JP2007016002A - Periodontal disease bacterium protease inhibitor, oral composition given by using the same, and food - Google Patents

Periodontal disease bacterium protease inhibitor, oral composition given by using the same, and food Download PDF

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JP2007016002A
JP2007016002A JP2005201356A JP2005201356A JP2007016002A JP 2007016002 A JP2007016002 A JP 2007016002A JP 2005201356 A JP2005201356 A JP 2005201356A JP 2005201356 A JP2005201356 A JP 2005201356A JP 2007016002 A JP2007016002 A JP 2007016002A
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periodontal disease
protease inhibitor
protein
oryzasistatin
rice
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Masayuki Taniguchi
正之 谷口
Masao Shinpo
正雄 新保
Naoto Murohashi
直人 室橋
Seinosuke Shimada
清之助 島田
Yoshiki Aoyanagi
芳喜 青柳
Takaaki Ogasawara
貴哲 小笠原
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SHIMADA KAGAKU KOGYO KK
Niigata University NUC
Bourbon Corp
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SHIMADA KAGAKU KOGYO KK
Niigata University NUC
Bourbon Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a periodontal disease bacterium protease inhibitor excellent in safety and useful for treating and preventing periodontal diseases, to provide an oral composition given by using the same, and to provide a foodstuff. <P>SOLUTION: This periodontal disease bacterium protease inhibitor contains a protein derived from a gramineous plant as an active ingredient, wherein the protein does not cause an anxiety about a side effect, etc., is used from old times, and comprises a natural product, and therefore the inhibitor is excellent in the safety. Further, the inhibitor inhibits gingipain, so that it is useful for treating and preventing the periodontal disease. Furthermore, the periodontal disease bacterium protease inhibitor is not only used for a medical purpose, but also as a component of a functional food. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、イネ科植物由来のタンパク質を有効成分とする歯周病菌プロテアーゼ阻害剤、並びにこれを用いた口腔組成物及び食料品に関する。   TECHNICAL FIELD The present invention relates to a periodontal disease protease inhibitor comprising a protein derived from a gramineous plant as an active ingredient, and an oral composition and a food product using the same.

歯周病菌であるＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓは成人性歯肉炎の最も重要な病原菌として認知され、幅広い研究が行われている（非特許文献１）。Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓは嫌気性のグラム陰性桿菌であり、タンパク質分解酵素（プロテアーゼ）の産生能が非常に高い。Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓが産生するプロテアーゼには複数の分子種が存在するが、特に、トリプシン様システインプロテアーゼであるアルギニン特異的システインプロテアーゼ（Ｒｇｐ）とリジン特異的システインプロテアーゼ（Ｋｇｐ）は本菌種が産生する主要酵素であり、様々な研究が行われてきた（非特許文献２及び３）。これらの研究を通して、ＲｇｐやＫｇｐがヒト・トランスフェリンを分解し、Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの増殖とヒドロキシルラジカルの産生を促進すること（非特許文献４）、ＲｇｐやＫｇｐがヒト血清アルブミン存在下でのＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの増殖に重要な役割を果たしていること（非特許文献５）、Ｋｇｐに対する特異抗体がＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ感染からマウスを保護すること（非特許文献６）、ＫｇｐがＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの鉄獲得に関わっていること（非特許文献７）、ＲｇｐのペプチドドメインがマウスにおいてＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ感染に対する免疫性を付与すること（非特許文献８）、歯周病患者においてジンジパインに対する抗体の産生が認められること（非特許文献９）などが報告されている。これらの知見はいずれも、歯周組織環境におけるＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの増殖と病原性にＲｇｐとＫｇｐが大きな役割を果たしていることを指し示しており、ＲｇｐとＫｇｐが歯周病の治療・予防の重要な標的因子であることは疑う余地がないと言える。   Porphyromonas gingivalis, a periodontal disease bacterium, has been recognized as the most important pathogen of adult gingivitis, and extensive research has been conducted (Non-patent Document 1). Porphyromonas gingivalis is an anaerobic gram-negative bacilli and has a very high proteolytic enzyme (protease) production ability. There are several molecular species in the protease produced by Porphyromonas gingivalis. In particular, arginine-specific cysteine protease (Rgp) and lysine-specific cysteine protease (Kgp), which are trypsin-like cysteine proteases, are the main species produced by this strain. It is an enzyme and various studies have been conducted (Non-patent Documents 2 and 3). Through these studies, Rgp and Kgp degrade human transferrin to promote the growth of Porphyromonas gingivalis and the production of hydroxyl radicals (Non-patent Document 4). Rgp and Kgp can also be used in Porphyromonas gingivalis in the presence of human serum albumin. It plays an important role in proliferation (Non-Patent Document 5), a specific antibody against Kgp protects mice against Porphyromonas gingivalis infection (Non-Patent Document 6), and Kgp is involved in iron acquisition by Porphyromonas gingivalis ( Non-Patent Document 7) that the peptide domain of Rgp confers immunity against Porphyromonas gingivalis infection in mice ( Non-patent document 8), production of antibodies against gingipain in patients with periodontal disease is reported (non-patent document 9). These findings all indicate that Rgp and Kgp play a major role in the growth and pathogenicity of Porphyromonas gingivalis in the periodontal tissue environment, and Rgp and Kgp are important targets for the treatment and prevention of periodontal disease. There can be no doubt that it is a factor.

Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの感染生理におけるＲｇｐやＫｇｐの役割が、上述のように明らかになるにつれて、歯周病の治療や予防を目的に、ＲｇｐやＫｇｐの阻害剤を開発しようとする試みが実施されている。合成薬剤や抗生物質としては、１−（３−フェニルプロピオニル）ピペラジン−３（Ｒ，Ｓ）−カルボン酸−[４−アミノ−１（Ｓ）−（ベンゾチアゾール−２−カルボニル）ブチル]アミド（非特許文献１０）、ベンザミジン誘導体（非特許文献１１）、テトラサイクリンとその誘導体（非特許文献１２）が報告されている。   As the role of Rgp and Kgp in the infection physiology of Porphyromonas gingivalis becomes clear as described above, attempts have been made to develop inhibitors of Rgp and Kgp for the purpose of treating and preventing periodontal disease. . Synthetic drugs and antibiotics include 1- (3-phenylpropionyl) piperazine-3 (R, S) -carboxylic acid- [4-amino-1 (S)-(benzothiazole-2-carbonyl) butyl] amide ( Non-patent document 10), benzamidine derivatives (non-patent document 11), tetracycline and its derivatives (non-patent document 12) have been reported.

