JP6243777B2 - Hyaluronic acid synthesis accelerator - Google Patents
Hyaluronic acid synthesis accelerator Download PDFInfo
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- JP6243777B2 JP6243777B2 JP2014068897A JP2014068897A JP6243777B2 JP 6243777 B2 JP6243777 B2 JP 6243777B2 JP 2014068897 A JP2014068897 A JP 2014068897A JP 2014068897 A JP2014068897 A JP 2014068897A JP 6243777 B2 JP6243777 B2 JP 6243777B2
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- casein
- hyaluronic acid
- degradation product
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Description
本発明は、カゼイン分解物を含有するヒアルロン酸合成促進剤に関する。 The present invention relates to a hyaluronic acid synthesis accelerator containing a casein degradation product.
ヒアルロン酸は、酸性ムコ多糖の一種であり、軟骨、滑膜、関節液、皮膚、臍帯、眼硝子体、その他の結合組織に広く存在し、水分を大量に保持し、ゲル状の形態を呈するという性質を有するため、生体内において重要な役割を果たしている。 Hyaluronic acid is a kind of acidic mucopolysaccharide that is widely present in cartilage, synovium, joint fluid, skin, umbilical cord, ophthalmic vitreous, and other connective tissues, retains a large amount of water, and exhibits a gel-like form Therefore, it plays an important role in vivo.
生体内のヒアルロン酸は、加齢により徐々にこれら皮膚や関節液等から減少することが知られているので、ヒアルロン酸と加齢との関係について多くの研究が行われている。
例えば、加齢によって皮膚に存在するヒアルロン酸が質的・量的に減少すると、皮膚のシワやタルミの形成、皮膚の弾力性やハリの低下といった皮膚の変化、いわゆる皮膚の老化が生じる。
また、関節液や滑膜等に存在するヒアルロン酸が質的・量的に減少すると、加齢による変形性関節症等の関節疾患を招く一因になると考えられている。加齢による変形性関節症の患者数は、今後、社会の高齢化に伴ってさらに増えることが懸念されている。
Since it is known that hyaluronic acid in a living body gradually decreases from these skin, joint fluid, and the like with aging, many studies have been conducted on the relationship between hyaluronic acid and aging.
For example, when the hyaluronic acid present in the skin decreases qualitatively and quantitatively due to aging, skin changes such as the formation of wrinkles and talmi on the skin, and the elasticity and firmness of the skin decrease, so-called skin aging occurs.
In addition, it is considered that a decrease in hyaluronic acid present in synovial fluid, synovium, etc. qualitatively and quantitatively may cause joint diseases such as osteoarthritis due to aging. There is concern that the number of patients with osteoarthritis due to aging will further increase with the aging of society in the future.
このことから、生体内におけるヒアルロン酸の合成を促進させるための種々の物質が検討されている。
例えば、特許文献１には、コラーゲン又はゼラチンのコラゲナーゼによる分解物であり、そのアミノ酸配列が、（Ｇｌｙ−Ｘ−Ｙ）（式中、Ｇｌｙは、グリシン残基を表し、Ｘ，Ｙはアミノ酸残基を表す）で表されるコラーゲントリペプチドからなるコラーゲンペプチド（Ｇｌｙ−Ｘ−Ｙ）ｎ（ｎは、正の整数）を含有するヒアルロン酸産生促進剤及びこれらを含有する皮膚外用剤、飲食品を提供することが開示されている。
また、特許文献２には、ソウジュツ（Atractylodis Lanceae Rhizoma）の溶媒抽出物を有効成分として含有するＨＡＳ２ｍＲＮＡ発現促進剤、又はヒアルロン酸産生促進剤、また、これを配合してなる皮膚外用剤、又は抗老化剤が開示されている。
For this reason, various substances for promoting the synthesis of hyaluronic acid in vivo have been studied.
For example, Patent Document 1 discloses a degradation product of collagen or gelatin by collagenase, the amino acid sequence of which is (Gly-XY) (wherein Gly represents a glycine residue, and X and Y represent amino acid residues). Hyaluronic acid production promoter containing collagen peptide (Gly-XY) n (n is a positive integer) consisting of collagen tripeptide represented by the following formula: Is disclosed.
Patent Document 2 discloses that a HAS2 mRNA expression promoter, hyaluronic acid production promoter containing a solvent extract of Atractylodis Lanceae Rhizoma as an active ingredient, a skin external preparation comprising the same, or an anti-antibody An aging agent is disclosed.
ここで、本技術に関連のある乳成分や乳由来ペプチド、乳分解物等について、これまでに知られている生理機能について、説明する。
例えば、特許文献３には、κ−カゼイン及び／又はκ−カゼインの加水分解物を有効成分とする動脈硬化防止剤が開示されている。
また、特許文献４には、Ｍｅｔ−Ｌｙｓ−Ｐｒｏからなるペプチドを有効成分として含有するアンジオテンシン変換酵素阻害活剤又は血圧降下剤が開示されている。
また、特許文献５には、獣乳カゼインを平均鎖長がアミノ酸残基数として２．１以下に加水分解して得た、ペプチドＸａａ−Ｐｒｏ又はＸａａ−Ｐｒｏ−Ｐｒｏを多く含むカゼイン加水分解物を有効成分として含有する経口摂取用関節リウマチ抑制剤が開示されている。
Here, the physiological functions known so far about milk components, milk-derived peptides, milk-decomposed products and the like related to the present technology will be described.
For example, Patent Document 3 discloses an arteriosclerosis inhibitor containing κ-casein and / or a hydrolyzate of κ-casein as an active ingredient.
Patent Document 4 discloses an angiotensin converting enzyme inhibitor or an antihypertensive agent containing a peptide composed of Met-Lys-Pro as an active ingredient.
Patent Document 5 discloses casein hydrolyzate containing a large amount of peptide Xaa-Pro or Xaa-Pro-Pro obtained by hydrolyzing animal milk casein to an average chain length of 2.1 or less as the number of amino acid residues. A rheumatoid arthritis inhibitor for oral intake containing as an active ingredient is disclosed.
本発明は、副作用が少なく、安全性に優れたヒアルロン酸合成促進剤、当該ヒアルロン酸合成促進剤を含む医薬、及びヒアルロン酸合成促進用食品組成物を提供することにある。 An object of the present invention is to provide a hyaluronic acid synthesis promoter having few side effects and excellent safety, a medicament containing the hyaluronic acid synthesis promoter, and a food composition for promoting hyaluronic acid synthesis .
本発明者は、ヒアルロン酸合成促進作用を有する物質について鋭意検討した結果、意外にも食品として長年使用されてきた乳由来の成分であるカゼインの分解物に、ヒアルロン酸合成酵素の遺伝子発現促進作用及びヒアルロン酸合成促進作用を有することを突き止めた。そして、カゼインの分解物がヒアルロン酸合成促進剤として利用できることを見出し、本発明を完成させるに至った。
上述のように、従来、乳成分や乳由来ペプチド、乳分解物等における生理機能が見出されている（例えば、前記特許文献１〜３参照）が、カゼイン分解物にヒアルロン酸合成酵素の遺伝子発現促進作用があることは全くの意外であった。
As a result of diligent research on a substance having a hyaluronic acid synthesis promoting action, the present inventor unexpectedly has a hyaluronic acid synthase gene expression promoting action on a degradation product of casein, which is a milk-derived component that has been used for many years as a food. It was also found that it has a hyaluronic acid synthesis promoting effect. And it discovered that the decomposition product of casein can be used as a hyaluronic acid synthesis promoter, and came to complete this invention.
As described above, conventionally, physiological functions in milk components, milk-derived peptides, milk-decomposed products, and the like have been found (see, for example, Patent Documents 1 to 3). It was completely unexpected that there was an expression promoting action.
すなわち、本発明は、カゼイン分解物を有効成分とするヒアルロン酸合成促進剤、当該ヒアルロン酸促進剤及び薬学的に許容される賦形剤を含む医薬、及びカゼイン分解物を有効成分とするヒアルロン酸合成促進用食品組成物を提供するものである。 That is, the present invention is hyaluronic acid synthesis promoting agent comprising as an active ingredient casein hydrolyzate, medicaments comprising those said hyaluronic acid promoter and a pharmaceutically acceptable excipient, and hyaluronic that casein hydrolyzate as an active ingredient A food composition for promoting acid synthesis is provided.
本発明は、副作用が少なく、安全性に優れたヒアルロン酸合成促進剤、当該ヒアルロン酸合成促進剤を含む医薬、及びヒアルロン酸合成促進用食品組成物を提供することができる。 INDUSTRIAL APPLICABILITY The present invention can provide a hyaluronic acid synthesis promoter having few side effects and excellent safety, a pharmaceutical containing the hyaluronic acid synthesis promoter, and a food composition for promoting hyaluronic acid synthesis .
本開示の好ましい実施形態について詳細に説明する。ただし、本開示は以下の好ましい実施形態に限定されず、本開示の範囲内で自由に変更することができるものである。 Preferred embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the following preferred embodiments, and can be freely changed within the scope of the present disclosure.
本開示のカゼイン分解物は、カゼインを加水分解したものが好適である。カゼインの分解処理としては、特に限定されないが、例えば、酵素処理、酸処理、アルカリ処理、熱処理等が挙げられ、これらから１種又は適宜２種以上の処理を組み合わせてもよい。 The casein hydrolyzate of the present disclosure is preferably a hydrolyzed casein. Although it does not specifically limit as a decomposition process of casein, For example, an enzyme process, an acid process, an alkali process, heat processing, etc. are mentioned, From these, you may combine 1 type or 2 or more types of processes suitably.
前記カゼイン分解物の非蛋白態窒素比率は、好ましくは５％以上、より好ましくは１０％以上、さらに好ましくは２０％以上である。 The non-protein nitrogen ratio of the casein degradation product is preferably 5% or more, more preferably 10% or more, and still more preferably 20% or more.
＜非蛋白態窒素比率の算出方法＞
ケルダール法日本食品工業学会編、「食品分析法」、第１０２ページ、株式会社光琳、昭和５９年）により試料の全窒素量を測定した。また、ラッパポート（Rappaport）−梅田変法（臨床検査、第９巻、第５３４乃至５３７頁、１９６５年）に基づく測定キット（商品名：ＮＰＮ−テストワコー；和光純薬工業社製）を使用し、該測定キットの説明書に従って試料の非蛋白態窒素量を測定し、得られた値に６．３８を乗じて非蛋白態窒素化合物量を算出した。これらの測定値から非蛋白態窒素比率（％）を次式により算出する。
非蛋白態窒素比率（％）＝（非蛋白態窒素化合物量／全窒素量）×１００
<Calculation method of non-protein nitrogen ratio>
The total nitrogen content of the sample was measured according to the Kjeldahl method edited by the Japan Food Industry Association, “Food Analysis Method”, page 102, Korin Co., Ltd. (1984). In addition, a measurement kit (trade name: NPN-Test Wako; manufactured by Wako Pure Chemical Industries, Ltd.) based on Rappaport-Modified Umeda (Clinical Laboratory, Vol. 9, pp. 534 to 537, 1965) is used. Then, the amount of non-protein nitrogen of the sample was measured according to the instructions of the measurement kit, and the amount obtained was multiplied by 6.38 to calculate the amount of non-protein nitrogen compound. From these measured values, the non-protein nitrogen ratio (%) is calculated by the following formula.
