JP2018058781A - Food compositions and oral pharmaceutical compositions for preventing, alleviating or treating arthropathy symptoms - Google Patents
Food compositions and oral pharmaceutical compositions for preventing, alleviating or treating arthropathy symptoms Download PDFInfo
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
Description
本発明は、特定のプロテオグリカンを含有する、関節症の症状を予防、軽減若しくは治療するための食品組成物又は経口医薬組成物に関する。 The present invention relates to a food composition or an oral pharmaceutical composition containing a specific proteoglycan for preventing, reducing or treating symptoms of arthropathy.
関節疾患の中でも最も患者数の多い疾患が変形性関節症である。当該疾患では加齢が軟骨変性の原因の一つと考えられており、高齢化社会の到来により今後更に患者数が増えることが懸念されている。その治療としては、軟骨変性や軟骨下骨破壊に伴う痛みを除去する目的で、鎮痛消炎剤の貼付やヒアルロン酸の注入が行われているが、いずれも十分な効果が挙げられているとは言えない。
また近年では、プロテオグリカンを関節内注入する治療も行われている（特許文献１）。
Among joint diseases, osteoarthritis is the most common disease. In this disease, aging is considered to be one of the causes of cartilage degeneration, and there is a concern that the number of patients will increase further in the future due to the arrival of an aging society. For the treatment, analgesic anti-inflammatory agents and hyaluronic acid injections have been performed for the purpose of removing pain associated with cartilage degeneration and subchondral bone destruction, but all have sufficient effects. I can not say.
Moreover, in recent years, a treatment in which proteoglycan is injected into the joint is also performed (Patent Document 1).
プロテオグリカンは、１本のコアタンパク質に数本から数十本の直鎖状の糖鎖が共有結合している、非常に複雑かつ多種の構造を有する糖タンパク質の総称であり、軟骨組織に存在するプロテオグリカンに含まれる糖鎖の代表的なものがコンドロイチン硫酸である。 Proteoglycan is a general term for glycoproteins with very complex and various structures in which several to several tens of linear sugar chains are covalently bonded to one core protein, and is present in cartilage tissue. A typical sugar chain contained in proteoglycan is chondroitin sulfate.
コンドロイチン硫酸は、保湿性、生体適合性或いは潤滑性に優れる等の高い有用性から産業上注目されている成分であり、天然資源からの効果的な回収、製造法が種々開発されている。 Chondroitin sulfate is a component that has attracted industry attention due to its high usefulness such as moisture retention, biocompatibility, and lubricity, and various effective recovery and production methods from natural resources have been developed.
軟骨組織においては、コンドロイチン硫酸はそれ自体単独では存在せず、タンパク質と共有結合を介して形成された複合体すなわちプロテオグリカンの形で存在している。しかし、プロテオグリカンをそのまま抽出することは糖タンパク質複合体という複雑な構造故に困難な場合が多く、そのためプロテオグリカンのコアタンパク質部分を徹底的に分解してコンドロイチン硫酸のみを抽出しようという方法が主に採用されてきた。この方法の製造物はコンドロイチン硫酸等のムコ多糖類である。 In cartilage tissue, chondroitin sulfate does not exist by itself, but exists in the form of a complex formed through covalent bonding with a protein, ie, proteoglycan. However, it is often difficult to extract proteoglycan as it is because of the complex structure of glycoprotein complex. Therefore, the method of extracting only chondroitin sulfate by thoroughly degrading the core protein part of proteoglycan is mainly adopted. I came. The product of this method is a mucopolysaccharide such as chondroitin sulfate.
一方、コンドロイチン硫酸としてではなく、プロテオグリカンそのままを回収、製造し利用する試みもなされている。特に、魚類、鳥類及び哺乳類の軟骨組織には、コンドロイチン硫酸を主要糖鎖とするプロテオグリカンが含まれている一方、これらの軟骨組織は通常廃棄処分となっていたことから、廃棄物の有効利用を兼ねた軟骨組織からのプロテオグリカンの製造法が幾つか提唱されている。 On the other hand, attempts have been made to collect, produce and use proteoglycan as it is, not as chondroitin sulfate. In particular, the cartilage tissue of fish, birds and mammals contains proteoglycans containing chondroitin sulfate as the main sugar chain. Several methods for producing proteoglycan from cartilage tissue which have also been proposed have been proposed.
例えば、軟骨組織からのプロテオグリカンの製造法として、特許文献２には、プロテオグリカンを含有する軟骨組織等の生物学的試料を界面活性剤の水溶液に浸漬し、浸漬後の溶液を回収することを含む、プロテオグリカンの製造方法が記載されている。 For example, as a method for producing proteoglycan from cartilage tissue, Patent Document 2 includes immersing a biological sample such as cartilage tissue containing proteoglycan in an aqueous solution of a surfactant and collecting the solution after immersion. A method for producing proteoglycans is described.
上述したように、変形性関節症の治療としては、鎮痛消炎剤の貼付、ヒアルロン酸の注入、プロテオグリカンの関節内注入などが行われているが、鎮痛消炎剤の貼付は、対症療法的に痛みを除くものであるし、また、ヒアルロン酸の注入及びプロテオグリカンの関節内注入は、侵襲的な処置方法であり、患者、特に高齢の患者の身体に負担をかけるものである。
したがって、患者、特に高齢の患者の身体に負担をかけずに非侵襲的に、関節症の症状を予防、緩和、又は治療できる方法が望まれている。
As described above, osteoarthritis treatment includes the application of analgesic / anti-inflammatory drugs, hyaluronic acid injection, and proteoglycan intra-articular injection, but the application of analgesic anti-inflammatory drugs is symptomatically painful. In addition, hyaluronic acid injection and proteoglycan intra-articular injection are invasive methods of treatment, which burden the body of patients, particularly elderly patients.
Therefore, there is a need for a method that can prevent, alleviate, or treat arthropathy symptoms non-invasively without burdening the body of a patient, particularly an elderly patient.
本発明者らは、上記課題に鑑み鋭意検討を行ったところ、特定の方法により製造されるプロテオグリカンを経口摂取することにより、関節症、特に変形性関節症の症状が、効果的に予防、緩和又は治療できることを見出し、本発明を完成するに至った。 The present inventors have conducted an intensive study in view of the above problems, and by taking orally proteoglycan produced by a specific method, the symptoms of arthropathy, particularly osteoarthritis, can be effectively prevented and alleviated. Or it discovered that it could treat and came to complete this invention.
したがって、本発明は、以下のものを提供する。
〔１〕プロテオグリカンを含有する生物学的試料を界面活性剤の水溶液に浸漬する工程、及び浸漬後の溶液を回収する工程を含む、プロテオグリカンの製造方法によって得られたプロテオグリカンを含有する、関節症の症状を予防、軽減若しくは治療するための食品組成物又は経口医薬組成物。
〔２〕界面活性剤が、サポニン、レシチン、スークロース脂肪酸エステルからなる群から選択される少なくとも１種である、〔１〕に記載の組成物。
〔３〕プロテオグリカンを含有する生物学的試料が、魚類、軟体動物、鳥類及び哺乳類の軟骨組織、筋肉繊維並びに皮からなる群から選択される少なくとも１種である、〔１〕又は〔２〕に記載の組成物。
〔４〕プロテオグリカンを含有する生物学的試料が、魚類、鳥類又は哺乳類の軟骨組織からなる群から選択される少なくとも１種である、〔３〕に記載の組成物。
〔５〕関節症が変形性関節症である、〔１〕〜〔４〕のいずれかに記載の組成物。
〔６〕関節症における炎症を低減するための、〔１〕〜〔５〕のいずれかに記載の組成物。
Accordingly, the present invention provides the following.
[1] An arthritic disease containing proteoglycan obtained by a method for producing proteoglycan, comprising a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant and a step of recovering the solution after immersion. A food composition or oral pharmaceutical composition for preventing, reducing or treating symptoms.
[2] The composition according to [1], wherein the surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid ester.