また、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．ＦＡ−７０株が産生するアンチパインアナログ（ＦＡ−７０Ｃ１）がＲｇｐを強く阻害するとの結果も見出されている（非特許文献１３）。ＦＡ−７０Ｃ１がＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓの病原性を抑えるという知見も同時に得られており、このことからもジンジパイン阻害剤が歯周病の予防に有効であるのは確実である。   In addition, Streptomyces sp. It has also been found that an antipine analog (FA-70C1) produced by the FA-70 strain strongly inhibits Rgp (Non-patent Document 13). The finding that FA-70C1 suppresses the pathogenicity of Porphyromonas gingivalis has been obtained at the same time, and it is certain that a gingipain inhibitor is effective in preventing periodontal disease.

従来、安全性の観点から、植物成分からジンジパイン阻害剤を見出そうとする試みも行われている。例えば、特許文献１には、カテキン又はカテキン混合物を有効成分とする病原性システインプロテアーゼ活性阻害剤が開示されており、このカテキン又はカテキン混合物がアルギニン特異的システインプロテアーゼ（Ｒｇｐ）やリジン特異的システインプロテアーゼ（Ｋｇｐ）の活性を阻害することが記載されている。また、特許文献２には、赤霊芝及び／又は黒霊芝がジンジパイン阻害剤として記載されている。さらに、特許文献３には、黄杞葉、緑茶、ウコン、ヨモギ、カリン、刺梨、ギムネマ、ルイボス茶、サンザシ、ラカンカ、シリマリン、枸杞子、紫玄米、エレウテロコック、月桃葉、ドクダミ、大棗及び霊芝がジンジパイン阻害剤として記載されている。
特願２００４−１４３１２７号公報 特願２００５−３５９０９号公報 特願２００３−３３５６４８号公報 Biology of the species Porphyromonas gingivalis, CRCPress, Inc., Florida（１９９３） Clin. Infect. Dis. ２８：４５６−４６５（１９９９） Oral Microbiol. Immunol. １３：２６３−２７０（１９９８） Infect. Immun. ７２：４３５１−４３５６（２００４） Infect. Immun. ６９：５１６６−５１７２（２００１） Infect. Immun. ６９：２９７２−２９７９（２００１） Acta Biochim．Polonica ５１：２５３−２６２（２００４） Infect. Immun. ６６：４１０８−４１１４（１９９８） Infect. Immun. ７２：１３７４−１３８２（２００４） Infect. Immun. ７０：６９６８−６９７５（２００２） Biol. Chem．３８３：１１９３−１１９８（２００２） Antimicrob. Agents Chemother．４５：２８７１−２８７６（２００１） Biol. Chem．３８４：９１１−９２０（２００３） Conventionally, from the viewpoint of safety, attempts have been made to find a gingipain inhibitor from plant components. For example, Patent Document 1 discloses a pathogenic cysteine protease activity inhibitor containing catechin or a catechin mixture as an active ingredient. This catechin or catechin mixture is an arginine-specific cysteine protease (Rgp) or a lysine-specific cysteine protease. It has been described to inhibit the activity of (Kgp). In Patent Document 2, red ganoderma and / or black ganoderma are described as gingipain inhibitors. Further, Patent Document 3 includes yellow cocoon leaves, green tea, turmeric, mugwort, karin, ginger pear, gymnema, rooibos tea, hawthorn, kankan, silymarin, eggplant, purple brown rice, eleutherococcus, moon peach leaf, dokudami, daikon and ganoderma. Has been described as a gingipain inhibitor.
Japanese Patent Application No. 2004-143127 Japanese Patent Application No. 2005-35909 Japanese Patent Application No. 2003-335648 Biology of the species Porphyromonas gingivalis, CRCPress, Inc., Florida (1993) Clin. Infect. Dis. 28: 456-465 (1999) Oral Microbiol. Immunol. 13: 263-270 (1998) Infect. Immun. 72: 4351-4356 (2004) Infect. Immun. 69: 5166-5172 (2001) Infect. Immun. 69: 2972-2979 (2001) Acta Biochim. Polonica 51: 253-262 (2004) Infect. Immun. 66: 4108-4114 (1998) Infect. Immun. 72: 1374-1382 (2004) Infect. Immun. 70: 6968-6975 (2002) Biol. Chem. 383: 1193-1198 (2002) Antimicrob. Agents Chemother. 45: 2871-2876 (2001) Biol. Chem. 384: 911-920 (2003)

しかしながら、合成薬物は医薬品としての利用には適しているものの、食品を介した歯周病の予防目的には適していない。また、上記特許文献２や３に挙げられた植物の中には、日常的な摂食が健康に及ぼす影響については必ずしも明らかになっていないものも含まれており、安全性の点で不安が残る。従って、食品から医薬品までの幅広い利用が可能なジンジパイン阻害剤は限られており、歯周病予防に適した様々な機能性食品を具現化するためには、安全性に優れた天然物由来の新たな阻害剤が求められている。   However, although synthetic drugs are suitable for use as pharmaceuticals, they are not suitable for the purpose of preventing periodontal disease through food. In addition, some of the plants listed in Patent Documents 2 and 3 above include those that are not necessarily clarified about the effects of daily eating on health, and are worried about safety. Remain. Therefore, gingipain inhibitors that can be widely used from foods to pharmaceuticals are limited, and in order to realize various functional foods suitable for periodontal disease prevention, they are derived from natural products with excellent safety. There is a need for new inhibitors.

そこで、本発明は、安全性に優れ、且つ歯周病の治療や予防に有用である歯周病菌プロテアーゼ阻害剤、並びにこれを用いた口腔組成物及び食料品を提供することを目的とする。   Therefore, an object of the present invention is to provide a periodontal bacterial protease inhibitor that is excellent in safety and useful for the treatment and prevention of periodontal disease, and an oral composition and food product using the same.

上記課題に鑑みて鋭意検討した結果、イネ科植物由来のタンパク質がジンジパイン阻害活性を有することを見出し、本発明に想到した。   As a result of intensive studies in view of the above problems, the present inventors have found that a protein derived from a gramineous plant has gingipaine inhibitory activity, and have arrived at the present invention.

本発明における請求項１の歯周病菌プロテアーゼ阻害剤は、イネ科植物由来のタンパク質を有効成分として含有することを特徴とする。   The periodontal disease protease inhibitor of claim 1 in the present invention is characterized by containing a protein derived from a grass family plant as an active ingredient.

