JP5307019B2 - Bone resorption inhibitor and beverages, foods and pharmaceuticals containing the same - Google Patents
Bone resorption inhibitor and beverages, foods and pharmaceuticals containing the same Download PDFInfo
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- JP5307019B2 JP5307019B2 JP2009534368A JP2009534368A JP5307019B2 JP 5307019 B2 JP5307019 B2 JP 5307019B2 JP 2009534368 A JP2009534368 A JP 2009534368A JP 2009534368 A JP2009534368 A JP 2009534368A JP 5307019 B2 JP5307019 B2 JP 5307019B2
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- bone resorption
- barley
- fraction
- enzyme
- bone
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Abstract
Description
本発明は、骨吸収抑制剤、並びにこれを含有する飲料、食品及び医薬品に関する。 The present invention relates to a bone resorption inhibitor and beverages, foods and pharmaceuticals containing the same.
近年、高齢化社会の進展や生活習慣の変化に伴って骨粗鬆症患者が増加しており、日本における骨粗鬆症患者の数は１０００万人を超えている。骨粗鬆症患者は、全体の７割を女性が占め、５０歳以上の女性に多く発症している。このため、特に女性は、骨量が最大に達する３０歳くらいまでの間に、骨量をできるだけ高めておくことが骨粗鬆症の予防に重要であるとされている。 In recent years, with the progress of an aging society and changes in lifestyle habits, osteoporosis patients are increasing, and the number of osteoporosis patients in Japan exceeds 10 million. Women account for 70% of osteoporosis patients, and the disease often occurs in women over 50 years old. For this reason, especially for women, it is said that it is important for prevention of osteoporosis to keep the bone mass as high as possible until about 30 years old when the bone mass reaches the maximum.
しかしながら、最近では、運動不足や栄養不足が原因で若年期における潜在的な骨粗鬆症患者が増加している。骨粗鬆症の予防又は改善法に関しては、医学的なアプローチに加え、遺伝学的・栄養学的観点からの様々な研究も行われている（非特許文献１）。 Recently, however, there are an increasing number of potential osteoporosis patients in early life due to lack of exercise and nutrition. Regarding the prevention or improvement of osteoporosis, in addition to a medical approach, various studies from a genetic / nutritional viewpoint have been conducted (Non-patent Document 1).
骨代謝においては、破骨細胞が骨吸収を担い、骨芽細胞が骨形成を担っている。骨粗鬆症は、骨吸収と骨形成とのバランスが崩れ、骨吸収が骨形成を上回ることが原因であると考えられている。また、骨吸収の亢進は、骨粗鬆症以外にも、慢性関節リウマチ等の骨疾患に深く関与している。このことから、骨吸収を担う破骨細胞は、骨粗鬆症、慢性関節リウマチを始めとする骨疾患を予防又は改善（治療若しくは症状緩和）する際の標的になると考えられている。 In bone metabolism, osteoclasts are responsible for bone resorption and osteoblasts are responsible for bone formation. Osteoporosis is thought to be caused by an imbalance between bone resorption and bone formation and bone resorption exceeding bone formation. In addition to osteoporosis, increased bone resorption is deeply involved in bone diseases such as rheumatoid arthritis. Thus, osteoclasts responsible for bone resorption are considered to be targets for preventing or ameliorating (treating or alleviating symptoms) bone diseases including osteoporosis and rheumatoid arthritis.
骨吸収に関与するプロテアーゼとしては、カテプシンＢ、Ｈ、Ｋ及びＬが知られている。中でもカテプシンＫは、破骨細胞に特異的に局在し、カテプシンＫの阻害によって骨吸収を抑制できることが報告されている（非特許文献２及び３）。このことから、カテプシンＫは、骨粗鬆症、慢性関節リウマチを始めとする骨疾患を予防又は改善（治療若しくは症状緩和）する際の標的分子として注目を集めている。
Cathepsins B, H, K, and L are known as proteases involved in bone resorption. Among them, it has been reported that cathepsin K is specifically localized in osteoclasts and can suppress bone resorption by inhibiting cathepsin K (
カテプシンＫ阻害剤としては、例えば、ジペプチジルケトン阻害剤、ジアミノピリジノン阻害剤、ジアシルカルボヒドラジド阻害剤、ピリドキサルプロピオネート誘導体、環状ケトン誘導体、アリルアミノエチルアミド誘導体、４−アミノ−アゼパン−３−オン阻害剤、３−アリルアミノ−アゼチジン−２−オン阻害剤及びケトアミド誘導体が開発されている。 Examples of cathepsin K inhibitors include dipeptidyl ketone inhibitors, diaminopyridinone inhibitors, diacyl carbohydrazide inhibitors, pyridoxalpropionate derivatives, cyclic ketone derivatives, allylaminoethylamide derivatives, 4-amino-azepane. -3-one inhibitors, 3-allylamino-azetidin-2-one inhibitors and ketoamide derivatives have been developed.
天然物を起源とするカテプシンＫ阻害剤としては、乳又は乳由来原料に含まれる塩基性タンパク質組成物（特許文献１）や、イネ科植物に含まれるオリザシスタチン−Ｉ及びオリザシスタチン−ＩＩ（特許文献２）が報告されている。 Cathepsin K inhibitors originating from natural products include basic protein compositions contained in milk or milk-derived raw materials (Patent Document 1), oryzastatin-I and oryzasistatin-II contained in gramineous plants (patents) Reference 2) has been reported.
また、健康機能食品に利用されている素材としては、例えば、ＣＰＰ（カゼインホスホペプチド）、ＣＣＭ（クエン酸リンゴ酸カルシウム）、大豆イソフラボン、フラクトオリゴ糖、ポリグルタミン酸、ＭＢＰ（乳塩基性タンパク質）及びビタミンＫ２（メナキノン−７）が知られている（非特許文献４）。In addition, examples of materials used in health functional foods include CPP (casein phosphopeptide), CCM (calcium malate citrate), soy isoflavone, fructooligosaccharide, polyglutamic acid, MBP (milk basic protein) and vitamins. K 2 (menaquinone-7) is known (Non-patent Document 4).
しかしながら、現在広く一般的に使用されているカテプシンＫ阻害剤は、人工的に化学合成された化合物であるため、医師の診断を受けることなく摂取するのは安全性の点で問題がある。 However, since cathepsin K inhibitors that are currently widely used are artificially chemically synthesized compounds, there is a problem in terms of safety when taken without receiving a doctor's diagnosis.
また、カテプシンＫ阻害剤は、カテプシンＫによる骨の基質成分（タンパク質成分）の分解は抑制するが、基質成分の周囲でセメントの役割を担う無機成分（ハイドロキシアパタイト）の分解を抑制することはできない。このため、カテプシンＫ阻害剤が投与されても、無機成分の分解が進行し、カテプシンＫ以外のプロテアーゼ（例えば、ＭＭＰ−９）による基質成分の分解を効果的に食い止めることができない。 Cathepsin K inhibitors inhibit the degradation of bone matrix components (protein components) by cathepsin K, but cannot inhibit the degradation of inorganic components (hydroxyapatite) that play a role of cement around the matrix components. . For this reason, even if a cathepsin K inhibitor is administered, the decomposition of the inorganic component proceeds, and the decomposition of the substrate component by a protease other than cathepsin K (for example, MMP-9) cannot be effectively prevented.
そこで、本発明は、人体への安全性に優れ、医薬品のみならず食品への応用も可能な、カテプシンＫ阻害剤とは異なるメカニズムの骨吸収抑制剤であって、骨粗鬆症、慢性関節リウマチを始めとする骨疾患の予防及び改善（治療、症状緩和）を可能とする骨吸収抑制剤を提供することを目的とする。 Therefore, the present invention is a bone resorption inhibitor having a mechanism different from that of cathepsin K inhibitors, which is excellent in safety to the human body and can be applied not only to pharmaceuticals but also to foods, including osteoporosis and rheumatoid arthritis. It is an object of the present invention to provide a bone resorption inhibitor capable of preventing and improving (treating and relieving symptoms) bone diseases.
本発明者らは、一定の大麦抽出物、当該大麦抽出物を分画して得られる画分、及び前記大麦抽出物若しくは画分を酵素処理して得られる酵素処理物が、カテプシンＫを阻害することなく、破骨細胞により引き起こされる骨の無機成分の分解を抑制することを見出し、本発明を完成させた。 The present inventors inhibit cathepsin K by a certain barley extract, a fraction obtained by fractionating the barley extract, and an enzyme-treated product obtained by enzymatic treatment of the barley extract or fraction. The present invention has been completed by finding that the decomposition of the inorganic components of bone caused by osteoclasts is suppressed.
