JP2013126951A - Muscular atrophy inhibitor - Google Patents
Muscular atrophy inhibitor Download PDFInfo
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- JP2013126951A JP2013126951A JP2010044113A JP2010044113A JP2013126951A JP 2013126951 A JP2013126951 A JP 2013126951A JP 2010044113 A JP2010044113 A JP 2010044113A JP 2010044113 A JP2010044113 A JP 2010044113A JP 2013126951 A JP2013126951 A JP 2013126951A
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- delphinidin
- expression
- atrophy
- murf1
- muscle
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Abstract
Description
本発明は、筋萎縮を阻害する効果を有する組成物に関する。 The present invention relates to a composition having an effect of inhibiting muscle atrophy.
骨格筋は人体で最大の組織であり、エネルギー代謝、糖取込み、運動において重要な役割を果たす。骨格筋の量と質はその機能に重要であり、筋肉タンパク質の量と質は絶え間ない合成と分解によるタンパク質代謝回転により維持されている。また骨格筋は身体活動の程度やホルモン、成長因子、ストレスや栄養状態によってその筋量を調節している。骨格筋量が減少すると、身体的な活動を低下させることによりQOL(Quality of Life：生活の質)を低下させ、また筋力を低下させ疲労抵抗性が低下することから、高齢化の進む日本において筋萎縮を防ぐことは意義深いと考えられる。 Skeletal muscle is the largest tissue in the human body and plays an important role in energy metabolism, glucose uptake, and exercise. The amount and quality of skeletal muscle is important for its function, and the amount and quality of muscle protein is maintained by protein turnover through constant synthesis and degradation. Skeletal muscle regulates muscle mass according to the level of physical activity, hormones, growth factors, stress and nutritional status. When skeletal muscle mass decreases, QOL (Quality of Life) decreases by reducing physical activity, and muscle strength decreases and fatigue resistance decreases. Preventing muscle atrophy is considered significant.
骨格筋萎縮は様々な要因によって起こることが知られており、加齢や筋肉を使用せずにいることといった活動量の低下により起こるが、一方で慢性閉塞性肺疾患、重度のやけどなどにおける悪液質、代謝性疾患、ガン、神経変性病など様々な病態によっても生じることが知られている。（Ann Surg., 233, 9-17(2001); Pharmacol Ther., 113, 461-487(2007); N Engl J Med., 335, 1897-1905(1996)）。また特にいくつかの悪液質では、グルココルチコイドの一種であるコルチゾールのレベルが上昇することが筋萎縮に関係していると考えられている（J. Am. Coll. Cardiol., 30, 997-1001(1997); J. Am. Coll. Surg., 188,98-103.(1999); Am. J. Physiol., 264, E668-E676.(1993)）。 Skeletal muscle atrophy is known to be caused by a variety of factors, and it is caused by a decrease in activity such as aging or not using muscles, but it is worse in chronic obstructive pulmonary disease, severe burns, etc. It is known to occur due to various pathological conditions such as liquid quality, metabolic diseases, cancer, neurodegenerative diseases. (Ann Surg., 233, 9-17 (2001); Pharmacol Ther., 113, 461-487 (2007); N Engl J Med., 335, 1897-1905 (1996)). In particular, in some cachexia, elevated cortisol, a type of glucocorticoid, is thought to be related to muscle atrophy (J. Am. Coll. Cardiol., 30, 997- 1001 (1997); J. Am. Coll. Surg., 188, 98-103. (1999); Am. J. Physiol., 264, E668-E676. (1993)).
こういった筋萎縮において、タンパク合成と分解の代謝回転が分解に傾くことがその原因のひとつであるとされている。骨格筋の構成タンパク質を分解する経路の一つにユビキチン化タンパク質を分解するユビキチン・プロテアソーム経路がある。ユビキチン・プロテアソームタンパク質分解経路では、ユビキチン活性化酵素、ユビキチン結合酵素とユビキチンリガーゼの酵素群からなるユビキチン化システムにより分解すべきタンパク質にユビキチンという小さなタンパク質を次々に結合させ、26Sプロテアソームによってポリユビキチンを認識し、タンパク質を分解する。またその経路の中では基質の特異性を決定するユビキチンリガーゼの発現がこの経路の律速段階であると考えられている。 One of the causes of such muscle atrophy is that the turnover of protein synthesis and degradation tends to degradation. One of the pathways that degrade skeletal muscle constituent proteins is the ubiquitin-proteasome pathway that degrades ubiquitinated proteins. In the ubiquitin-proteasome proteolysis pathway, small proteins called ubiquitin are successively bound to the protein to be degraded by the ubiquitination system consisting of ubiquitin-activating enzyme, ubiquitin-conjugating enzyme and ubiquitin ligase enzymes, and polyubiquitin is recognized by the 26S proteasome. And break down the protein. In the pathway, the expression of ubiquitin ligase, which determines the specificity of the substrate, is considered to be the rate-limiting step of this pathway.
ユビキチンリガーゼの中でもMuRF1 (muscle RING finger protein-1)やatorogin-1/MAFbx-1 (muscle atrophy F-box protein-1)は骨格筋や心筋に発現し、またこれらの遺伝子をノックアウトしたマウスが筋萎縮に抵抗性を示したことにより、筋萎縮原因遺伝子（atrogenes）と示され注目されている（Science 294, 1704(2001)）。これらのMuRF1とatrogin-1はグルココルチコイド処理、筋肉の不使用や酸化ストレスなど、少なくとも13種類の筋萎縮で発現が上昇し、萎縮現象のマーカーであるとされている（Science 294, 1704(2001); FEBS Lett. 544, 214-217(2003); Cell Biol., 37, 1974-1984(2005); Am. J. Physiol. Endocrinol. Metab., 289, E969-E980(2005); J. Biol. Chem. 280, 2737-2744(2005); J. Am. Soc. Nephrol., 15, 1537-1545(2004); Am. J. Physiol. Cell Physiol., 285, C806-C812(2003); FASEB J., 19, 362-370(2005); Cell, 117, 399-412(2004); Int. J. Biochem. Cell Biol., 35, 698-705(2003)）。 Among ubiquitin ligases, MuRF1 (muscle RING finger protein-1) and atorogin-1 / MAFbx-1 (muscle atrophy F-box protein-1) are expressed in skeletal muscle and myocardium. Due to its resistance to atrophy, it has been noted as atrophy gene (acigenes) (Science 294, 1704 (2001)). The expression of these MuRF1 and atrogin-1 is increased by at least 13 types of muscle atrophy such as glucocorticoid treatment, muscle nonuse and oxidative stress, and is considered to be a marker of atrophy (Science 294, 1704 (2001). FEBS Lett. 544, 214-217 (2003); Cell Biol., 37, 1974-1984 (2005); Am. J. Physiol. Endocrinol. Metab., 289, E969-E980 (2005); J. Biol Chem. 280, 2737-2744 (2005); J. Am. Soc. Nephrol., 15, 1537-1545 (2004); Am. J. Physiol. Cell Physiol., 285, C806-C812 (2003); FASEB J., 19, 362-370 (2005); Cell, 117, 399-412 (2004); Int. J. Biochem. Cell Biol., 35, 698-705 (2003)).
またグルココルチコイドは筋肉分解の重要なメディエーターであり、骨格筋においてユビキチン・プロテアソーム経路依存性のタンパク分解を引き起こす（Crit. Care. Med., 35, S602-S608(2007)）。また合成グルココルチコイドの一種であるデキサメタゾンは骨格筋においてMuRF1およびatrogin-1の発現を上昇させることが明らかになっている（J. Cell. Biochem., 105, 353-364(2008)）。グルココルチコイド処理した培養筋管細胞は筋萎縮のモデルとして多くの研究で用いられてきた。デキサメタゾンによってもたらされる代謝的な変化は、動物や患者の筋萎縮において見られる変化と似ており、デキサメタゾン処理された筋管は筋萎縮のメカニズムを解析するために良く用いられている。 Glucocorticoids are important mediators of muscle degradation and cause ubiquitin / proteasome pathway-dependent proteolysis in skeletal muscle (Crit. Care. Med., 35, S602-S608 (2007)). Dexamethasone, a kind of synthetic glucocorticoid, has been shown to increase the expression of MuRF1 and atrogin-1 in skeletal muscle (J. Cell. Biochem., 105, 353-364 (2008)). Glucocorticoid-treated cultured myotubes have been used in many studies as a model for muscle atrophy. The metabolic changes brought about by dexamethasone are similar to those seen in animal and patient muscle atrophy, and dexamethasone-treated myotubes are often used to analyze the mechanism of muscle atrophy.