本発明における請求項２の歯周病菌プロテアーゼ阻害剤は、請求項１において、前記イネ科植物由来のタンパク質が、イネに含まれるタンパク質、イネ由来のオリザシスタチン又はそれらの部分ペプチドのうちの少なくとも１つからなることを特徴とする。   The periodontal disease protease inhibitor according to claim 2 of the present invention is characterized in that, in claim 1, the protein derived from the Gramineae plant is at least one of a protein contained in rice, oryzacystatin derived from rice, or a partial peptide thereof. It consists of one.

本発明における請求項３の口腔用組成物は、請求項１又は２記載の歯周病菌プロテアーゼ阻害剤を含有することを特徴とする。   The composition for oral cavity of Claim 3 in this invention contains the periodontal disease protease inhibitor of Claim 1 or 2 characterized by the above-mentioned.

本発明における請求項４の食料品は、請求項１又は２記載の歯周病菌プロテアーゼ阻害剤を含有することを特徴とする。   The foodstuff of Claim 4 in this invention contains the periodontal disease protease inhibitor of Claim 1 or 2, It is characterized by the above-mentioned.

本発明における請求項１の歯周病菌プロテアーゼ阻害剤によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the periodontal disease protease inhibitor of claim 1 in the present invention, since it has been used for a long time without worrying about side effects and contains a protein derived from a Gramineae plant which is a natural product, it is safe. And is useful for the treatment and prevention of periodontal disease because it inhibits gingipain.

本発明における請求項２の歯周病菌プロテアーゼ阻害剤によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the periodontal disease protease inhibitor of claim 2 in the present invention, since it has been used for a long time without worrying about side effects and contains a protein derived from the grass family, which is a natural product, it is safe. And is useful for the treatment and prevention of periodontal disease because it inhibits gingipain.

本発明における請求項３の口腔用組成物によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the composition for oral cavity of claim 3 in the present invention, it has been used for a long time without worrying about side effects, and since it contains a natural product of a grass-derived protein as an active ingredient, it is excellent in safety. In addition, since it inhibits gingipain, it is useful for the treatment and prevention of periodontal disease.

本発明における請求項４の食料品によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the food product of claim 4 in the present invention, it has been used for a long time without worrying about side effects, and since it contains a protein derived from a grass family plant that is a natural product, it is excellent in safety, and Since gingipain is inhibited, it is useful for the treatment and prevention of periodontal disease.

本発明の歯周病菌プロテアーゼ阻害剤は、イネ科植物由来のタンパク質を有効成分として含有するものである。   The periodontal disease protease inhibitor of the present invention contains a protein derived from a grass family plant as an active ingredient.

本発明の歯周病菌プロテアーゼ阻害剤の有効成分の原料として用いられるイネ科植物とは、植物分類学上のイネ科に属する植物をいい、例えば、イネ、コムギ、オオムギ、トウモロコシ、アワ、ヒエなどが挙げられ、特にイネ（Ｏｒｙｚａ ｓａｔｉｖa）を用いるのが好ましい。ここで、産業廃棄物である米糠や、イネ刈り後にイネ株から発生する再生イネ等の利用価値のないイネを用いれば、極めて安価に本発明の歯周病菌プロテアーゼ阻害剤の有効成分の原料を得ることができる。   The grass family plant used as a raw material for the active ingredient of the periodontal disease protease inhibitor of the present invention refers to a plant belonging to the family Gramineae in plant taxonomy, such as rice, wheat, barley, corn, millet, millet, etc. Especially, it is preferable to use rice (Oryza sativa). Here, when using rice bran, which is an industrial waste, or non-useful rice such as regenerated rice generated from rice after cutting, the raw material of the active ingredient of the periodontal disease protease inhibitor of the present invention can be obtained at a very low cost. Obtainable.

本発明において用いられるイネ科植物由来のタンパク質とは、イネ科植物に含まれるタンパク質をいい、例えばイネに含まれるタンパク質、イネ由来のオリザシスタチン又はそれらの部分ペプチドが含まれる。ここでいう「部分ペプチド」とは、本発明の有効成分であるイネ科植物由来のタンパク質の全アミノ酸配列の一部からなるペプチドであって、イネ科植物由来のタンパク質のアミノ酸配列において１若しくは複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつジンジパイン阻害活性機能の活性部位の領域を少なくとも含むタンパク質をいう。この「部分ペプチド」は、有効成分であるタンパク質の製造過程において得られるか、又は、得られたタンパク質を慣用のアミノ酸配列切断手段によって切断し、後述する活性測定試験により活性の有無を確認することによって得ることができる。   The protein derived from the grass family used in the present invention refers to a protein contained in the grass family plant, and includes, for example, a protein contained in rice, oryzacystatin derived from rice, or a partial peptide thereof. The “partial peptide” as used herein is a peptide consisting of a part of the entire amino acid sequence of a protein derived from the gramineous plant that is the active ingredient of the present invention, and one or more of the amino acid sequences of the protein derived from the grass A protein consisting of an amino acid sequence in which one amino acid is deleted, substituted or added, and including at least a region of the active site of the gingipain inhibitory activity function. This “partial peptide” is obtained in the process of producing an active ingredient protein, or the obtained protein is cleaved by a conventional amino acid sequence cleaving means, and the presence or absence of activity is confirmed by an activity measurement test described later. Can be obtained by:

なお、イネ（Ｏｒｙｚａ ｓａｔｉｖa）由来のオリザシスタチンは、これまでに３種類の存在が報告されており、それぞれオリザシスタチン−I（阿部ら：J.BIo.Chem, 262, 16793-16797(1987)）、オリザシスタチン−II（近藤ら：J.BIo.Chem, 265, 15832-15837(1990)）、オリザシスタチン−III（World Rice Research Conference 2004, Abstract p.117 (2004)、J. Agric. Food Chem. Web release 07-Jun-2005 (2005)）と命名されている。また、このオリザシスタチン−Iは、Ｎ末端のメチオニンよりＣ末端のアラニンまでの合計１０２残基のアミノ酸より構成されており、オリザシスタチン−IIは、Ｎ末端のメチオニンよりＣ末端のアラニンまでの合計１０７残基のアミノ酸より構成されている。また、オリザシスタチン−IIIは、Ｎ末端のメチオニンからＣ末端のアラニンまでの合計１０８残基のアミノ酸より構成され、分子量１２，０００のタンパク質である。   In addition, three kinds of oryzasistatin derived from rice (Oryza sativa) have been reported so far, and each of oryzasistatin-I (Abe et al .: J.BIo.Chem, 262, 16793-16797 (1987)). Oryzasistatin-II (Kondo et al .: J. BIo. Chem, 265, 15832-15837 (1990)), Oryzastatin-III (World Rice Research Conference 2004, Abstract p.117 (2004), J. Agric. Food Chem Web release 07-Jun-2005 (2005)). This oryzasistatin-I is composed of a total of 102 amino acids from the N-terminal methionine to the C-terminal alanine, and the oryzasistatin-II is a total from the N-terminal methionine to the C-terminal alanine. It consists of 107 amino acids. Oryzasistatin-III is a protein with a molecular weight of 12,000, consisting of a total of 108 amino acids from N-terminal methionine to C-terminal alanine.