[3] The biological sample containing proteoglycan is at least one selected from the group consisting of fish, mollusc, avian and mammalian cartilage tissue, muscle fiber, and skin, [1] or [2] The composition as described.
[4] The composition according to [3], wherein the biological sample containing proteoglycan is at least one selected from the group consisting of fish, birds or mammalian cartilage tissue.
[5] The composition according to any one of [1] to [4], wherein the arthropathy is osteoarthritis.
[6] The composition according to any one of [1] to [5] for reducing inflammation in arthropathy.
本発明の組成物を経口摂取することにより、患者、特に高齢の患者の身体に負担をかけずに非侵襲的に、関節症の症状を予防、緩和、又は治療できる。また、本発明の組成物は、生物由来のものであり毒性が低い。更には、本発明で使用されるプロテオグリカンは、通常廃棄されるような原料から製造することができることから、本発明は、資源の有効活用に寄与し得、医療費の上昇をも抑制し得るものである。 By taking the composition of the present invention orally, the symptoms of arthropathy can be prevented, alleviated or treated non-invasively without imposing a burden on the body of a patient, particularly an elderly patient. The composition of the present invention is derived from a living organism and has low toxicity. Furthermore, since the proteoglycans used in the present invention can be produced from raw materials that are normally discarded, the present invention can contribute to effective utilization of resources and can suppress an increase in medical costs. It is.
本発明で使用するプロテオグリカン（以下、「本発明のプロテオグリカン」ともいう。）は、非常に複雑かつ多種の構造を有する糖タンパク質であり、その構造を構造式等で表現することは不可能であるかおよそ実際的ではないため、本発明では、製造方法により当該プロテオグリカンを特定する。 The proteoglycan used in the present invention (hereinafter also referred to as “the proteoglycan of the present invention”) is a glycoprotein having a very complex and various structure, and its structure cannot be expressed by a structural formula or the like. In the present invention, the proteoglycan is specified by the production method because it is not practical.
本発明のプロテオグリカンは、特開２０１４−９１６４号公報に記載される方法に準じて製造することができ、その方法を以下に説明する。 The proteoglycan of the present invention can be produced according to the method described in JP 2014-9164 A, and the method will be described below.
本発明のプロテオグリカンを製造する方法は、プロテオグリカンを含有する生物学的試料を界面活性剤の水溶液に浸漬する工程、及び浸漬後の溶液を回収する工程を含む。 The method for producing a proteoglycan of the present invention includes a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after immersion.
プロテオグリカンを含む生物学的試料としては、魚類、軟体動物、鳥類及び哺乳類の軟骨組織、筋肉繊維並びに皮が挙げられるが、中でも、軟骨組織が好ましい。軟骨組織としては、魚類、鳥類及び哺乳類の軟骨組織や、当該軟骨組織の廃棄部位のいずれも、利用することができる。ここで、軟骨組織とは、軟骨単独或いは軟骨の周辺部位、例えば骨、筋肉繊維、皮等を含む組織のいずれも包含する。魚類の軟骨組織としては、一般に流通しているヨシキリザメ、アオザメなどの軟骨魚類のヒレ、軟骨部分はもちろん、特に、サケの頭部にその平均重量で約６％含まれている、氷頭とよばれる鼻軟骨組織が好ましい。北海道沿岸部で漁獲されたサケ（大半はシロサケ）が、様々な加工品として処理される際、その頭部は不要とされることが多く、そのため切断された頭部は、一部魚粉に加工され利用されてはいるものの、その大半は産業廃棄物として廃棄処理されており、氷頭は、その様な廃棄物から簡便、安価かつ安定的に入手することができる。
本発明では、氷頭の他、エイの軟骨組織、サメの軟骨組織等の魚類由来の軟骨組織、ニワトリの軟骨組織等の鳥類の軟骨組織、更にはウシの喉軟骨や気管支軟骨、クジラの軟骨等の哺乳動物由来の軟骨組織も使用することができる。更に、軟体動物であるイカやタコの表皮・軟骨組織にもプロテオグリカンが存在することが知られており、これら軟体動物の表皮・軟骨等も本発明で使用することができる。また、哺乳動物血液中には硫酸をほとんど含まないコンドロイチン・タンパク質複合体（ビクニン）が存在しており、このコンドロイチンは、哺乳動物血液中のムコ多糖のほぼ１００％を占めることが報告されている（Salier J.P., Rouet P., Raguenez G., Daveau M.: The inter-alpha-inhibitor family: from structure to regulation Biochem. J., 315, 1-9, 1996）ことから、哺乳動物の血液由来コンドロイチンも、本発明におけるプロテオグリカンを含む生物学的試料として使用できる。上記のプロテオグリカンを含む生物学的試料の多くは産業廃棄物であり、その入手は容易である。これらの原料は、界面活性剤水溶液への浸漬に先だって、表面積を増加させてプロテオグリカンの抽出量を上げるために、可能な限り細かく破砕すること、脱脂することなどの前処理を行なうことが好ましい。
Biological samples containing proteoglycans include fish, mollusk, avian and mammalian cartilage tissue, muscle fibers and skin, with cartilage tissue being preferred. As the cartilage tissue, any of the cartilage tissue of fish, birds and mammals, and the disposal site of the cartilage tissue can be used. Here, the cartilage tissue includes any of cartilage alone or a tissue including a peripheral portion of the cartilage, such as bone, muscle fiber, and skin. As the cartilage tissue of fish, not only fins and cartilage parts of cartilaginous fish such as blue sharks and gray sharks that are generally distributed, but especially ice salmon heads, which contain about 6% in average weight in salmon heads. Nasal cartilage tissue is preferred. When salmon (mostly salmon) caught in the coastal area of Hokkaido are processed as various processed products, the head is often unnecessary, so the cut head is partially processed into fishmeal. Although most of them are disposed of as industrial waste, ice heads can be obtained simply, inexpensively and stably from such waste.
In the present invention, in addition to ice head, fish cartilage tissue such as ray cartilage tissue, shark cartilage tissue, avian cartilage tissue such as chicken cartilage tissue, bovine throat cartilage, bronchial cartilage, whale cartilage It is also possible to use a mammal-derived cartilage tissue such as Furthermore, it is known that proteoglycan is also present in the epidermis / cartilage tissue of squid and octopus, which are molluscs, and the epidermis / cartilage of these molluscs can also be used in the present invention. In addition, chondroitin protein complex (bikunin) containing almost no sulfate is present in mammalian blood, and this chondroitin is reported to occupy almost 100% of mucopolysaccharide in mammalian blood. (Salier JP, Rouet P., Raguenez G., Daveau M .: The inter-alpha-inhibitor family: from structure to regulation Biochem. J., 315, 1-9, 1996) Can also be used as a biological sample containing a proteoglycan in the present invention. Many of the biological samples containing the above-mentioned proteoglycans are industrial waste and are readily available. Prior to the immersion in the surfactant aqueous solution, these raw materials are preferably subjected to pretreatment such as crushing and degreasing as finely as possible in order to increase the surface area and increase the extracted amount of proteoglycan.
界面活性剤としては、陰イオン界面活性剤、陽イオン界面活性剤、両面界面活性剤及び非イオン界面活性剤等の任意の界面活性剤が使用できる。中でも、食品添加物又は食品用乳化剤として承認されている、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、プロピレングリコール脂肪酸エステル、スークロース脂肪酸エステル、ソルビタン脂肪酸エステル、大豆リン脂質（レシチン）、サポニン等が好ましく、サポニン、レシチン或いはスークロース脂肪酸エステル単体或いはこれらいずれかの混合物が特に好ましい。 As the surfactant, any surfactant such as an anionic surfactant, a cationic surfactant, a double-sided surfactant and a nonionic surfactant can be used. Among them, glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, soybean phospholipid (lecithin), saponin, etc., which are approved as food additives or food emulsifiers, are preferable. Lecithin or sucrose fatty acid ester alone or a mixture of any of these is particularly preferred.