一方、ポリフェノールはその抗酸化活性などの機能性によって注目されている食品成分である。中でもアントシアニン類はパソコン作業による疲労を軽減させるなどの活性から注目されている（Altern. Med. Rev., 5, 553-562(2000)）。また緑茶に含まれるポリフェノールであるカテキンは様々な生理機能を有する。中でも茶葉に含まれるカテキンの約半分量を占めるエピガロカテキンガレート(Epigallocatechin-3-O-gallate ：EGCG)はその生理活性に抗酸化作用、抗ガン作用などが知られている。 On the other hand, polyphenols are food ingredients that are attracting attention due to their functionality such as antioxidant activity. Among these, anthocyanins are attracting attention because of their activity to reduce fatigue caused by personal computer work (Altern. Med. Rev., 5, 553-562 (2000)). Catechin, which is a polyphenol contained in green tea, has various physiological functions. Among these, epigallocatechin gallate (EGCG), which accounts for about half of the catechin contained in tea leaves, is known to have antioxidant and anticancer effects on its physiological activity.
骨格筋の筋萎縮に関するポリフェノールの作用については、酸化ストレスの関与が示唆されている廃用性筋萎縮の抑制を目的として、果実ポリフェノール（具体的にはりんごポリフェノール）を有効成分として含有することを特徴とする廃用性筋萎縮抑制組成物が報告されている（特許文献１：特開２００１−８９３８７）。りんごポリフェノールの主成分はプロシアニジンやプロアントシアニジンである。また、プロアントシアニジンを有効成分と含有する筋肉萎縮抑制剤（特許文献２：特開２００２−３３８４６４）、果実由来ポリフェノールを有効成分とする廃用性筋萎縮時の筋繊維タイプの移行を抑制する筋繊維タイプ移行抑制剤（特許文献３：特開２００６−３２８０３１）、カテキン類を有効成分とする筋機能低下抑制剤（特許文献４：特開２００８−１３４７３）も開示されている。 Regarding the action of polyphenols on muscular atrophy of skeletal muscle, it contains fruit polyphenols (specifically apple polyphenols) as an active ingredient for the purpose of suppressing disuse muscle atrophy, which is suggested to be involved in oxidative stress. A characteristic disuse muscular atrophy inhibitor composition has been reported (Patent Document 1: JP-A-2001-89387). The main components of apple polyphenol are procyanidins and proanthocyanidins. Moreover, the muscle atrophy inhibitor which contains a proanthocyanidin and an active ingredient (patent document 2: Unexamined-Japanese-Patent No. 2002-338464), the muscle which suppresses a transition of the muscle fiber type at the time of a disuse muscle atrophy which uses fruit origin polyphenol as an active ingredient A fiber type migration inhibitor (Patent Document 3: JP-A 2006-328031) and a muscle function decrease inhibitor (Patent Document 4: JP-A 2008-13473) containing catechins as active ingredients are also disclosed.
しかしながら、これらの文献において、筋萎縮原因遺伝子やユビキチンリガーゼに対するポリフェノールの作用に関する言及はない。 However, in these documents, there is no mention regarding the action of polyphenols on a gene causing muscular atrophy or ubiquitin ligase.
筋萎縮原因遺伝子の発現阻害作用を有するポリフェノールを特定し、骨格筋萎縮の防止を目的とする新たな用途を提供することが本発明の課題である。 It is an object of the present invention to specify a polyphenol having an inhibitory action on the expression of a gene causing muscular atrophy and to provide a new use for the purpose of preventing skeletal muscle atrophy.
本発明者らは、ストレスホルモンであるグルココルチコイドの合成薬剤であるデキサメタゾンによる骨格筋萎縮において、筋萎縮原因遺伝子の発現に対するポリフェノールの作用を検討した。そして、デルフィニジン、デルフィニジングルコシド、EGCGの各ポリフェノールが筋萎縮を阻害する可能性を持つことを見出し、本発明を完成するに至った。 The present inventors examined the effect of polyphenols on the expression of genes causing muscular atrophy in skeletal muscle atrophy caused by dexamethasone, which is a synthetic agent for glucocorticoid, which is a stress hormone. And it discovered that each polyphenol of delphinidin, delphinidin glucoside, and EGCG had the possibility of inhibiting muscle atrophy, and came to complete this invention.
本発明の要旨は以下の通りである。
（１）デルフィニジン、デルフィニジングルコシド及びEGCGから選択されるポリフェノールを含有する、筋萎縮阻害作用を有する組成物。
The gist of the present invention is as follows.
(1) A composition having a muscle atrophy inhibitory effect, comprising a polyphenol selected from delphinidin, delphinidin glucoside and EGCG.
（２）筋萎縮原因遺伝子の発現を抑制することにより、筋萎縮を阻害する作用を有する、（１）の組成物。
（３）デルフィニジンまたはデルフィニジングルコシドを含有する、（１）または（２）の組成物。
(2) The composition according to (1), which has an action of inhibiting muscle atrophy by suppressing expression of a gene causing muscle atrophy.
(3) The composition of (1) or (2), comprising delphinidin or delphinidin glucoside.
（４）筋萎縮原因遺伝子がMuRF1である、（２）の組成物。
（５）組成物が食品、栄養補助食品、機能性食品または医薬品である、（１）から（４）のいずれかに記載の組成物。
(4) The composition according to (2), wherein the muscle atrophy-causing gene is MuRF1.
(5) The composition according to any one of (1) to (4), wherein the composition is a food, a dietary supplement, a functional food, or a pharmaceutical.
本発明により、食品由来の機能性成分として知られるアントシアニンおよび緑茶カテキンを、骨格筋萎縮阻害を目的とした食品、栄養補助食品、機能性食品または医薬品などの組成物に利用することが可能となった。特に、飲食品による筋萎縮阻害が可能になるので、病者や高齢者のQOL改善に資する新たな手段を提供することができる。 The present invention makes it possible to use anthocyanins and green tea catechins, which are known as food-derived functional ingredients, in compositions such as foods, nutritional supplements, functional foods or pharmaceuticals for the purpose of inhibiting skeletal muscle atrophy. It was. In particular, since muscle atrophy can be inhibited by food and drink, it is possible to provide a new means for improving the quality of life of sick and elderly people.
骨格筋はグルココルチコイド長期投与・ガン・糖尿病・エイズなどの病気、加齢、栄養不足、あるいはギプス固定や寝たきりによって骨格筋を長い間使用しない場合などによって萎縮し、骨格筋機能が低下することが知られ、その結果、生活の質の低下がもたらされる。 Skeletal muscle may atrophy due to long-term administration of glucocorticoid, diseases such as cancer, diabetes, AIDS, aging, nutritional deficiencies, or when skeletal muscle is not used for a long time due to cast fixation or bedridden, and skeletal muscle function may decrease As a result, the quality of life is reduced.
この骨格筋萎縮にはユビキチン・プロテアソーム系が関与しており、特にユビキチンリガーゼであるmuscle RING finger protein-1 (MuRF1)やatrogin-1といった遺伝子が注目されている。MuRF1やatrogin-1は筋萎縮原因遺伝子であり、筋萎縮において初期に誘導されるなど重要な遺伝子であり、様々な筋萎縮に関与していることが知られている。 This skeletal muscle atrophy involves the ubiquitin / proteasome system. In particular, genes such as muscle RING finger protein-1 (MuRF1) and atrogin-1 which are ubiquitin ligases are attracting attention. MuRF1 and atrogin-1 are genes that cause muscle atrophy and are important genes such as those induced early in muscle atrophy, and are known to be involved in various muscle atrophy.
一方、ストレス誘導ホルモンであるコルチゾールはグルココルチコイドの一種であり、異化の働きを持つことにより筋萎縮との関連が考えられている。またデキサメタゾンは合成グルココルチコイドの一種であり、in vitroおよびin vivoにおいて骨格筋萎縮を招くため、デキサメタゾン処理された筋管細胞は筋萎縮のメカニズムを検討するために用いられる評価系である。 On the other hand, cortisol, which is a stress-inducing hormone, is a kind of glucocorticoid and is considered to be related to muscle atrophy by having a function of catabolism. Dexamethasone is a kind of synthetic glucocorticoid and causes skeletal muscle atrophy in vitro and in vivo. Therefore, dexamethasone-treated myotube cells are an evaluation system used to study the mechanism of muscle atrophy.
本発明者らは、機能性食品因子として知られるポリフェノールに着目し、ポリフェノールが筋萎縮原因遺伝子の発現に及ぼす作用を検討した。具体的には、骨格筋細胞株においてデキサメタゾン処理により誘導されたMuRF1発現に対して、ポリフェノールが及ぼす影響を評価した。ポリフェノールとしては、各種アントシアニン及びEGCGを用いた。 The present inventors focused on polyphenols known as functional food factors, and examined the effect of polyphenols on the expression of muscle atrophy-causing genes. Specifically, the effect of polyphenols on MuRF1 expression induced by dexamethasone treatment in skeletal muscle cell lines was evaluated. Various anthocyanins and EGCG were used as polyphenols.