イネ科植物由来のタンパク質の製造方法は、イネ科植物の葉、茎、皮、根、種子などの抽出物から慣用の方法に従って、タンパク質画分を分離して得ることができる。ここで、前記抽出物の製造方法は、特に限定されないが、例えば、イネ科植物の葉、茎、皮、根、種子などの植物体部分を、水又はリン酸緩衝液のような溶媒で磨砕した後、ろ過又は遠心分離により上清を集め、これを必要に応じて溶媒で希釈することにより得ることができる。   A method for producing a protein derived from a gramineous plant can be obtained by separating a protein fraction from an extract of leaves, stems, skin, roots, seeds, etc. of a gramineous plant according to a conventional method. Here, the method for producing the extract is not particularly limited. For example, plant parts such as leaves, stems, skins, roots and seeds of gramineous plants are polished with a solvent such as water or a phosphate buffer. After crushing, the supernatant can be collected by filtration or centrifugation, and this can be obtained by diluting with a solvent if necessary.

また、抽出物からのタンパク質画分の分離精製は、特に限定されないが、例えば、前記抽出物に硫酸アンモニウムを加えて、析出したタンパク質を遠心分離により集め、さらにリン酸緩衝液などを用いて溶解させて、これを透析やゲルろ過などの操作を行い、低分子画分等を取り除くことにより分離精製を行うことができる。   The separation and purification of the protein fraction from the extract is not particularly limited. For example, ammonium sulfate is added to the extract, the precipitated protein is collected by centrifugation, and further dissolved using a phosphate buffer or the like. Then, separation and purification can be performed by performing operations such as dialysis and gel filtration to remove low molecular fractions.

さらに、前記抽出物を加熱処理してもよい。加熱方法としては、電気やガスなどの熱源を用いた方法や、マイクロ波などが利用可能であり、方法は特に限定しない。加熱処理の温度範囲としては特に限定しないが、好ましくは６０〜１２０℃の温度範囲で加熱処理を行う。   Furthermore, you may heat-process the said extract. As a heating method, a method using a heat source such as electricity or gas, a microwave, or the like can be used, and the method is not particularly limited. Although it does not specifically limit as a temperature range of heat processing, Preferably heat processing is performed in the temperature range of 60-120 degreeC.

また、本発明において、ジンジパイン阻害活性機能を有する有効成分には、上述したように所望のタンパク質を精製したものを用いてもよいが、有効成分であるタンパク質を有効量含むのであれば、植物体組織またはその乾燥品、粉砕品、抽出物をそのまま有効成分として使用してもよい。なお、乾燥品や粉砕品の調製方法も本発明による有効成分を含んで得ることができる限り、特に限定されない。   In the present invention, the active ingredient having a gingipaine inhibitory activity function may be obtained by purifying a desired protein as described above, but if it contains an effective amount of the protein as an active ingredient, the plant body The tissue or its dried product, pulverized product, or extract may be used as it is as an active ingredient. In addition, the preparation method of a dried product or a pulverized product is not particularly limited as long as the active ingredient according to the present invention can be obtained.

本発明の口腔組成物は、前記本発明の歯周病菌プロテアーゼ阻害剤を含んでおればよく、口腔組成物の種類は特に限定されず、例えば歯磨きやマウスウォッシュのように口腔内で用いられているものを含み、具体的には、練り状、液体状、粉末状の歯磨き剤、マウススプレーなどの口中清涼剤、トローチ剤、うがい剤、シロップ剤等の医薬品又は医薬部外品が挙げられる。   The oral composition of the present invention only needs to contain the periodontal disease protease inhibitor of the present invention, and the type of oral composition is not particularly limited. For example, it is used in the oral cavity such as toothpaste and mouthwash. Specific examples thereof include pastes, liquids and powders of toothpastes, mouth fresheners such as mouse sprays, troches, gargles, syrups, and other drugs or quasi drugs.

また、本発明の食料品は、前記本発明の歯周病菌プロテアーゼ阻害剤を含んでおればよく、食料品の種類は特に限定されず、例えば、飴、チューインガム、トローチ、ジャム、飲料等が挙げられる。   Moreover, the foodstuff of this invention should just contain the periodontal disease protease inhibitor of the said this invention, and the kind of foodstuff is not specifically limited, For example, salmon, chewing gum, troche, jam, a drink, etc. are mentioned. It is done.

また、本発明の口腔組成物及び食料品において、本発明の歯周病菌プロテアーゼ阻害剤の配合割合は、口腔組成物、食料品の種類及び該口腔組成物や食料品に配合される他の成分の種類や量に応じて適宜選択することができる。また、歯周病菌プロテアーゼ阻害剤の剤型は特に限定されず任意である。   Further, in the oral composition and food product of the present invention, the blending ratio of the periodontal disease protease inhibitor of the present invention is the oral composition, the type of food product, and other components blended in the oral composition and food product. It can select suitably according to the kind and quantity. Further, the dosage form of the periodontal disease protease inhibitor is not particularly limited and is arbitrary.

本発明の歯周病菌プロテアーゼ阻害剤によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the periodontal disease protease inhibitor of the present invention, it has been used for a long time without worrying about side effects and the like, and since it contains a natural product of a gramineous plant-derived protein as an active ingredient, it is excellent in safety, and Since gingipain is inhibited, it is useful for the treatment and prevention of periodontal disease.

本発明の口腔組成物及び食料品によれば、副作用等の心配のない古くから用いられ、天然物であるイネ科植物由来のタンパク質を有効成分として含有しているため、安全性に優れ、且つジンジパインを阻害するので歯周病の治療や予防に有用である。   According to the oral composition and food of the present invention, since it has been used for a long time without worrying about side effects, etc., it contains a protein derived from the grass family plant, which is a natural product, as an active ingredient. Since gingipain is inhibited, it is useful for the treatment and prevention of periodontal disease.

以下に本発明の実施例によって、本発明を詳細に説明するが、本発明はこれらの実施例により何ら制限されるものではない。   EXAMPLES The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited to these examples.