すなわち、本発明は、大麦を粉砕した大麦粉を水若しくは緩衝液で抽出した大麦抽出物又はこれを分画した画分或いは前記大麦抽出物又は前記画分の酵素処理物、を有効成分として含有する骨吸収抑制剤を提供する。 That is, the present invention contains, as an active ingredient, a barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer solution, a fraction obtained by fractionating the same, or an enzyme-treated product of the barley extract or the fraction. A bone resorption inhibitor is provided.
大麦は、古くから主要な穀物として人類の食用に供されてきたものであり、大麦抽出物由来の骨吸収抑制剤は人体に対する安全性に優れている。また、本発明の骨吸収抑制剤は、骨吸収においてまず初めに進行する骨の無機成分（ハイドロキシアパタイト）の分解を抑制するため、基質成分（タンパク質成分）の分解のみを抑制するカテプシンＫ阻害剤とは異なるメカニズムを介して骨吸収を効果的に抑制することができる。 Barley has been used for human consumption as a major cereal since ancient times, and a bone resorption inhibitor derived from barley extract is excellent in safety to the human body. In addition, the bone resorption inhibitor of the present invention is a cathepsin K inhibitor that suppresses only the decomposition of the substrate component (protein component) in order to suppress the decomposition of the bone inorganic component (hydroxyapatite) that proceeds first in bone resorption. Bone resorption can be effectively suppressed through a different mechanism.
上記大麦抽出物の画分は、上記大麦抽出物を陽イオン交換樹脂に接触させ、次いで、樹脂に吸着した成分を当該樹脂から分離して得られる塩基性画分であることが好ましい。このような塩基性画分を使用すれば、破骨細胞により引き起こされる骨の無機成分の分解を顕著に抑制することができる。 The barley extract fraction is preferably a basic fraction obtained by bringing the barley extract into contact with a cation exchange resin and then separating the components adsorbed on the resin from the resin. If such a basic fraction is used, the decomposition | disassembly of the inorganic component of the bone caused by the osteoclast can be suppressed notably.
上記大麦抽出物の画分としては、例えば、上記塩基性画分を更に７５％飽和硫安で分画して得られる沈殿画分を使用することもできる。また、例えば、上記沈殿画分を更に限外濾過膜で分画して得られる塩基性タンパク質画分（例えば、分子量５０００〜５００００のタンパク質の画分）を使用することもできる。 As a fraction of the barley extract, for example, a precipitate fraction obtained by further fractionating the basic fraction with 75% saturated ammonium sulfate can be used. In addition, for example, a basic protein fraction (for example, a protein fraction having a molecular weight of 5,000 to 50,000) obtained by further fractionating the precipitate fraction with an ultrafiltration membrane can be used.
上記大麦抽出物又は画分の酵素処理物は、上記大麦抽出物又は画分（好ましくは、塩基性画分、沈殿画分又は塩基性タンパク質画分）を、ペプシン、パンクレアチン及びトリプシンのうちの少なくとも１種の酵素で酵素処理（消化）して得られる酵素処理物であることが好ましい。 The barley extract or the enzyme-treated product of the fraction is the barley extract or fraction (preferably, the basic fraction, the precipitated fraction or the basic protein fraction) of pepsin, pancreatin and trypsin. An enzyme-treated product obtained by enzymatic treatment (digestion) with at least one kind of enzyme is preferable.
上記酵素処理物は、胃液、腸液等の消化液中の酵素（例えば、ペプシン、パンクレアチン、トリプシン）で消化して得られるものでもよく、上記大麦抽出物又は画分は、経口で摂取された場合にも骨吸収を抑制することができる。 The enzyme-treated product may be obtained by digestion with an enzyme (eg, pepsin, pancreatin, trypsin) in digestive fluid such as gastric juice or intestinal juice, and the barley extract or fraction was taken orally. In some cases, bone resorption can be suppressed.
上記塩基性タンパク質画分及びその酵素処理物は、本発明の骨吸収抑制剤の有効成分として使用することができる。すなわち、本発明はまた、大麦抽出物の塩基性画分に含まれるタンパク質又はその酵素処理物、を有効成分として含有する骨吸収抑制剤であって、前記塩基性画分は、大麦を粉砕した大麦粉を水又は緩衝液で抽出する工程と、得られた抽出物を陽イオン交換樹脂に接触させる工程と、前記樹脂に吸着した成分を当該樹脂から分離する工程と、をこの順に実施することによって得られる画分である骨吸収抑制剤を提供する。 The basic protein fraction and the enzyme-treated product thereof can be used as an active ingredient of the bone resorption inhibitor of the present invention. That is, the present invention is also a bone resorption inhibitor containing, as an active ingredient, a protein contained in the basic fraction of barley extract or an enzyme-treated product thereof, wherein the basic fraction pulverizes barley. The step of extracting barley flour with water or a buffer, the step of bringing the obtained extract into contact with a cation exchange resin, and the step of separating the components adsorbed on the resin from the resin are performed in this order. The bone resorption inhibitor which is a fraction obtained by is provided.
本発明の骨吸収抑制剤は、骨吸収抑制活性及び安全性に優れることから、飲料、食品、飲食品添加物、飼料、医薬品等の成分として使用することができる。 Since the bone resorption inhibitor of this invention is excellent in bone resorption suppression activity and safety | security, it can be used as components, such as a drink, a foodstuff, food-drinks additives, feed, and a pharmaceutical.
本発明によれば、人体に対する安全性に優れた骨吸収抑制剤が提供される。また、本発明によれば、骨吸収においてまず初めに進行する骨の無機成分（ハイドロキシアパタイト）の分解を抑制し、骨の基質成分（タンパク質成分）の分解とは異なるメカニズムにより骨吸収を効果的に抑制することが可能な骨吸収抑制剤が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the bone resorption inhibitor excellent in the safety | security with respect to a human body is provided. In addition, according to the present invention, the degradation of the bone inorganic component (hydroxyapatite) that progresses first in the bone resorption is suppressed, and the bone resorption is effectively performed by a mechanism different from the degradation of the bone matrix component (protein component). A bone resorption inhibitor capable of being suppressed is provided.
本発明の骨吸収抑制剤は、胃液、腸液等の消化液中の酵素（例えば、ペプシン、パンクレアチン、トリプシン）で酵素処理されても活性が維持されるため、経口で摂取した場合にも骨吸収を抑制することができる。 The bone resorption inhibitor of the present invention maintains its activity even if it is treated with enzymes in digestive juices such as gastric juice and intestinal juice (eg, pepsin, pancreatin, trypsin). Absorption can be suppressed.
以下、本発明の好適な実施形態について説明する。 Hereinafter, preferred embodiments of the present invention will be described.
本発明の骨吸収抑制剤は、大麦を粉砕した大麦粉を水若しくは緩衝液で抽出した大麦抽出物、又はこれを分画した画分、或いは前記大麦抽出物又は前記画分を酵素処理して得られる酵素処理物、を有効成分として含有する。 The bone resorption inhibitor of the present invention is a barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer solution, a fraction obtained by fractionating the same, or the barley extract or the fraction obtained by enzyme treatment. The obtained enzyme-treated product is contained as an active ingredient.
上記大麦としては、二条、六条、裸、皮等、任意の種類の大麦を使用することができ、より具体的には、例えば、はるな二条、イチバンボシ、ＣＤＣ Ｆｉｂａｒが挙げられる。また、全粒、精麦粒、糠等、大麦種子に由来する任意の組織を使用することができる。例えば、大麦種子の内層部や外層部を単独で使用してもよく、また、麦芽を使用してもよい。更に、例えば、Ｂホルディン欠失系統やプロテインＺ４欠失系統の大麦を使用することもできる。 As the barley, any kind of barley such as Nijo, Rojo, bare, and leather can be used, and more specifically, for example, Haruna Nijo, Ichibanboshi, and CDC Fibar. In addition, any tissue derived from barley seeds such as whole grains, wheat grains, and straw can be used. For example, the inner layer or outer layer of barley seeds may be used alone, or malt may be used. Furthermore, for example, barley of a B hordin deletion line or a protein Z4 deletion line can be used.
大麦を粉砕して大麦粉を得る工程では、大麦を効率的に微粉状に粉砕できればよく、例えば、ピンミル、ハンマーミル、ボールミル、ミキサー等の粉砕機を用いることができる。 In the step of obtaining barley flour by pulverizing barley, it is sufficient if barley can be efficiently pulverized into fine powders. For example, a pulverizer such as a pin mill, a hammer mill, a ball mill, or a mixer can be used.