Non-protein nitrogen ratio (%) = (non-protein nitrogen compound amount / total nitrogen amount) × 100
前記カゼイン分解物のアミノ酸遊離率は、好ましくは５０％以下、より好ましくは４５％以下である。 The amino acid release rate of the casein degradation product is preferably 50% or less, more preferably 45% or less.
＜アミノ酸遊離率の算定方法＞
トリプトファン、システイン及びメチオニン以外のアミノ酸については、試料を６規定の塩酸で１１０℃、２４時間加水分解し、トリプトファンについては、水酸化バリウムで１１０℃、２２時間アルカリ分解し、システイン及びメチオニンについては、過ギ酸処理後、６規定の塩酸で１１０℃、１８時間加水分解し、それぞれアミノ酸自動分析機（日立製作所製。８３５型）により分析し、アミノ酸の質量を測定した。
上記の方法により試料中の各アミノ酸の組成を測定し、これを合計して試料中の全アミノ酸の質量を算出する。次いで、スルホサリチル酸で試料を除蛋白し、残留する各遊離アミノ酸の質量を上記の方法により測定し、これを合計して試料中の全遊離アミノ酸の質量を算出する。これらの値から、試料中の遊離アミノ酸含有率を次式により算出した。
アミノ酸遊離率（％）＝（全遊離アミノ酸の質量／全アミノ酸の質量）×１００
<Calculation method of amino acid release rate>
For amino acids other than tryptophan, cysteine and methionine, the sample was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and for tryptophan, the sample was alkali-degraded with barium hydroxide at 110 ° C. for 22 hours. For cysteine and methionine, After treatment with performic acid, hydrolysis was performed with 6 N hydrochloric acid at 110 ° C. for 18 hours, and each was analyzed with an amino acid automatic analyzer (manufactured by Hitachi, Ltd. Model 835) to measure the mass of amino acids.
The composition of each amino acid in the sample is measured by the above method, and these are summed to calculate the mass of all amino acids in the sample. Next, the sample is deproteinized with sulfosalicylic acid, the mass of each free amino acid remaining is measured by the above method, and the total is calculated to calculate the mass of all free amino acids in the sample. From these values, the free amino acid content in the sample was calculated by the following equation.
Amino acid release rate (%) = (mass of all free amino acids / mass of all amino acids) × 100
前記カゼイン分解物の分子量は、好ましくは２０００ダルトン（以下、「Ｄａ」とする）以下又は未満、より好ましくは１０００Ｄａ以下、さらにより好ましくは５００Ｄａ以下である。 The molecular weight of the casein decomposition product is preferably 2000 daltons (hereinafter referred to as “Da”) or less, more preferably 1000 Da or less, and even more preferably 500 Da or less.
＜分子量の算定方法＞
本開示におけるカゼイン分解物の分子量は、以下の数平均分子量の概念により求めるものである。
数平均分子量（Number Average of Molecular Weight）は、例えば文献（社団法人高分子学会編、「高分子科学の基礎」、第１１６〜１１９頁、株式会社東京化学同人、１９７８年）に記載されているとおり、高分子化合物の分子量の平均値を次のとおり異なる指標に基づき示すものである。
すなわち、タンパク質加水分解物等の高分子化合物は不均一な物質であり、かつ分子量に分布があるため、タンパク質加水分解物の分子量は、物理化学的に取り扱うためには、平均分子量で示す必要があり、数平均分子量（以下、Ｍｎと略記することがある。）は、分子の個数についての平均であり、ペプチド鎖ｉの分子量がＭｉであり、その分子数をＮｉとすると、次の式により定義される。
<Method for calculating molecular weight>
The molecular weight of the casein degradation product in the present disclosure is determined by the following concept of number average molecular weight.
The number average molecular weight (Number Average of Molecular Weight) is described in, for example, the literature (edited by the Society of Polymer Science, “Basics of Polymer Science”, pages 116 to 119, Tokyo Kagaku Dojin, 1978). As shown, the average value of the molecular weight of the polymer compound is shown based on different indicators as follows.
That is, high molecular weight compounds such as protein hydrolysates are heterogeneous substances and have a distribution in molecular weight. Therefore, the molecular weight of protein hydrolysates must be expressed as an average molecular weight in order to handle them physicochemically. The number average molecular weight (hereinafter sometimes abbreviated as Mn) is the average of the number of molecules, the molecular weight of the peptide chain i is Mi, and the number of molecules is Ni. Defined.
前記カゼイン分解物における出発原料のカゼインは、例えば、市販品の各種カゼイン、乳酸カゼイン、塩酸カゼイン等の酸カゼイン；ナトリウムカゼイネイト、カリウムカゼイネイト、カルシウムカゼイネイト等のカゼイネイト等から選ばれる１種のもの又は２種以上の混合物が挙げられる。当該混合物は、任意の割合で混合すればよい。また、前記カゼインは、ウシ由来、ヤギ由来、ヒツジ由来、ウマ由来等の獣由来のものが好ましく、ウシ由来のものがより好ましい。具体的には、乳、脱脂乳、全脂粉乳、脱脂粉乳等から公知の方法によって単離されたカゼインであることが好ましい。
すなわち、本発明に用いられるカゼイン分解物は、蛋白質であるカゼインを出発原料として加水分解された分解物であることが好適である。
The casein as the starting material in the casein decomposition product is, for example, one kind selected from commercially available casein, lactic acid casein, acid casein such as casein hydrochloride; sodium caseinate, potassium caseinate, calcium caseinate, and the like. Or a mixture of two or more. What is necessary is just to mix the said mixture in arbitrary ratios. Further, the casein is preferably derived from animals such as cattle, goat, sheep, or horse, and more preferably derived from cattle. Specifically, casein isolated from milk, skim milk, whole milk powder, skim milk or the like by a known method is preferable.
That is, the casein decomposition product used in the present invention is preferably a hydrolysis product obtained by hydrolysis using casein, which is a protein, as a starting material.
原料カゼインを水又は温湯に分散し、溶解してカゼイン水溶液を調製する。溶解時には、炭酸カリウム、水酸化ナトリウム等の食品上使用可能な塩類でｐＨを調整することができる。当該カゼイン水溶液の濃度は、特に限定されないが、通常、蛋白質濃度として２％以上、さらに５〜１５質量％程度の濃度範囲に設定するのが好適である。 Raw material casein is dispersed in water or hot water and dissolved to prepare a casein aqueous solution. At the time of dissolution, the pH can be adjusted with salts usable on food such as potassium carbonate and sodium hydroxide. The concentration of the casein aqueous solution is not particularly limited, but it is usually preferable to set the protein concentration to a concentration range of 2% or more, and further about 5 to 15% by mass.
次いで、前記カゼイン水溶液を、加水分解処理する。当該加水分解処理として、例えば酸処理、アルカリ処理、熱処理及び酵素処理等が挙げられるが、酵素処理による加水分解が好ましい。また、適宜２種以上の処理を組み合わせてもよい。 Next, the casein aqueous solution is hydrolyzed. Examples of the hydrolysis treatment include acid treatment, alkali treatment, heat treatment and enzyme treatment, and hydrolysis by enzyme treatment is preferred. Moreover, you may combine 2 or more types of processes suitably.
本開示における蛋白質分解酵素は、例えば、植物由来、動物由来、微生物由来等が挙げられ、これらから１種又は２種以上組み合わせて使用できる。当該蛋白質分解酵素としては、エンドプロテアーゼが好適である。 Examples of the proteolytic enzyme in the present disclosure include plant-derived, animal-derived, and microorganism-derived enzymes, and these can be used alone or in combination of two or more. An endoprotease is suitable as the proteolytic enzyme.
前記エンドプロテア−ゼとして、例えば、セリンプロテアーゼ、メタロプロテアーゼ、システインプロテアーゼ、アスパラギン酸プロテイナーゼが挙げられ、これらを１種又は２種以上選択して用いることができる。このうち、セリンプロテアーゼ及び/又はメタロプロテアーゼを用いるのが好適である。前記セリンプロテアーゼとして、例えば、トリプシンやパンクレアチン（すい臓由来酵素、トリプシン、キモトリプシン、リパーゼなどを含む）等が挙げられる。
また、プロテアーゼは、アルカリ性プロテアーゼ、中性プロテアーゼ及び酸性プロテアーゼに分類される。このうち中性プロテアーゼを用いるのが好適である。
Examples of the endoprotease include serine protease, metalloprotease, cysteine protease, and aspartic proteinase, and one or more of these can be selected and used. Among these, it is preferable to use serine protease and / or metalloprotease. Examples of the serine protease include trypsin and pancreatin (including pancreatic enzyme, trypsin, chymotrypsin, lipase, etc.).
Proteases are classified into alkaline proteases, neutral proteases and acidic proteases. Of these, neutral protease is preferably used.
前記蛋白質分解酵素は、市販品を用いることができる。前記蛋白質分解酵素の例示として、例えば、ビオプラーゼ（長瀬生化学工業社製）、プロレザー（天野エンザイム社製）、プロテアーゼＳ（天野エンザイム社製）、プロテアーゼＡ（天野エンザイム社製）、パンクレアチン（天野エンザイム社製）、ＰＴＮ６．０Ｓ（ノボザイムズ社製）、サビナーゼ（ノボザイムズ社製）、ＧＯＤＯ Ｂ．Ａ．Ｐ（合同酒精社製）、プロテアーゼＮ（天野エンザイム社製）、ＧＯＤＯ Ｂ．Ｎ．Ｐ（合同酒精社製）、ニュートラーゼ（ノボザイムズ社製）、アルカラーゼ（ノボザイムズ社製）、トリプシン（ノボザイムズ社製）、キモトリプシン（ノボザイムズ社製）、ズブチリシン（ノボザイムズ社製）、パパイン（天野エンザイム社製）、ブロメライン（天野エンザイム社製）等が挙げられ、これらから１種又は２種以上の酵素を選択して用いてもよい。
このうち、スブチリシン（subtilisin：例えば、ビオプラーゼ）、トリプシン（trypsin：例えばＰＴＮ６．０Ｓ）、バシロシン（bachillolysin：例えばプロテアーゼＮ）、セリンプロテアーゼ（例えば、パンクレアチン）から選ばれる１種又は２種以上のものが好適であり、これらは中性のプロテアーゼである。さらに、これらのうちで、スブチリシン及びトリプシン及びバシロシンの３種の組み合わせ、又はプロテアーゼＡ及びパンクレアチンの２種の組み合わせの何れかを使用することが好適である。
A commercially available product can be used as the proteolytic enzyme. Examples of the proteolytic enzyme include, for example, bioprase (manufactured by Nagase Seikagaku Corporation), pro leather (manufactured by Amano Enzyme), protease S (manufactured by Amano Enzyme), protease A (manufactured by Amano Enzyme), pancreatin ( Amano Enzyme), PTN 6.0S (Novozymes), Sabinase (Novozymes), GODO B. A. P (manufactured by Godo Shusei Co., Ltd.), protease N (manufactured by Amano Enzyme), GODO B.I. N. P (manufactured by Godo Shusei Co., Ltd.), Neutase (manufactured by Novozymes), Alcalase (manufactured by Novozymes), trypsin (manufactured by Novozymes), chymotrypsin (manufactured by Novozymes), subtilisin (manufactured by Novozymes), papain (manufactured by Amano Enzymes) ), Bromelain (manufactured by Amano Enzyme), etc., and one or more enzymes may be selected from these.
