buffer) and then 0.125 mL of 0.2% NP-40 (PBS-containing 1 mM MgCl ₂ ). Lysed in A) and cultivated at 37 ° C. for 2 hours to lyse the cells, and then centrifuged at 10,000 rpm for 5 minutes. The obtained supernatant was acid mucopolysaccharide using an acid mucopolysaccharide measurement kit (Hokudo). The polysaccharide was quantified. The results are shown in Table 1. In addition, the numerical value in a table | surface is computed as the light absorbency of a chondrocin addition group when the light absorbency (650 nm) of a chondrosin addition group is set to 100% as activity%. The concentration is the chondrocin concentration (μg) per 1 mL of the reaction mixture in each well.
As can be seen from the table, chondrosin increases ATDC5 coloration and has a promoting effect on proteoglycan production using the acidic mucopolysaccharide of chondrocytes as an index.
[0020]
[Table 1]
Table 1. Promoting proteoglycan production by chondrocytes

[0021]
【The invention's effect】
According to the present invention, a nutritional supplement containing chondrosin as an active ingredient is provided. This nutritional supplement has a chondrocyte proteoglycan production promoting action.
Since chondrocin is a low molecular weight compound, it is more digestible and absorbable than mucopolysaccharides such as chondroitin sulfate. Moreover, since it has the basic skeleton of chondroitin sulfate constituting proteoglycan, it is expected that biosynthesis to chondroitin sulfate and proteoglycan is performed faster than glucosamine and chondroitin sulfate after absorption.
Furthermore, chondrosin can be obtained stably in large quantities, and the nutritional supplement containing chondrosin has no side effects and is highly safe.
Chondrosin is also effective for the prevention or treatment of cartilage disorders and may be used for tissue cell culture.

Claims (2)

A chondrocytic proteoglycan production promoter comprising chondrosin or a physiologically acceptable salt thereof as an active ingredient. A prophylactic or therapeutic agent for cartilage disorders, comprising chondrosin or a physiologically acceptable salt thereof as an active ingredient.