大麦粉を水又は緩衝液で抽出して大麦抽出物を得る工程では、大麦粉を水又は緩衝液中で攪拌し、大麦に含有される水溶性のタンパク質を抽出する。水又は緩衝液の温度は、０℃超３０℃以下が好ましく、０℃超１０℃以下がより好ましく、２℃以上８℃以下（特に３℃以上７℃以下）が更に好ましい。 In the step of extracting barley flour with water or a buffer solution to obtain a barley extract, the barley flour is stirred in water or a buffer solution to extract water-soluble proteins contained in the barley. The temperature of water or the buffer is preferably more than 0 ° C. and not more than 30 ° C., more preferably more than 0 ° C. and not more than 10 ° C., more preferably 2 ° C. to 8 ° C. (particularly 3 ° C. to 7 ° C.).
また、抽出に用いる緩衝液は、中性から弱酸性のｐＨとなるように緩衝作用を発揮するものが好ましく、緩衝液のｐＨとしては、４．０〜７．０が好ましく、５．０〜６．０がより好ましい。緩衝剤としては、例えば、リン酸、クエン酸、酢酸、フタル酸、コハク酸、炭酸、ＨＥＰＥＳ、ＭＥＳ、トリスが挙げられ、例えば、０．１Ｍ 酢酸ナトリウム緩衝液（ｐＨ５．５）、２０ｍＭ 酢酸ナトリウム緩衝液（ｐＨ５．０）、２０ｍＭ ＭＥＳ緩衝液（ｐＨ６．０）が特に好ましい。 In addition, the buffer solution used for extraction preferably exhibits a buffering action so as to have a neutral to slightly acidic pH, and the pH of the buffer solution is preferably 4.0 to 7.0, preferably 5.0 to 6.0 is more preferable. Examples of the buffer include phosphoric acid, citric acid, acetic acid, phthalic acid, succinic acid, carbonic acid, HEPES, MES, and Tris. For example, 0.1 M sodium acetate buffer (pH 5.5), 20 mM sodium acetate A buffer solution (pH 5.0) and a 20 mM MES buffer solution (pH 6.0) are particularly preferable.
大麦抽出物を分画して画分を得る工程では、まず、上記大麦抽出物を陽イオン交換樹脂に接触させる。次いで、樹脂に吸着しなかった成分を回収すれば、酸性画分が得られる。また、樹脂に吸着した成分を当該樹脂から分離すれば、塩基性画分が得られる。大麦抽出物と陽イオン交換樹脂との接触は、例えば、大麦抽出物を陽イオン交換樹脂に通液することにより行うことができる。また、陽イオン交換樹脂に吸着した成分を当該樹脂から分離するには、例えば、樹脂に溶離液を通液して吸着成分を溶出させればよい。陽イオン交換樹脂としては、例えば、Ｈｉｔｒａｐ ＳＰ ＦＦ、ＨｉＴｒａｐ ＳＰ ＨＰ、ＨｉＴｒａｐ ＣＭ ＦＦ、ＨｉＴｒａｐ １６／１０ ＳＰ ＸＬ、ＨｉＴｒａｐ １６／１０ ＳＰ ＦＦ、ＨｉＴｒａｐ １６／１０ ＣＭ ＦＦ、Ｒｅｓｏｕｒｃｅ Ｓ、Ｓｏｕｒｃｅ １５Ｓ、Ｓｏｕｒｃｅ ３０Ｓ、Ｍｏｎｏ Ｓ、Ｍｉｎｉ Ｓ、ＳＰ Ｓｅｐｈａｒｏｓｅ ＨＰ、ＳＰ Ｓｅｐｈａｒｏｓｅ ＸＬ、ＳＰ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ、ＣＭ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ（以上、ＧＥヘルスケアバイオサイエンス社）、ＡＧ ５０Ｗ Ｒｅｓｉｎｓ、ＡＧ ＭＰ−５０ Ｒｅｓｉｎ、Ｂｉｏ−Ｒｅｘ ７０ Ｒｅｓｉｎ、Ｍａｃｒｏ−Ｐｒｅｐ ｈｉｇｈ Ｑ、Ｍａｃｒｏ−Ｐｒｅｐ ｈｉｇｈ Ｓ、Ｍａｃｒｏ−Ｐｒｅｐ ＣＭ（以上、Ｂｉｏ−Ｒａｄ社）、ゲル型 ダイヤイオン（登録商標） ＳＫシリーズ、ポーラス型 ダイヤイオン（登録商標） ＰＫシリーズ、水処理用ゲル型均一粒径 ダイヤイオン（登録商標）、工業クロマト分離用陽イオン交換樹脂 ダイヤイオン（登録商標）ＵＢＫ５００シリーズ（以上、三菱化学社）、Ａｓａｈｉｐａｋ Ｃｏｌｕｍｎ ＥＳ−５０２Ｃ ７Ｃ、Ｓｈｏｄｅｘ Ｃｏｌｕｍｎ ＩＥＣ ＣＭ−８２５、Ｓｈｏｄｅｘ Ｃｏｌｕｍｎ ＩＥＣ ＳＰ−８２５、Ｓｈｏｄｅｘ Ｃｏｌｕｍｎ ＩＥＣ ＳＰ−４２０Ｎ（以上、昭和電工社）、ＳＰ−Ｔｏｙｏｐｅａｒｌ（東ソー社）が挙げられる。陽イオン交換樹脂の中では、強酸性型のイオン交換樹脂が好ましい。 In the step of fractionating the barley extract to obtain a fraction, the barley extract is first brought into contact with a cation exchange resin. Subsequently, an acidic fraction can be obtained by collecting the components that have not been adsorbed on the resin. Moreover, a basic fraction can be obtained by separating the component adsorbed on the resin from the resin. The contact between the barley extract and the cation exchange resin can be performed, for example, by passing the barley extract through the cation exchange resin. In order to separate the component adsorbed on the cation exchange resin from the resin, for example, the adsorbed component may be eluted by passing an eluent through the resin. Examples of the cation exchange resin include Hitrap SP FF, HiTrap SP HP, HiTrap CM FF, HiTrap 16/10 SP XL, HiTrap 16/10 SP FF, HiTrap 16/10 CM FF, Resource S, Source S30, Source S30. , Mono S, Mini S, SP Sepharose HP, SP Sepharose XL, SP Sepharose Fast Flow, CM Sepharose Fast Flow (above GE Healthcare Biosciences), AG 50W Resins, AG MP-50R , Macro-Prep high Q, Macro-Prep high S, Macro Prep CM (above, Bio-Rad), gel type Diaion (registered trademark) SK series, porous type Diaion (registered trademark) PK series, gel type uniform particle size for water treatment Diaion (registered trademark), industrial chromatography Cation exchange resin for separation Diaion (registered trademark) UBK500 series (Mitsubishi Chemical Corporation), Asahipak Column ES-502C 7C, Shodex Column IEC CM-825, Shodex Column IEC SP-825, Shodex Column IEC (825) As mentioned above, Showa Denko Co., Ltd.) and SP-Toyopearl (Tosoh Corporation) can be mentioned. Of the cation exchange resins, strongly acidic ion exchange resins are preferred.
上記大麦抽出物の画分としては、上記大麦抽出物を陽イオン交換樹脂に接触させ、次いで、樹脂に吸着した成分を当該樹脂から分離させて得られる塩基性画分が好ましい。このような塩基性画分を使用すれば、骨吸収の際の骨の無機成分（ハイドロキシアパタイト）の分解を顕著に抑制することができる。 The fraction of the barley extract is preferably a basic fraction obtained by bringing the barley extract into contact with a cation exchange resin and then separating the components adsorbed on the resin from the resin. If such a basic fraction is used, the decomposition | disassembly of the inorganic component (hydroxyapatite) of the bone in the case of bone resorption can be suppressed notably.
上記大麦抽出物の画分としては、例えば、上記塩基性画分を更に７５％飽和硫安で分画して得られる沈殿画分を使用することもできる。また、例えば、上記沈殿画分を更に限外濾過膜で分画して得られる塩基性タンパク質画分（例えば、分子量５０００〜５００００のタンパク質の画分）を使用することもできる。 As a fraction of the barley extract, for example, a precipitate fraction obtained by further fractionating the basic fraction with 75% saturated ammonium sulfate can be used. In addition, for example, a basic protein fraction (for example, a protein fraction having a molecular weight of 5,000 to 50,000) obtained by further fractionating the precipitate fraction with an ultrafiltration membrane can be used.