サポニンとしては、キラヤサポニン、大豆サポニン、茶サポニンが挙げられる。中でも、キラヤサポニンが好ましい。 Examples of saponins include Kiraya saponin, soybean saponin, and tea saponin. Of these, Kirayasaponin is preferable.
レシチンとしては、植物レシチン、分別レシチン、卵黄レシチン、酵素処理レシチン、酵素分解レシチンが挙げられる。中でも、ＨＬＢ ３〜４の植物性レシチンが好ましい。 Examples of lecithin include plant lecithin, fractionated lecithin, egg yolk lecithin, enzyme-treated lecithin, and enzyme-decomposed lecithin. Among them, plant lecithin having HLB 3 to 4 is preferable.
スークロース脂肪酸エステルとしては、スークロース１分子に脂肪酸１分子が付加してできるショ糖脂肪酸モノエステル、脂肪酸２分子が付加してできるジエステル、脂肪酸３分子が付価してできるトリエステルが挙げられる。理論上は、スークロース脂肪酸エステルとしては、脂肪酸８分子の付加したオクタエステルまで存在し得るが、食品用に適するのは、モノエステル、ジエステル、トリエステル及びこれらいずれかの混合物である。中でも、エマルジョンの乳化安定性の観点から、ＨＬＢ １３〜１９、特に１４〜１９、最適にはＨＬＢ １９のスークロース脂肪酸エステルが好ましい。エマルジョンコストの低減の観点も加えれば、モノエステルとジエステルとトリエステルの混合物（以下、「スークロース脂肪酸混合エステル」ともいう）が望ましく、特に、ＨＬＢ １４〜１６のスークロース脂肪酸混合エステルとＨＬＢ １９のスークロース脂肪酸モノエステルとの混合物が望ましい。脂肪酸としては、炭素数１０〜２２の飽和又は不飽和脂肪酸が好ましく、炭素数１２〜１８の飽和又は不飽和脂肪酸が更に好ましい。 Examples of the sucrose fatty acid ester include a sucrose fatty acid monoester formed by adding one molecule of fatty acid to one molecule of sucrose, a diester formed by adding two molecules of fatty acid, and a triester formed by adding three molecules of fatty acid. Theoretically, sucrose fatty acid esters may exist up to octaesters with 8 molecules of fatty acid, but those suitable for food are monoesters, diesters, triesters and mixtures of any of these. Among these, from the viewpoint of emulsion stability of the emulsion, sucrose fatty acid esters of HLB 13 to 19, particularly 14 to 19, and most preferably HLB 19 are preferred. From the viewpoint of reducing emulsion cost, a mixture of monoester, diester and triester (hereinafter also referred to as “sucrose fatty acid mixed ester”) is desirable, and in particular, sucrose fatty acid mixed ester of HLB 14-16 and sucrose of HLB 19 Mixtures with fatty acid monoesters are preferred. As the fatty acid, a saturated or unsaturated fatty acid having 10 to 22 carbon atoms is preferable, and a saturated or unsaturated fatty acid having 12 to 18 carbon atoms is more preferable.
本発明で使用する界面活性剤の中でも、プロテオグリカンの抽出効率、後処理の簡便さ等から、スークロース脂肪酸エステル類が好ましく、特に、炭素数１２の飽和脂肪酸であるラウリン酸のスークロース脂肪酸エステルであるスークロースラウリン酸エステルが好ましい。 Among the surfactants used in the present invention, sucrose fatty acid esters are preferred from the standpoint of proteoglycan extraction efficiency, ease of post-treatment, etc., and in particular, sucrose fatty acid esters of lauric acid, which is a saturated fatty acid having 12 carbon atoms. Clauslaurate is preferred.
界面活性剤水溶液中の界面活性剤の濃度は、好ましくは０．０１〜２０重量％、特に好ましくは０．１〜１０重量％、更に好ましくは０．５〜５重量％である。浸漬時間は、必要に応じて適宜設定できるが、例えば０．１〜２重量％の界面活性剤、例えばサポニン、レシチン若しくはスークロース脂肪酸エステル、又はこれらいずれかの混合物の水溶液を用いる場合の浸漬時間は、１０時間以上とすることが好ましい。また、２〜５重量％の界面活性剤、例えばサポニン、レシチン若しくはスークロース脂肪酸エステル又はこれらいずれかの混合物の溶液を用いる場合の浸漬時間は、１２時間程度とすることが好ましい。これら条件により、より高い分子量のプロテオグリカンを効率的に回収し、製造することができる。 The concentration of the surfactant in the aqueous surfactant solution is preferably 0.01 to 20% by weight, particularly preferably 0.1 to 10% by weight, and more preferably 0.5 to 5% by weight. The immersion time can be appropriately set according to need. For example, when using an aqueous solution of 0.1 to 2% by weight of a surfactant, for example, saponin, lecithin or sucrose fatty acid ester, or a mixture of any of these, 10 hours or more is preferable. In addition, the immersion time when using a solution of 2 to 5% by weight of a surfactant, such as saponin, lecithin, sucrose fatty acid ester, or a mixture of any of these, is preferably about 12 hours. Under these conditions, higher molecular weight proteoglycans can be efficiently recovered and produced.
界面活性剤水溶液への生物学的試料の浸漬は、好ましくは３０℃〜１００℃、より好ましくは４０℃〜８０℃、特に好ましくは５０℃〜７０℃で行う。特に、浸漬温度を５０℃〜６０℃とすることにより、プロテオグリカンは、ほとんど分解されず、高分子の糖タンパク質複合体として抽出することができる。 The immersion of the biological sample in the aqueous surfactant solution is preferably performed at 30 ° C to 100 ° C, more preferably 40 ° C to 80 ° C, and particularly preferably 50 ° C to 70 ° C. In particular, by setting the immersion temperature to 50 ° C. to 60 ° C., the proteoglycan is hardly decomposed and can be extracted as a polymer glycoprotein complex.
浸漬は、軟骨１重量部に対して界面活性剤水溶液を、好ましくは１〜５０重量部、より好ましくは２〜３０重量部、更に好ましくは４〜２０重量部、特に好ましくは６〜１５重量部用いて行う。ミキサー又はスターラーなどを用いて攪拌しながら浸漬するのが好ましい。 The immersion is preferably 1 to 50 parts by weight, more preferably 2 to 30 parts by weight, still more preferably 4 to 20 parts by weight, particularly preferably 6 to 15 parts by weight, based on 1 part by weight of cartilage. To do. It is preferable to immerse with stirring using a mixer or a stirrer.
軟骨組織からのプロテオグリカンの抽出は、例えばカルバゾール法（Johnら、ANALYTICAL BIOCHEMISTRY、1967年、第19巻、第119-132頁）によってウロン酸量を検出ないし定量することでモニターすることができるが、その他公知の方法によってウロン酸を検出し、モニターしてもよい。 Extraction of proteoglycan from cartilage tissue can be monitored by detecting or quantifying the amount of uronic acid by, for example, the carbazole method (John et al., ANALYTICAL BIOCHEMISTRY, 1967, Vol. 19, pp. 119-132). In addition, uronic acid may be detected and monitored by a known method.
浸漬が終了した界面活性剤水溶液は、プロテオグリカンを抽出した後の残渣を多く含むので、これらを濾過、遠心分離その他の方法で取り除くことが好ましい。 The surfactant aqueous solution that has been soaked contains a large amount of residue after extraction of proteoglycan, and it is preferable to remove these by filtration, centrifugation, or other methods.
プロテオグリカンを含む抽出液はそのまま製品として利用してもよいが、プロテオグリカンの各種用途に対して求められる純度にまで適当な方法でプロテオグリカンを分離ないし精製することが好ましい。 Although the extract containing proteoglycan may be used as a product as it is, it is preferable to separate or purify proteoglycan by an appropriate method up to the purity required for various uses of proteoglycan.
本発明において、プロテオグリカンの精製方法には公知の精製方法を使用することができる。好ましい精製方法としては、遠心分離法を挙げることができる。遠心分離法によって、細かい固形物を沈殿残渣として簡便に除去することができる。 In the present invention, a known purification method can be used as a method for purifying proteoglycans. As a preferred purification method, a centrifugal separation method can be exemplified. By the centrifugal separation method, a fine solid can be easily removed as a precipitation residue.