Among these, one or more selected from subtilisin (for example, bioplase), trypsin (for example, PTN6.0S), bacilosin (for example, protease N), and serine protease (for example, pancreatin) Are preferred and these are neutral proteases. Furthermore, among these, it is preferable to use any one of three combinations of subtilisin and trypsin and basilosine, or two combinations of protease A and pancreatin.
前記カゼインに対するエンドプロテア−ゼの使用量は、特に限定されず、基質濃度、酵素力価、反応温度及び反応時間等により適宜調整すればよいが、一般的には、カゼイン中の蛋白質１ｇ当り２０００〜１１０００活性単位の割合で添加することが好ましい。 The amount of endoprotease used for the casein is not particularly limited, and may be appropriately adjusted depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, etc. Generally, 2000 g per 1 g of protein in casein. It is preferable to add at a rate of ˜11000 active units.
前記蛋白質分解酵素による加水分解は、所望の条件（例えば、分子量の範囲内等）になるように行うのが望ましい。これにより、本開示で用いるカゼイン分解物を得ることができる。 The hydrolysis by the proteolytic enzyme is desirably performed so as to satisfy desired conditions (for example, within a molecular weight range). Thereby, the casein decomposition product used by this indication can be obtained.
前記蛋白質分解酵素による加水分解前に前記原料乳蛋白質溶液のｐＨを、炭酸カリウム、水酸化ナトリウム等の食品上使用可能な塩類で使用酵素の至適ｐＨに調整することもできる。前記原料乳蛋白質溶液のｐＨは、好ましくは５〜１０、より好ましくは７〜１０に調整する。 Prior to hydrolysis by the proteolytic enzyme, the pH of the raw milk protein solution can be adjusted to the optimum pH of the enzyme used with salts that can be used on food such as potassium carbonate and sodium hydroxide. The pH of the raw milk protein solution is preferably adjusted to 5 to 10, more preferably 7 to 10.
前記蛋白質分解酵素の反応温度は、使用酵素の最適温度の範囲で行うことが望ましく、好ましくは３０〜６０℃、より好ましくは４０〜６０℃で行う。 The reaction temperature of the proteolytic enzyme is desirably within the optimum temperature range of the enzyme used, preferably 30 to 60 ° C, more preferably 40 to 60 ° C.
前記蛋白質分解酵素の反応保持時間は、前記特定の非蛋白態窒素比率になるように適宜調整すればよく、例えば０．５〜２４時間で行うことが可能であり、好ましくは１〜１５時間、より好ましくは３〜１０時間である。 The reaction holding time of the proteolytic enzyme may be appropriately adjusted so as to be the specific non-protein nitrogen ratio, and can be performed, for example, in 0.5 to 24 hours, preferably 1 to 15 hours, More preferably, it is 3 to 10 hours.
前記蛋白質分解酵素による加水分解は、当該酵素を加熱して失活させて終了させればよい。例えば、１００℃以上（好適には１１０〜１３０℃）で失活させる場合には１〜３秒間、１００℃未満６０℃以上で失活させる場合には３〜４０分間で行うことが好適である。
加水分解終了後、必要に応じて分解液のｐＨを、好ましくは６〜８、より好ましくは７．０±０．５、さらに好ましくは７．０±０．３とするのが好適である。
The hydrolysis by the proteolytic enzyme may be terminated by heating and deactivating the enzyme. For example, when deactivating at 100 ° C. or higher (preferably 110-130 ° C.) for 1 to 3 seconds, when deactivating at less than 100 ° C. and 60 ° C. or higher, it is preferable to perform for 3 to 40 minutes. .
After completion of hydrolysis, the pH of the decomposition solution is preferably 6 to 8, more preferably 7.0 ± 0.5, and even more preferably 7.0 ± 0.3, if necessary.
なお、本開示のカゼイン分解物の製造において、カルシウム濃度未調整の溶液を加水分解した場合には、得られた分解液を、前記のような脱塩処理し、カルシウム濃度を調整してもよい。次いで、常法により加熱して酵素を失活させる。反応加熱温度と反応保持時間は使用した酵素の熱安定性を配慮し、十分に失活できる条件を適宜設定することができる。加熱失活後、常法により冷却し、そのまま利用することもでき、必要に応じて濃縮して濃縮液を得ることもでき、更に濃縮液を乾燥し、粉末製品を得ることも可能である。 In the production of the casein degradation product of the present disclosure, when the calcium concentration unadjusted solution is hydrolyzed, the obtained decomposition solution may be desalted as described above to adjust the calcium concentration. . Next, the enzyme is inactivated by heating by a conventional method. The reaction heating temperature and the reaction holding time can be set as appropriate under conditions that allow for sufficient deactivation in consideration of the thermal stability of the enzyme used. After heat deactivation, it can be cooled by a conventional method and used as it is, or it can be concentrated as necessary to obtain a concentrate, and the concentrate can be dried to obtain a powder product.
また、前記カゼイン水溶液を酸処理又はアルカリ処理にて加水分解する際には、カゼイン水溶液のｐＨを調整して処理すればよい。当該ｐＨ調整による処理の場合には、カゼイン水溶液のｐＨが、好ましくはｐＨ５以下又はｐＨ９以上であり、より好ましくはｐＨ４以下又はｐＨ１０以上である。このようにｐＨ処理された水溶液は、室温にて数分以上、好ましくは５分〜１時間、放置又は撹拌することによって、酸処理又はアルカリ処理の加水分解物を得ることができる。ここで、「室温」とは、４〜４０℃程度であるが、１０〜３０℃が好適である。 Moreover, what is necessary is just to adjust and adjust the pH of casein aqueous solution, when hydrolyzing the casein aqueous solution by acid treatment or alkali treatment. In the case of the treatment by pH adjustment, the pH of the casein aqueous solution is preferably pH 5 or lower or pH 9 or higher, more preferably pH 4 or lower or pH 10 or higher. The pH-treated aqueous solution can be left or stirred at room temperature for several minutes or more, preferably 5 minutes to 1 hour, to obtain an acid-treated or alkali-treated hydrolyzate. Here, “room temperature” is about 4 to 40 ° C., but 10 to 30 ° C. is preferable.
また、前記カゼイン水溶液を、熱処理にて加水分解してもよい。このカゼイン水溶液は、ｐＨ未調整でもよく、またｐＨ調整（具体的には、酸性（ｐＨ５以下）、中性（ｐＨ６〜８）、アルカリ性（ｐＨ８以上））してもよい。熱処理は、４〜１００℃程度で、上記酸アルカリ処理のような条件にて行えばよい。 The casein aqueous solution may be hydrolyzed by heat treatment. This aqueous casein solution may be pH-unadjusted, or may be pH-adjusted (specifically, acidic (pH 5 or lower), neutral (pH 6-8), alkaline (pH 8 or higher)). The heat treatment may be performed at about 4 to 100 ° C. under the conditions such as the acid-alkali treatment.
得られたカゼイン分解物は、未精製のままの状態で使用しても効能を発揮することが可能である。さらに、効能を向上させるために適宜本開示の効能を有する成分又はこの成分を有する画分を公知の分離精製を行うことで得ることも可能である。
例えば、得られたカゼイン分解物に対して分子量分画を行い、本開示の効能を高めることも可能である。これにより、カゼイン分解物を分子量２０００Ｄａ未満又は以下にすることが好ましく、さらに分子量１０００Ｄａ以下にすることが好ましい。
The obtained casein degradation product can exert its effects even when used in an unpurified state. Furthermore, in order to improve efficacy, it is also possible to obtain a component having the efficacy of the present disclosure or a fraction having this component by performing known separation and purification as appropriate.
For example, molecular weight fractionation can be performed on the obtained casein degradation product to enhance the efficacy of the present disclosure. Thereby, it is preferable to make a casein decomposition product into the molecular weight less than 2000 Da or less, and also it is preferable to make it molecular weight 1000 Da or less.
分子量分画として、例えば、限外濾過、ゲル濾過等の方法が採用でき、これにより不要な分子量のペプチドの除去率を高めることができる。
限外濾過の場合には、所望の限外濾過膜を使用すればよく、ゲル濾過の場合には、所望のサイズ排除クロマトグラフィーに用いるゲルろ過剤を使用すればよい。
さらに、脱塩や不純物を除去したり、純度を高めたりするために、公知の分離精製方法（例えば、イオン交換樹脂等）を用いてもよい。
As the molecular weight fractionation, for example, methods such as ultrafiltration and gel filtration can be employed, whereby the removal rate of unnecessary molecular weight peptides can be increased.
In the case of ultrafiltration, a desired ultrafiltration membrane may be used, and in the case of gel filtration, a gel filtration agent used for desired size exclusion chromatography may be used.
Furthermore, a known separation and purification method (for example, an ion exchange resin or the like) may be used for desalting, removing impurities, and increasing the purity.
本開示のカゼイン分解物は、後記実施例に示すように、ヒアルロン酸合成酵素の遺伝子発現促進作用又はヒアルロン酸合成促進作用を有する。従って、本開示のカゼイン分解物は、ヒアルロン酸合成酵素の遺伝子発現促進用又はヒアルロン酸合成促進用の各製剤として使用することができる。さらに、当該ヒアルロン酸合成酵素の遺伝子発現促進用又はヒアルロン酸合成促進作用を有することにより、生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少に関わる様々な状態を、予防、改善又は治療することができる。 The casein degradation product of the present disclosure has a gene expression promoting action or a hyaluronic acid synthesis promoting action of hyaluronic acid synthase, as shown in Examples below. Therefore, the casein degradation product of the present disclosure can be used as a preparation for promoting gene expression of hyaluronic acid synthase or promoting hyaluronic acid synthesis. Furthermore, by having the hyaluronic acid synthase for promoting gene expression or promoting hyaluronic acid synthesis, it can prevent and improve various conditions related to a decrease in the biosynthetic ability of hyaluronic acid in vivo or a decrease in hyaluronic acid in vivo. Or it can be treated.
ところで、生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少に関わる様々な状態とは、例えば、皮膚老化及び加齢による関節症等が挙げられる。
前記皮膚老化として、例えば、皮膚のシワやタルミの形成、皮膚の弾力性低下、皮膚のハリの低下抑制剤、光（例えば紫外線等）又は酸化成分（例えば活性酸素等）による皮膚老化等が挙げられる。
前記加齢による関節症として、例えば、加齢による変形性関節症、関節軟骨の加齢変性、機械的ストレス（関節の摩耗等）による軟骨変性等が挙げられる。
By the way, various states related to a decrease in the biosynthetic ability of in vivo hyaluronic acid or a decrease in in vivo hyaluronic acid include, for example, skin aging and aging-related arthropathy.
Examples of the skin aging include formation of skin wrinkles and talmi, skin elasticity reduction, skin elasticity reduction inhibitor, skin aging due to light (for example, ultraviolet rays) or an oxidizing component (for example, active oxygen). It is done.
Examples of the arthropathy due to aging include osteoarthritis due to aging, age-related degeneration of articular cartilage, and cartilage degeneration due to mechanical stress (such as joint wear).