上記大麦抽出物又は画分の酵素処理物は、上記大麦抽出物又は画分（好ましくは塩基性画分、沈殿画分又は塩基性タンパク質画分）を、ペプシン、パンクレアチン及びトリプシンのうちの少なくとも１種の酵素で酵素処理（消化）して得られる酵素処理物であることが好ましい。 The barley extract or the enzyme-treated product of the fraction is the barley extract or fraction (preferably the basic fraction, the precipitated fraction or the basic protein fraction), which is at least one of pepsin, pancreatin and trypsin. An enzyme-treated product obtained by enzyme treatment (digestion) with one kind of enzyme is preferable.
ペプシン、パンクレアチン及びトリプシンは、胃液、腸液等の消化液に含有される消化酵素であり、試薬メーカーから容易に入手することができる。上記大麦抽出物又は画分を酵素で処理する条件（酵素量、温度、時間等）は、当該酵素の至適条件であれば特に制限されない。入手可能なペプシン、パンクレアチン及びトリプシンとしては、例えば、ブタ胃粘膜由来ペプシン、ブタ膵臓由来パンクレアチン、ウシ膵臓由来トリプシン、ブタ膵臓由来トリプシンが挙げられる。酵素処理は、例えば、上記大麦抽出物又は画分に対して体積比が１０％となるように酵素を加え、３７℃で少なくとも２時間反応させることによって行うことができる。 Pepsin, pancreatin, and trypsin are digestive enzymes contained in digestive fluids such as gastric juice and intestinal fluid, and can be easily obtained from reagent manufacturers. Conditions for treating the barley extract or fraction with an enzyme (enzyme amount, temperature, time, etc.) are not particularly limited as long as they are optimum conditions for the enzyme. Examples of the available pepsin, pancreatin and trypsin include porcine gastric mucosa-derived pepsin, porcine pancreatic pancreatin, bovine pancreatic trypsin, and porcine pancreatic trypsin. The enzyme treatment can be performed, for example, by adding an enzyme such that the volume ratio is 10% with respect to the barley extract or fraction and reacting at 37 ° C. for at least 2 hours.
骨吸収抑制活性は、例えば、次のように測定することができる。すなわち、まず、骨の無機成分（ハイドロキシアパタイト）で被膜された培養プレートの各ウェルに破骨細胞前駆細胞を播種し、細胞がプレートに完全に接着するまで培養する。その後、骨吸収抑制剤の候補品を加えて３７℃で７〜１０日間培養し、骨吸収抑制剤を添加していないコントロールのウェルにおいて骨吸収痕跡の形成が一面に確認できた時点で培養をストップする。そして、各ウェルの表面の様子をデジタルマイクロスコープで観察し、骨吸収面積又は骨吸収痕跡数を計測する。骨吸収痕跡の形成の阻害は、骨吸収が抑制されたことを意味し、その阻害の程度によって骨吸収抑制活性の強さを判定することができる。 The bone resorption suppressing activity can be measured, for example, as follows. That is, first, osteoclast precursor cells are seeded in each well of a culture plate coated with an inorganic component of bone (hydroxyapatite) and cultured until the cells are completely adhered to the plate. Thereafter, a candidate for bone resorption inhibitor is added and cultured at 37 ° C. for 7 to 10 days. When the formation of bone resorption traces can be confirmed in one well in a control well to which no bone resorption inhibitor is added, the culture is performed. Stop. Then, the state of the surface of each well is observed with a digital microscope, and the bone resorption area or the number of bone resorption traces is measured. The inhibition of the formation of the bone resorption trace means that the bone resorption is suppressed, and the strength of the bone resorption suppressing activity can be determined by the degree of the inhibition.
本発明の骨吸収抑制剤は、例えば、骨粗鬆症、慢性関節リウマチ、変形性関節症、歯周病疾患、骨パジェット病、微小重力環境下での骨量減少・骨折に対して予防及び改善（治療、症状緩和）効果を有しており、特に、骨粗鬆症及び慢性関節リウマチの予防及び改善に適している。 The bone resorption inhibitor of the present invention prevents and improves (treats, for example, osteoporosis, rheumatoid arthritis, osteoarthritis, periodontal disease, Paget's disease of bone, and bone loss / fracture in a microgravity environment. In particular, it is suitable for prevention and improvement of osteoporosis and rheumatoid arthritis.
本発明の骨吸収抑制剤は、骨粗鬆症、慢性関節リウマチ等の骨疾患の予防や改善（治療、症状緩和）を目的として飲料及び食品に添加でき、また、これらの骨疾患の予防又は改善（治療若しくは症状緩和）剤（医薬品）の成分として使用できる。本発明の骨吸収抑制剤は、固体、液体（水溶性又は脂溶性の溶液又は懸濁液）、ペースト等のいずれの形状でもよく、また、散剤、顆粒剤、錠剤、シロップ剤、トローチ剤、カプセル剤、注射剤等のいずれの剤形を取ってもよい。また、放出制御製剤の形態を取ることもできる。上記飲料、食品及び医薬品は、大麦を粉砕した大麦粉を水若しくは緩衝液で抽出した大麦抽出物、又はこれを分画した画分（好ましくは前述の塩基性画分、沈殿画分若しくは塩基性タンパク質画分）、或いは当該大麦抽出物又は画分を酵素処理して得られる酵素処理物（好ましくは、ペプシン、パンクレアチン及びトリプシンのうちの少なくとも１種の酵素で酵素処理して得られる酵素処理物）、のみからなっていてもよい。 The bone resorption inhibitor of the present invention can be added to beverages and foods for the purpose of prevention and improvement (treatment, alleviation of symptoms) of bone diseases such as osteoporosis and rheumatoid arthritis, and prevention or improvement (treatment of these bone diseases) Alternatively, it can be used as a component of a symptom alleviating agent (pharmaceutical). The bone resorption inhibitor of the present invention may be in any form of solid, liquid (water-soluble or fat-soluble solution or suspension), paste, etc., and powder, granule, tablet, syrup, troche, Any dosage form such as a capsule or an injection may be used. It can also take the form of a controlled release formulation. The above beverages, foods and pharmaceuticals are barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer, or a fraction obtained by fractionating the same (preferably the aforementioned basic fraction, precipitated fraction or basic fraction). A protein fraction), or an enzyme-treated product obtained by subjecting the barley extract or fraction to an enzyme treatment (preferably an enzyme treatment obtained by subjecting at least one enzyme of pepsin, pancreatin and trypsin to an enzyme treatment) Thing), may consist only of.
なお、本発明の骨吸収抑制剤は、特定保健用食品、特殊栄養食品、栄養補助食品、健康食品、機能性食品、病者用食品等の飲食品に配合することもできる。また、飼料に配合することもできる。 In addition, the bone resorption inhibitor of this invention can also be mix | blended with food-drinks, such as food for specific health, special nutrition food, nutritional supplement food, health food, functional food, and food for sick people. Moreover, it can also mix | blend with feed.
本発明の飲料、食品及び飼料は、当該分野で通常使用される添加物を更に含有してもよい。このような添加物としては、例えば、リンゴファイバー、大豆ファイバー、肉エキス、黒酢エキス、ゼラチン、コーンスターチ、蜂蜜、動植物油脂；グルコース等の単糖類；スクロース、フルクトース、マンニトール等の二糖類；デキストロース、デンプン等の多糖類；エリスリトール、キシリトール、ソルビトール等の糖アルコール類；ビタミンＣ等のビタミン類、が挙げられる。これらの添加物は、単独で用いても、複数種を組み合わせて用いてもよい。 The beverage, food and feed of the present invention may further contain additives usually used in the art. Examples of such additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats; monosaccharides such as glucose; disaccharides such as sucrose, fructose and mannitol; dextrose, Examples thereof include polysaccharides such as starch; sugar alcohols such as erythritol, xylitol, and sorbitol; vitamins such as vitamin C. These additives may be used alone or in combination of two or more.