また、遠心分離法によって回収されるプロテオグリカンを含む液相を、更にフィルターペーパー又は適当な分画分子量を有する限外濾過膜分離装置などを用いて濾過してもよい。排除分子量としては概ね５万〜１００万の範囲であればよい。この操作において、分画分子量５０万以上のものを使用すれば、液相からコラーゲンも除去することができ、プロテオグリカンの純度を上げることが可能である。また、プロテオグリカンを含む液相に水を加えて液相の粘度を下げることで、膜の通過を容易にするとともに、これを繰り返すことで、わずかに生じる原料由来の特異臭を除去することも可能である。
更に、得られた濃縮液を食塩飽和エタノールに加えることで、ゲル状のプロテオグリカンを回収することもできる。このゲル状プロテオグリカンは真空凍結乾燥機を用いて固形物にしてもよいし、スプレードライヤーで乾燥させ、粉末状固形物としてもよい。
Further, the liquid phase containing proteoglycan recovered by the centrifugal separation method may be further filtered using a filter paper or an ultrafiltration membrane separation device having an appropriate fractional molecular weight. The excluded molecular weight may be in the range of about 50,000 to 1,000,000. In this operation, if a fractional molecular weight of 500,000 or more is used, collagen can also be removed from the liquid phase, and the purity of proteoglycan can be increased. In addition, by adding water to the liquid phase containing proteoglycan to reduce the viscosity of the liquid phase, it is possible to facilitate passage through the membrane, and by repeating this, it is also possible to remove the slightly specific odor originating from the raw material It is.
Furthermore, gel-like proteoglycan can also be collect | recovered by adding the obtained concentrate to salt saturated ethanol. This gel-like proteoglycan may be made into a solid using a vacuum freeze dryer, or may be dried with a spray dryer to make a powdered solid.
本発明のプロテオグリカンの分子量は、本発明の目的が達成できる限り特段限定されるものではないが、例えば１００万以上、好ましくは１５０万以上、更に好ましくは１８０万以上が好ましく、上限は特段制限されないが、例えば３００万程度以下が好ましい。
分子量の測定方法としては、高速液体クロマトグラフィ装置を用いる方法が挙げられる。
The molecular weight of the proteoglycan of the present invention is not particularly limited as long as the object of the present invention can be achieved. For example, it is 1 million or more, preferably 1.5 million or more, more preferably 1.8 million or more, and the upper limit is not particularly limited. However, for example, about 3 million or less is preferable.
Examples of the method for measuring the molecular weight include a method using a high performance liquid chromatography apparatus.
本発明の組成物には、必要に応じて、健康食品等の食品組成物の製造や経口医薬組成物の製造において公知ないし周知の不活性成分を含めてもよいし、また、作用の増強や他の作用を期待して、本発明のプロテオグリカン以外の有効成分を含めてもよい。 The composition of the present invention may contain, as necessary, known or well-known inactive ingredients in the production of food compositions such as health foods and oral pharmaceutical compositions, In anticipation of other actions, an active ingredient other than the proteoglycan of the present invention may be included.
本発明の組成物に含められ得る不活性成分としては、本発明の効果が達成される限り特段制限されるものではないが、例えば、食品又は医薬として許容される担体、例えば、賦形剤、結合剤、増量剤、崩壊剤、界面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、嬌味剤、香料、被膜剤、担体、希釈剤などが挙げられる。 The inactive ingredient that can be included in the composition of the present invention is not particularly limited as long as the effects of the present invention are achieved, but for example, a food or pharmaceutically acceptable carrier such as an excipient, Examples include binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, and diluents.
本発明の組成物に含められ得る、本発明のプロテオグリカン以外の有効成分としては、特段限定されるものではないが、骨強化の点から、例えば、カルシウムやビタミンＤなどが挙げられる。 The active ingredient other than the proteoglycan of the present invention that can be included in the composition of the present invention is not particularly limited, but examples thereof include calcium and vitamin D from the viewpoint of bone strengthening.
本発明の組成物の具体的な摂取量や摂取間隔は、対象の体重、性別、年齢、状態又はその他の要因に従って変動し得る。具体的な摂取量や摂取間隔は、当業者により適宜決定され得る。しかしながら、非限定的な具体例を挙げるとすれば、通常、成人１人（６０ｋｇ）に対して、本発明のプロテオグリカンの１日摂取量は、例えば５ｍｇ以上、好ましくは１０ｍｇ以上、より好ましくは２０ｍｇ以上、更に好ましくは３０ｍｇ以上であって、好ましくは１０００ｍｇ以下、より好ましくは５００ｍｇ以下、更に好ましくは２００ｍｇ、より更に好ましくは１００ｍｇ以下、なお更により好ましくは８０ｍｇ以下であり、そして、特に好ましくは４０ｍｇである。本発明ではこのような量を、１日に１回〜複数回で摂取するのが好ましい。 The specific intake and interval of the composition of the present invention may vary according to the subject's weight, gender, age, condition or other factors. Specific intake and intake intervals can be appropriately determined by those skilled in the art. However, if a non-limiting specific example is given, the daily intake of the proteoglycan of the present invention is usually 5 mg or more, preferably 10 mg or more, more preferably 20 mg per adult (60 kg). More preferably, it is 30 mg or more, preferably 1000 mg or less, more preferably 500 mg or less, still more preferably 200 mg, still more preferably 100 mg or less, still more preferably 80 mg or less, and particularly preferably 40 mg. It is. In the present invention, such an amount is preferably taken once to several times a day.
摂取期間は特に限定されないが、反復又は連続して摂取することが好ましく、５日間以上連続して投与又は摂取することがより好ましく、１５日間以上連続して投与又は摂取することが更に好ましい。 The intake period is not particularly limited, but it is preferably taken repeatedly or continuously, more preferably administered or ingested continuously for 5 days or more, and further preferably administered or ingested continuously for 15 days or more.
本発明の組成物の摂取対象としては、特段限定されるものではないが、例えば、マウス、ラット、ハムスター、モルモット、ウサギ、ネコ、イヌ、ウシ、ウマ、ヒツジ、サル、ヒト等の哺乳動物が挙げられる。特に、本発明の組成物は、関節症の自覚があるヒト対象に有効である。 The ingestion target of the composition of the present invention is not particularly limited, and examples thereof include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, and humans. Can be mentioned. In particular, the composition of the present invention is effective for human subjects who are aware of arthropathy.
本発明の組成物の具体的な形状としては、本発明の効果が達成される限り特段の制限はない。食品組成物又は経口医薬組成物の形状としては、例えば、錠剤、カプセル剤、顆粒剤、散剤、トローチ剤、液剤、シロップ剤、懸濁剤などが挙げられる。また、食品組成物の形状には、清涼飲料水、茶系飲料、コーヒー飲料、果汁飲料、炭酸飲料、乳飲料、ゼリー、ウエハース、ビスケット、パン、麺、ソーセージなども挙げられる。 The specific shape of the composition of the present invention is not particularly limited as long as the effects of the present invention are achieved. Examples of the shape of the food composition or oral pharmaceutical composition include tablets, capsules, granules, powders, troches, solutions, syrups, suspensions, and the like. Examples of the shape of the food composition include soft drinks, tea beverages, coffee beverages, fruit juice beverages, carbonated beverages, milk beverages, jelly, wafers, biscuits, bread, noodles, sausages and the like.
本発明の食品組成物又は経口医薬組成物の形状としては、カプセル剤、顆粒剤、散剤、トローチ剤等の固形製剤が好ましく、簡便に摂取ができることから、カプセル剤及び錠剤が更に好ましく、錠剤が特に好ましい。 As the shape of the food composition or oral pharmaceutical composition of the present invention, solid preparations such as capsules, granules, powders, troches and the like are preferable, and capsules and tablets are more preferable because tablets can be easily ingested. Particularly preferred.