近年、皮膚老化及び加齢による関節症に対する対策として、生体内ヒアルロン酸が減少している箇所に、外因性のヒアルロン酸を注入することが行われている。しかし、生体内分解等により注入した箇所のヒアルロン酸が減少するため、注射等の侵襲行為にて再注入を数週間程度で行う必要があり、また所定の医療機関に赴いて処置を行う必要がある。このため、費用負担以外にも、身体的負担等の様々な負担を希望者にかけることになる。 In recent years, exogenous hyaluronic acid is injected into a site where hyaluronic acid in the body is decreasing as a countermeasure against arthropathy due to skin aging and aging. However, since hyaluronic acid at the site of injection is reduced due to biodegradation, etc., it is necessary to perform reinjection in several weeks by an invasive action such as injection, and it is necessary to visit a predetermined medical institution for treatment. is there. For this reason, in addition to the cost burden, various burdens such as a physical burden are placed on the applicant.
本開示のカゼイン分解物は、上述した皮膚老化及び加齢による関節症等を、予防、改善又は治療することができる。また、本開示のカゼイン分解物は、上述した皮膚老化の抑制作用及び加齢による関節症等の病変抑制作用を有する。
すなわち、本開示のカゼイン分解物は、皮膚老化抑制、シワ・タルミ形成抑制、皮膚弾力低下抑制、ハリ低下抑制等の各製剤の有効成分として含有させることができる。また、本開示のカゼイン分解物は、加齢による関節症、加齢による変形性関節症、関節軟骨の加齢変性、機械的ストレスによる軟骨変性等の予防、治療又は改善のための各製剤の有効性分として含有させることができる。
また、本開示のカゼイン分解物は、上述した各製剤として使用することができ、また製造するために使用することもできる。
The casein degradation product of the present disclosure can prevent, ameliorate, or treat the above-described skin aging and arthropathy due to aging. In addition, the casein degradation product of the present disclosure has the above-described skin aging-inhibiting effect and lesion-inhibiting effect such as arthropathy due to aging.
That is, the casein degradation product of the present disclosure can be contained as an active ingredient of each preparation such as skin aging inhibition, wrinkle / tarmi formation inhibition, skin elasticity reduction inhibition, and elasticity reduction inhibition. In addition, the casein degradation product of the present disclosure includes each formulation for prevention, treatment or improvement of arthropathy due to aging, osteoarthritis due to aging, age-related degeneration of articular cartilage, cartilage degeneration due to mechanical stress, etc. It can be contained as an effective component.
Moreover, the casein decomposition product of this indication can be used as each formulation mentioned above, and can also be used in order to manufacture.
上記使用とは、適用対象であるヒト若しくは非ヒト動物における使用であり得、また治療的使用であっても非治療的使用であってもよい。
本明細書において、「非治療的」とは、医療行為、すなわち治療による人体への処置行為を含まない概念である。
また、本明細書において、「改善」とは、疾患、症状又は状態の好転；疾患、症状又は状態の悪化の防止、遅延若しくは疾患又は症状の進行の逆転、防止又は遅延をいう。
また、本明細書において、「予防」とは、適用対象における疾患若しくは症状の発症の防止又は遅延、或いは適用対象の疾患若しくは症状の発症の危険性を低下させることをいう。
The above-mentioned use may be a use in a human or non-human animal to be applied, and may be a therapeutic use or a non-therapeutic use.
In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by therapy.
In the present specification, “improvement” refers to improvement of a disease, symptom or condition; prevention, delay or deterioration of progression of the disease, symptom or condition, reversal, prevention or delay of progression of the disease or symptom.
In the present specification, “prevention” means prevention or delay of the onset of the disease or symptom in the application target, or reduction of the risk of the onset of the disease or symptom of the application target.
以上、従って、本開示のカゼイン分解物及びこれを有効成分として含有する上述の各種製剤（以下、「本技術の各種製剤」ともいう）は、ヒトを含む動物に摂取又は投与して、上述の、生体内ヒアルロン酸の生合成能の低下が関与する疾病、疾患や症状の改善等を図るための方法に使用することができる。 Accordingly, the casein degradation product of the present disclosure and the above-described various preparations containing this as an active ingredient (hereinafter also referred to as “various preparations of the present technology”) are ingested or administered to animals including humans, In addition, it can be used in a method for improving diseases, diseases, and symptoms associated with a decrease in biosynthesis ability of hyaluronic acid in vivo.
また、本開示のカゼイン分解物及びこれを有効成分として含有する上述の本技術の各種製剤は、上述のような生体内ヒアルロン酸の生合成能の低下等が関与する疾病、疾患や症状のための、又は上述した皮膚老化又は加齢による関節症等の予防、改善及び／又は治療のための、ヒト用若しくは動物用の医薬品、医薬部外品、皮膚外用剤、化粧品及び食品等の有効成分としてこれらに配合して使用可能である。また、本開示のカゼイン分解物は、これら各種製剤の製造のために使用可能である。 In addition, the casein degradation product of the present disclosure and the various preparations of the present technology containing the same as an active ingredient are for diseases, diseases, and symptoms that involve a decrease in the biosynthetic ability of in vivo hyaluronic acid as described above. Active ingredients such as pharmaceuticals for humans or animals, quasi-drugs, external preparations for skin, cosmetics and foods for the prevention, improvement and / or treatment of the above-mentioned skin aging or arthropathy due to aging It can be used by blending with these. Moreover, the casein degradation product of the present disclosure can be used for the production of these various preparations.
医薬品に配合する場合、経口投与剤や非経口投与剤等とすることができる。
また、食品に配合する場合には、上述の生体内ヒアルロン酸の生合成能の低下等によって引き起こされる各種疾患等の予防、改善又は治療をコンセプトとする機能性食品、病者用食品、特定保健用食品等に応用できる。また、本開示のカゼイン分解物は、これら食品等の製造のために使用可能である。
When blended in a pharmaceutical product, it can be an orally administered agent or a parenterally administered agent.
In addition, when formulated in foods, functional foods based on the concept of prevention, improvement or treatment of various diseases caused by the above-mentioned decrease in biosynthetic ability of in vivo hyaluronic acid, foods for the sick, specified health It can be applied to food products. Moreover, the casein decomposition product of this indication can be used for manufacture of these foodstuffs.
本開示のカゼイン分解物及びこれを有効成分として含有する上述の本技術の各種製剤は、経口投与及び非経口投与の何れでもよいが、経口投与が望ましい。非経口投与として、静注、直腸投与、吸入等が挙げられる。経口投与の剤形として、錠剤、カプセル剤、トローチ剤、シロップ剤、顆粒剤、散剤、軟膏等が挙げられる。
製剤化に際しては、本開示のカゼイン分解物の他に、通常製剤化に用いられている賦形剤、ｐＨ調整剤、着色剤、矯味剤等の成分を用いることができる。また、公知の又は将来的に見出される上述のヒアルロン酸合成促進作用を有する医薬；ヒアルロン酸、コンドロイチン等のグリコサミノグリカン及びこれら多糖の構成糖（例えば、グルコサミン、ウロン酸等）等を併用することも可能である。
The casein degradation product of the present disclosure and the various preparations of the present technology containing this as an active ingredient may be either oral administration or parenteral administration, but oral administration is desirable. Parenteral administration includes intravenous injection, rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders and ointments.
In formulating, in addition to the casein degradation product of the present disclosure, components such as excipients, pH adjusters, colorants, and corrigents that are usually used for formulation can be used. In addition, a known or future-discovered pharmaceutical having the above-mentioned hyaluronic acid synthesis promoting action; glycosaminoglycans such as hyaluronic acid and chondroitin, and constituent sugars of these polysaccharides (for example, glucosamine, uronic acid, etc.) are used in combination It is also possible.
また、本開示のカゼイン分解物を有効成分として食品中に含有させ、本開示のカゼイン分解物及びこれを有効成分として含有する上述の各種製剤の一態様として、上述のヒアルロン酸の合成促進作用等を有する食品として加工することも可能である。
このような食品として、液状、ペースト状、固体、粉末等の形態を問わず、錠菓、流動食、飼料（ペット用を含む）等のほか、例えば、小麦粉製品、即席食品、農産加工品、水産加工品、畜産加工品、乳・乳製品、油脂類、基礎調味料、複合調味料・食品類、冷凍食品、菓子類、飲料類、これら以外の市販食品等が挙げられる。
In addition, the casein degradation product of the present disclosure is contained in a food as an active ingredient, and the casein degradation product of the present disclosure and an aspect of the above-described various preparations containing the degradation product as an active ingredient include the above-described hyaluronic acid synthesis promoting action It is also possible to process it as a food having
Examples of such foods include liquids, pastes, solids, powders, etc. In addition to tablet confectionery, liquid foods, feeds (including for pets), etc., for example, flour products, instant foods, processed agricultural products, Processed marine products, processed livestock products, milk / dairy products, fats and oils, basic seasonings, compound seasonings / foods, frozen foods, confectionery, beverages, commercial foods other than these, and the like.
例えば、前記小麦粉製品として、パン、マカロニ、スパゲッティ、めん類、ケーキミックス、から揚げ粉、パン粉等が挙げられる。
前記即席食品類として、即席めん、カップめん、レトルト・調理食品、調理缶詰め、電子レンジ食品、即席スープ・シチュー、即席みそ汁・吸い物、スープ缶詰め、フリーズ・ドライ食品、その他の即席食品等が挙げられる。
例えば、前記農産加工品として、 農産缶詰め、果実缶詰め、ジャム・マーマレード類、漬物、煮豆類、農産乾物類、シリアル（穀物加工品）等が挙げられる。
前記水産加工品として、水産缶詰め、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、つくだ煮類等が挙げられる。
前記畜産加工品として、畜産缶詰め・ペースト類、畜肉ハム・ソーセージ等が挙げられる。
For example, examples of the flour product include bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread crumbs and the like.
Examples of the instant foods include instant noodles, cup noodles, retort / cooked food, cooking canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, and other instant foods.
Examples of the processed agricultural products include canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (cereal processed products), and the like.
Examples of the processed fishery products include canned fishery products, fish hams and sausages, fish paste products, marine delicacies, and tsukudani.
Examples of the processed livestock products include canned livestock, pastes, livestock ham, sausages and the like.
例えば、前記乳・乳製品として、加工乳、乳飲料、ヨーグルト類、乳酸菌飲料類、チーズ、アイスクリーム類、調製粉乳類、クリーム、その他の乳製品等が挙げられる。
前記油脂類として、バター、マーガリン類、植物油等が挙げられる。
例えば、前記基礎調味料として、しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類等が挙げられる。
前記複合調味料・食品類として、調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類、その他の複合調味料等が挙げられる。
例えば、前記冷凍食品として、素材冷凍食品、半調理冷凍食品、調理済冷凍食品等が挙げられる。
Examples of the milk / dairy products include processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheese, ice creams, prepared powdered milks, creams, and other dairy products.
Examples of the fats and oils include butter, margarines, and vegetable oils.
For example, examples of the basic seasoning include soy sauce, miso, sauces, processed tomato seasonings, mirin, and vinegar.
Examples of the complex seasonings and foods include cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings.
Examples of the frozen food include raw material frozen food, semi-cooked frozen food, cooked frozen food, and the like.