その結果、デルフィニジン、デルフィニジングルコシド、EGCGの各ポリフェノールが、筋萎縮原因遺伝子であるMuRF1の発現を抑制することを見出した。
以上の結果より、これらアントシアニンやEGCGを、筋萎縮原因遺伝子の発現を抑制するための組成物の有効成分として利用することが可能であることが分かった。
As a result, it was found that each polyphenol of delphinidin, delphinidin glucoside and EGCG suppresses the expression of MuRF1, which is a gene causing muscle atrophy.
From the above results, it was found that these anthocyanins and EGCG can be used as an active ingredient of a composition for suppressing the expression of a gene causing muscle atrophy.
筋萎縮原因遺伝子の具体例としては、MuRF1やatrogin-1が挙げられる。MuRF1は多くの筋萎縮で発現が上昇し、また骨格筋において構成タンパク質の分解に関わるため、上述の組成物は骨格筋の筋萎縮を阻害するために利用することができると考えられる。 Specific examples of muscle atrophy-causing genes include MuRF1 and atrogin-1. Since the expression of MuRF1 increases with many muscle atrophys and is involved in the degradation of constituent proteins in skeletal muscles, it is considered that the above composition can be used to inhibit muscle atrophy of skeletal muscles.
すなわち、本発明は、哺乳動物において筋萎縮原因遺伝子の発現の抑制により骨格筋の筋萎縮を阻害する、ポリフェノールを含有する組成物を提供する。
本発明の組成物は、デルフィニジン、デルフィニジングルコシド、またはEGCGを含有する食品やサプリメント（栄養補助食品）、あるいはこれらポリフェノールを利用した筋萎縮阻害剤の形態であることができる。
That is, the present invention provides a composition containing polyphenol, which inhibits muscular atrophy of skeletal muscle by suppressing the expression of a gene causing muscular atrophy in mammals.
The composition of the present invention can be in the form of delphinidin, delphinidin glucoside or EGCG-containing food or supplement (nutritional supplement), or a muscle atrophy inhibitor using these polyphenols.
これらのポリフェノールは食品由来の成分であることから、特に、経口投与あるいは経口摂取により筋萎縮予防効果を発揮する医薬用組成物あるいは食品としての利用が好ましい。 Since these polyphenols are components derived from food, it is particularly preferable to use them as pharmaceutical compositions or foods that exhibit the effect of preventing muscle atrophy by oral administration or ingestion.
経口投与剤としての形態に特に制限はなく、例えば、散剤、顆粒剤、カプセル剤、丸剤、錠剤などの固形製剤、水剤、懸濁剤、乳剤などの液剤その他に適宜製剤化し得る。製剤化にあたっては、有効成分であるデルフィニジン、デルフィニジングルコシド、またはEGCGの他に、経口投与剤に一般に用いられる賦形剤、結合剤、崩壊剤、滑沢剤、コーティング剤、基剤、懸濁化剤、乳化剤、保湿剤、保存剤、安定剤、界面活性剤、矯味剤などを適宜添加し、常法に従って製造できる。 There is no restriction | limiting in particular in the form as an oral administration agent, For example, it can formulate suitably, for example, solid preparations, such as a powder, a granule, a capsule, a pill, and a tablet, liquids, such as a liquid agent, a suspension agent, and an emulsion. In formulation, in addition to the active ingredients delphinidin, delphinidin glucoside, or EGCG, excipients, binders, disintegrants, lubricants, coating agents, bases, suspensions commonly used for oral administration An agent, an emulsifier, a humectant, a preservative, a stabilizer, a surfactant, a corrigent and the like can be added as appropriate, and production can be performed according to a conventional method.
デルフィニジン、デルフィニジングルコシドまたはEGCGを含有する食品としては、例えば、その摂取により筋萎縮の予防効果が期待できる旨の表示を付した飲食品、例えば、病者用食品、高齢者用食品、特定保健用食品などの特別用途飲食品や機能性食品またはサプリメントとしての利用が考えられる。 Examples of foods containing delphinidin, delphinidin glucoside or EGCG include, for example, foods and drinks with a label indicating that the effect of preventing muscle atrophy can be expected by ingestion thereof, for example, foods for the sick, foods for the elderly, and special health uses It can be used as a special-purpose food or drink such as food, a functional food, or a supplement.
食品の形態には特に制限はなく、飲料、ヨーグルト、ジャムなどの液状若しくはペースト状食品、麺類、パン、キャンディー、ゼリー、クッキー、ガム、豆腐などの固形状食品、あるいは粉茶、ふりかけ調味料、粉末状スープなどの粉末状食品などいかなる形態でもよい。デルフィニジン、デルフィニジングルコシドまたはEGCGは、食品製造時に原料の一部として添加あるいは食品製造終了後に添加できる。 There is no particular limitation on the form of the food, liquid or pasty food such as beverage, yogurt, jam, solid food such as noodles, bread, candy, jelly, cookies, gum, tofu, or powdered tea, sprinkle seasoning, Any form such as powdered food such as powdered soup may be used. Delphinidin, delphinidin glucoside or EGCG can be added as part of the raw material during food production or added after food production is complete.
デルフィニジン、デルフィニジングルコシド、EGCGは、公知の方法により適当な原料から抽出される粗精製物、生成物あるいは市販品のいずれを用いても良い。
デルフィニジンおよびデルフィニジングルコシドは、例えばサクランボ、ビルベリー、ブルーベリー、クロフサスグリ、カーラント、ブドウ、ツルコケモモ、イチゴ、茶品種「サンルージュ」の抽出物から調製できる。精製処理の方法として、順相又は逆相クロマトグラフィー、イオン交換クロマトグラフィー、ゲルろ過等が挙げられる。これらの方法を組み合わせて用いることもできる。上記の果汁又は抽出物からデルフィニジンおよびデルフィニジングルコシドを単離精製する方法は特に限定されないが、例えばＨＰＬＣ、合成吸着剤クロマトグラフィー、イオン交換クロマトグラフィー、ゲルろ過等があり、特に合成吸着剤クロマトグラフィーが好ましい。この場合、溶出条件としては、例えば１０〜５０％エタノール溶液を用いて溶出することが好ましい。またさらに、デルフィニジンおよびデルフィニジングルコシドは酸性条件下で安定化するため、この溶出液に塩酸又は酢酸などを加え酸性にすることが特に好ましい。
Delphinidin, delphinidin glucoside, and EGCG may be any of a crude product, a product or a commercially available product extracted from an appropriate raw material by a known method.
Delphinidin and delphinidin glucoside can be prepared, for example, from extracts of cherries, bilberries, blueberries, black currants, currants, grapes, cranberries, strawberries, tea variety “San Rouge”. Examples of the purification treatment include normal phase or reverse phase chromatography, ion exchange chromatography, gel filtration and the like. A combination of these methods can also be used. The method for isolating and purifying delphinidin and delphinidin glucoside from the above juice or extract is not particularly limited, but there are, for example, HPLC, synthetic adsorbent chromatography, ion exchange chromatography, gel filtration, etc., especially synthetic adsorbent chromatography. preferable. In this case, as elution conditions, it is preferable to elute using, for example, a 10-50% ethanol solution. Furthermore, since delphinidin and delphinidin glucoside are stabilized under acidic conditions, it is particularly preferable to acidify the eluate by adding hydrochloric acid or acetic acid.