本発明の医薬品は、薬学的に許容される添加物を更に含有してもよい。この添加物としては、例えば、グルコース等の単糖類；スクロース、フルクトース、マンニトール等の二糖類；デキストロース、デンプン等の多糖類；エリスリトール、キシリトール、ソルビトール等の糖アルコール類；ビタミンＣ等のビタミン類；アカシアゴム、リン酸カルシウム、アルギン酸塩、珪酸カルシウム、微結晶性セルロース、ポリビニルピロリドン、セルロース誘導体、トラガカント、ゼラチン、シロップ、ヒドロキシ安息香酸メチル、タルク、ステアリン酸マグネシウム、水、鉱油が挙げられる。これらの添加物は、単独で用いても、複数種を組み合わせて用いてもよい。 The pharmaceutical product of the present invention may further contain a pharmaceutically acceptable additive. Examples of the additive include monosaccharides such as glucose; disaccharides such as sucrose, fructose and mannitol; polysaccharides such as dextrose and starch; sugar alcohols such as erythritol, xylitol and sorbitol; vitamins such as vitamin C; Acacia gum, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil. These additives may be used alone or in combination of two or more.
本発明の骨吸収抑制剤は、ヒトに投与されても、非ヒト哺乳動物に投与されてもよい。投与方法は、経口投与、直腸投与、静脈内投与、筋肉内投与、皮下投与、槽内投与、膣内投与、腹腔内投与、膀胱内投与、吸入投与等の全身投与、及び軟膏剤、ゲル剤、クリーム剤等による局所投与のいずれでもよい。投与量及び投与方法は、投与される個体の状態、年齢等に応じて適宜決定することができる。好適な投与方法としては、例えば、経口投与が挙げられる。 The bone resorption inhibitor of the present invention may be administered to humans or non-human mammals. Administration methods include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intracisternal administration, intravaginal administration, intraperitoneal administration, intravesical administration, inhalation administration, etc., and ointments and gels Any of topical administration using a cream or the like may be used. The dosage and administration method can be appropriately determined according to the condition, age, etc. of the individual to be administered. Suitable administration methods include, for example, oral administration.
本発明の骨吸収抑制剤は、胃液、腸液等の消化液中の酵素（例えば、ペプシン、パンクレアチン、トリプシン）で酵素処理されても活性が維持される。そのため、本発明の骨吸収抑制剤を含有する飲料、食品、飼料、医薬品等は、これらを経口で摂取した場合にも骨吸収を効果的に抑制することができる。 The bone resorption inhibitor of the present invention maintains its activity even when it is treated with an enzyme (eg, pepsin, pancreatin, trypsin) in digestive fluid such as gastric juice and intestinal juice. Therefore, beverages, foods, feeds, pharmaceuticals and the like containing the bone resorption inhibitor of the present invention can effectively inhibit bone resorption even when these are taken orally.
以下、実施例を挙げて本発明をより具体的に説明する。但し、本発明は、これらの実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
〔実施例１：大麦抽出物の骨吸収抑制活性〕
（大麦抽出物の調製）
十分に乾燥した大麦種子（品種：はるな二条）をミキサー（ラボミルサー、岩谷産業社）で約１０分間粉砕して微粉状態の大麦粉を得た。得られた大麦粉２ｇを乳鉢に移し、そこにホモジナイズ用緩衝液（０．１Ｍ 酢酸ナトリウム、ｐＨ５．５、５ｍＭ ＥＤＴＡ、０．１％ Ｔｗｅｅｎ−２０、５ｍＭ ＤＴＴ、１錠のコンプリート・ＥＤＴＡフリー・プロテアーゼインヒビターカクテル錠（ロシュ社））１０ｍＬを加えて、乳棒で懸濁した。[Example 1: Bone resorption inhibitory activity of barley extract]
(Preparation of barley extract)
The fully dried barley seeds (variety: Haruna Nijo) were pulverized with a mixer (Lab Miller, Iwatani Corp.) for about 10 minutes to obtain finely baked barley flour. 2 g of the obtained barley flour was transferred to a mortar, where it was buffered for homogenization (0.1 M sodium acetate, pH 5.5, 5 mM EDTA, 0.1% Tween-20, 5 mM DTT, 1 tablet complete, EDTA free, 10 mL of protease inhibitor cocktail tablets (Roche) were added and suspended with a pestle.
その後、大麦粉の懸濁液を、４℃で１０分間、１００００×ｇで遠心分離し、その上清をＭｉｒａｃｌｏｔｈフィルター（Ｃａｌｂｉｏｃｈｅｍ社）で濾過した。得られた濾液をＰＤ−１０カラム（ＧＥ Ｈｅａｌｔｈｃａｒｅ社）にアプライし、２０ｍＭ ＭＥＳ（ｐＨ６．０）にバッファー交換して、大麦抽出物を得た。 Thereafter, the barley flour suspension was centrifuged at 10,000 × g for 10 minutes at 4 ° C., and the supernatant was filtered through a Miracloth filter (Calbiochem). The obtained filtrate was applied to a PD-10 column (GE Healthcare), and buffer exchanged with 20 mM MES (pH 6.0) to obtain a barley extract.
（Ｐｉｔ Ｆｏｒｍａｔｉｏｎアッセイ）
得られた大麦抽出物の骨吸収抑制活性をＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイで測定した。(Pit formation assay)
Bone resorption inhibitory activity of the obtained barley extract was measured by Pit Formation assay.
具体的には、まず、ラット破骨前駆細胞（１×１０６個）を、ラット破骨細胞培養キット（セルガレージ社）に含まれる分化誘導用培地（５ｍＬ）（培地組成：α−ＭＥＭ、１０％ＦＢＳ、５０ｎｇ／ｍＬ ＲＡＮＫＬ＆Ｍ−ＣＳＦ）に懸濁し、これを、リン酸カルシウムで被膜されたＯＡＡＳプレート（オスコテック社）の各ウェルに１００μＬ（２×１０４個／ウェル）ずつ分注し、細胞がプレートに完全に接着するまでＣＯ２インキュベーター内で培養した。Specifically, first, rat osteoclast progenitor cells (1 × 10 6 cells) were added to a differentiation-inducing medium (5 mL) (medium composition: α-MEM, included in a rat osteoclast culture kit (Cell Garage)). 10% FBS, 50 ng / mL RANKL & M-CSF), and 100 μL (2 × 10 4 cells / well) was dispensed into each well of a calcium phosphate-coated OAAS plate (Oscotec). The cells were cultured in a CO 2 incubator until they completely adhered to the plate.
その後、各ウェルに所定タンパク質濃度の大麦抽出物を加え、３７℃のＣＯ２インキュベーター内で７〜１０日間培養し、コントロールのウェル（大麦抽出物の代わりに等量の滅菌水を添加したウェル）において骨吸収痕跡の形成が一面に確認された時点で、各ウェルの表面の様子をデジタルマイクロスコープ（ＶＨＸ−１００Ｆ、キーエンス社）で観察し、骨吸収面積を計測した。なお、大麦抽出物の骨吸収抑制活性の強さは、骨吸収痕跡形成の阻害の程度によって判定することができる。Thereafter, a barley extract having a predetermined protein concentration was added to each well and cultured in a CO 2 incubator at 37 ° C. for 7 to 10 days. Control wells (wells to which an equal amount of sterile water was added instead of barley extract) When the formation of bone resorption traces was confirmed on the whole surface, the state of the surface of each well was observed with a digital microscope (VHX-100F, Keyence), and the bone resorption area was measured. In addition, the intensity | strength of the bone resorption suppression activity of a barley extract can be determined with the grade of inhibition of bone resorption trace formation.
図１は、大麦抽出物の存在下で行われたＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイにおける骨吸収面積を示すグラフである。図１において、骨吸収面積は、コントロールの骨吸収面積に対する相対値（％）で示されている。また、濃度（μｇ／ｍＬ）は、培地中のタンパク質の濃度を示す。 FIG. 1 is a graph showing bone resorption area in a Pit Formation assay performed in the presence of barley extract. In FIG. 1, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.
図１から明らかなように、大麦抽出物は、タンパク質濃度が１μｇ／ｍＬ以上の場合に骨吸収をほぼ完全に抑制し、０．２μｇ／ｍＬの場合にも骨吸収を顕著に抑制した。 As is clear from FIG. 1, the barley extract almost completely suppressed bone resorption when the protein concentration was 1 μg / mL or more, and markedly suppressed bone resorption when the protein concentration was 0.2 μg / mL.
実施例１により、大麦抽出物が、骨吸収抑制活性、特に、骨の無機成分（ハイドロキシアパタイト）の分解を抑制する活性を有することが示された。 Example 1 showed that the barley extract has an activity of suppressing bone resorption, particularly an activity of suppressing the decomposition of the bone inorganic component (hydroxyapatite).