米糠タンパク質の抽出。コシヒカリ玄米を９０％まで精米した際に生じる米糠１ｋｇを原料として、これに４倍量の２５ｍＭリン酸ナトリウム緩衝液（０．１５Ｍ ＮａＣｌを含む、ｐＨ６．０）を加えて、４℃で１時間浸漬した。次いで、磨砕処理を行い、固形分を除いた後、８０℃で１０分間加熱処理し、タンパク質分解酵素の活性を失活させた。生じた沈殿物を除去し、抽出物に３０％飽和になるように硫酸アンモニウムを添加して沈殿する画分を除去し、その上清に７０％飽和になるように硫酸アンモニウムを追加して沈殿する画分を米糠タンパク質画分として分離した。本画分は、リン酸ナトリウム緩衝液（０．１５Ｍ ＮａＣｌを含む、ｐＨ６．０）に再溶解し、４℃で透析後、凍結保存した。   Extraction of rice bran protein. 4 kg of 25 mM sodium phosphate buffer solution (containing 0.15 M NaCl, pH 6.0) is added to 1 kg of rice bran that is produced when 90% of Koshihikari brown rice is polished, and 1 hour at 4 ° C. Soaked. Next, grinding treatment was performed to remove the solid content, and then heat treatment was performed at 80 ° C. for 10 minutes to deactivate the activity of the proteolytic enzyme. The precipitate formed is removed, ammonium sulfate is added to the extract to 30% saturation to remove the precipitated fraction, and ammonium sulfate is added to the supernatant to precipitate 70% to precipitate. The fraction was separated as a rice bran protein fraction. This fraction was redissolved in sodium phosphate buffer (containing 0.15 M NaCl, pH 6.0), dialyzed at 4 ° C., and stored frozen.

組換えオリザシスタチン−Ｉ, −II, −IIIの調製。
（オリザシスタチン−Ｉならびにオリザシスタチン−IIIをコードするｃＤＮＡの単離）コシヒカリのカルスから抽出したＲＮＡを用いて、オリザシスタチン−Ｉとオリザシスタチン−IIIをコードするｃＤＮＡをＰＣＲクローニングにより取得した。増幅反応にはTaKaRa One Step RNA PCR Kit（タカラバイオ製）を利用した。オリザシスタチン-I ｃＤＮＡのクローニングに用いたプライマーは５’−ＡＴＧＴＣＧＡＧＣＧＡＣＧＧＡＧＧＧＣＣ−３’と５’−ＧＡＴＧＧＧＣＣＴＴＡＧＧＣＡＴＴＴＧＣ−３’、オリザシスタチン−III ｃＤＮＡ用のプライマーは、５’−ＡＴＧＧＣＣＧＡＧＧＡＧＧＣＧＣＡＧＡＧ−３’と５’−ＣＴＡＴＧＴＡＣＧＴＴＴＡＧＧＣＧＧＴＧ−３’である。ＰＣＲ増幅産物は、TA cloning Kit（インビトロジェン製）によりｐＣＲ２．１にサブクローニングした。 Preparation of recombinant oryzastatin-I, -II, -III.
(Isolation of cDNAs encoding oryzasistatin-I and oryzasistatin-III) Using RNA extracted from calli of Koshihikari, cDNAs encoding oryzasistatin-I and oryzasistatin-III were obtained by PCR cloning. For the amplification reaction, TaKaRa One Step RNA PCR Kit (manufactured by Takara Bio Inc.) was used. Primers used for cloning of oryzasistatin-I cDNA were 5'-ATGTCGGACGCACGGAGGGGCC-3 'and 5'-GATGGGCCCTTAGGGCATTTGC-3', and primers for oryzastatin-III cDNA were 5'-ATGGCCGAGGAGGCGCAGAG-3 ' -3 ′. The PCR amplification product was subcloned into pCR2.1 using TA cloning Kit (Invitrogen).

（オリザシスタチン−Ｉならびにオリザシスタチン−IIIの発現プラスミドの構築）オリザシスタチン−Ｉならびにオリザシスタチン−IIIは、グルタチオンＳ-トランスフェラーゼ（ＧＳＴ）配列をＮ末に有し、ＧＳＴ配列とオリザシスタチン配列の間にFactor Xaによる切断部位を持つ融合タンパク質として発現させた。Factor Xa切断部位（Ｉｌｅ−Ｇｌｕ−Ｇｌｙ−Ａｒｇ）は、ＰＣＲ反応を利用して、オリザシスタチン−Ｉならびにオリザシスタチン−IIIの翻訳シグナルの直上に付加した。そのために用いたプライマーは、オリザシスタチン−Ｉ用が５’−ＡＴＣＧＡＡＧＧＴＣＧＴＡＴＧＴＣＧＡＧＣＧＡＣＧＧＡＧＧＧＣＣ−３’と５’−ＧＡＴＧＧＧＣＣＴＴＡＧＧＣＡＴＴＴＧＣ−３’、オリザシスタチン−III用が５’−ＧＧＡＴＣＣＡＴＣＧＡＡＧＧＴＣＧＴＡＴＧＧＣＣＧＡＧＧＡＧＧＣＧＣＡＧＣＡ−３’と５’−ＴＴＡＧＧＣＧＧＴＧＧＣＧＴＣＧＴＣＧＡＧＧＧＧＣＴＴＧＡＡＡＴＣＣ−３’である。ＰＣＲ増幅産物は、TA cloning Kit（インビトロジェン製）によりｐＣＲ２．１にサブクローニングした。制限酵素で切断後、Factor Xa認識部位を付加したオリザシスタチン−Ｉ配列はpGEX-4T-2（アマシャムバイオサイエンス製）、Factor Xa認識部位を付加したオリザシスタチン−III配列はpGEX-6P-2（アマシャムバイオサイエンス製）の、それぞれＧＳＴコーディング領域の下流に挿入した。
（オリザシスタチン−IIをコードする発現プラスミドの構築）Factor Xa認識部位をN末に付加したオリザシスタチン-II ｃＤＮＡを、オリザシスタチン-III ｃＤＮＡをテンプレートとするＰＣＲ反応により合成した。そのために用いたプライマーは５’− ＧＧＡＴＣＣＡＴＣＧＡＡＧＧＴＣＧＴＡＴＧＧＣＣＧＡＧＧＡＧＧＣＧＣＡＧＡＧＣＣＡＣＧＣＧＣＧＴＧＡＡＧＧＴＧＧＧＣＧＧＣＡＴＣＣＡＣＧＡＣＡＧＣＣＧＧＣＣＧＧＧＣＧＣＧＡＧＡ−３’と５’− ＴＴＡＧＧＣＧＧＴＧＧＣＧＴＣＧＴＣＧＡＧＧＧＧＣＴＴＧＡＡＡＴＣＣ−３’である。ＰＣＲ増幅断片はTA cloning Kit（インビトロジェン製）によりｐＣＲ２．１にサブクローニングした。制限酵素で切断後、Factor Xa認識部位を付加したオリザシスタチン−II配列はpGEX-6P-2（アマシャムバイオサイエンス製）のＧＳＴコーディング領域の下流に挿入した。 (Construction of expression plasmids of oryzasistatin-I and oryzastatin-III) Oryzasistatin-I and oryzastatin-III have a glutathione S-transferase (GST) sequence at the N-terminus, and between the GST sequence and the oryzastatin sequence. Was expressed as a fusion protein having a cleavage site by Factor Xa. The Factor Xa cleavage site (Ile-Glu-Gly-Arg) was added directly above the translation signals of oryzasistatin-I and oryzastatin-III using a PCR reaction. Primers used for this purpose were 5'-ATCGAAGGTCCGTATGCTCGAGCGCGCGGGTGCGCGGGTGCGTCGGGCTGCGCGTGCGTCGG It is. The PCR amplification product was subcloned into pCR2.1 using TA cloning Kit (Invitrogen). Oryzasistatin-I sequence with Factor Xa recognition site added after cleavage with restriction enzymes is pGEX-4T-2 (manufactured by Amersham Biosciences), and oryzastatin-III sequence with Factor Xa recognition site added is pGEX-6P-2 ( Each of Amersham Biosciences), downstream of the GST coding region.
(Construction of an expression plasmid encoding oryzasistatin-II) Oryzasistatin-II cDNA having a Factor Xa recognition site added to the N-terminal was synthesized by PCR reaction using oryzastatin-III cDNA as a template. The primers used for this were 5′-GGATCCCATGAAGGTCGTATGGCCGAGGAGGCGGCAGAGCCACCGCGCGGAGAGGTGGGCGGGCATCCACGAGCGCGGCCGGGGCGCGGATG-3CTGGTGCGTGTGCGTG The PCR amplified fragment was subcloned into pCR2.1 using TA cloning Kit (Invitrogen). After cleaving with a restriction enzyme, the oryzasistatin-II sequence added with Factor Xa recognition site was inserted downstream of the GST coding region of pGEX-6P-2 (Amersham Biosciences).