本発明の組成物を錠剤とする場合には、結晶セルロース及び滑沢剤を含めることが好ましい。
錠剤中における結晶セルロースの含有量は、１〜８０重量％、好ましくは３〜７５重量％である。錠剤中における結晶セルロースの含有割合が高すぎると、スティッキングが発生しやすくなる。逆に、結晶セルロースの含有割合が低すぎると、錠剤の硬度が低下する。
錠剤中に含める滑沢剤としては、ステアリン酸カルシウム、二酸化ケイ素、カルボキシメチルセルロースカルシウム、ショ糖脂肪酸エステル、ケイ酸カルシウム、ケイ酸マグネシウム、タルク及びシリカヒドロゲルなどの一般的に使用されている滑沢剤が挙げられるが、中でも、ステアリン酸カルシウム、二酸化ケイ素、カルボキシメチルセルロースカルシウムが好ましい。錠剤中における滑沢剤の含有量は、０．５〜５質量％、好ましくは０．５〜３質量％である。錠剤中における滑沢剤の含有割合が少なすぎると、錠剤の製造過程でスティッキングやキャッピングなどの障害が発生しやすくなる。逆に、滑沢剤の含有割合が多すぎると錠剤の成形性が悪くなり、十分な錠剤硬度が得られない。
When the composition of the present invention is used as a tablet, it is preferable to include crystalline cellulose and a lubricant.
The content of crystalline cellulose in the tablet is 1 to 80% by weight, preferably 3 to 75% by weight. If the content of crystalline cellulose in the tablet is too high, sticking tends to occur. On the contrary, if the content ratio of crystalline cellulose is too low, the hardness of the tablet is lowered.
Lubricants included in tablets include commonly used lubricants such as calcium stearate, silicon dioxide, carboxymethylcellulose calcium, sucrose fatty acid ester, calcium silicate, magnesium silicate, talc and silica hydrogel. Among them, calcium stearate, silicon dioxide, and carboxymethylcellulose calcium are preferable. The content of the lubricant in the tablet is 0.5 to 5% by mass, preferably 0.5 to 3% by mass. If the content of the lubricant in the tablet is too small, obstacles such as sticking and capping are likely to occur during the tablet production process. On the other hand, if the content of the lubricant is too large, the tablet moldability deteriorates and sufficient tablet hardness cannot be obtained.
錠剤は、原料を同時に又は順次配合して均一に混合し、圧縮成形することにより製造することができる。圧縮成形法とは、一般的に打錠による製造法をいい、原料混合粉末から顆粒を造粒した後、これを圧縮成形する顆粒打錠法と、原料混合粉末をそのまま直接圧縮成形する直接打錠法とを含む。本発明の錠剤は打錠性が良好であるため、その製造法としては、顆粒打錠法及び直接打錠法のいずれを用いることもできるが、工程が簡便であるという点で直接打錠法が好ましい。打錠は、単発打錠機や、ロータリー打錠機等の連続打錠機を利用して、常法に従って行うことができる。打錠の圧力は、後述する所望される錠剤の大きさや硬度を考慮して適宜設定することができるが、例えば、２０００〜４０００ｋｇｆに設定することができる。 Tablets can be produced by blending the raw materials simultaneously or sequentially, mixing them uniformly, and compressing them. The compression molding method generally refers to a production method by tableting. The granulation tableting method in which granules are granulated from a raw material mixed powder and then compression molded, and the direct compression in which the raw material mixed powder is directly compression molded. Including the lock method. Since the tablet of the present invention has good tableting properties, either the granule tableting method or the direct tableting method can be used as its production method, but the direct tableting method is used in that the process is simple. Is preferred. Tableting can be performed according to a conventional method using a single tableting machine or a continuous tableting machine such as a rotary tableting machine. The tableting pressure can be appropriately set in consideration of the desired tablet size and hardness, which will be described later, but can be set to 2000 to 4000 kgf, for example.
錠剤の大きさは、一般的な大きさ、例えば直径５〜１２ｍｍ程度、質量１００〜４００ｍｇ程度であればよいが、飲みやすさを考慮すると、直径５〜１０ｍｍ程度、質量２００〜３００ｍｇ程度であることが好ましい。錠剤の硬度は、一般的な錠剤の硬度、例えば６ｋｇｆ以上であればよいが、割れや欠け等の破損の抑制や、表面の平滑性、摂取後の崩壊性などを考慮すると、６．５〜１５ｋｇｆ程度であることが好ましい。錠剤の硬度は、汎用の錠剤硬度計（例えば、フロイントの錠剤硬度計）を用いて測定することができる。 The size of the tablet may be a general size, for example, a diameter of about 5 to 12 mm and a mass of about 100 to 400 mg, but considering the ease of drinking, the diameter is about 5 to 10 mm and the mass of about 200 to 300 mg. It is preferable. The hardness of the tablet may be a general tablet hardness, for example, 6 kgf or more. However, considering the suppression of breakage such as cracking and chipping, surface smoothness, disintegration after ingestion, etc., 6.5 to It is preferably about 15 kgf. The hardness of the tablet can be measured using a general-purpose tablet hardness meter (for example, Freund's tablet hardness meter).
以下に製造例及び実施例を用いて本発明を更に詳細に説明するが、本発明はこれらの例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to production examples and examples, but the present invention is not limited to these examples.
〔製造例１〕
−４０℃で冷凍保管したシロサケの頭部から摘出した鼻軟骨を電動のミートチョッパーで細かく破砕しミンチ状にした。ミンチ状にした鼻軟骨から脱脂及び脱水を行った。処理後の鼻軟骨を通風又は減圧乾燥したもの２４ｇを出発原料とした。５リットルの抽出用容器にあらかじめ５℃に冷却しておいた蒸留水２９７０ｇを入れ、更に製造例１で使用した固形のスークロース脂肪酸エステル３０ｇを投入し、総量３０００ｇ（１重量％）のスークロース脂肪酸エステル水溶液を準備した。この抽出容器に出発原料２４ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、鼻軟骨を除去して、プロテオグリカンを含む抽出液を回収した。
抽出液を日立ｈｉｍａｃＣＦ７Ｄ２型の遠心分離機を用いて３０００rpm、２０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。
更に、この液相をフィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＢＩＯＭＡＸ １００Ｋ ＰＯＬＹＥＴＨＥＲＳＵＬＦＯＮＥ（分画分子量１０万）を用いて分画と濃縮を同時に行った。
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、２４ｇの出発原料から、換算値でその２２．２１％に相当する５．５ｇの乾燥固形分を得ることができた。
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、ガランボス法によりウロン酸量を定量してプロテオグリカン量を計算した。更に高速液体クロマトグラフィ装置（島津製作所、カラムＴＳＫ−ＧＥＬ Ｇ４０００ＰＷＸＬ）を用いて、プロテオグリカンの分子量を測定した。
これらの分析の結果、固形分中にタンパク質１４．８％、灰分１８．４％、炭水化物６４．８％、脂質０％が存在することが判った。また、プロテオグリカンの分子量は約１３０万であった。
[Production Example 1]
The nasal cartilage extracted from the head of the chum salmon stored frozen at −40 ° C. was finely crushed with an electric meat chopper to form a mince. Degreasing and dehydration were performed from minced nasal cartilage. 24 g of the nasal cartilage that had been treated and ventilated or dried under reduced pressure was used as a starting material. 2970 g of distilled water cooled to 5 ° C. in advance in a 5 liter extraction container, and 30 g of the solid sucrose fatty acid ester used in Production Example 1 are added, and the total amount of sucrose fatty acid ester is 3000 g (1 wt%). An aqueous solution was prepared. 24 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
After the immersion, the contents were transferred to another container set with a 1 mm square stainless steel strainer, the nasal cartilage was removed, and the extract containing proteoglycan was collected.
The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himacCF7D2 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycan was recovered.
Furthermore, this liquid phase is filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water is added to the filtrate, and then fractionated using BIOMAX 100K POLYETHERSULFONE (fraction molecular weight 100,000) manufactured by Nihon Millipore. And concentrating at the same time.