例えば、前記菓子類として、キャラメル、キャンディー、チューインガム、チョコレート、クッキー、ビスケット、ケーキ、パイ、スナック、クラッカー、和菓子、米菓子、豆菓子、デザート菓子、その他の菓子等が挙げられる。
例えば、前記飲料類として、炭酸飲料、天然果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、野菜系飲料、豆乳、豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料、その他の嗜好飲料等が挙げられる。
例えば、上記以外の市販食品として、ベビーフード、ふりかけ、お茶潰けのり等が挙げられる。
Examples of the confectionery include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, and other confectionery.
For example, the beverages include carbonated beverages, natural fruit juices, fruit juice beverages, soft drinks with fruit juices, fruit drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks Sports drinks, nutritional drinks, alcoholic drinks, other taste drinks, and the like.
For example, examples of commercially available foods other than the above include baby foods, sprinkles, and tea paste.
本技術の各種製剤において、本開示のカゼイン分解物の含有量は、製剤の最終組成物に対し、少なくとも０．００１質量％であることが好ましい。
本開示のカゼイン分解物の摂取量又は投与量は、年齢、症状等により異なるが、通常、０．００１〜３０００ｍｇ／ｋｇ体重／日、好ましくは０．０１〜２００ｍｇ／ｋｇ体重／日であり、１日１回から３回に分けて投与してもよい。ヒトに対する摂取量又は投与量は、好ましくは１０ｍｇ／ｋｇ体重／日である。
In various preparations of the present technology, the content of the casein degradation product of the present disclosure is preferably at least 0.001% by mass with respect to the final composition of the preparation.
The intake or dose of the casein degradation product of the present disclosure varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / kg body weight / day, preferably 0.01 to 200 mg / kg body weight / day, It may be administered once to three times a day. The intake or dose for humans is preferably 10 mg / kg body weight / day.
前記カゼイン分解物のヒトへの投与の際の用量は、『体表面積に基づく動物からのＨＥＤ（Human Equivalent Dose）交換』（例えば、参考文献１を参照）による換算式から算出することができる。
ＨＥＤ＝［動物への投与量（ｍｇ／ｋｇ体重）］×｛［動物の体重（ｋｇ）］÷［ヒトの体重（ｋｇ）］｝０．３３
ヒトの体重：６０ｋｇ
マウスの体重：２０ｇ
参考文献１：Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, V. STEP 2:HUMAN EQUIVALENT DOSE CALCULATION, July 2005, Pharmacology and Toxicology, p.6-7 / U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER)
The dose at the time of administration of the casein degradation product to humans can be calculated from a conversion formula by “HED (Human Equivalent Dose) exchange from animals based on body surface area” (for example, see Reference 1).
HED = [dosage to animal (mg / kg body weight)] × {[animal body weight (kg)] ÷ [human body weight (kg)]} 0.33
Human weight: 60kg
Mouse weight: 20g
Reference 1: Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, V. STEP 2: HUMAN EQUIVALENT DOSE CALCULATION, July 2005, Pharmacology and Toxicology, p.6-7 / US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER)
本技術は、以下の構成を採用することも可能である。
〔１〕 カゼイン分解物を有効成分として含有するヒアルロン酸合成促進剤。
〔２〕 前記カゼイン分解物が蛋白質加水分解酵素による加水分解物である前記〔１〕記載のヒアルロン酸合成促進剤。
〔３〕 前記カゼイン分解物の分子量が１０００Ｄａ以下である前記〔１〕又は〔２〕記載のヒアルロン酸合成促進剤。
〔４〕 前記蛋白質加水分解酵素が、エンドプロテアーゼである前記〔２〕又は〔３〕記載のヒアルロン酸合成促進剤。
The present technology can also employ the following configurations.
[1] A hyaluronic acid synthesis accelerator containing a casein degradation product as an active ingredient.
[2] The hyaluronic acid synthesis promoter according to [1], wherein the casein degradation product is a hydrolysis product of a protein hydrolase.
[3] The hyaluronic acid synthesis accelerator according to [1] or [2], wherein the casein decomposition product has a molecular weight of 1000 Da or less.
[4] The hyaluronic acid synthesis promoter according to the above [2] or [3], wherein the protein hydrolase is an endoprotease.
〔５〕 ヒアルロン酸合成促進剤又は本技術の製剤の製造のための、カゼイン分解物の使用。
〔６〕 ヒアルロン酸合成促進用食品の製造のための、カゼイン分解物の使用。
〔７〕 カゼイン分解物の、ヒアルロン酸合成促進用食品への使用。
[5] Use of a casein degradation product for the production of a hyaluronic acid synthesis accelerator or a preparation of the present technology.
[6] Use of casein degradation product for the production of hyaluronic acid synthesis promoting food.
[7] Use of a casein degradation product in a food for promoting hyaluronic acid synthesis.
〔８〕 生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少が関与する疾病、疾患や症状の予防、改善又治療のための、カゼイン分解物。
〔９〕 生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少が関与する疾病、疾患や症状の予防、改善又治療における使用のための、カゼイン分解物。
〔１０〕 生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少が関与する疾病、疾患や症状の予防、改善又治療のための、カゼイン分解物の使用。
〔１１〕 カゼイン分解物を有効成分として摂取又は投与する、生体内のヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少が関与する疾病、疾患や症状の予防、改善又治療方法。
〔１２〕 前記〔８〕〜〔１１〕記載の生体内ヒアルロン酸の生合成能の低下又は生体内ヒアルロン酸の減少が関与する疾病、疾患や症状が、皮膚のシワやタルミの形成、皮膚の弾力性低下、皮膚のハリの低下、光（例えば紫外線等）又は酸化成分（例えば活性酸素等）による皮膚老化、変形性関節症、関節軟骨の加齢変性、機械的ストレス（関節の摩耗等）による軟骨変性である、カゼイン分解物又はカゼイン分解物の使用。
[8] A casein degradation product for the prevention, amelioration, or treatment of a disease, disorder or symptom associated with a decrease in the biosynthetic ability of in vivo hyaluronic acid or a decrease in in vivo hyaluronic acid.
[9] A casein degradation product for use in the prevention, amelioration, or treatment of a disease, disorder or symptom associated with a decrease in the biosynthetic ability of in vivo hyaluronic acid or a decrease in in vivo hyaluronic acid.
[10] Use of a casein degradation product for the prevention, amelioration, or treatment of a disease, disorder or symptom associated with a decrease in the biosynthetic ability of in vivo hyaluronic acid or a decrease in in vivo hyaluronic acid.
[11] A method for preventing, ameliorating, or treating a disease, disorder or symptom associated with a decrease in the biosynthesis ability of hyaluronic acid in vivo or a decrease in in vivo hyaluronic acid, ingesting or administering a casein degradation product as an active ingredient.
[12] The disease, disease or symptom associated with a decrease in the biosynthetic ability of in vivo hyaluronic acid or a decrease in in vivo hyaluronic acid according to the above [8] to [11] is caused by formation of skin wrinkles or tarmi, Reduced elasticity, reduced skin firmness, skin aging due to light (for example, ultraviolet rays) or oxidizing components (for example, active oxygen), osteoarthritis, age-related degeneration of articular cartilage, mechanical stress (for example, joint wear) Use of casein degradation product or casein degradation product, which is cartilage degeneration caused by the above.
〔１３〕 前記〔５〕〜〔１２〕の何れかに記載のカゼイン分解物は、熱処理、酸処理、アルカリ処理、蛋白質分解酵素処理から選ばれる１種又は２種以上の処理による分解物であるのが好適である。
〔１４〕 前記〔１３〕に記載のカゼイン分解物は、カゼインの蛋白質加水分解酵素による加水分解物であるのが好適である。
〔１５〕 前記〔５〕〜〔１４〕の何れかに記載のカゼイン分解物の分子量が１０００Ｄａ以下であるのが好適である。
〔１６〕前記〔５〕〜〔１５〕の何れかに記載のカゼインが牛由来のカゼインであるのが好適である。
[13] The casein degradation product according to any one of [5] to [12] is a degradation product resulting from one or more treatments selected from heat treatment, acid treatment, alkali treatment, and proteolytic enzyme treatment. Is preferred.
[14] The casein degradation product according to [13] is preferably a hydrolysis product of casein by a protein hydrolase.
[15] The casein degradation product according to any one of [5] to [14] preferably has a molecular weight of 1000 Da or less.
[16] The casein according to any one of [5] to [15] is preferably cow-derived casein.
〔製造例１〕
市販のカゼイン（ニュージーランドデーリーボード社製）１００ｍｇに水９００ｍｇを加え、よく分散させ、水酸化ナトリウムを添加して溶液のｐＨを７．０に調整し、カゼインを完全に溶解し、濃度約１０％のカゼイン水溶液を調製した。
該カゼイン水溶液を８５℃で１０分間加熱殺菌し、５０℃に温度調整し、水酸化ナトリウムを添加してｐＨを９．０に調整した後、パンクレアチン２ｍｇ（天野エンザイム社製）、プロテアーゼＡを４ｍｇ（天野エンザイム社製）を添加して、加水分解反応を開始した。８時間後に８０℃で６分間加熱して酵素を失活させて酵素反応を停止し、１０℃に冷却した。
この加水分解液を分画分子量１０００の限外ろ過膜（日本ポール社製）で限外ろ過し、濃縮後凍結乾燥し、カゼイン分解物Ａを８５ｍｇを得た。
[Production Example 1]
Add 900 mg of water to 100 mg of commercially available casein (made by New Zealand Daily Board), disperse well, adjust the pH of the solution to 7.0 by adding sodium hydroxide, completely dissolve casein, concentration about 10% A casein aqueous solution was prepared.
The casein aqueous solution was sterilized by heating at 85 ° C. for 10 minutes, adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.0, pancreatin 2 mg (manufactured by Amano Enzyme), protease A 4 mg (manufactured by Amano Enzyme) was added to initiate the hydrolysis reaction. After 8 hours, the enzyme reaction was stopped by heating at 80 ° C. for 6 minutes to stop the enzyme reaction and cooled to 10 ° C.
This hydrolyzed solution was ultrafiltered through an ultrafiltration membrane (manufactured by Nippon Pole Co., Ltd.) having a molecular weight cut off of 1000, concentrated and freeze-dried to obtain 85 mg of casein degradation product A.
カゼイン分解物Ａは、アミノ酸遊離率３８％、分子量２２０Ｄａであった。これらは上述の＜アミノ酸遊離率の算定方法＞、＜分子量の算定方法＞にて算定した。 Casein degradation product A had an amino acid release rate of 38% and a molecular weight of 220 Da. These were calculated by the above <Method for calculating amino acid release rate> and <Method for calculating molecular weight>.