（組換えオリザシスタチン−Ｉ、オリザシスタチン−IIならびにオリザシスタチン−IIIの調製）ＧＳＴ−オリザシスタチン（−Ｉ/−II/−III）発現プラスミドを導入した大腸菌ＢＬ２１株（アマシャムバイオサイエンス製）を、１００μｇ／ｍｌのアンピシリンを含むＬＢ培地で、３７℃で培養した。培養開始から３時間後にＩＰＴＧ（isopropyl-1-thio-β-D-galactoside）を１．０ｍＭの濃度になるように添加し、更に６時間の培養を行った。培養終了後、集菌した菌体を１% Triton X-100を含むＰＢＳ（１０ｍＭ Ｎａ２ＨＰＯ４/１．８ｍＭ ＫＨ２ＰＯ４-１４０ｍＭ ＮａＣｌ-２．７ｍＭ ＫＣｌ）に懸濁し、超音波処理にて菌体を破壊した。次いで、固形分を遠心分離によって除去した菌体破砕液から、GSTrap HPカラム（アマシャムバイオサイエンス製）を用いて融合タンパク質をアフィニティー精製した。融合タンパク質を含む画分を回収したあと、５０ｍＭ Ｔｒｉｓ−ＨＣｌ (ｐＨ８．０)-１００ｍＭ ＮａＣｌ−５ｍＭ ＣａＣｌ２で透析し、更に５Ｕ／ｍｌの濃度になるようにFactor Xa（ノバジェン製）を加えて、２５℃で２〜３時間の切断反応を行った。反応液からHiTrap Benzamidine FFカラム（アマシャムバイオサイエンス製）でFactor Xaを吸着除去した後、GSTrap HPカラムクロマトグラフィーによって切断されたＧＳＴを除去し、オリザシスタチン−Ｉ、オリザシスタチン−IIならびにオリザシスタチン−IIIの精製標品を得た。 (Preparation of Recombinant Oryzasistatin-I, Oryzasistatin-II and Oryzastatin-III) E. coli BL21 strain (manufactured by Amersham Bioscience) into which a GST-oryzastatin (-I / -II / -III) expression plasmid was introduced The cells were cultured at 37 ° C. in LB medium containing 100 μg / ml ampicillin. Three hours after the start of the culture, IPTG (isopropyl-1-thio-β-D-galactoside) was added to a concentration of 1.0 mM, and the culture was further performed for 6 hours. After completion of the culture, the collected cells are suspended in PBS containing 1% Triton X-100 (10 mM Na 2 HPO 4 /1.8 mM KH 2 PO 4 -140 mM NaCl-2.7 mM KCl) and subjected to ultrasonic treatment. Destroyed the cells. Subsequently, the fusion protein was affinity purified from the cell disruption solution from which the solid content was removed by centrifugation using a GSTrap HP column (manufactured by Amersham Biosciences). After collecting the fraction containing the fusion protein, dialyzed with 50 mM Tris-HCl (pH 8.0) -100 mM NaCl-5 mM CaCl 2 and further added Factor Xa (manufactured by Novagen) to a concentration of 5 U / ml. The cleavage reaction was performed at 25 ° C. for 2 to 3 hours. After the Factor Xa was adsorbed and removed from the reaction solution using a HiTrap Benzamidine FF column (Amersham Biosciences), GST cleaved by GSTrap HP column chromatography was removed, and oryzasistatin-I, oryzasistatin-II and oryzastatin-III A purified sample was obtained.

ジンジパイン阻害活性の測定。
（酵素標品の調製）Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ ＪＣＭ８５２５を、５％ウシ胎児血清を添加したＫＧＢ培地で波長６００ｎｍにおける吸光度が２．１に達するまで培養し、遠心分離（１０,０００ｒｐｍ、１０分間）で菌体を集めた。次いで、菌体を５０ｍＭ Ｔｒｉｓ−ＨＣｌ（１ｍＭ ＣａＣｌ２を含む、ｐＨ７．４）に懸濁し、超音波処理によって菌体を破砕した後、固形分を遠心分離で除去した。回収した破砕上清をＲｇｐ（アルギニン特異的システインプロテアーゼ）並びにＫｇｐ（リジン特異的システインプロテアーゼ）測定の酵素標品として使用した。 Measurement of gingipain inhibitory activity.
(Preparation of enzyme preparation) Porphyromonas gingivalis JCM8525 was cultured in KGB medium supplemented with 5% fetal calf serum until the absorbance at a wavelength of 600 nm reached 2.1, and the cells were centrifuged (10,000 rpm, 10 minutes). Collected. Next, the cells were suspended in 50 mM Tris-HCl (containing 1 mM CaCl 2 , pH 7.4), and the cells were crushed by ultrasonication, and then the solid content was removed by centrifugation. The recovered crushed supernatant was used as an enzyme preparation for measuring Rgp (arginine-specific cysteine protease) and Kgp (lysine-specific cysteine protease).