A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, 5.5 g of dry solids corresponding to 22.21% in terms of conversion value could be obtained from 24 g of starting material.
In addition, the amount of amino acid in the concentrated solution is measured using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the galambos method. The amount of proteoglycan was calculated by quantification. Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column TSK-GEL G4000PWXL).
As a result of these analyses, it was found that 14.8% protein, 18.4% ash, 64.8% carbohydrate, and 0% lipid were present in the solid content. The molecular weight of proteoglycan was about 1.3 million.
〔製造例２〕
−４０℃で冷凍保管したシロサケの頭部から摘出した鼻軟骨を電動のミートチョッパーで細かく破砕しミンチ状にした。ミンチ状にした鼻軟骨から脱脂及び脱水を行った。処理後の鼻軟骨を通風又は減圧乾燥したもの１７．９ｇを出発原料とした。５リットルの抽出用容器にあらかじめ５℃に冷却しておいた蒸留水２４９５ｇを入れ、更に固形のキラヤサポニン５ｇを投入し、総量２５００ｇ（０．２重量％）のサポニン水溶液を準備した。この抽出容器に出発原料１７．９ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、鼻軟骨を除去して、プロテオグリカンを含む抽出液を回収した。
抽出液を日立ｈｉｍａｃＣＦ７Ｄ２型の遠心分離機を用いて３０００rpm、２０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。
更に、この液相をフィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＢＩＯＭＡＸ １００Ｋ ＰＯＬＹＥＴＨＥＲＳＵＬＦＯＮＥ（分画分子量１０万）を用いて分画と濃縮を同時に行った。
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、１７．９ｇの出発原料から換算値でその１８．４１％に相当する３．２９ｇの乾燥固形分を得ることができた。
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、ガランボス法によりウロン酸量を定量してプロテオグリカン量を計算した。更に高速液体クロマトグラフィ装置（島津製作所、カラムＴＳＫ−ＧＥＬ Ｇ４０００ＰＷＸＬ）を用いて、プロテオグリカンの分子量を測定した。
これらの分析の結果、固形分中にタンパク質１７％、灰分２２．４％、炭水化物６０．６％、脂質０％が存在することが判った。また、プロテオグリカンの分子量は約１２０万であった。
[Production Example 2]
The nasal cartilage extracted from the head of the chum salmon stored frozen at −40 ° C. was finely crushed with an electric meat chopper to form a mince. Degreasing and dehydration were performed from minced nasal cartilage. After treatment, 17.9 g of nasal cartilage that had been ventilated or dried under reduced pressure was used as a starting material. Distilled water (2495 g) that had been cooled to 5 ° C. in advance was placed in a 5-liter extraction container, and 5 g of solid Quillaja saponin was added to prepare a total amount of 2500 g (0.2 wt%) of a saponin aqueous solution. 17.9 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
After the immersion, the contents were transferred to another container set with a 1 mm square stainless steel strainer, the nasal cartilage was removed, and the extract containing proteoglycan was collected.
The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himacCF7D2 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycan was recovered.
Furthermore, this liquid phase is filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water is added to the filtrate, and then fractionated using BIOMAX 100K POLYETHERSULFONE (fraction molecular weight 100,000) manufactured by Nihon Millipore. And concentrating at the same time.
A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, 3.29 g of dry solid content corresponding to 18.41% of the converted value was obtained from 17.9 g of the starting material.
In addition, the amount of amino acid in the concentrated solution is measured using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the galambos method. The amount of proteoglycan was calculated by quantification. Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column TSK-GEL G4000PWXL).
As a result of these analyses, it was found that 17% protein, 22.4% ash, 60.6% carbohydrate, and 0% lipid were present in the solid content. The molecular weight of proteoglycan was about 1.2 million.
〔製造例３〕
−４０℃で冷凍保管したアオザメから摘出した軟骨を電動のミートチョッパーで細かく破砕しミンチ状にしたものを２００ｇ用意し、出発原料とした。５リットルの抽出用容器にあらかじめ０℃に冷却しておいた蒸留水２３７６ｇを入れ、更に固形のスークロース脂肪酸エステル（Ｃ１２；ＨＬＢ１４ 〜１８）２４ｇを投入し、総量２４００ｇ（１重量％）のスークロース脂肪酸エステル水溶液を準備した。この抽出用容器に出発原料２００ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、プロテオグリカンを含む抽出液を回収した。
抽出液をＩＷＡＫＩ ＣＦＳ−４００型の遠心分離機を用いて１５００×ｇ、３０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。
更にこの液相を、フィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＰＲＥＰ／ＳＣＡＬＥ ＴＦＦ膜（分画分子量１０万）を用いて分画と濃縮を同時に行った。
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、２００ｇの出発原料から、換算値でその２．７５％に相当する５．５ｇの乾燥固形分を得ることができた。
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、カルバゾール法によりウロン酸量を定量してプロテオグリカン量を計算した。
更に高速液体クロマトグラフィ装置（島津製作所、カラムＡｓａｈｉｐａｋ）を用いて、プロテオグリカンの分子量を測定した。
これらの分析の結果、固形分中にタンパク質３５％、灰分２１．５％、炭水化物４２．９％、脂質０．６％が存在することが判った。特許文献２によれば、プロテオグリカンのコアタンパク質の重量比は約７％と記載されており、従って本発明におけるプロテオグリカンは、炭水化物が約４２．９％であることから計算して、純度は約４６％と推定される。またプロテオグリカンの分子量は約２００万であった。
[Production Example 3]
200 g of minced cartilage extracted from a gray shark stored frozen at −40 ° C. with an electric meat chopper was prepared as a starting material. Add 2376 g of distilled water that has been cooled to 0 ° C. in a 5 liter extraction container, and then add 24 g of solid sucrose fatty acid ester (C12; HLB14-18) to a total amount of 2400 g (1% by weight) of sucrose fatty acid. An aqueous ester solution was prepared. 200 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
After the immersion, the contents were transferred to another container in which a 1 mm square stainless steel strainer was set, and the extract containing proteoglycan was collected.
The extract was centrifuged at 1500 × g for 30 minutes using an IWAKI CFS-400 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycans was recovered.
Further, this liquid phase was filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water was added to the filtrate, and then a PREP / SCALE TFF membrane (manufactured by Nippon Millipore) (molecular weight cut off 100,000) was used. Fractionation and concentration were performed simultaneously.
A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, 5.5 g of dry solids corresponding to 2.75% in terms of conversion value could be obtained from 200 g of starting material.
In addition, the amount of amino acid in the concentrated solution is measured by using an amino acid automatic analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the carbazole method. The amount of proteoglycan was calculated by quantification.
Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column Asahipak).
As a result of these analyses, it was found that 35% protein, 21.5% ash, 42.9% carbohydrate, and 0.6% lipid were present in the solid content. According to Patent Document 2, the weight ratio of the proteoglycan core protein is described as about 7%, and thus the proteoglycan in the present invention has a purity of about 46, as calculated from about 42.9% carbohydrate. %It is estimated to be. The molecular weight of proteoglycan was about 2 million.
上記の製造例３に示す操作において、スークロース脂肪酸エステル溶液の濃度を０．１％、５％、１０％に変えて２４時間浸漬、抽出を行ったときのプロテオグリカンの回収量（ウロン酸量）の経時変化を調べた結果、ほぼ同様な結果が得られた。
また、上記の製造例１に示す操作において、スークロース脂肪酸エステルを０．６６６Ｎの酢酸に変えて２４時間浸漬、抽出を行ったときのプロテオグリカンの回収量（ウロン酸量）の経時変化を調べてみたところ、製造例３に比べて劣っていた。
In the operation shown in Production Example 3 above, the recovered amount of proteoglycan (the amount of uronic acid) when the concentration of the sucrose fatty acid ester solution was changed to 0.1%, 5%, 10% for 24 hours and extracted. As a result of examining the change with time, almost the same result was obtained.