〔実施例２：塩基性画分及び酸性画分の骨吸収抑制活性〕
（塩基性画分及び酸性画分の調製）
実施例１と同様にして大麦抽出物を得た。得られた大麦抽出物をＨｉＴｒａｐ ＳＰ ＦＦ（強陽イオン交換カラム、ＧＥ Ｈｅａｌｔｈｃａｒｅ社）にアプライし、陽イオン交換樹脂に非吸着の成分を開始バッファー（２０ｍＭ ＭＥＳバッファー、ｐＨ６．０）５ｍＬで溶出して、大麦抽出物の酸性画分を得た。引き続き、陽イオン交換樹脂に吸着した成分を溶出バッファー（２０ｍＭ ＭＥＳバッファー、ｐＨ６．０、１Ｍ ＮａＣｌ）５ｍＬで溶出して、大麦抽出物の塩基性画分を得た。なお、塩基性画分には、タンパク質、アミノ酸及び核酸が含有されていた。[Example 2: Bone resorption inhibitory activity of basic fraction and acidic fraction]
(Preparation of basic and acidic fractions)
A barley extract was obtained in the same manner as in Example 1. The obtained barley extract was applied to HiTrap SP FF (strong cation exchange column, GE Healthcare), and components that were not adsorbed to the cation exchange resin were eluted with 5 mL of the starting buffer (20 mM MES buffer, pH 6.0). Thus, an acidic fraction of the barley extract was obtained. Subsequently, the component adsorbed on the cation exchange resin was eluted with 5 mL of elution buffer (20 mM MES buffer, pH 6.0, 1M NaCl) to obtain a basic fraction of barley extract. The basic fraction contained proteins, amino acids and nucleic acids.
（Ｐｉｔ Ｆｏｒｍａｔｉｏｎアッセイ）
得られた塩基性画分及び酸性画分の骨吸収抑制活性を、実施例１と同様にしてＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイで試験した。(Pit formation assay)
The bone resorption inhibitory activity of the obtained basic fraction and acidic fraction was tested by the Pit Formation assay in the same manner as in Example 1.
図２は、大麦抽出物の塩基性画分又は酸性画分の存在下で行われたＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイにおける骨吸収面積を示すグラフである。図２において、骨吸収面積は、コントロールの骨吸収面積に対する相対値（％）で示されている。また、濃度（μｇ／ｍＬ）は、培地中のタンパク質の濃度を示す。 FIG. 2 is a graph showing the bone resorption area in a Pit Formation assay performed in the presence of a basic fraction or an acidic fraction of barley extract. In FIG. 2, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.
図２から明らかなように、塩基性画分は、タンパク質濃度が１μｇ／ｍＬ以上の場合に骨吸収をほぼ完全に抑制し、０．２μｇ／ｍＬの場合にも骨吸収を顕著に抑制した。また、酸性画分も、塩基性画分ほど強くはないものの、骨吸収抑制活性を示した。 As is apparent from FIG. 2, the basic fraction almost completely suppressed bone resorption when the protein concentration was 1 μg / mL or more, and markedly suppressed bone resorption when the protein concentration was 0.2 μg / mL. The acidic fraction also showed bone resorption inhibitory activity, although not as strong as the basic fraction.
実施例２により、大麦抽出物の塩基性画分及び酸性画分が骨吸収抑制活性、特に、骨の無機成分（ハイドロキシアパタイト）の分解を抑制する活性を有することが示された。また、塩基性画分が、特に強い骨吸収抑制活性を有することが示された。 Example 2 showed that the basic fraction and the acidic fraction of the barley extract have bone resorption inhibitory activity, in particular, the activity of inhibiting the decomposition of bone inorganic components (hydroxyapatite). Moreover, it was shown that a basic fraction has especially strong bone resorption suppression activity.
〔参考例１：大麦抽出物のカテプシンＫ阻害活性〕
実施例１と同様にして大麦抽出物を得た。得られた大麦抽出物のカテプシンＫ阻害活性を次のように試験した。[Reference Example 1: Cathepsin K inhibitory activity of barley extract]
A barley extract was obtained in the same manner as in Example 1. The barley extract obtained was tested for cathepsin K inhibitory activity as follows.
まず、９６ウェルプレートの各ウェルに酵素反応用緩衝液（１００ｍＭ ＮａＯＡｃ、２０ｍＭ Ｌ−Ｃｙｓｔｅｉｎ、５ｍＭ ＥＤＴＡ、ｐＨ５．５）１３０μＬと４μｇ／ｍＬのカテプシンＫ（ヒトリコンビナント・プロカテプシンＫ、カルビオケム社）溶液１０μＬとを加え、そこに、所定タンパク質濃度の上記大麦抽出物１０μＬ、又は１μｇ／ｍＬのシスタチンＣ（ヒト尿由来シスタチンＣ、カルビオケム社）溶液１０μＬ、又は上記酵素反応用緩衝液（コントロール）１０μＬ、を更に加え、３７℃で３０分間、プレインキュベートした。 First, a buffer solution for enzyme reaction (100 mM NaOAc, 20 mM L-Cystein, 5 mM EDTA, pH 5.5) 130 μL and 4 μg / mL cathepsin K (human recombinant procathepsin K, Calbiochem) solution in each well of a 96-well plate 10 μL of the barley extract having a predetermined protein concentration, or 10 μL of a 1 μg / mL cystatin C (human urine-derived cystatin C, Calbiochem) solution, or 10 μL of the enzyme reaction buffer (control), Was further added and pre-incubated at 37 ° C. for 30 minutes.
次いで、蛍光基質溶液（２０μＭ Ｚ−Ｐｈｅ−Ａｒｇ−ＭＣＡ（Ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ−Ｌ−ｐｈｅｎｙｌａｌａｎｙｌ−Ｌ−ａｒｇｉｎｉｎｅ−４−ｍｅｔｈｙｌｃｏｕｍａｒｙｌ−７−ａｍｉｄｅ）、ペプチド研究所）５０μＬを加え、３７℃で酵素反応を行った。蛍光強度の増加度をマイクロプレートリーダーＭＴＰ−８００ＡＦＣ（励起波長３５０ｎｍ、吸収波長４５０ｎｍ、コロナ社）で測定して、カテプシンＫ活性を評価した。 Subsequently, 50 μL of a fluorescent substrate solution (20 μM Z-Phe-Arg-MCA (Benzyloxycarbonyl-L-phenylalanyl-L-argineline-4-methylcoumeric-7-amide), peptide laboratory) was added, and an enzyme reaction was performed at 37 ° C. . The degree of increase in fluorescence intensity was measured with a microplate reader MTP-800AFC (excitation wavelength 350 nm, absorption wavelength 450 nm, Corona) to evaluate cathepsin K activity.
図３は、大麦抽出物及びシスタチンＣのカテプシンＫ活性を示すグラフである。図３において、大麦抽出物及びシスタチンＣのカテプシンＫ活性は、コントロールに対する相対値（％）で示されている。また、濃度（μｇ／ｍＬ）は、反応液中のタンパク質の濃度を示す。 FIG. 3 is a graph showing the cathepsin K activity of barley extract and cystatin C. In FIG. 3, the cathepsin K activities of barley extract and cystatin C are shown as a relative value (%) to the control. The concentration (μg / mL) indicates the concentration of the protein in the reaction solution.
図３から明らかなように、大麦抽出物は、タンパク質濃度が５μｇ／ｍＬの場合にもカテプシンＫの活性を阻害しなかった。他方、カテプシンＫ阻害剤であるシスタチンＣは、１μｇ／ｍＬでもカテプシンＫの活性を完全に抑制した。 As is clear from FIG. 3, the barley extract did not inhibit the activity of cathepsin K even when the protein concentration was 5 μg / mL. On the other hand, cystatin C, which is a cathepsin K inhibitor, completely suppressed cathepsin K activity even at 1 μg / mL.
参考例１により、大麦抽出物がカテプシンＫ阻害活性を有しないこと、すなわち、大麦抽出物の骨吸収抑制作用が、カテプシンＫ阻害剤とは異なるメカニズムを介するものであることが示された。 Reference Example 1 shows that the barley extract does not have cathepsin K inhibitory activity, that is, the bone resorption inhibitory action of the barley extract is via a mechanism different from that of the cathepsin K inhibitor.
〔実施例３：大麦抽出物の骨吸収抑制活性〕
２種の大麦品種イチバンボシ及びはるな二条の大麦種子から、実施例１と同様にして大麦抽出物を得た。そして、得られた各大麦抽出物の骨吸収抑制活性を、実施例１と同様にしてＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイで試験した。[Example 3: Bone resorption inhibitory activity of barley extract]
A barley extract was obtained in the same manner as in Example 1 from two barley varieties Ichibanboshi and Haruna Nijo barley seeds. And the bone resorption inhibitory activity of each obtained barley extract was tested by the Pit Formation assay similarly to Example 1.