（Ｒｇｐ阻害活性）測定用の緩衝液には、０．２Ｍ Ｔｒｉｓ−ＨＣｌ／０．１Ｍ ＮａＣｌ／５ｍＭ ＣａＣｌ２／１０ｍＭ Ｌ−システイン（ｐＨ７．６）を使用した。酵素標品として前述のＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ菌体抽出液（２．７８μｇタンパク質／ｍｌ）と所定濃度の測定サンプル（イネタンパク質標品）を加え、４０℃で５分間のプレインキュベーションを行った。その後、酵素基質となるＢｚ−Ａｒｇ−ＭＣＡ（ペプチド研究所製）を５０μＭの濃度になるように添加し、酵素反応を開始した。蛍光分光光度計（Ｓｈｉｍａｄｚｕ ＲＦ−５３００ＰＣ；（株）島津製作所社製、励起波長３８０ｎｍ、吸収波長４４０ｎｍ）を用いて、反応の進行に伴う蛍光強度の増加をモニタリングし、酵素反応の強さは基質から遊離するＡＭＣ量で評価した。３０〜７０％硫安分画によって調製した米糠タンパク質標品のＲｇｐ阻害活性を図１に示す。 The (Rgp inhibitory activity) buffer for measurement was used 0.2M Tris-HCl / 0.1M NaCl / 5mM CaCl 2 / 10mM L- cysteine (pH 7.6). As the enzyme preparation, the aforementioned Porphyromonas gingivalis cell extract (2.78 μg protein / ml) and a measurement sample (rice protein preparation) having a predetermined concentration were added, and preincubation was performed at 40 ° C. for 5 minutes. Thereafter, Bz-Arg-MCA (Peptide Laboratories) as an enzyme substrate was added to a concentration of 50 μM to start the enzyme reaction. Using a fluorescence spectrophotometer (Shimadzu RF-5300PC; manufactured by Shimadzu Corporation, excitation wavelength 380 nm, absorption wavelength 440 nm), the increase in fluorescence intensity accompanying the progress of the reaction was monitored. It was evaluated by the amount of AMC released from FIG. 1 shows the Rgp inhibitory activity of rice bran protein preparations prepared by 30-70% ammonium sulfate fractionation.

図１に示したように、２．０ｍｇ／ｍｌ以下の濃度で強いＲｇｐ阻害活性を認めることができた。また、９０℃、３０分間の加熱処理によってＲｇｐ阻害活性が増強されたことから、米糠タンパク質標品の阻害活性に対する加熱処理の有効性が明らかとなった。これらの結果より、米糠タンパク質がジンジパイン阻害剤として、さらには、歯周病予防因子として利用できることが明らかとなった。   As shown in FIG. 1, strong Rgp inhibitory activity could be recognized at a concentration of 2.0 mg / ml or less. Moreover, since the Rgp inhibitory activity was enhanced by the heat treatment at 90 ° C. for 30 minutes, the effectiveness of the heat treatment on the inhibitory activity of the rice bran protein preparation was clarified. From these results, it became clear that rice bran protein can be used as a gingipain inhibitor and further as a preventive factor for periodontal disease.

（Ｋｇｐ阻害活性）測定用の緩衝液には、０．２Ｍ Ｔｒｉｓ−ＨＣｌ／０．１Ｍ ＮａＣｌ／５ｍＭ ＣａＣｌ２／１０ｍＭ Ｌ−システイン（ｐＨ７．６）を使用した。酵素標品として前述のＰｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ菌体抽出液（２１．５μｇタンパク質／ｍｌ）と所定濃度の測定サンプル（イネタンパク質標品又はオリザシスタチン−Ｉ，−II，−III）を加え、４０℃で５分間のプレインキュベーションを行った。その後、酵素基質となるＢｏｃ−Ｖａｌ−Ｌｅｕ−Ｌｙｓ−ＭＣＡ（ペプチド研究所製）を５０μＭの濃度になるように添加し、酵素反応を開始した。蛍光分光光度計（Ｓｈｉｍａｄｚｕ ＲＦ−５３００ＰＣ；（株）島津製作所社製、励起波長３８０ｎｍ、吸収波長４４０ｎｍ）を用いて、反応の進行に伴う蛍光強度の増加をモニタリングし、酵素反応の強さは基質から遊離するＡＭＣ量で評価した。オリザシスタチン−Ｉ，−II，及び−IIIのＫｇｐ阻害活性を図２に示し、３０〜７０％硫安分画によって調製した米糠タンパク質標品のＫｇｐ阻害活性を図３に示す。 The (Kgp inhibitory activity) buffer for measurement was used 0.2M Tris-HCl / 0.1M NaCl / 5mM CaCl 2 / 10mM L- cysteine (pH 7.6). As the enzyme preparation, the aforementioned Porphyromonas gingivalis cell extract (21.5 μg protein / ml) and a measurement sample (rice protein preparation or oryzastatin-I, -II, -III) at a predetermined concentration are added, and 5 ° C. at 5 ° C. A pre-incubation for minutes was performed. Thereafter, Boc-Val-Leu-Lys-MCA (Peptide Laboratories) as an enzyme substrate was added to a concentration of 50 μM to start the enzyme reaction. Using a fluorescence spectrophotometer (Shimadzu RF-5300PC; manufactured by Shimadzu Corporation, excitation wavelength 380 nm, absorption wavelength 440 nm), the increase in fluorescence intensity accompanying the progress of the reaction was monitored. It was evaluated by the amount of AMC released from The Kgp inhibitory activity of oryzasistatin-I, -II, and -III is shown in FIG. 2, and the Kgp inhibitory activity of the rice bran protein preparation prepared by 30-70% ammonium sulfate fraction is shown in FIG.