〔製造例２〕
市販のカゼイン（ニュージーランドデーリーボード社製）１００ｍｇに水９００ｍｇを加え、よく分散させ、水酸化ナトリウムを添加して溶液のｐＨを７．０に調整し、カゼインを完全に溶解し、濃度約１０％のカゼイン水溶液を調製した。
該カゼイン水溶液を８５℃で１０分間加熱殺菌し、５０℃に温度調整し、水酸化ナトリウムを添加してｐＨを９．５に調整した後、ビオプラーゼｓｐ−２０（長瀬生化学工業社製）１００活性単位（蛋白質１ｇ当たり１２００活性単位）、プロテアーゼＮ（天野エンザイム社製）１７０活性単位（蛋白質１ｇ当たり２０００活性単位）、及びＰＴＮ６．０Ｓ（ノボザイムズ・ジャパン社製）６００活性単位（蛋白質１ｇ当たり７０００活性単位）を添加して、加水分解反応を開始した。８時間後に８０℃で６分間加熱して酵素を失活させて酵素反応を停止し、１０℃に冷却した。
この加水分解液を分画分子量１０００の限外ろ過膜（日本ポール社製）で限外ろ過し、濃縮後凍結乾燥し、カゼイン分解物Ｂを８５ｍｇを得た。
[Production Example 2]
Add 900 mg of water to 100 mg of commercially available casein (made by New Zealand Daily Board), disperse well, adjust the pH of the solution to 7.0 by adding sodium hydroxide, completely dissolve casein, concentration about 10% A casein aqueous solution was prepared.
The aqueous casein solution was sterilized by heating at 85 ° C. for 10 minutes, adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.5, and then biopulase sp-20 (manufactured by Nagase Seikagaku Corporation) 100 Activity units (1200 active units per gram of protein), protease N (manufactured by Amano Enzyme) 170 active units (2000 active units per gram of protein), and PTN 6.0S (manufactured by Novozymes Japan) 600 active units (7,000 per gram of protein) Activity unit) was added to initiate the hydrolysis reaction. After 8 hours, the enzyme reaction was stopped by heating at 80 ° C. for 6 minutes to stop the enzyme reaction and cooled to 10 ° C.
This hydrolyzed solution was ultrafiltered with an ultrafiltration membrane (manufactured by Nippon Pole Co., Ltd.) having a fractional molecular weight of 1000, concentrated and freeze-dried to obtain 85 mg of casein degradation product B.
カゼイン分解物Bは、非蛋白態窒素比率２５％、アミノ酸遊離率５％、分子量３４０Ｄａであった。これらは上述の＜非蛋白態窒素比率の算出方法＞、＜アミノ酸遊離率の算定方法＞、＜分子量の算定方法＞にて算定した。 Casein degradation product B had a non-protein nitrogen ratio of 25%, an amino acid release rate of 5%, and a molecular weight of 340 Da. These were calculated by the above <Method for calculating non-protein nitrogen ratio>, <Method for calculating amino acid release rate>, and <Method for calculating molecular weight>.
〔試験例１：ヒアルロン酸合成酵素（ＨＡＳ１）遺伝子発現量解析〕
本開示のカゼイン分解物による生体内でのヒアルロン酸合成促進作用を確認するため、製造例１にて得られたカゼイン分解物Ａを用いて細胞培養をし、ヒアルロン酸合成酵素ＨＡＳ１の遺伝子発現量の測定を以下の方法で行った。
[Test Example 1: Hyaluronic acid synthase (HAS1) gene expression level analysis]
To confirm the hyaluronic acid synthesis promoting action in vivo by the casein degradation product of the present disclosure, cell culture was performed using the casein degradation product A obtained in Production Example 1, and the gene expression level of hyaluronic acid synthase HAS1 Was measured by the following method.
（１）細胞培養
１０％ＦＢＳ〔Fetal Bovin Serum（ウシ胎児血清）、ＧＩＢＣＯ社製〕及び１％抗生物質（PSN Antibiotic Mixtuire、ＧＩＢＣＯ社製）を含むＦ−１２培地（Ham’s F-12 Nutrient Mixture、ＧＩＢＣＯ社製）を用いて、白ウサギの膝関節滑膜組織由来の細胞株であるＨＩＧ−８２細胞（大日本住友製薬株式会社製）を１００ｍｍ径シャーレに１．５×１０６個になるように播種し、５％ＣＯ２、３７℃で２４時間培養し、培地交換を行った後さらに２４時間、合計４８時間培養した。その後、培地を除去し、製造例１で得られたカゼイン分解物Ａを５０μｇ／ｍＬ含有するＦ−１２培地を添加し、３時間培養した。また、カゼイン分解物Ａを添加せずに、Ｆ−１２培地で３時間培養したサンプルを調製し、これを対照試料とした。
(1) Cell culture F-12 medium (Ham's F-12 Nutrient Mixture, containing 10% FBS [Fetal Bovin Serum (Fetal Bovine Serum), manufactured by GIBCO)] and 1% antibiotics (PSN Antibiotic Mixtuire, manufactured by GIBCO), HIB-82 cells (manufactured by Dainippon Sumitomo Pharma Co., Ltd.), which is a cell line derived from the knee joint synovial tissue of white rabbits, are made into 1.5 × 10 6 cells in a 100 mm diameter petri dish using GIBCO). And then cultured at 5% CO 2 and 37 ° C. for 24 hours. After changing the medium, the cells were further cultured for 24 hours for a total of 48 hours. Thereafter, the medium was removed, and an F-12 medium containing 50 μg / mL of casein degradation product A obtained in Production Example 1 was added and cultured for 3 hours. Moreover, the sample which culture | cultivated for 3 hours by F-12 culture medium without adding casein degradation product A was prepared, and this was made into the control sample.
（２）ＲＮＡの抽出
培養終了後、常法によりＲＮＡを抽出した。すなわち、上清を除去したシャーレに１ｍＬの細胞溶解液（TRIzol Reagent、invitrogen社製）を加えて細胞を溶解させ、これをチューブに回収してクロロホルムを添加して攪拌した後、遠心分離して、ＲＮＡを含む上層を回収した。この回収液に、０．５ｍＬのイソプロピルアルコールを添加して攪拌し、常温で１０分間静置した。その後、さらに遠心分離して上清を除去した後、７５％エタノールで沈殿物を洗浄した。得られた沈殿物を風乾し、３９．５μＬのＤＥＰＣＨ２Ｏ（0.1%Diethyl Dicarbonate、和光純薬工業株式会社）に懸濁した。さらに、残存するＤＮＡを除去するために、ＤＮＡ除去キット（DNase treatment removal reagent、Ambion社製）を使用し、当該キットの使用方法にしたがって残存ＤＮＡを除去した上清を回収し、これをＲＮＡ溶液とした。
(2) Extraction of RNA After completion of the culture, RNA was extracted by a conventional method. That is, 1 mL of cell lysate (TRIzol Reagent, manufactured by Invitrogen) was added to the petri dish from which the supernatant had been removed to lyse the cells. This was collected in a tube, added with chloroform, stirred, and then centrifuged. The upper layer containing RNA was collected. To this recovered solution, 0.5 mL of isopropyl alcohol was added and stirred, and allowed to stand at room temperature for 10 minutes. Then, after further centrifuging to remove the supernatant, the precipitate was washed with 75% ethanol. The obtained precipitate was air-dried and suspended in 39.5 μL of DEPCH 2 O (0.1% Diethyl Dicarbonate, Wako Pure Chemical Industries, Ltd.). Furthermore, in order to remove the remaining DNA, a DNA removal kit (DNase treatment removal reagent, manufactured by Ambion) was used, and the supernatant from which the remaining DNA was removed was collected according to the method of using the kit, and this was recovered as an RNA solution. It was.
（３）ｃＤＮＡ合成及びリアルタイムＰＣＲによる遺伝子発現量の解析
上記抽出によって得られたＲＮＡ溶液から、逆転写反応によりｃＤＮＡの合成を行った。ｃＤＮＡの合成は、ｃＤＮＡ合成キット（Prime Script RT-PCR Kit、タカラバイオ株式会社製）を用いて、当該キットの使用方法に従って行った。インターカレーターとしては、Sybr Green I（タカラバイオ株式会社製）を使用した。ＨＡＳ１の遺伝子特異性プライマーとしては下記の配列を有するプライマー（タカラバイオ株式会社製）を使用した。
Ｆｏｒｗａｒｄ：５’−ＧＴＧＴＣＣＴＧＣＡＴＣＡＧＴＧＧＴＣＣＴＣ−３’
Ｒｅｖｅｒｓｅ：５’−ＧＴＧＣＣＣＡＧＧＡＡＣＴＴＣＴＧＧＴＴＧＴＡ−３’
遺伝子発現量は、増幅させたＧＡＰＤＨで標準化し、ＧＡＰＤＨ遺伝子増幅用プライマーとしては、下記の配列を有するプライマー（タカラバイオ株式会社製）を使用した。
Ｆｏｒｗａｒｄ：５’−ＧＧＣＡＣＡＧＴＣＡＡＧＧＣＴＧＡＧＡＡＴＧ−３’
Ｒｅｖｅｒｓｅ：５’−ＡＴＧＧＴＧＧＴＧＡＡＧＡＣＧＣＣＡＧＴＡ−３’
(3) cDNA synthesis and analysis of gene expression level by real-time PCR From the RNA solution obtained by the above extraction, cDNA was synthesized by reverse transcription reaction. cDNA synthesis was performed using a cDNA synthesis kit (Prime Script RT-PCR Kit, manufactured by Takara Bio Inc.) according to the method of using the kit. Sybr Green I (manufactured by Takara Bio Inc.) was used as an intercalator. As a gene-specific primer for HAS1, a primer (manufactured by Takara Bio Inc.) having the following sequence was used.
Forward: 5'-GTGTCCTGCATCATGGTGCCTC-3 '
Reverse: 5'-GTGCCCAGGAACTTCTGGTTGTA-3 '
The gene expression level was standardized with the amplified GAPDH, and a primer (manufactured by Takara Bio Inc.) having the following sequence was used as a GAPDH gene amplification primer.
Forward: 5′-GGCACAGTCAAGGCTGGAATG-3 ′
Reverse: 5′-ATGGTGGTGAAGACGCCAGTA-3 ′
表１に、ＨＡＳ１の遺伝子発現量の測定結果を示す。これらの数値は、ｎ＝３における測定値の平均を示す。対照試料を用いた場合の発現量平均値を１．００としたとき、カゼイン分解物Ａで３時間刺激した培地上清のＨＡＳ１の発現量平均値は、２．８３となり、対照試料と比較して遺伝子発現量が有意に増加した（ｔ検定による解析結果、Ｐ＜０．０１で有意差あり）。 Table 1 shows the measurement results of the gene expression level of HAS1. These numerical values show the average of the measured values at n = 3. When the average expression level when using the control sample was 1.00, the average expression level of HAS1 in the culture supernatant stimulated with casein degradation product A for 3 hours was 2.83, which was compared with the control sample. Thus, the gene expression level was significantly increased (analysis result by t test, P <0.01, there is a significant difference).
ところで、ヒアルロン酸を合成する酵素には、ＨＡＳ１、ＨＡＳ２、ＨＡＳ３が存在するが、その中でも、ＨＡＳ１は高分子ヒアルロン酸を合成する酵素と言われている。そこで、試験例２において、ヒアルロン酸分子の高分子化を確認するため、培地上清中のヒアルロン酸の分子量測定を行った。 By the way, there are HAS1, HAS2, and HAS3 as enzymes that synthesize hyaluronic acid. Among them, HAS1 is said to be an enzyme that synthesizes polymeric hyaluronic acid. Therefore, in Test Example 2, the molecular weight of hyaluronic acid in the culture supernatant was measured in order to confirm that the hyaluronic acid molecules were polymerized.