In addition, in the operation shown in Production Example 1 above, the change over time in the recovered amount of proteoglycan (uronic acid amount) when sucrose fatty acid ester was changed to 0.666N acetic acid and immersed for 24 hours was extracted. However, it was inferior to Production Example 3.
〔実施例〕
プラセボ対照ランダム化二重盲検並行群間比較法により、本発明の組成物の効果を試験した。
〔Example〕
The effect of the composition of the present invention was tested by a placebo-controlled randomized double-blind parallel group comparison method.
試験の概略Outline of the test
群分け及び割付方法
統計解析責任者がスクリーニング検査時の性別、年齢、ＪＯＡスコア、ＪＫＯＭスコアに有意差を示さないように２群にランダム化し、コントローラーが２群を以下の摂取群に割り付けた。
・ 本発明組成物摂取群
・ 対照組成物摂取群
Grouping and allocation method The statistical analysis manager randomized the two groups so that there was no significant difference in gender, age, JOA score, and JKOM score at the screening test, and the controller allocated the two groups to the following intake groups.
-Group of the present invention intake group-Control composition intake group
試験組成物の処方
以下の実施例では、製造例１で製造したプロテオグリカン（以下、「プロテオグリカン複合体」ともいう。）を使用し、以下の処方の試験組成物を使用した。
Formulation of Test Composition In the following examples, the proteoglycan produced in Production Example 1 (hereinafter also referred to as “proteoglycan complex”) was used, and the test composition having the following formulation was used.
１錠が上記の組成を有するようになる割合で、各原料を混合し、得られた混合物を直接打錠して錠剤を製造した。 Each raw material was mixed in such a ratio that one tablet had the above composition, and the obtained mixture was directly tableted to produce a tablet.
投与方法
１日１回１錠を、夕食後に水又は湯とともに被験者に摂取させた。
Method of administration One tablet once a day was ingested by the subject together with water or hot water after dinner.
被験者
以下の者を被験者として選択した。
４０歳以上７０歳未満の男女であって、膝関節に違和感がある者
It was selected subject to the following persons as subjects.
Men and women between the ages of 40 and 70 who have a discomfort in the knee joint
検査及び調査
スクリーニング検査（前間接期間前）、摂取０週、４週、８週、１２週に下記を実施し、有効性を確認した。
１．ＪＯＡスコア
２．ＪＫＯＭスコア
Tests and survey screening tests (before the previous indirect period), intake 0 weeks, 4 weeks, 8 weeks, and 12 weeks, the following was performed to confirm the effectiveness.
1. 1. JOA score JKOM score
有効性
１．ＪＯＡスコア
ＪＯＡスコアの解析結果を図１、ＪＯＡスコアの経時的変化を図２に示す。
Ｉ疼痛・歩行能力 右では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に高い値を示し、対照組成物摂取群においては、摂取後４週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に高い値を示した。
Ｉ疼痛・歩行能力 左では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に高い値を示した。
Ｉ疼痛・歩行能力では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に高い値を示し、摂取後４、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に高い値を示した。
ＪＯＡ 右では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に高い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に高い値を示した。
ＪＯＡでは、本発明組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に高い値を示し、摂取後４、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に高い値を示した。摂取後８週においては本発明組成物摂取群と対照組成物摂取群の間に有意な差（ｐ＝０．０７３）は認められなかったが、これは８週時に現地の気温が高い値を示したことにより、膝関節の症状が全体的に緩和されたためと考えられた。
Effectiveness
1. JOA Score FIG. 1 shows the analysis result of the JOA score, and FIG. 2 shows the change over time of the JOA score.
I Pain / walking ability On the right, in the group receiving the composition of the present invention, the value is significantly higher at the start of intake at 12 weeks after the intake, and at the start of intake at 4 weeks after the intake in the control composition intake group In 12 weeks after ingestion, the group taking the composition of the present invention showed a significantly higher value than the group taking the control composition.
I Pain / walking ability On the left, in the group taking the composition of the present invention, the value was significantly higher at 12 weeks after ingestion than at the start of ingestion.
In the I-pain / walking ability, the group taking the composition of the present invention showed a significantly higher value at the start of the intake at 12 weeks after the intake, and the group taking the composition of the present invention at the 4th and 12th week after the intake The value was significantly higher than.
On the right side, in the group taking the composition of the present invention, the value of the present invention composition was significantly higher at 12 weeks after ingestion than at the start of the ingestion, and at 12 weeks after ingestion, the group of the present invention ingested was significantly higher than the control composition ingestion group. The value is shown.
In JOA, in the group ingesting the composition of the present invention, the value is significantly higher at the start of ingestion at 8 and 12 weeks after ingestion, and in the group of ingestion of the present invention at 4 and 12 weeks after ingestion, the group ingesting the composition of the present invention is compared with the control composition ingestion group. The value was significantly higher. In 8 weeks after ingestion, no significant difference (p = 0.073) was observed between the group ingested with the composition of the present invention and the group ingested with the control composition. This suggests that the symptoms of the knee joint were alleviated as a whole.
２．ＪＫＯＭスコア
ＪＫＯＭスコアの解析結果を図３、ＪＫＯＭスコアの経時的変化を図４に示す。
ＪＫＯＭについては、表３に記載するように、質問番号と質問項目とが対応する。
2. JKOM Score FIG. 3 shows the analysis result of the JKOM score, and FIG. 4 shows the change over time of the JKOM score.
As for JKOM, as described in Table 3, the question number corresponds to the question item.
Ｉ 膝の痛みの程度では、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
II Ｑ１では、本発明組成物摂取群において、摂取後４、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示した。
II Ｑ２では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
II Ｑ３では、本発明組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後１２週で摂取開始時にくらべ有意に低い値を示し、摂取後８週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
II Ｑ４では、本発明組成物摂取群、対照組成物摂取群ともに摂取後８、１２週で摂取開始時にくらべ有意に低い値を示した。
II Ｑ５、Ｑ６では、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示した。
II Ｑ７、Ｑ８では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示した。
II 膝の痛みやこわばりでは、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
III Ｑ９では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
III Ｑ１０では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後１２週で摂取開始時にくらべ有意に低い値を示した。
III Ｑ１１では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
III Ｑ１２では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示した。
III Ｑ１３では、本発明組成物摂取群において、摂取後１２週で摂取開始時にくらべ有意に低い値を示した。
III Ｑ１４では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。変化量において、摂取後１２週で本発明組成物摂取群では対照組成物摂取群にくらべ有意な減少が認められた。
III Ｑ１６では、本発明組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後８、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
III Ｑ１７では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
III Ｑ１８では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後８、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。変化量において、摂取後８週で本発明組成物摂取群では対照組成物摂取群にくらべ有意な減少が認められた。
III 日常生活の状態では、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
IV Ｑ２０では、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示した。
IV Ｑ２１では、本発明組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後１２週で摂取開始時にくらべ有意に低い値を示し、摂取後８週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
IV Ｑ２２では、対照組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示した。
IV ふだんの生活などでは、本発明組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示した。
Ｖ Ｑ２４では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後４週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
Ｖ Ｑ２５では、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群においては、摂取後８週で摂取開始時にくらべ有意に低い値を示し、摂取後４、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
Ｖ 健康状態についてでは、本発明組成物摂取群において、摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、対照組成物摂取群において、摂取後８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後４、１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。変化量において、摂取後４週で本発明組成物摂取群では対照組成物摂取群にくらべ有意な減少が認められた。
ＪＫＯＭでは、本発明組成物摂取群、対照組成物摂取群ともに摂取後４、８、１２週で摂取開始時にくらべ有意に低い値を示し、摂取後１２週で本発明組成物摂取群は対照組成物摂取群にくらべ有意に低い値を示した。
I In the degree of knee pain, the group taking the composition of the present invention and the group taking the control composition showed significantly lower values at the beginning of the intake at 4, 8, and 12 weeks after ingestion, and the composition of the present invention at 12 weeks after the ingestion. The intake group showed a significantly lower value than the control composition intake group.