EGCGは「べにふうき」、「べにふじ」、「べにほまれ」、「やえほ」、「するがわせ」、「ゆたかみどり」、「かなやみどり」、「やぶきた」、「さやまかおり」、「さえみどり」、「あさつゆ」、「おおいわせ」、「おくむさし」、「めいりょく」、「ふうしゅん」、「おくゆたか」、「青心大パン」、「青心烏龍」、「大葉烏龍」、「鳳凰単叢」、「鳳凰水仙」、「白葉単叢水仙」、「黄枝香」、「武夷水仙」、「紅花」、「べにひかり」、「やまかい」、「やまとみどり」、「からべに」、「香駿」、「おくみどり」及び「サンルージュ」からなる群から選ばれる緑茶葉の抽出液から調製できる。精製処理の方法として、順相又は逆相クロマトグラフィー、イオン交換クロマトグラフィー、ゲルろ過等が挙げられる。これらの方法を組み合わせて用いることもできる。緑茶葉からEGCGを単離精製する方法は特に限定されないが、例えばＨＰＬＣ、合成吸着剤クロマトグラフィー、イオン交換クロマトグラフィー、ゲルろ過等があり、特に合成吸着剤クロマトグラフィーが好ましい。この場合、溶出条件としては、例えば１０〜５０％エタノール溶液を用いて溶出することが好ましい。またさらに、EGCGは酸性条件下で安定化するため、この溶出液に塩酸又は酢酸などを加え酸性にすることが特に好ましい。 EGCG is "Benifuuki", "Benifuuji", "Benihomare", "Yaeho", "Surugaze", "Yutaka Midori", "Kanayama Midori", "Yabukita", "Sayaka Kaori" ”,“ Samidori ”,“ Asatsuyu ”,“ Ooisase ”,“ Okumusashi ”,“ Meiryoku ”,“ Fushun ”,“ Okuyutaka ”,“ Blue heart large bread ”,“ Blue hearted dragon ”,“ Ooba Soryu ”,“ Kyo Monosplex ”,“ Suisuisen ”,“ Shiraba Monosou Daffodil ”,“ Koueda Incense ”,“ Wushu Daffodil ”,“ Safflower ”,“ Benihikari ”,“ Yamakai ”, It can be prepared from an extract of green tea leaves selected from the group consisting of “Yamato Midori”, “Karabeni”, “Kouen”, “Okumidori” and “Saint Rouge”. Examples of the purification treatment include normal phase or reverse phase chromatography, ion exchange chromatography, gel filtration and the like. A combination of these methods can also be used. A method for isolating and purifying EGCG from green tea leaves is not particularly limited, and examples thereof include HPLC, synthetic adsorbent chromatography, ion exchange chromatography, gel filtration, and the like, and synthetic adsorbent chromatography is particularly preferable. In this case, as elution conditions, it is preferable to elute using, for example, a 10 to 50% ethanol solution. Furthermore, since EGCG is stabilized under acidic conditions, it is particularly preferable to add hydrochloric acid or acetic acid to the eluate to make it acidic.
本発明に用いるデルフィニジン、デルフィニジングルコシドまたはEGCGの有効投与量は、成人一日用量として０．１ｍｇ〜２０００ｍｇ、または、０．１ｍｇ〜１０００ｍｇ、または、０．１ｍｇ〜２００ｍｇ、好ましくは、０．１ｍｇ〜１００ｍｇを使用する。本発明においては、ポリフェノールの有効投与量が前記範囲となるように、組成物中に適宜配合すればよい。 The effective dosage of delphinidin, delphinidin glucoside or EGCG used in the present invention is 0.1 mg to 2000 mg, or 0.1 mg to 1000 mg, or 0.1 mg to 200 mg, preferably 0.1 mg to 100 mg is used. In this invention, what is necessary is just to mix | blend suitably in a composition so that the effective dosage of polyphenol may become the said range.
具体的な実施態様において、本発明の組成物中の有効成分として、デルフィニジン及びデルフィニジングルコシドから選択されるアントシアニンが好適に使用される。
本発明の組成物は、活動量の低下により起こる筋萎縮（廃用性筋萎縮）、あるいは様々な病態による筋萎縮のいずれの要因による筋萎縮の抑制にも用いられる。言い換えると、本発明の組成物は、ユビキチン・プロテアソーム系が関与する、あらゆる要因による筋萎縮の予防、抑制または改善を目的として用いることができる。そのような筋萎縮の抑制においてはユビキチン・プロテアソーム系を阻害することが有益であるので、例えば、MuRF1またはatrogin-1の発現を阻害することにより、筋萎縮阻害作用を得ることができる。あるいは、本発明の組成物は、グルココルチコイドにより誘導される筋萎縮、またはコルチゾールのレベルが上昇することで特徴付けられる病態に伴う筋萎縮の予防、抑制または改善を目的として用いることができる。
In a specific embodiment, an anthocyanin selected from delphinidin and delphinidin glucoside is preferably used as an active ingredient in the composition of the present invention.
The composition of the present invention can also be used to suppress muscle atrophy caused by any of the causes of muscle atrophy (disuse muscle atrophy) caused by a decrease in the amount of activity or muscle atrophy caused by various pathological conditions. In other words, the composition of the present invention can be used for the purpose of preventing, suppressing or improving muscle atrophy caused by any factor involving the ubiquitin-proteasome system. In order to suppress such muscle atrophy, it is beneficial to inhibit the ubiquitin / proteasome system. For example, by inhibiting the expression of MuRF1 or atrogin-1, a muscle atrophy inhibiting action can be obtained. Alternatively, the composition of the present invention can be used for the purpose of prevention, suppression or improvement of muscular atrophy induced by glucocorticoid or muscular atrophy associated with a pathological condition characterized by an increase in the level of cortisol.
[実験材料および実験方法]
１）デキサメタゾンによる骨格筋萎縮誘導作用の評価
骨格筋萎縮誘導作用の評価に用いたマウス骨格筋細胞株C2C12(ATCC)は10% ウシ胎児血清(FCS)(BIOLOGICAL INDUSTRIES)添加DMEMにて37℃、水蒸気飽和した5% CO2条件下で継代、維持した。細胞は対数増殖期で培養維持した。培養に使用したDMEM培地は、dH2O 1 Lあたり、ダルベッコMEM培地(コスモ・バイオ株式会社、東京)13.38 g、HEPES(和光純薬工業株式会社、大阪) 5.958 g、注射用ペニシリン G カリウム20万単位(明治製菓株式会社、東京) 0.5 vial、硫酸ストレプトマイシン注射用1g(明治製菓株式会社、東京) 0.1 vial、NaHCO3(nacalai tesque、京都)3.7 gを懸濁した後、フィルター滅菌した。そして、ウシ胎児血清(FCS)をDMEM培地に添加し、細胞培養に使用した。細胞の継代の際は、PBSで洗浄した後、トリプシン溶液で細胞をはがした。PBSはdH2O 1LあたりNaCl (nacalai tesque、京都)8.0 g、KCl (nacalai tesque、京都)0.2g、Na2HPO4(和光純薬工業株式会社、大阪)1.15 g、KH2PO4(nacalai tesque、京都)0.2 gを懸濁し、オートクレーブ滅菌した。トリプシン溶液は、100 mL PBSあたりEDTA・2Na (和光純薬工業株式会社、大阪)0.05 g、トリプシン(nacalai tesque、京都)0.02 gを懸濁し、フィルター滅菌した。また、細胞の継代・維持には10 mL接着dish(nunc TM、東京)を使用した。
[Experimental materials and methods]
1) Evaluation of skeletal muscle atrophy induction by dexamethasone The mouse skeletal muscle cell line C2C12 (ATCC) used for evaluation of skeletal muscle atrophy induction was 37 ° C in DMEM supplemented with 10% fetal calf serum (FCS) (BIOLOGICAL INDUSTRIES). It was subcultured and maintained under steam saturated 5% CO 2 conditions. Cells were maintained in culture in the logarithmic growth phase. The DMEM medium used for the culture was 13.38 g of Dulbecco MEM medium (Cosmo Bio Inc., Tokyo), 5.958 g of HEPES (Wako Pure Chemical Industries, Ltd., Osaka), 20 mg of penicillin G for injection per 1 L of dH 2 O. Ten thousand units (Meiji Seika Co., Ltd., Tokyo) 0.5 vial, 1 g of streptomycin sulfate for injection (Meiji Seika Co., Ltd., Tokyo) 0.1 vial, NaHCO 3 (nacalai tesque, Kyoto) 3.7 g were suspended and then filter sterilized. Fetal calf serum (FCS) was added to the DMEM medium and used for cell culture. When the cells were passaged, the cells were washed with PBS and then detached with a trypsin solution. PBS is 8.0 g of NaCl (nacalai tesque, Kyoto) per liter of dH 2 O, 0.2 g of KCl (nacalai tesque, Kyoto), Na 2 HPO 4 (Wako Pure Chemical Industries, Ltd., Osaka), 1.15 g, KH 2 PO4 (nacalai tesque , Kyoto) 0.2 g was suspended and autoclaved. The trypsin solution was prepared by suspending 0.05 g of EDTA · 2Na (Wako Pure Chemical Industries, Ltd., Osaka) and 0.02 g of trypsin (nacalai tesque, Kyoto) per 100 mL PBS, and sterilizing the filter. In addition, a 10 mL adhesive dish (nunc ™ , Tokyo) was used for cell passage and maintenance.