〔試験例２：ヒアルロン酸分子量の測定〕
試験例１（１）の細胞培養と同様の手順で、カゼイン分解物Ａを添加したＦ−１２培地で３時間培養し、培養上清を回収して凍結乾燥した試料中のヒアルロン酸の分子量を以下の方法により測定した。また、製造例２で得られたカゼイン分解物Ｂ、及び、参考例として未分解のカゼイン（参考例１、ニュージーランドデーリーボード社製）、ヒアルロン酸の構成成分であるグルコサミン（参考例２、甲陽ケミカル社製）を用い、これらを添加した培地上清におけるヒアルロン酸分子量も測定し、試料を添加しないＦ−１２培地で培養した培地上清を対照試料とした。カゼイン分解物Ａ、カゼイン分解物Ｂ、及び、グルコサミンについては、添加量の影響を確認するために、Ｆ−１２培地への添加量を５０μｇ／ｍＬ、５００μｇ／ｍＬとした２種類の濃度で試験を実施し、未分解のカゼインの添加量は５００μｇ／ｍＬとした。
[Test Example 2: Measurement of hyaluronic acid molecular weight]
The molecular weight of hyaluronic acid in the sample obtained by culturing in F-12 medium supplemented with casein degradation product A for 3 hours and recovering the culture supernatant and lyophilized in the same procedure as in the cell culture of Test Example 1 (1). It measured by the following method. In addition, casein degradation product B obtained in Production Example 2, undegraded casein (Reference Example 1, manufactured by New Zealand Daily Board) as a reference example, and glucosamine as a constituent of hyaluronic acid (Reference Example 2, Koyo Chemical) The molecular weight of hyaluronic acid in the medium supernatant to which these were added was also measured, and the medium supernatant cultured in F-12 medium without the sample was used as a control sample. For casein degradation product A, casein degradation product B, and glucosamine, in order to confirm the effect of the addition amount, the addition amount to the F-12 medium was tested at two concentrations of 50 μg / mL and 500 μg / mL. The amount of undegraded casein added was 500 μg / mL.
（１）ヒアルロン酸の精製
細胞培養した培地上清１８ｍＬ分の凍結乾燥物に、４．５ｍＬの超純水を加え、４．５ｍＬのアクチナーゼ緩衝液（100mM Tris-HCL,5.0mM CaCl2,ｐＨ７．８）を加えた。溶液を沸騰水浴で１０分間加熱した後、１０ｍｇのチモール（和光純薬工業社製）と３．０ｍｇのアクチナーゼＥ（科研製薬社製）を加えた。その後、５０℃の恒温槽で２４時間振とうしてタンパク質を分解した後、４．５ｍＬの３０％トリクロロ酢酸を加え、４℃で１時間静置してタンパク質を沈殿した。０℃、９０００ｒｐｍで遠心分離し、上清を蒸留水で透析処理した後、凍結乾燥した。この凍結乾燥物を５０μＬの超純水に懸濁したものを１検体分のヒアルロン酸分子量測定用試料とした。
(1) Purification of hyaluronic acid 4.5 mL of ultrapure water was added to 18 mL of lyophilized supernatant of cell culture medium, and 4.5 mL of actinase buffer (100 mM Tris-HCL, 5.0 mM CaCl 2 , pH 7). .8) was added. After heating the solution in a boiling water bath for 10 minutes, 10 mg of thymol (manufactured by Wako Pure Chemical Industries, Ltd.) and 3.0 mg of actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) were added. Thereafter, the protein was decomposed by shaking in a constant temperature bath at 50 ° C. for 24 hours, 4.5 mL of 30% trichloroacetic acid was added, and the mixture was allowed to stand at 4 ° C. for 1 hour to precipitate the protein. Centrifugation was performed at 9000 rpm at 0 ° C., and the supernatant was dialyzed with distilled water and then freeze-dried. A suspension of this lyophilized product in 50 μL of ultrapure water was used as a sample for measuring hyaluronic acid molecular weight for one sample.
（２）アガロースゲル電気泳動によるヒアルロン酸分子量の測定
アガロース(ＧＩＢＣＯ社製)を０．１Ｍピリジン−０．２Ｍギ酸緩衝液（ｐＨ３．５）に、１％になるように加熱溶解した。その後３分間撹拌冷却し、アガロース溶液３０ｍＬをトレー(縦１００ｍｍ×横１１０ｍｍ×高さ１０ｍｍ)に流し込み、コームを立てて、凝固するまで室温で３０分間静置した。（１）で得たヒアルロン酸分子量測定用の懸濁液に、１μＬの６×Loading Buffer（タカラバイオ株式会社製）を混合して全量をゲルに添加した。４℃にて５０Ｖ、２００ｍＡの定電流で泳動した。４時間の泳動後、ゲルを染色液(ステインズオール５．６ｍｇ、ジオキサン５ｍＬ、超純水９０ｍＬ、１Ｍ酢酸０．１ｍＬ、０．０１Ｍアスコルビン酸５ｍＬ)にて染色した。
分子量１５４０ｋＤａ、２１０ｋＤａ、２９ｋＤａのヒアルロン酸（R&D Systems社製）をそれぞれ１．０ｇ/ｍＬになるように調製し、６× Loading Bufferで希釈した試料を分子量マーカーとして、得られたゲル中に泳動されたヒアルロン酸のバンドの濃淡の分析を行い、ヒアルロン酸の分子量を測定した。
(2) Measurement of hyaluronic acid molecular weight by agarose gel electrophoresis Agarose (manufactured by GIBCO) was dissolved in 0.1 M pyridine-0.2 M formic acid buffer (pH 3.5) by heating to 1%. The mixture was then stirred and cooled for 3 minutes, and 30 mL of agarose solution was poured into a tray (length 100 mm × width 110 mm × height 10 mm), a comb was set up, and the mixture was allowed to stand at room temperature for 30 minutes until solidified. To the suspension for measuring hyaluronic acid molecular weight obtained in (1), 1 μL of 6 × Loading Buffer (manufactured by Takara Bio Inc.) was mixed and the whole amount was added to the gel. Electrophoresis was performed at 4 ° C. with a constant current of 50 V and 200 mA. After the electrophoresis for 4 hours, the gel was stained with a staining solution (Stainsall 5.6 mg, dioxane 5 mL, ultrapure water 90 mL, 1 M acetic acid 0.1 mL, 0.01 M ascorbic acid 5 mL).
Molecular weight 1540 kDa, 210 kDa, 29 kDa hyaluronic acid (manufactured by R & D Systems) was prepared at 1.0 g / mL, and the sample diluted with 6 × Loading Buffer was used as a molecular weight marker and migrated into the resulting gel. The concentration of hyaluronic acid bands was analyzed to determine the molecular weight of hyaluronic acid.
測定した結果、各試料中のヒアルロン酸の分子量（検出されたバンド幅の最大値に対応する値）は、表２のとおりになった。数値は、各培養系での３検体分の測定値の平均を示す。この結果から、細胞培養過程において、カゼイン分解物Ａ及びＢを添加して３時間刺激した後の培地上清に含まれるヒアルロン酸は、カゼイン分解物を添加しなかった対照試料と比べて、有意に増加（高分子化）していることが確認された（ｔ検定において、P＜０．０１で有意差あり）。また、未分解のカゼイン、グルコサミンの添加と比較しても、同濃度のカゼイン分解物を添加したほうが顕著に高分子化されていることが確認された。培地中への添加量が５００μｇ／ｍＬにおける未分解のカゼインとの比較では、カゼイン分解物Ａ及びＢでは、ｔ検定においてｐ＜０．０５で有意に高分子化した。そして、ヒアルロン酸分子は、カゼイン分解物の濃度が高いほど高分子化する傾向であった。この結果から、試験例１で確認されたＨＡＳ１の遺伝子発現量の増加は、産生されるヒアルロン酸の高分子化に関与していることが明らかとなった。 As a result of the measurement, the molecular weight of hyaluronic acid in each sample (value corresponding to the maximum value of the detected bandwidth) was as shown in Table 2. A numerical value shows the average of the measured value for 3 samples in each culture system. From this result, in the cell culture process, hyaluronic acid contained in the culture supernatant after addition of casein degradation products A and B and stimulation for 3 hours was significantly more significant than the control sample to which no casein degradation product was added. (In the t test, P <0.01, there is a significant difference). In addition, it was confirmed that the addition of the casein degradation product at the same concentration significantly increased the polymer compared with the addition of undegraded casein and glucosamine. In comparison with undegraded casein at an addition amount in the medium of 500 μg / mL, casein degradation products A and B were significantly polymerized at p <0.05 in the t-test. And hyaluronic acid molecules tended to be polymerized as the concentration of casein degradation products increased. From this result, it became clear that the increase in the gene expression level of HAS1 confirmed in Test Example 1 is involved in the polymerization of the produced hyaluronic acid.
生体内ヒアルロン酸の分子量は千オーダー〜１００万オーダーといわれ、１０万〜１００万オーダーのような分子量の高いヒアルロン酸が高粘性等の点から、生体内において有益な機能を発揮していると考えられている。
本開示のカゼイン分解物は、高分子のヒアルロン酸が合成されやすいと考えられる。すなわち、本開示のカゼイン分解物は、ヒアルロン酸の合成が促進しやすいと考えられる。このようなヒアルロン酸の合成促進効果が、生体内でのヒアルロン酸の減少に関与する症状に与える影響を確認するため、ヒアルロン酸減少が関与する疾患の一例として、本開示のカゼイン分解物の摂取が、変形性関節症に与える影響を以下の試験例３において確認した。
The molecular weight of hyaluronic acid in vivo is said to be in the order of 1,000 to 1,000,000, and hyaluronic acid having a high molecular weight such as 100,000 to 1,000,000 is exhibiting a useful function in vivo from the point of high viscosity. It is considered.
It is considered that the casein degradation product of the present disclosure is likely to synthesize high-molecular hyaluronic acid. That is, the casein degradation product of the present disclosure is considered to facilitate the synthesis of hyaluronic acid. In order to confirm the effect of such hyaluronic acid synthesis promoting effect on symptoms related to the decrease in hyaluronic acid in vivo, as an example of a disease associated with a decrease in hyaluronic acid, ingestion of the casein degradation product of the present disclosure However, the effect on osteoarthritis was confirmed in Test Example 3 below.
〔試験例３；変形性関節症モデルマウスに対するカゼイン分解物の投与効果の検証〕
（１）病理学的スコアの算出
病態動物として、雄性ＣＤ１（ＩＣＲ）マウス（外科的処置により片脚の内側半月板を切除した変形性関節症モデル、日本チャールス・リバー社）を実験動物として用いた。
実験動物は、８週齢で入荷後、ラボＭＲストック飼料（ノーサン工業社製）および水道水を自由摂取させた。１週間の馴化後、病態動物をカゼイン投与群（未分解カゼイン５００ｍｇ／ｋｇ体重投与）、カゼイン分解物Ａ投与群（カゼイン分解物Ａを５００ｍｇ／ｋｇ体重投与）、カゼイン分解物Ｂ投与群（カゼイン分解物Ｂを５００ｍｇ／ｋｇ体重投与）およびグルコサミン投与群（甲陽ケミカル社製グルコサミンを５００ｍｇ／ｋｇ体重投与）の４群（ｎ＝６／群）に分け、カゼイン投与群を対照群として、各マウスに１日１回経口ゾンデを用いた経口投与を８週間継続した。最終投与終了後に実験動物を１６時間絶食させ、その後イソフルラン麻酔下で処置脚の大腿より下部を摘出し、剥皮した後中性ホルマリン緩衝液中で浸漬固定した。固定した検体は、ＥＤＴＡにて脱灰し、膝関節前額断を包埋面としたパラフィンブロックを作製した。
[Test Example 3; Verification of administration effect of casein degradation product on osteoarthritis model mice]
(1) Pathological score calculation Male CD1 (ICR) mice (osteoarthritis model with the internal meniscus of one leg excised by surgical treatment, Charles River, Japan) are used as experimental animals. It was.