図２及び図３に示すように、オリザシスタチン−IIIは１０μＭ以下の低い濃度でＫｇｐに対して阻害活性を示し、米糠タンパク質標品も２ｍｇ／ｍｌ以下の濃度で強いＫｇｐ活性を示した。オリザシスタチン−IIIと米糠タンパク質標品のいずれの場合においても、加熱処理はＫｇｐ阻害活性を増強した。以上の結果からも、オリザシスタチン−IIIや米糠タンパク質標品というイネ由来のタンパク質が、ジンジパイン阻害剤として、更には、歯周病予防因子として利用できることが明らかとなった。   As shown in FIGS. 2 and 3, oryzastatin-III showed inhibitory activity against Kgp at a low concentration of 10 μM or less, and rice bran protein preparation also showed strong Kgp activity at a concentration of 2 mg / ml or less. In both cases of oryzasistatin-III and rice bran protein preparation, the heat treatment enhanced Kgp inhibitory activity. From the above results, it was revealed that rice-derived proteins such as oryzasistatin-III and rice bran protein preparation can be used as a gingipain inhibitor and further as a preventive factor for periodontal disease.

（練歯磨の調製）以下の処方により常法に従って練歯磨を調製した。 (Preparation of toothpaste) A toothpaste was prepared according to a conventional method according to the following formulation.

炭酸カルシウム５０．００重量％、グリセリン２０．００重量％、カラギーナン０．５０重量％、カルボキシメチルセルロース１．００重量％、ラウリル硫酸ナトリウム２．００重量％、香料１．００重量％、サッカリン０．１０重量％、イネ由来タンパク質画分２．００重量％、水２３．４０重量％。   Calcium carbonate 50.00 wt%, glycerin 20.00 wt%, carrageenan 0.50 wt%, carboxymethylcellulose 1.00 wt%, sodium lauryl sulfate 2.00 wt%, fragrance 1.00 wt%, saccharin 0.10 % By weight, rice-derived protein fraction 2.00% by weight, water 23.40% by weight.

（寒天ゼリーの調製）以下の処方により常法に従って寒天ゼリーを調製した。 (Preparation of agar jelly) An agar jelly was prepared according to a conventional method according to the following formulation.

寒天１５ｇ、水あめ２５０ｇ、砂糖６００ｇ、水４５０ｍｌ、香料・洋酒 少量、オリザシスタチン−ＩＩＩ５ｇを均一に過熱・攪拌し、型に入れ、冷却し寒天ゼリーを得た。   15 g of agar, 250 g of syrup, 600 g of sugar, 450 ml of water, a small amount of perfume / sake, and 5 g of oryzasistatin-III were uniformly heated and stirred, placed in a mold, and cooled to obtain an agar jelly.

米糠タンパク質標品のＲｇｐ阻害活性試験の結果を示すグラフである。It is a graph which shows the result of the Rgp inhibitory activity test of a rice bran protein sample. オリザシスタチン−Ｉ，−II，及び−IIIのＫｇｐ阻害活性試験の結果を示すグラフである。It is a graph which shows the result of the Kgp inhibitory activity test of oryzastatin-I, -II, and -III. 米糠タンパク質標品のＫｇｐ阻害活性試験の結果を示すグラフである。It is a graph which shows the result of the Kgp inhibitory activity test of a rice bran protein sample.

Claims (4)

イネ科植物由来のタンパク質を有効成分として含有することを特徴とする歯周病菌プロテアーゼ阻害剤。   A periodontal disease protease inhibitor comprising a protein derived from a grass family as an active ingredient. 前記イネ科植物由来のタンパク質が、イネに含まれるタンパク質、イネ由来のオリザシスタチン又はそれらの部分ペプチドのうちの少なくとも１つからなることを特徴とする請求項１記載の歯周病菌プロテアーゼ阻害剤。   The periodontal disease protease inhibitor according to claim 1, wherein the protein derived from the grass family comprises at least one of a protein contained in rice, oryzacystatin derived from rice, or a partial peptide thereof. 請求項１又は２記載の歯周病菌プロテアーゼ阻害剤を含有することを特徴とする口腔用組成物。   A composition for oral cavity comprising the periodontal disease protease inhibitor according to claim 1 or 2. 請求項１又は２記載の歯周病菌プロテアーゼ阻害剤を含有することを特徴とする食料品。   A food product comprising the periodontal disease protease inhibitor according to claim 1 or 2.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009084161A (en) * 2007-09-27 2009-04-23 Niigata Prefecture Protease inhibitor and antibacterial agent
JP2011101634A (en) * 2009-11-12 2011-05-26 Nippon Suisan Kaisha Ltd Method for enhancing gel strength of fish paste and additive for enhancing gel strength
WO2011115225A1 (en) * 2010-03-17 2011-09-22 国立大学法人 鹿児島大学 Periodontal-disease-specific peptide, and treatment and diagnosis of periodontal disease using same
WO2012113037A1 (en) * 2011-02-25 2012-08-30 The University Of Melbourne Method for inhibiting proteins
JP2018538534A (en) * 2015-12-09 2018-12-27 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Method for directly measuring affinity of human IgG1 binding to multimeric antigen
WO2020167152A1 (en) * 2019-02-15 2020-08-20 RAPAK, Andrzej Marek Inhibitors of cysteine peptidases isolated from natural raw materials and use of the inhibitors in medicine and veterinary medicine

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JP2004143127A (en) * 2002-08-30 2004-05-20 Ito En Ltd Pathogenic cysteine protease activity inhibitor
WO2004106541A1 (en) * 2003-05-30 2004-12-09 Kyushu Tlo Company, Limited Periodontal disease marker

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004143127A (en) * 2002-08-30 2004-05-20 Ito En Ltd Pathogenic cysteine protease activity inhibitor
WO2004106541A1 (en) * 2003-05-30 2004-12-09 Kyushu Tlo Company, Limited Periodontal disease marker

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009084161A (en) * 2007-09-27 2009-04-23 Niigata Prefecture Protease inhibitor and antibacterial agent
JP2011101634A (en) * 2009-11-12 2011-05-26 Nippon Suisan Kaisha Ltd Method for enhancing gel strength of fish paste and additive for enhancing gel strength
WO2011115225A1 (en) * 2010-03-17 2011-09-22 国立大学法人 鹿児島大学 Periodontal-disease-specific peptide, and treatment and diagnosis of periodontal disease using same
WO2012113037A1 (en) * 2011-02-25 2012-08-30 The University Of Melbourne Method for inhibiting proteins
JP2018538534A (en) * 2015-12-09 2018-12-27 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Method for directly measuring affinity of human IgG1 binding to multimeric antigen
WO2020167152A1 (en) * 2019-02-15 2020-08-20 RAPAK, Andrzej Marek Inhibitors of cysteine peptidases isolated from natural raw materials and use of the inhibitors in medicine and veterinary medicine

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