図４は、イチバンボシ又ははるな二条の大麦抽出物の存在下で行われたＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイにおける骨吸収面積を示すグラフである。図４において、骨吸収面積は、コントロールの骨吸収面積に対する相対値（％）で示されている。また、濃度（μｇ／ｍＬ）は、培地中のタンパク質の濃度を示す。 FIG. 4 is a graph showing the bone resorption area in a Pit Formation assay performed in the presence of Ichibanboshi or Haruna Nijo barley extract. In FIG. 4, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.
図４から明らかなように、イチバンボシ及びはるな二条のいずれも、顕著な骨吸収抑制活性を示した。 As is clear from FIG. 4, both Ichibanboshi and Haruna Nijo showed remarkable bone resorption inhibitory activity.
〔実施例４：酵素処理物の骨吸収抑制活性〕
大麦抽出物を人工胃液及び人工腸液で処理して得た酵素処理物について、骨吸収抑制活性を試験した。[Example 4: Bone resorption inhibitory activity of enzyme-treated product]
Bone resorption inhibitory activity was tested on enzyme-treated products obtained by treating barley extract with artificial gastric juice and artificial intestinal fluid.
実施例１と同様にして大麦抽出物を得た。所定タンパク質濃度の大麦抽出物に対して体積比が１０％となるように人工胃液（２％ ＮａＣｌ、１０ｋＵ／ｍＬ ペプシン、０．６Ｎ ＨＣｌ）を加え、３７℃で２時間反応させ、水酸化ナトリウム水溶液を加えて中和した。 A barley extract was obtained in the same manner as in Example 1. Artificial gastric juice (2% NaCl, 10 kU / mL pepsin, 0.6N HCl) was added to the barley extract with a predetermined protein concentration so that the volume ratio was 10%, and the mixture was reacted at 37 ° C. for 2 hours. An aqueous solution was added for neutralization.
引き続き、体積比が１０％となるように人工腸液（４％ パンクレアチン、４％ トリプシン、１Ｍ ＮａＨＣＯ３）を加え、３７℃で２時間反応させ、９０℃で２分間の熱処理を行って、大麦抽出物の酵素処理物を得た。Subsequently, artificial intestinal fluid (4% pancreatin, 4% trypsin, 1M NaHCO 3 ) was added so that the volume ratio would be 10%, reacted at 37 ° C. for 2 hours, and heat-treated at 90 ° C. for 2 minutes. An enzyme-treated product of the extract was obtained.
得られた大麦抽出物の酵素処理物について、実施例１と同様にしてＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイを行った。 Pit formation assay was performed on the enzyme-treated product of the obtained barley extract in the same manner as in Example 1.
図５は、大麦抽出物の酵素処理物の存在下で行われたＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイにおける骨吸収面積を示すグラフである。図５において、骨吸収面積は、コントロールの骨吸収面積に対する相対値（％）で示されている。また、濃度（μｇ／ｍＬ）は、培地中のタンパク質の濃度を示す。 FIG. 5 is a graph showing the bone resorption area in a Pit formation assay performed in the presence of an enzyme-treated product of barley extract. In FIG. 5, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.
図５から明らかなように、大麦抽出物を人工胃液及び人工腸液で処理して得られる酵素処理物は、タンパク質濃度が１μｇ／ｍＬ以上の場合に骨吸収を完全に抑制し、０．２μｇ／ｍＬの場合にも骨吸収を顕著に抑制した。 As is clear from FIG. 5, the enzyme-treated product obtained by treating the barley extract with artificial gastric juice and artificial intestinal fluid completely inhibits bone resorption when the protein concentration is 1 μg / mL or more, and 0.2 μg / mL. In the case of mL, bone resorption was remarkably suppressed.
実施例４により、大麦抽出物の酵素処理物が骨吸収抑制活性、特に、骨の無機成分（ハイドロキシアパタイト）の分解を抑制する活性を有することが示された。 Example 4 showed that the enzyme-treated product of barley extract has an activity to suppress bone resorption, particularly an activity to suppress the decomposition of bone mineral components (hydroxyapatite).
〔比較例１：カテプシンＫ阻害剤の骨吸収抑制活性〕
カテプシンＫ阻害剤であるシスタチンＣの骨吸収抑制活性を、実施例１と同様にしてＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイで試験した。[Comparative Example 1: Bone resorption inhibitory activity of cathepsin K inhibitor]
The bone resorption inhibitory activity of cystatin C, which is a cathepsin K inhibitor, was tested in the same manner as in Example 1 by the Pit Formation assay.
図６は、シスタチンＣの存在下で行われたＰｉｔ Ｆｏｒｍａｔｉｏｎアッセイにおける骨吸収面積を示すグラフである。図６において、濃度（μｇ／ｍＬ）は、培地中のシスタチンＣの濃度を示す。 FIG. 6 is a graph showing the bone resorption area in a Pit formation assay performed in the presence of cystatin C. In FIG. 6, the concentration (μg / mL) indicates the concentration of cystatin C in the medium.
図６から明らかなように、培地中にシスタチンＣが１０μｇ／ｍＬ含有されている場合にも、骨吸収面積はコントロールとほぼ同等であり、シスタチンＣの添加によって骨吸収はほとんど抑制されなかった。 As is apparent from FIG. 6, even when cystatin C was contained at 10 μg / mL in the medium, the bone resorption area was almost the same as that of the control, and the bone resorption was hardly suppressed by the addition of cystatin C.
比較例１により、シスタチンＣは、骨の無機成分（ハイドロキシアパタイト）の分解を抑制する活性を有せず、カテプシンＫが関与しない骨吸収の系では骨吸収を抑制しないことが示された。 Comparative Example 1 shows that cystatin C does not have the activity of suppressing the decomposition of bone inorganic component (hydroxyapatite), and does not suppress bone resorption in a bone resorption system that does not involve cathepsin K.
〔実施例５：塩基性タンパク質画分の骨吸収抑制活性〕
７週齢の雌性ｄｄｙマウス（日本エスエルシー社）を用いて、以下のようにして、大麦抽出物の塩基性画分の骨吸収抑制活性を確認した。以下の試験において、マウスは、１週間の馴化飼育後、一般状態が良好であった２４匹を選抜し、無作為に、シャム（偽手術）群、コントロール群、大麦タンパク質０．５％群及び大麦タンパク質１．０％群の４群（各群６匹）に分けた。なお、馴化飼育期間及び試験期間を通じて、マウスは、温度２３±１℃、湿度６０±５％、明暗サイクル１２時間（明期：８時〜２０時；暗期：２０時〜８時）の条件で飼育した。[Example 5: Bone resorption inhibitory activity of basic protein fraction]
Using a 7-week-old female ddy mouse (Japan SLC), the bone resorption inhibitory activity of the basic fraction of the barley extract was confirmed as follows. In the following test, mice were selected from 24 animals with good general condition after one week of acclimatization, and randomly selected sham (sham surgery) group, control group, barley protein 0.5% group and Divided into 4 groups of barley protein 1.0% group (6 animals in each group). During the acclimation breeding period and the test period, the mice were kept at a temperature of 23 ± 1 ° C., a humidity of 60 ± 5%, and a light / dark cycle of 12 hours (light period: 8 hours to 20 hours; dark period: 20 hours to 8 hours). Reared in
（塩基性タンパク質画分の調製）
十分に乾燥した大麦種子（品種：りょうふう）をミキサー（やまびこ号Ｌ−Ｓ型、国光社）で約１０分間粉砕して微粉状態の大麦粉を得た。得られた大麦粉５．４ｋｇを２０ｍＭ 酢酸ナトリウム水溶液（ｐＨ５．０）２７Ｌに一晩浸漬し、４℃で２０分間、８０００ｒｐｍで遠心分離して、大麦抽出物（上清）を得た。得られた大麦抽出物を強陽イオン交換樹脂（ＳＰ−Ｔｏｙｏｐｅａｒｌ、東ソー社）に通液し、樹脂に吸着した成分を０．５Ｍ 塩化ナトリウム水溶液で溶出して、大麦抽出物の塩基性画分を得た。(Preparation of basic protein fraction)
Fully dried barley seeds (variety: Ryofu) were pulverized for about 10 minutes with a mixer (Yamabiko No. L-S, Kokuko) to obtain finely baked barley flour. 5.4 kg of the obtained barley flour was immersed in 27 L of 20 mM aqueous sodium acetate solution (pH 5.0) overnight and centrifuged at 8000 rpm for 20 minutes at 4 ° C. to obtain a barley extract (supernatant). The obtained barley extract was passed through a strong cation exchange resin (SP-Toyopearl, Tosoh Corp.), and the components adsorbed on the resin were eluted with a 0.5 M aqueous sodium chloride solution to obtain a basic fraction of the barley extract. Got.