骨格筋萎縮誘導作用の評価にはデキサメタゾンによるMuRF1の発現上昇を用いた。マウス骨格筋細胞株C2C12を1x104cells/mLに調整して2 mL dish (nunc TM、東京)に播種し、10% FCS含有DMEMにて24時間培養した。その後、0.5% FCS含有DMEM（分化培地）に培地交換することにより分化誘導をかけた。分化培地は誘導開始48時間後に培地交換した。誘導開始96時間後、デキサメタゾン(Sigma)終濃度0、0.1、1 μMを含む分化培地に培地交換し24時間後処理した。デキサメタゾンはジメチルスルホキシド(DMSO) (Nacalai tesque, Inc. Kyoto, Japan)にて10 mMとなるように溶解し、-30℃にて保存した。使用に際しては適宜解凍して用いた。その後、上清を除去し、PBS 1 mLにて洗浄し、TRIzolTMReagent (invitogen、東京)800 μLによって細胞を回収し、室温で5分間放置した。次に、クロロホルム(nacalai tesque、京都)200 μLを添加・撹拌し、室温で3分間放置後、4℃で15分間遠心(12,000 x g)した。その後、上層を取り出し、2-プロパノール(nacalai tesque、京都)500 μLを添加・撹拌し、室温で10分間放置後、4℃で10分間遠心(12000 x g)した。その後、上清を除去し、75% EtOH(nacalai tesque、京都) in DEPE水(DEPC (SIGMA-ALDRICH 、東京)1 mLをdH2O 1 Lに溶かし、オートクレーブ滅菌したもの)を1 mL添加・撹拌後、4℃で5分間遠心(12000 x g)した。その上清を完全に除去し、DEPC水を20から25 μL入れて懸濁した。その後、PrimixScript RT reagent kit (TaKaRa)を用いてcDNAを合成した。調製したcDNAは-20℃で保存した。その後リアルタイムPCRによってMuRF1およびGAPDHの発現を検討した。リアルタイムPCRにはThermal Cycler Dice Real Time System TP800 (TaKaRa)を用いた。PCR反応液の組成は、1サンプルにつきdH2O 8.5 μL、SYBR Premix Ex Taq II (TaKaRa) 12.5 μL、Forward primer (10 μM) 1 μL、Reverse primer (10 μM) 1 μL、鋳型2 μLとした。PCR条件は初期変性を95℃10秒間行い、その後95℃5 秒間、60℃20 秒間で50サイクルとした。プライマーはMuRF1は
Forward : 5'-TGAGGTGCCTACTTGCTCCT-3'
Reverse : 5'-TCACCTGGTGGCTATTCTCC-3'
を用いた。プライマーは株式会社ジーンネット(福岡)に合成を委託した。GAPDHのプライマーはタカラバイオから購入した。各プライマーはTE bufferによって溶解・保存した。TE bufferは、500 mLあたり、10mM Tris(nacalai tesque) 0.605 g、EDTA・2Na (和光純薬工業株式会社)0.186 g を約350 mLのdH2Oに溶解してHCl(和光純薬)によってpH 8.0に調整後、500 mLにフィルアップし、オートクレーブ滅菌をした。実験結果の統計処理にはStudent’s t検定を用いた。
The evaluation of skeletal muscle atrophy induction was based on the increased expression of MuRF1 by dexamethasone. Mouse skeletal muscle cell line C2C12 was adjusted to 1 × 10 4 cells / mL, seeded in a 2 mL dish (nunc ™ , Tokyo), and cultured in DMEM containing 10% FCS for 24 hours. Thereafter, differentiation was induced by exchanging the medium with DMEM (differentiation medium) containing 0.5% FCS. The differentiation medium was changed 48 hours after the start of induction. 96 hours after the start of induction, the medium was changed to a differentiation medium containing dexamethasone (Sigma) final concentrations of 0, 0.1, and 1 μM, followed by 24 hours of post-treatment. Dexamethasone was dissolved in dimethyl sulfoxide (DMSO) (Nacalai tesque, Inc. Kyoto, Japan) to a concentration of 10 mM and stored at −30 ° C. In use, it was appropriately thawed. Thereafter, the supernatant was removed, washed with 1 mL of PBS, and the cells were collected with 800 μL of TRIzol ™ Reagent (invitogen, Tokyo) and left at room temperature for 5 minutes. Next, 200 μL of chloroform (nacalai tesque, Kyoto) was added and stirred, allowed to stand at room temperature for 3 minutes, and then centrifuged (12,000 × g) at 4 ° C. for 15 minutes. Thereafter, the upper layer was taken out, 500 μL of 2-propanol (nacalai tesque, Kyoto) was added and stirred, allowed to stand at room temperature for 10 minutes, and then centrifuged (12000 × g) at 4 ° C. for 10 minutes. After removing the supernatant, add 1 mL of 75% EtOH (nacalai tesque, Kyoto) in DEPE water (1 mL of DEPC (SIGMA-ALDRICH, Tokyo) dissolved in dH 2 O 1 L and autoclaved). After stirring, the mixture was centrifuged (12000 xg) at 4 ° C for 5 minutes. The supernatant was completely removed, and 20 to 25 μL of DEPC water was added and suspended. Thereafter, cDNA was synthesized using PrimixScript RT reagent kit (TaKaRa). The prepared cDNA was stored at -20 ° C. Thereafter, the expression of MuRF1 and GAPDH was examined by real-time PCR. Thermal Cycler Dice Real Time System TP800 (TaKaRa) was used for real-time PCR. The composition of the PCR reaction solution was dH 2 O 8.5 μL, SYBR Premix Ex Taq II (TaKaRa) 12.5 μL, Forward primer (10 μM) 1 μL, Reverse primer (10 μM) 1 μL, and template 2 μL per sample. . PCR conditions were such that initial denaturation was performed at 95 ° C. for 10 seconds, followed by 50 cycles of 95 ° C. for 5 seconds and 60 ° C. for 20 seconds. Primer is MuRF1
Forward: 5'-TGAGGTGCCTACTTGCTCCT-3 '
Reverse: 5'-TCACCTGGTGGCTATTCTCC-3 '
Was used. The primer was commissioned to Genenet Co., Ltd. (Fukuoka). GAPDH primers were purchased from Takara Bio. Each primer was dissolved and stored in TE buffer. TE buffer is dissolved in approximately 350 mL of dH 2 O by dissolving 0.605 g of 10 mM Tris (nacalai tesque) 0.605 g and EDTA-2Na (Wako Pure Chemical Industries, Ltd.) in 500 mL of pH, and then pH is adjusted with HCl (Wako Pure Chemical). After adjusting to 8.0, it was filled up to 500 mL and sterilized by autoclave. Student's t test was used for statistical processing of the experimental results.
２）アントシアニン及びEGCGの筋萎縮阻害作用の検討（mRNAレベル）
アントシアニン及びEGCGのデキサメタゾン誘導性ユビキチンリガーゼ発現に対する評価を行った。C2C12を2x104cell/mLにて2 mL dishに播種し、分化誘導した。分化培地は誘導開始から48時間後に交換した。誘導開始から72時間後にアントシアニンであるCyanidin-3-O-galactoside、Cyanidin-3-O-glucoside、Cyanidin、Delphinidin-glucoside、DelphinidinおよびEGCG各終濃度5 μM含有分化培地に置換し、24時間前処理した。その後、各被験物5 μMおよびデキサメタゾン1 μMを含有する分化培地にて24時間処理した。
2) Examination of anthocyanins and EGCG in inhibiting muscle atrophy (mRNA level)
We evaluated dexamethasone-induced ubiquitin ligase expression of anthocyanins and EGCG. C2C12 was seeded in a 2 mL dish at 2 × 10 4 cells / mL to induce differentiation. The differentiation medium was changed 48 hours after the start of induction. 72 hours after the start of induction, anthocyanins Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, Cyanidin, Delphinidin-glucoside, Delphinidin, and EGCG were each replaced with a differentiation medium containing 5 μM final treatment for 24 hours. did. Thereafter, each test article was treated with a differentiation medium containing 5 μM and dexamethasone 1 μM for 24 hours.
Cyanidin-3-O-galactoside、Cyanidin-3-O-glucoside、Cyanidin、Delphinidin-3-glucoside、DelphinidinはEXTRASYNTHESEから購入した。各々5 mMになるように、Cya-gal、Cya-glu、Del-gluは超純水に、Cyanidin、DelphinidinはDMSOにそれぞれ溶解し、-30℃で保存した。EGCGはSigmaより購入し、5 mMとなるように超純水に溶解し、-15℃で凍結保存した。各試薬とも用いる際は適宜解凍して用いた。 Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, Cyanidin, Delphinidin-3-glucoside and Delphinidin were purchased from EXTRASYNTHESE. Cya-gal, Cya-glu and Del-glu were dissolved in ultrapure water, and Cyanidin and Delphinidin were dissolved in DMSO so as to be 5 mM each, and stored at −30 ° C. EGCG was purchased from Sigma, dissolved in ultrapure water to 5 mM, and stored frozen at -15 ° C. When each reagent was used, it was appropriately thawed.