After arrival at the age of 8 weeks, laboratory animals were allowed to freely ingest lab MR stock feed (Nosan Kogyo Co., Ltd.) and tap water. After acclimatization for 1 week, the diseased animals were treated with a casein (undegraded casein 500 mg / kg body weight), casein degradation product A administration group (casein degradation product A 500 mg / kg body weight administration), casein degradation product B administration group (casein). Each of the mice was divided into 4 groups (n = 6 / group) of the degradation product B (500 mg / kg body weight administered) and the glucosamine administration group (Glucosamine manufactured by Koyo Chemical Co., Ltd., 500 mg / kg body weight). Oral administration once a day using an oral sonde was continued for 8 weeks. After completion of the final administration, the experimental animals were fasted for 16 hours, and then the lower part of the thigh of the treated leg was removed under anesthesia with isoflurane, peeled, and immersed and fixed in a neutral formalin buffer. The fixed specimen was decalcified with EDTA to prepare a paraffin block with the knee joint forehead as an embedded surface.
当該標本を用いて、大腿骨の軟骨及び脛骨の軟骨それぞれについて、軟骨基質の変化〔軟骨のひ薄化、サフラニンＯ（Ｓ．Ｏ．）染色性の低下、軟骨基質の裂隙(クラック)形成〕と軟骨細胞の変化〔軟骨細胞の消失（染色性の低下を含む）、軟骨細胞の集塊（クラスター）形成〕を所見として採取した。Ｓ．Ｏ．染色は、常法によりパラフィン切片を作製し、染色後に光学顕微鏡下の観察によって病理組織学的評価を行った。そして、当該所見について、以下〔１〕〜〔５〕のとおり４段階のグレードを定義し、大腿骨及び脛骨のそれぞれの軟骨において採取した所見に合致するスコア（０〜３点）を付与した。各マウス毎のスコアの合計値を病理学的スコアとした。 Using the specimen, changes in the cartilage matrix (thinning of cartilage, decrease in safranin O (SO) staining, formation of cracks in the cartilage matrix) for the cartilage of the femur and the cartilage of the tibia And chondrocyte changes (disappearance of chondrocytes (including decreased staining), chondrocyte cluster formation) were collected as findings. S. O. For staining, paraffin sections were prepared by a conventional method, and histopathological evaluation was performed by observation under an optical microscope after staining. And about the said finding, the grade of four steps was defined as [1]-[5] below, and the score (0-3 points) corresponding to the observation extract | collected in each cartilage of a femur and a tibia was provided. The total score for each mouse was taken as the pathological score.
＜各所見グレード及びスコア＞
〔１〕軟骨ひ薄化〔０点：変化なし、１点：正常の２分の１以上の厚さ、２点：正常の２分の１以下の厚さ、３点：軟骨層なし〕
〔２〕Ｓ．Ｏ．染色性低下〔０点：変化なし、１点：表層のみ低下、２点：中層まで低下、３点：深層まで低下〕
〔３〕クラック形成〔０点：変化なし、１点：表層内、２点：中層まで達する裂隙、３点：深層まで達する裂隙、または軟骨表面と並行な列隙がある〕
〔４〕クラスター形成〔０点：変化なし、１点：一個、２点：二〜三個、３点：多数〕
〔５〕軟骨細胞消失〔０点：変化なし、１点：狭いエリア、２点：やや広いエリア、３点：広範囲のエリア〕
<Each finding grade and score>
[1] Cartilage thinning [0 point: no change, 1 point: thickness more than half of normal, 2 points: thickness less than half of normal, 3 points: no cartilage layer]
[2] S. O. Decrease in dyeability [0 point: no change, 1 point: only the surface layer decreases, 2 points: decreases to the middle layer, 3 points: decreases to the deep layer]
[3] Crack formation [0 point: no change, 1 point: in the surface layer, 2 points: fissure reaching the middle layer, 3 points: fissure reaching the deep layer, or a line parallel to the cartilage surface]
[4] Cluster formation [0 point: no change, 1 point: 1 piece, 2 points: 2 to 3 pieces, 3 points: many]
[5] Chondrocyte disappearance [0 point: no change, 1 point: narrow area, 2 points: slightly wide area, 3 points: wide area]
各投与群における病理学的スコアは表３のとおりとなった。カゼイン分解物Ａ、Ｂ投与群は、いずれも対照群（未分解カゼイン投与群）、グルコサミン投与群よりも病理学的スコアが低下する傾向が認められた。特に、カゼイン分解物Ｂは、大腿骨の病理学的スコアを顕著に低下させる傾向が確認された。 The pathological scores in each administration group are shown in Table 3. In the casein degradation product A and B administration groups, the pathological score tended to be lower than the control group (undegraded casein administration group) and the glucosamine administration group. In particular, the casein degradation product B was confirmed to tend to significantly reduce the pathological score of the femur.
（２）ＩＩ型コラーゲン代謝マーカー及び血清ヒアルロン酸濃度の測定
さらに、病態動物の血液中のＩＩ型コラーゲン代謝マーカー及び血清ヒアルロン酸濃度の測定を行った。ＩＩ型コラーゲンは、軟骨においてプロテオグリカンとともに細胞外マトリックスを形成し、軟骨の機能維持において重要な役割を果たしている。そのため、関節液中や血液中におけるＩＩ型コラーゲンの分解マーカーであるＣ２Ｃ（Collagen Type II Cleavage）、合成マーカーであるＣＰ２（Procollagen TypeII C-propeptide）、及びこれらの比（Ｃ２Ｃ／ＣＰ２）は、軟骨における代謝状態を反映するマーカーとして知られている。また、ヒアルロン酸は、炎症の修復過程で血中濃度が増加するため、関節炎の症状の進展度とよく相関することが知られている。そこで、（１）で使用した病態マウスのカゼイン投与群とカゼイン分解物Ａ投与群の血液を解剖前に採取し、ＩＩ型コラーゲンの代謝マーカーであるＣ２ＣをCollagen Type II Cleavage ELISA（IBEX Pharmaceuticals社製）、ＣＰ２をProcollagen TypeII C-propeptide ELISA（IBEX Pharmaceuticals社製）によりそれぞれ測定し、血清ヒアルロン酸濃度は、QnE Hyaluronic Acid ELISA Assay（IBEX Pharmaceuticals社製）により測定した。測定は、各キットの使用方法に従って行った。
(2) Measurement of Type II Collagen Metabolic Marker and Serum Hyaluronic Acid Concentration Further, type II collagen metabolic marker and serum hyaluronic acid concentration in the blood of diseased animals were measured. Type II collagen forms an extracellular matrix together with proteoglycans in cartilage and plays an important role in maintaining the function of cartilage. Therefore, C2C (Collagen Type II Cleavage) which is a degradation marker of type II collagen in synovial fluid and blood, CP2 (Procollagen Type II C-propeptide) which is a synthetic marker, and a ratio thereof (C2C / CP2) It is known as a marker that reflects the metabolic state in. Hyaluronic acid is known to correlate well with the degree of progression of arthritis symptoms because its blood concentration increases during the repair of inflammation. Therefore, the blood of the casein administration group and the casein degradation product A administration group of the pathological mouse used in (1) was collected before dissection, and C2C, which is a metabolic marker of type II collagen, was collected by Collagen Type II Cleavage ELISA (manufactured by IBEX Pharmaceuticals). CP2 was measured by Procollagen Type II C-propeptide ELISA (manufactured by IBEX Pharmaceuticals), and serum hyaluronic acid concentration was measured by QnE Hyaluronic Acid ELISA Assay (manufactured by IBEX Pharmaceuticals). The measurement was performed according to the usage method of each kit.
Ｃ２Ｃ、ＣＰ２及びヒアルロン酸濃度の測定結果（ｎ＝６／群の平均値）を表４に示し、Ｃ２Ｃ／ＣＰ２を表５に示す。カゼイン投与群よりもカゼイン分解物Ａ投与群の方が、ＩＩ型コラーゲン分解マーカーであるＣ２Ｃが低く、Ｃ２Ｃ／ＣＰ２比も低くなる傾向が確認された。また、カゼイン分解物Ａ投与群では、血清ヒアルロン酸濃度も未分解カゼイン投与群よりも低下し、炎症が抑制されている傾向が確認された。これらの傾向はいずれも、カゼイン分解物Ａ投与群は、軟骨中のＩＩ型コーラゲンの分解が、未分解のカゼイン投与群よりも抑制される傾向があることを示唆している。 The measurement results of C2C, CP2 and hyaluronic acid concentrations (n = 6 / group average value) are shown in Table 4, and C2C / CP2 is shown in Table 5. It was confirmed that C2C, which is a type II collagen degradation marker, was lower and the C2C / CP2 ratio tended to be lower in the casein degradation product A administration group than in the casein administration group. Moreover, in the casein degradation product A administration group, the serum hyaluronic acid concentration was also lower than in the undegraded casein administration group, confirming the tendency that inflammation was suppressed. Both of these tendencies suggest that the casein degradation product A administration group tends to suppress the degradation of type II cholergen in cartilage more than the undegraded casein administration group.
（１）及び（２）の結果から、カゼイン分解物投与群では、病態動物の変形性関節症に伴う症状が改善されている傾向が確認された。したがって、カゼイン分解物の投与は、ヒアルロン酸の減少が関与する疾患の一つである変形性関節症の治療に有効であることが示唆された。 From the results of (1) and (2), it was confirmed that in the casein degradation product administration group, the symptoms associated with osteoarthritis of pathological animals were improved. Therefore, it was suggested that administration of a casein degradation product is effective in the treatment of osteoarthritis, which is one of the diseases in which a decrease in hyaluronic acid is involved.
なお、試験例３で病態動物として使用した外科的処置により片脚の内側半月板を切除したマウスは、変形性関節症のモデルとして、例えば、参考文献２に記載されているように当該疾患モデルとして認められている。
参考文献２：The surgical destabilization of the medial meniscus(DMM) model of osteoarthritis in the 129/SvEv mouse, OsteoArthris and Cartilage(2007) Vol.15 No.9,1061-1069
In addition, a mouse in which the inner meniscus of one leg was excised by a surgical procedure used as a diseased animal in Test Example 3 is a model of the disease as described in Reference 2 as a model of osteoarthritis, for example. Is recognized as.
Reference 2: The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129 / SvEv mouse, OsteoArthris and Cartilage (2007) Vol.15 No.9,1061-1069
Claims (2)
A food composition for promoting hyaluronic acid synthesis comprising a casein degradation product having a molecular weight of 1000 daltons or less as an active ingredient.
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