そして、上記塩基性画分を更に７５％飽和硫安で分画し、得られた沈殿画分を更に限外濾過膜で分画して、塩基性タンパク質画分（分子量５０００〜５００００のタンパク質の画分）を得た。 Then, the basic fraction is further fractionated with 75% saturated ammonium sulfate, and the resulting precipitate fraction is further fractionated with an ultrafiltration membrane to obtain a basic protein fraction (a fraction of a protein having a molecular weight of 5000 to 50000). Min).
（飼料の調製）
大麦タンパク質０．５％群、大麦タンパク質１．０％群のマウスに投与する飼料は、大麦タンパク質の含有量がそれぞれ０．５％、１．０％となるように、粉末飼料ＡＩＮ９３Ｇに上記塩基性タンパク質画分を配合して調製した。シャム群、コントロール群のマウスについては、ＡＩＮ９３Ｇをそのまま使用した。(Feed preparation)
The feed administered to the mice of the barley protein 0.5% group and barley protein 1.0% group was prepared by adding the above base to the powdered feed AIN93G so that the barley protein content would be 0.5% and 1.0%, respectively. It was prepared by blending the sex protein fraction. For mice in the sham group and the control group, AIN93G was used as it was.
各飼料の組成を下記表１に示す。表中、各成分量の単位はｇ／ｋｇ飼料である。 The composition of each feed is shown in Table 1 below. In the table, the unit of each component amount is g / kg feed.
（飼料の投与）
上述の馴化飼育後、コントロール群、大麦タンパク質０．５％群及び大麦タンパク質１．０％群のマウスには卵巣摘出手術を、また、シャム群のマウスには偽手術を施し、次いで、４週間、各群のマウスに所定の飼料及び水を自由摂取させた。なお、卵巣が摘出されると、エストロゲンが欠乏し、骨量が減少することが知られている。(Food administration)
After the acclimation breeding described above, mice in the control group, barley protein 0.5% group and barley protein 1.0% group were subjected to ovariectomy, and mice in the sham group were subjected to sham surgery, and then for 4 weeks. Each group of mice was allowed to freely ingest a predetermined feed and water. It is known that when the ovaries are removed, estrogen is deficient and bone mass is reduced.
（骨密度の測定）
４週間の飼料投与後、各群のマウスを解剖し、ＤＣＳ６００−ＥＲ−Ｘ（アロカ社製）を用いて二重エネルギーＸ線吸収測定法（ＤＸＡ法）により大腿骨近位部の骨密度を測定した。結果（平均±標準偏差）を図７に示す。有意差検定は、Ｔｕｋｅｙ ＨＳＤ法により行った。図７において、各データ（グラフ）の上方に付されたアルファベットは、異なるアルファベットの付されたデータ間で有意差（ｐ＜０．０５）があることを示す。(Measurement of bone density)
After 4 weeks of feed administration, each group of mice was dissected and the bone density at the proximal part of the femur was measured by a dual energy X-ray absorption measurement method (DXA method) using DCS600-ER-X (Aloka). It was measured. The results (mean ± standard deviation) are shown in FIG. The significant difference test was performed by the Tukey HSD method. In FIG. 7, the alphabets attached above each data (graph) indicate that there is a significant difference (p <0.05) between the data with different alphabets.
図７から明らかなように、コントロール群では、シャム群と比較して骨密度が有意に低下した。そして、大麦タンパク質０．５％群、大麦タンパク質１．０％群では、コントロール群と比較して、大腿骨近位部の骨密度低下が有意に抑制された。 As is clear from FIG. 7, the bone density was significantly reduced in the control group compared to the sham group. And in the barley protein 0.5% group and the barley protein 1.0% group, the bone density fall of the femur proximal part was significantly suppressed compared with the control group.
（子宮重量の測定）
解剖した各群のマウスから子宮を摘出し、その重量を測定した。結果（平均±標準偏差）を図８に示す。(Measurement of uterine weight)
The uterus was removed from each group of dissected mice and weighed. The results (mean ± standard deviation) are shown in FIG.
図８から明らかなように、卵巣摘出手術を施した３群（コントロール群、大麦タンパク質０．５％群、大麦タンパク質１．０％群）では、シャム群と比較して子宮重量が有意に小さかった。また、卵巣摘出手術を施した３群の間では、子宮重量にほとんど差がなかった。 As is apparent from FIG. 8, the three groups (control group, barley protein 0.5% group, barley protein 1.0% group) that had undergone ovariectomy had a significantly lower uterine weight than the sham group. It was. In addition, there was almost no difference in uterine weight among the three groups subjected to ovariectomy.
実施例５により、大麦の塩基性タンパク質画分（塩基性画分中のタンパク質）、又はこれを消化液中の酵素で消化して得られる酵素処理物は、子宮等の生殖器官に影響することなく、骨吸収及びこれに伴う骨密度低下を抑制することが可能であることが示された。 According to Example 5, the basic protein fraction of barley (protein in the basic fraction) or the enzyme-treated product obtained by digesting this with the enzyme in the digestive fluid affects the reproductive organs such as the uterus. In other words, it was shown that it is possible to suppress bone resorption and the accompanying decrease in bone density.
以上の実施例、比較例及び参考例により、大麦抽出物、その塩基性画分、酸性画分及び塩基性タンパク質画分（塩基性画分中のタンパク質）、並びにこれらの酵素処理物は、骨の基質成分（タンパク質成分）の分解とは異なるメカニズムで骨吸収を抑制することが可能であることが明らかとなった。 According to the above examples, comparative examples and reference examples, barley extract, its basic fraction, acidic fraction and basic protein fraction (protein in the basic fraction), and these enzyme-treated products It was clarified that bone resorption can be suppressed by a mechanism different from the degradation of the substrate component (protein component).
Claims (5)
前記塩基性画分は、大麦を粉砕した大麦粉を水又は緩衝液で抽出する工程と、得られた抽出物を陽イオン交換樹脂に接触させる工程と、前記樹脂に吸着した成分を当該樹脂から分離する工程と、をこの順に実施することによって得られる画分である骨吸収抑制剤。 A bone resorption inhibitor containing a basic fraction of barley extract or an enzyme-treated product thereof as an active ingredient,
The basic fraction includes a step of extracting barley flour obtained by pulverizing barley with water or a buffer, a step of bringing the obtained extract into contact with a cation exchange resin, and a component adsorbed on the resin from the resin. The bone resorption inhibitor which is a fraction obtained by implementing the process to isolate | separate in this order.
前記塩基性画分は、大麦を粉砕した大麦粉を水又は緩衝液で抽出する工程と、得られた抽出物を陽イオン交換樹脂に接触させる工程と、前記樹脂に吸着した成分を当該樹脂から分離する工程と、をこの順に実施することによって得られる画分である骨吸収抑制剤。 A bone resorption inhibitor containing a protein contained in the basic fraction of barley extract or an enzyme-treated product thereof as an active ingredient,
The basic fraction includes a step of extracting barley flour obtained by pulverizing barley with water or a buffer, a step of bringing the obtained extract into contact with a cation exchange resin, and a component adsorbed on the resin from the resin. The bone resorption inhibitor which is a fraction obtained by implementing the process to isolate | separate in this order.
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| KR101659667B1 (en) * | 2013-11-25 | 2016-09-23 | 경희대학교 산학협력단 | Composition for Preventing or Treating Short Stature Comprising Hordeum vulgare Extract |
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| JP2002142723A (en) * | 2000-11-13 | 2002-05-21 | Japan Natural Laboratory Co Ltd | Raw material for dietary processed food and dietary processed food |
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| JPH0474127A (en) * | 1990-07-16 | 1992-03-09 | Takada Seiyaku Kk | Extract of boiled soybean soup having hypotensive action |
| JP2002142723A (en) * | 2000-11-13 | 2002-05-21 | Japan Natural Laboratory Co Ltd | Raw material for dietary processed food and dietary processed food |
| WO2004002978A1 (en) * | 2002-06-26 | 2004-01-08 | Suntory Limited | Refreshment capable of stimulating movement of digestive tract |
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