デキサメタゾン処理の後、細胞をTrizolにて回収後、cDNAを合成し、MuRF1およびGAPDHの発現レベルをリアルタイムPCRによって検討した。
３）アントシアニン及びEGCGの筋萎縮阻害作用の検討（タンパク質レベル）
次に、アントシアニン及びEGCGのデキサメタゾン誘導性ユビキチンリガーゼ発現に対する評価をタンパク質レベルで行った。C2C12を1x104cell/mLにて24 well plate (nunc TM、東京)に播種し、分化誘導した。分化培地は誘導開始から48 時間後に交換した。誘導開始から72時間後に、アントシアニンであるCyanidin-3-O-galactoside、Cyanidin-3-O-glucoside、Cyanidin、Delphinidin-glucoside、DelphinidinおよびEGCG各終濃度5 μM含有分化培地に置換し、24時間前処理した。その後、各被験物5 μMおよびデキサメタゾン1 μMを含有する分化培地にて24時間処理した。その後上清を除去し、PBS 1 mLで洗浄後、処理した細胞に細胞溶解バッファー(50 mM Tris (nacalai tesque)-HCl(和光純薬工業) (pH 7.5)、150 mM NaCl(和光純薬工業)、1% (v/v) Triton-X100(nacalai tesque)、1 mM EDTA (和光純薬工業)、50 mM NaF(nacalai tesque), 30 mM Na4P2O7(nacalai tesque)、1 mM Phenylmethylsulfonyl Fluoride(和光純薬)、2 μg/mL Aprotinin、1 mM pervanadate(和光純薬))を加え溶解した。その後、12000 x rpm で 10 min 遠心し、上清を回収した。これをサンプルとしてBicinchoninic Acid (BCA) Protein Assay Reagent (Rockford, IL)を用いてタンパク質の定量を行った。サンプルバッファー(0.5 M Tris-HCl (pH6.8)、10% (w/v) sodium dodecyl sulfate (SDS,和光純薬)，50% glycerol(nacalai tesque)、1 % (w/v) bromophenol blue(和光純薬)、0.65 M 2-mercaptoethanol(和光純薬工業))をサンプルに対して等量加えて、10分煮沸し熱変性を行った。これを6% polyacrylamideゲルに供し(acrylamide monomer (nacalai tesque)使用)、0.02 A 110分でSDS-polyacrylamide gel electrophoresis(SDS-PAGE)を行った。その後、ゲルを氷冷しながら、100 V 60分でエレクトロブロッティングを行い、ゲル中のタンパク質をニトロセルロース膜(Schleicher & Schuell , Keene , NH )に転写した。この膜を、2.5% Bovine serum albumin (BSA)(Roche)-TTBS (0.1% Tween20 (nacalai tesque)含有Tris buffered saline；20 mM Tris-HCl, pH7.6)にて室温で1時間ブロッキングを行った。ブロッキング後に、1次抗体を2.5% BSA-TTBSで希釈し4℃で一晩反応させ、2次抗体を室温で1時間反応させた。TTBSで4 回洗浄後、Chemi-Lumi Oneキット(nacalai tesque)またはECL キット(GE Healthcare)を用いて発色反応を行い、ChemiImagerTM 5500(Alpha Innotech)で検出を行った。
After treatment with dexamethasone, the cells were collected with Trizol, cDNA was synthesized, and the expression levels of MuRF1 and GAPDH were examined by real-time PCR.
3) Examination of muscle atrophy inhibitory action of anthocyanins and EGCG (protein level)
Next, dexamethasone-induced ubiquitin ligase expression of anthocyanins and EGCG was evaluated at the protein level. C2C12 was seeded on a 24-well plate (nunc ™ , Tokyo) at 1 × 10 4 cells / mL to induce differentiation. The differentiation medium was changed 48 hours after the start of induction. 72 hours after the start of induction, the anthocyanins Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, Cyanidin, Delphinidin-glucoside, Delphinidin, and EGCG were replaced with differentiation media containing 5 μM final concentrations, 24 hours before Processed. Thereafter, each test article was treated with a differentiation medium containing 5 μM and dexamethasone 1 μM for 24 hours. The supernatant was then removed, washed with 1 mL of PBS, and treated cells were treated with cell lysis buffer (50 mM Tris (nacalai tesque) -HCl (Wako Pure Chemical Industries, Ltd.) (pH 7.5), 150 mM NaCl (Wako Pure Chemical Industries, Ltd.). ), 1% (v / v) Triton-X100 (nacalai tesque), 1 mM EDTA (Wako Pure Chemical Industries), 50 mM NaF (nacalai tesque), 30 mM Na 4 P 2 O 7 (nacalai tesque), 1 mM Phenylmethylsulfonyl fluoride (Wako Pure Chemical), 2 μg / mL Aprotinin, 1 mM pervanadate (Wako Pure Chemical)) was added and dissolved. Thereafter, the mixture was centrifuged at 12000 x rpm for 10 min, and the supernatant was collected. Using this as a sample, protein was quantified using Bicinchoninic Acid (BCA) Protein Assay Reagent (Rockford, IL). Sample buffer (0.5 M Tris-HCl (pH 6.8), 10% (w / v) sodium dodecyl sulfate (SDS), 50% glycerol (nacalai tesque), 1% (w / v) bromophenol blue ( Wako Pure Chemical) and 0.65 M 2-mercaptoethanol (Wako Pure Chemical Industries) were added to the sample in an equal amount and boiled for 10 minutes for heat denaturation. This was subjected to 6% polyacrylamide gel (using acrylamide monomer (nacalai tesque)) and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 0.02 A for 110 minutes. Thereafter, electroblotting was performed at 100 V for 60 minutes while the gel was cooled on ice, and the protein in the gel was transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). This membrane was blocked with 2.5% Bovine serum albumin (BSA) (Roche) -TTBS (0.1% Tween20 (nacalai tesque) -containing Tris buffered saline; 20 mM Tris-HCl, pH 7.6) for 1 hour at room temperature. . After blocking, the primary antibody was diluted with 2.5% BSA-TTBS and reacted overnight at 4 ° C., and the secondary antibody was reacted at room temperature for 1 hour. After washing 4 times with TTBS, color reaction was performed using Chemi-Lumi One kit (nacalai tesque) or ECL kit (GE Healthcare), and detection was performed with ChemiImager ™ 5500 (Alpha Innotech).
1次抗体に用いた抗体は抗MuRF1抗体および抗β-Actin抗体である。Rabbit anti-MuRF1 monoclonal antibodyはSanta Cruz Biotechnologyより購入し、1000倍希釈して用いた。Mouse anti-β-Actin monoclonal antibodyはSigmaより購入し、10000倍希釈して用いた。Mouse anti-Horseradish peroxidase (HRP)-conjugated anti-Rabbit IgG antibody及びHRP-conjugated anti-Mouse IgG antibodyはROCKLANDより購入し、10000倍希釈して用いた。 The antibodies used as the primary antibody are anti-MuRF1 antibody and anti-β-Actin antibody. Rabbit anti-MuRF1 monoclonal antibody was purchased from Santa Cruz Biotechnology and diluted 1000 times. Mouse anti-β-Actin monoclonal antibody was purchased from Sigma and diluted 10,000 times. Mouse anti-Horseradish peroxidase (HRP) -conjugated anti-Rabbit IgG antibody and HRP-conjugated anti-Mouse IgG antibody were purchased from ROCKLAND and diluted 10,000 times.
[結果]
１）デキサメタゾンによる MuRF1 発現
C2C12細胞における筋萎縮原因遺伝子であるユビキチンリガーゼMuRF1の発現を誘導するデキサメタゾンの濃度を決定するために、分化させたC2C12筋管にデキサメタゾン処理を行った。
[result]
1) MuRF1 expression by dexamethasone
In order to determine the concentration of dexamethasone that induces the expression of ubiquitin ligase MuRF1, a gene causing muscular atrophy in C2C12 cells, the differentiated C2C12 myotube was treated with dexamethasone.
C2C12細胞を2 mL dishに2 x 104 cells/mLとなるように播種し、増殖培地である10% FCS-DMEMにて24 時間前培養後、分化誘導培地である0.5% FCS-DMEMに培地交換し分化誘導をかけた。分化培地は48時間後に一回交換した。分化４日後、デキサメタゾンを0（コントロール）、0.1、もしくは1 μM 含有する分化培地に置換し２４時間処理後、細胞を回収しリアルタイムPCRによってMuRF1の発現を検討した。 C2C12 cells are seeded at 2 x 10 4 cells / mL in a 2 mL dish, pre-cultured in 10% FCS-DMEM, which is a growth medium, for 24 hours, and then cultured in 0.5% FCS-DMEM, which is a differentiation-inducing medium. They were exchanged to induce differentiation. The differentiation medium was changed once after 48 hours. Four days after differentiation, dexamethasone was replaced with a differentiation medium containing 0 (control), 0.1, or 1 μM, treated for 24 hours, cells were collected, and MuRF1 expression was examined by real-time PCR.
その結果を図１に示す。デキサメタゾン0.1、1μMによってMuRF1の発現量は有意に増加した。C2C12細胞におけるデキサメタゾン処理によるユビキチンリガーゼ発現に関して、文献などでは1μMにてよく検討されているため、以後の検討にはデキサメタゾン1μMを用いることにした。 The result is shown in FIG. The expression level of MuRF1 was significantly increased by dexamethasone 0.1 and 1 μM. Since ubiquitin ligase expression by dexamethasone treatment in C2C12 cells has been well studied in the literature at 1 μM, we decided to use 1 μM dexamethasone for subsequent studies.