II Q1 shows significantly lower values at the start of ingestion at 4 and 12 weeks after ingestion in the composition ingestion group of the present invention, and significant at the start of ingestion at 8 and 12 weeks after ingestion in the control composition ingestion group Showed a low value.
II Q2 shows a significantly lower value at the start of ingestion at 12 weeks after ingestion in the composition intake group of the present invention, and significant at the start of ingestion at 4, 8, and 12 weeks after ingestion in the control composition intake group In 12 weeks after ingestion, the group ingesting the composition of the present invention showed a significantly lower value than the group ingesting the control composition.
II Q3 shows significantly lower values at the start of ingestion at 8 and 12 weeks after ingestion in the composition intake group of the present invention, and significantly lower at the start of ingestion at 12 weeks after ingestion in the control composition intake group 8 weeks after ingestion, the group ingesting the composition of the present invention showed a significantly lower value than the group ingesting the control composition.
In II Q4, both the group taking the composition of the present invention and the group taking the control composition showed significantly lower values than those at the start of ingestion at 8 and 12 weeks after ingestion.
II In Q5 and Q6, both the group taking the composition of the present invention and the group taking the control composition showed significantly lower values than those at the start of ingestion at 4, 8, and 12 weeks after ingestion.
II In Q7 and Q8, in the group taking the composition of the present invention, the value was significantly lower at the beginning of taking at 4, 8, and 12 weeks after taking, and in the group taking the control composition, taking at 8 and 12 weeks after taking The value was significantly lower than that at the beginning.
II In knee pain and stiffness, both the group taking the composition of the present invention and the group taking the control composition showed significantly lower values at the start of the intake at 4, 8, and 12 weeks after ingestion, and the composition of the present invention at 12 weeks after the ingestion. The intake group showed a significantly lower value than the control composition intake group.
III Q9 shows a significantly lower value at the start of ingestion at 12 weeks after ingestion in the composition intake group of the present invention, and significant at the start of ingestion at 4, 8, and 12 weeks after ingestion in the control composition intake group In 12 weeks after ingestion, the group ingesting the composition of the present invention showed a significantly lower value than the group ingesting the control composition.
III Q10 shows significantly lower values at the start of ingestion at 4, 8, and 12 weeks after ingestion in the composition intake group of the present invention, and significant at the start of ingestion at 12 weeks after ingestion in the control composition intake group Showed a low value.
III In Q11, the intake of the composition of the present invention showed significantly lower values at the start of intake at 4, 8, and 12 weeks after the intake, and the intake of the composition of the present invention at 12 weeks after the intake became the control composition intake group. The value was significantly lower than that.
In III Q12, in the group ingesting the composition of the present invention, the value was significantly lower than that at the start of ingestion at 4, 8, and 12 weeks after ingestion.
In III Q13, in the group ingesting the composition of the present invention, the value was significantly lower at the start of ingestion 12 weeks after ingestion.
III In Q14, in the group taking the composition of the present invention, the value was significantly lower at the beginning of the intake at 4, 8, and 12 weeks after the intake, and the group taking the composition of the present invention was changed to the control composition intake group at 12 weeks after the intake. The value was significantly lower than that. In the amount of change, a significant decrease was observed in the group receiving the composition of the present invention at 12 weeks after the intake compared to the group receiving the control composition.
III In Q16, the intake of the composition of the present invention showed a significantly lower value at the start of intake at 8 and 12 weeks after the intake, and the intake of the composition of the present invention became the control composition intake group at 8 and 12 weeks after the intake. The value was significantly lower than that.
III In Q17, the intake of the composition of the present invention showed a significantly lower value at the start of intake at 4, 8, and 12 weeks after intake, and the intake of the composition of the present invention at 12 weeks after intake became the control composition intake group. The value was significantly lower than that.
III In Q18, in the group taking the composition of the present invention, the value was significantly lower at the beginning of the intake at 4, 8, and 12 weeks after the intake, and the group taking the composition of the present invention ingested the control composition at 8 and 12 weeks after the intake. The value was significantly lower than that of the group. In the amount of change, a significant decrease was observed in the group receiving the composition of the present invention at 8 weeks after the intake compared to the group receiving the control composition.
III In daily life, the group taking the composition of the present invention and the group taking the control composition showed significantly lower values at the start of ingestion at 4, 8, and 12 weeks after ingestion, and ingested the composition at 12 weeks after ingestion. The group showed significantly lower values than the control composition intake group.
In IV Q20, both the group taking the composition of the present invention and the group taking the control composition showed significantly lower values at the start of ingestion at 4, 8, and 12 weeks after ingestion.
IV Q21 shows a significantly lower value at the start of ingestion at 8 and 12 weeks after ingestion in the composition ingestion group of the present invention, and significantly lower at the start of ingestion at 12 weeks after ingestion in the control composition ingestion group 8 weeks after ingestion, the group ingesting the composition of the present invention showed a significantly lower value than the group ingesting the control composition.
In IV Q22, the control composition ingestion group showed a significantly lower value at the start of ingestion at 8 and 12 weeks after ingestion.
In the daily life of IV, in the group ingesting the composition of the present invention, the value was significantly lower at the beginning of ingestion at 8 and 12 weeks, and in the control composition ingestion group at 4, 8, and 12 weeks after ingestion. The value was significantly lower than that at the start of ingestion.
VQ24 shows a significantly lower value at the start of ingestion at 4, 8, and 12 weeks after ingestion in the composition ingestion group of the present invention, and at the start of ingestion at 8 and 12 weeks after ingestion in the control composition ingestion group. Compared with the control composition intake group, the value of the present invention composition intake group was significantly lower than that of the control composition intake group.
VQ25 shows a significantly lower value at the start of ingestion at 4, 8, and 12 weeks after ingestion in the composition ingestion group of the present invention, and significant at the start of ingestion at 8 weeks after ingestion in the control composition ingestion group. In the 4th and 12th week after ingestion, the group ingesting the composition of the present invention showed a significantly lower value than the group ingesting the control composition.
V Regarding the health condition, in the group ingesting the composition of the present invention, the value was significantly lower at the start of ingestion at 4, 8, and 12 weeks after ingestion, and in the control composition ingestion group, ingestion started at 8 and 12 weeks after ingestion. The value of the present invention composition was significantly lower than that of the control composition intake group at 4 and 12 weeks after the intake. In the amount of change, a significant decrease was observed in the group receiving the composition of the present invention at 4 weeks after the intake compared to the group receiving the control composition.
In JKOM, both the group taking the composition of the present invention and the group taking the control composition showed significantly lower values at the start of the intake at 4, 8, and 12 weeks after ingestion. The value was significantly lower than the food intake group.
これら実施例の結果から、本発明の組成物が、経口摂取により、膝関節症に有効であることが確認された。 From the results of these examples, it was confirmed that the composition of the present invention was effective for knee arthropathy by ingestion.
Claims (6)
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Citations (3)
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| JP2013039087A (en) * | 2011-08-18 | 2013-02-28 | Fuji Agri Arsh:Kk | Drinking water containing proteoglycan and method for producing the same |
| JP2014009164A (en) * | 2012-06-27 | 2014-01-20 | Nihon Yakuhin Kk | Production method of proteoglycan |
| WO2014017570A1 (en) * | 2012-07-25 | 2014-01-30 | 国立大学法人弘前大学 | Composition for preventing or treating osteoarthrosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2013039087A (en) * | 2011-08-18 | 2013-02-28 | Fuji Agri Arsh:Kk | Drinking water containing proteoglycan and method for producing the same |
| JP2014009164A (en) * | 2012-06-27 | 2014-01-20 | Nihon Yakuhin Kk | Production method of proteoglycan |
| WO2014017570A1 (en) * | 2012-07-25 | 2014-01-30 | 国立大学法人弘前大学 | Composition for preventing or treating osteoarthrosis |
Non-Patent Citations (1)
| Title |
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| フレグランスジャーナル, 2014, VOL.42, NO.9, 広告ページ, JPN6020029982, ISSN: 0004324892 * |
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