２）デキサメタゾン誘導性MuRF1発現におよぼすアントシアニン類の影響
骨格筋細胞におけるデキサメタゾン誘導性MuRF1発現に及ぼすアントシアニン類の影響を見るために、分化させたC2C12にアントシアニン類を24時間添加し、その後デキサメタゾン処理した。
2) Effect of anthocyanins on dexamethasone-induced MuRF1 expression To see the effect of anthocyanins on dexamethasone-induced MuRF1 expression in skeletal muscle cells, anthocyanins were added to differentiated C2C12 for 24 hours and then treated with dexamethasone. .
結果を図２に示す。
図２Ａでは、C2C12を2 mL dishに2 x 104 cells/mLとなるように播種し、増殖培地である10% FCS-DMEMにて24時間前培養後、分化誘導培地である0.5% FCS-DMEMに培地交換し分化誘導をかけた。分化培地は48時間後に一回交換した。分化3日後、各アントシアニン5 mM含有分化培地に置換し、24時間前処理後、各成分を5 mMおよびデキサメタゾンを1 mM含有する分化培地にて24時間刺激した。その後細胞を回収しリアルタイムPCR によってMuRF1の発現(mRNA)を検討した。
The results are shown in FIG.
In FIG. 2A, C2C12 is seeded in a 2 mL dish at 2 × 10 4 cells / mL, pre-cultured for 10 hours in 10% FCS-DMEM, which is a growth medium, and then 0.5% FCS-, which is a differentiation-inducing medium. The medium was changed to DMEM to induce differentiation. The differentiation medium was changed once after 48 hours. Three days after differentiation, each medium was replaced with a differentiation medium containing 5 mM of anthocyanin. After pretreatment for 24 hours, each component was stimulated with a differentiation medium containing 5 mM and 1 mM of dexamethasone for 24 hours. Thereafter, the cells were collected, and MuRF1 expression (mRNA) was examined by real-time PCR.
シアニジンガラクトシド、シアニジングルコシド、シアニジンにはMuRF1発現低下作用は見られなかったが、デルフィニジングルコシド、デルフィニジンはデキサメタゾン誘導性MuRF1発現レベルを有意に低下させた(図２Ａ)。 Cyanidin galactoside, cyanidin glucoside, and cyanidin did not show any MuRF1 expression-reducing action, but delphinidin glucoside and delphinidin significantly reduced the dexamethasone-induced MuRF1 expression level (FIG. 2A).
図２Ｂでは、C2C12を24 well plateに1 x 104 cells/wellとなるよう播種し、増殖培地である10% FCS-DMEMにて24時間前培養後、分化誘導培地である0.5% FCS-DMEMに培地交換し分化誘導をかけた。分化培地は48時間後に一回交換した。分化3日後、各アントシアニン5 mM含有分化培地に置換し、24時間前処理後、各成分を5 mMおよびデキサメタゾンを1 mM含有する分化培地にて6日間刺激した。その後細胞を回収しウェスタンブロット解析によってMuRF1の発現(タンパク質)を検討した。 In FIG. 2B, C2C12 was seeded on a 24-well plate at 1 × 10 4 cells / well, pre-cultured for 24 hours in 10% FCS-DMEM as a growth medium, and then 0.5% FCS-DMEM as a differentiation induction medium. The medium was changed to induce differentiation. The differentiation medium was changed once after 48 hours. Three days after differentiation, each medium was replaced with a differentiation medium containing 5 mM of anthocyanin. After pretreatment for 24 hours, each component was stimulated for 6 days in a differentiation medium containing 5 mM and dexamethasone 1 mM. Thereafter, the cells were collected, and MuRF1 expression (protein) was examined by Western blot analysis.
タンパクレベルでは、シアニジングルコシドおよびシアニジンに若干の低下作用が、またデルフィニジングルコシドおよびデルフィニジンには明らかなMuRF1発現低下作用が見られた(図２Ｂ)。 At the protein level, cyanidin glucoside and cyanidin had a slight lowering effect, and delphinidin glucoside and delphinidin had a clear MuRF1 expression lowering effect (FIG. 2B).
３）デキサメタゾン誘導性MuRF1発現におよぼすEGCGの影響
骨格筋細胞におけるデキサメタゾン誘導性MuRF1発現に及ぼすEGCGの影響を見るために、分化させたC2C12にEGCGを24時間添加し、その後デキサメタゾン処理した。
3) Effect of EGCG on dexamethasone-induced MuRF1 expression In order to see the effect of EGCG on dexamethasone-induced MuRF1 expression in skeletal muscle cells, EGCG was added to differentiated C2C12 for 24 hours, and then treated with dexamethasone.
結果を図３に示す。
図３Ａでは、C2C12を2 mL dishに2 x 104 cells/mLとなるように播種し、増殖培地である10% FCS-DMEMにて24時間前培養後、分化誘導培地である0.5% FCS-DMEMに培地交換し分化誘導をかけた。分化培地は48時間後に一回交換した。分化3日後、EGCG 5 μM含有分化培地に置換し、24時間前処理後、EGCG 5 μMおよびデキサメタゾンを1 μM含有する分化培地にて24時間刺激した。その後細胞を回収し、リアルタイムPCRによってMuRF1 の発現(mRNA)を検討した。
The results are shown in FIG.
In FIG. 3A, C2C12 is seeded in a 2 mL dish at 2 × 10 4 cells / mL, pre-cultured with 10% FCS-DMEM as a growth medium for 24 hours, and then differentiated with 0.5% FCS− as a differentiation induction medium. The medium was changed to DMEM to induce differentiation. The differentiation medium was changed once after 48 hours. Three days after differentiation, the medium was replaced with a differentiation medium containing EGCG 5 μM, and after 24 hours of pretreatment, the cells were stimulated with a differentiation medium containing EGCG 5 μM and dexamethasone 1 μM for 24 hours. Thereafter, the cells were collected, and MuRF1 expression (mRNA) was examined by real-time PCR.
EGCGはデキサメタゾン誘導性MuRF1の発現を有意に阻害した(図３Ａ)。
図３Ｂでは、C2C12を24 well plateに1 x 104 cells/wellとなるよう播種し、増殖培地である10% FCS-DMEMにて24 時間前培養後、分化誘導培地である0.5% FCS-DMEMに培地交換し分化誘導をかけた。分化培地は48時間後に一回交換した。分化3日後、EGCG 5 mM含有分化培地に置換し、24時間前処理後、EGCG 5 μMおよびデキサメタゾン1 μMを含有する分化培地にて6日間刺激した。その後細胞を回収しウェスタンブロット解析によってMuRF1の発現(タンパク質)を検討した。
EGCG significantly inhibited dexamethasone-induced MuRF1 expression (FIG. 3A).
In FIG. 3B, C2C12 is seeded on a 24-well plate at 1 × 10 4 cells / well, pre-cultured for 24 hours in 10% FCS-DMEM as a growth medium, and then 0.5% FCS-DMEM as a differentiation induction medium. The medium was changed to induce differentiation. The differentiation medium was changed once after 48 hours. Three days after differentiation, the medium was replaced with a differentiation medium containing EGCG 5 mM, and after pretreatment for 24 hours, the cells were stimulated with a differentiation medium containing EGCG 5 μM and dexamethasone 1 μM for 6 days. Thereafter, the cells were collected, and MuRF1 expression (protein) was examined by Western blot analysis.
タンパク質レベルでも、MuRF1の発現誘導は顕著に阻害されていた（図３Ｂ）。
[考察]
デキサメタゾン誘導性のMuRF1発現に対し、シアニジン、シアニジンガラクトシド、シアニジングルコシドは影響を及ぼさなかったが、デルフィニジンおよびデルフィニジングルコシドはMuRF1発現低下作用を発揮した。また、緑茶カテキンの一種であるEGCGも顕著にMuRF1の発現誘導を阻害することが明らかになった。
Even at the protein level, the induction of MuRF1 expression was markedly inhibited (FIG. 3B).
[Discussion]
Cyanidin, cyanidin galactoside, and cyanidin glucoside had no effect on dexamethasone-induced MuRF1 expression, but delphinidin and delphinidin glucoside exerted MuRF1 expression-reducing action. In addition, EGCG, a kind of green tea catechin, was also found to significantly inhibit the induction of MuRF1 expression.
MuRF1は多くの筋萎縮で発現が上昇し、また骨格筋において構成タンパク質の分解に関わるため、デルフィニジン、デルフィニジングルコシド、EGCGは筋萎縮を防ぐ可能性が示された。 The expression of MuRF1 is increased by many muscle atrophys, and since it is involved in the degradation of constituent proteins in skeletal muscle, delphinidin, delphinidin glucoside, and EGCG have the potential to prevent muscle atrophy.
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Latres, E., Amini, A.R., Amini, A.A., Griffiths, J., Martin, F.J., Wei, Y., Lin, H.C., Yancopoulos, G.D., and Glass, D.J. Insulin-like growth factor-1 (IGF-1) inversely regulates atrophy-induced genes cia the phosphatidylinositol 3-kinase/akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, Journal of Biological Chemistry, 280, 2737-2744(2005)
Lee, S.W., Dai, G., Hu, Z., Wang, X., Du, J., and Mitch, W.E., Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitin-proteasome systems by phosphatidylinositol 3 kinase, Journal of American Society of Nephrology, 15, 1537-1545(2004)
Li, Y.-P., Chen, Y., Li, A.S., and Reid, M.B., Hydrogen peroxide stimulates ubiquitin conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes, American Journal of Physiology-Cell Phisiology, 285, C806-C812(2003)
Li, Y.-P., Chen, Y., John, J., Moylan, J., Jin, B., Mann, D.L., and Reid, M.B., TNF-{alpha} acts via p38 MAPK to stimulates expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. FASEB Journal, 19, 362-370(2005)
Menconi M, Fareed M, O Neal P, Poylin B. Wei W, Hasselgren PO., Role of glucocorticoids in the molecular regulation of muscle wasting, Critical Care Medicine, 35, S602-S608(2007)
Michael Menconi, Patricia Gonnella, Victoria Petkova, Stewart Lecker, and Per-Olof Hasselgren, Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12, Journal of Cellular Biochemistry, 105, 353-364(2008)
Nakaishi H.,, Matsumoto H., Tominaga S., Hirayama M., Effects of Black Currant Anthocyanoside Intake on Dark Adaptation and VDT Work-induced Transient Refractive Alteration in Healthy Humans, Alternative Medicine Review, 5, 553-562(2000)
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K., Schiaffino, S., Lecker, S.H., and Goldverg, A.L., Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy, Cell, 117, 399-412(2004)
Sue C.Bodine, et al., Identification of ubiquitin ligases required for skeletal muscle atrophy, Science 294, 1704-1708(2001)
Wing, S.S., and Goldberg, A.L., Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting, American Journal of Physiology, 264, E668-E676. (1993)
Wray, C.J., Mammen, J.M., Hershko, D.D., and Hasseigren, P. O., Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle, International Journal lf Biochemistry & Cell Biology, 35, 698-705(2003)
[References]
Anker, SD, Clark, AL, Kemp, M., Salsbury, C., Teixeira, MM, Hellewell, PG, and Coats, AJ, Tumor necrosis factor and steroidmetabolism in chronic heart failure: possible relation to muscle wasting, Journal of American College of Cardiology, 30, 997-1001. (1997)
Dehoux, MJM, van Beneden, RP, Fernandez-Celemin, L., Lause, PK, and Thissen, JP.M., Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal muscle after LPS injection, FEBS Letters, 544, 214 -217 (2003)
Glass, DJ Skeletal muscle hypertrophy and atrophy signaling pathways.The International Jounal of Biochemistry & Cell Biology, 37, 1974-1984 (2005)
Hasselgren, PO, Burns and metabolism, Journal of American College of Surgeons, 188, 98-103. (1999)
H. Fujino, A. Ishihara, S. Murakami, T. Yasuhara, H. Kondo, S. Mohri, I. Takeda and RRRoy, Protective effects of exercise preconditioning of hindlimb unloading-induced atrophy of rat soleus muscle, Acta Physiologica, 197 , 65-74 (2009)
Krawiec, BJ, Frost, RA, Vary, TC, Jefferson, LS, and Lang, CH, Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis, American Journal of Physiology-Endocrinology and metabolism, 289, E969-E980 (2005)
Latres, E., Amini, AR, Amini, AA, Griffiths, J., Martin, FJ, Wei, Y., Lin, HC, Yancopoulos, GD, and Glass, DJ Insulin-like growth factor-1 (IGF-1 ) inversely regulates atrophy-induced genes cia the phosphatidylinositol 3-kinase / akt / mammalian target of rapamycin (PI3K / Akt / mTOR) pathway, Journal of Biological Chemistry, 280, 2737-2744 (2005)
Lee, SW, Dai, G., Hu, Z., Wang, X., Du, J., and Mitch, WE, Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitin-proteasome systems by phosphatidylinositol 3 kinase, Journal of American Society of Nephrology, 15, 1537-1545 (2004)
Li, Y.-P., Chen, Y., Li, AS, and Reid, MB, Hydrogen peroxide stimulates ubiquitin conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes, American Journal of Physiology-Cell Phisiology , 285, C806-C812 (2003)
Li, Y.-P., Chen, Y., John, J., Moylan, J., Jin, B., Mann, DL, and Reid, MB, TNF- {alpha} acts via p38 MAPK to stimulates expression of the ubiquitin ligase atrogin1 / MAFbx in skeletal muscle. FASEB Journal, 19, 362-370 (2005)
Menconi M, Fareed M, O Neal P, Poylin B. Wei W, Hasselgren PO., Role of glucocorticoids in the molecular regulation of muscle wasting, Critical Care Medicine, 35, S602-S608 (2007)
Michael Menconi, Patricia Gonnella, Victoria Petkova, Stewart Lecker, and Per-Olof Hasselgren, Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12, Journal of Cellular Biochemistry, 105, 353-364 (2008)
Nakaishi H. ,, Matsumoto H., Tominaga S., Hirayama M., Effects of Black Currant Anthocyanoside Intake on Dark Adaptation and VDT Work-induced Transient Refractive Alteration in Healthy Humans, Alternative Medicine Review, 5, 553-562 (2000)
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K., Schiaffino, S., Lecker, SH, and Goldverg, AL, Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy, Cell, 117, 399-412 (2004)
Sue C. Bodine, et al., Identification of ubiquitin ligases required for skeletal muscle atrophy, Science 294, 1704-1708 (2001)
Wing, SS, and Goldberg, AL, Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting, American Journal of Physiology, 264, E668-E676. (1993)
Wray, CJ, Mammen, JM, Hershko, DD, and Hasseigren, PO, Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle, International Journal lf Biochemistry & Cell Biology, 35, 698-705 (2003)
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| JP2013091608A (en) * | 2011-10-25 | 2013-05-16 | Kao Corp | Myostatin/smad signal inhibitor |
| JP2013150558A (en) * | 2012-01-24 | 2013-08-08 | Kyushu Univ | Method for evaluating fatigue state and stress state |
| CN107048383A (en) * | 2012-10-04 | 2017-08-18 | 雅培制药有限公司 | For strengthen EGCg to slow down skeletal muscle loss effect method |
| WO2014065369A1 (en) * | 2012-10-25 | 2014-05-01 | 日本製紙株式会社 | Tea leaf extract |
| WO2014186441A1 (en) * | 2013-05-14 | 2014-11-20 | Kemin Industries, Inc. | The effect of a green and black tea extract formulation on exercise performance |
| EP3135293A4 (en) * | 2014-04-23 | 2018-02-28 | Nippon Paper Industries Co., Ltd. | Agent for preventing or ameliorating diabetes |
| US20170042924A1 (en) * | 2014-04-28 | 2017-02-16 | Suntory Holdings Limited | Muscle atrophy inhibitor containing quercetin glycoside |
| JP6664956B2 (en) * | 2015-02-27 | 2020-03-13 | キリンホールディングス株式会社 | Muscle differentiation promoting composition |
| US20180104269A1 (en) * | 2015-04-27 | 2018-04-19 | Suntory Holdings Limited | Composition for suppressing muscular fatty change |
| JP2018087175A (en) * | 2016-11-30 | 2018-06-07 | 花王株式会社 | Muscular atrophy inhibitor |
| WO2022052017A1 (en) * | 2020-09-11 | 2022-03-17 | Liu Hsuan Miao | Pharmaceutical compositions and uses thereof in treating muscle atrophy |
| JPWO2022131063A1 (en) * | 2020-12-18 | 2022-06-23 | ||
| WO2022139544A1 (en) * | 2020-12-24 | 2022-06-30 | 고려대학교 산학협력단 | Composition for preventing or treating muscle diseases containing aronia or houttuynia cordata extract as active ingredient |
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| JP2002338464A (en) * | 2001-05-14 | 2002-11-27 | Kikkoman Corp | Muscular atrophy inhibitor |
| JP2006328031A (en) * | 2005-05-30 | 2006-12-07 | Asahi Breweries Ltd | Muscular fiber type shift inhibitor |
| US8962678B2 (en) * | 2006-07-05 | 2015-02-24 | Kao Corporation | Senescence inhibitor |
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