JP2012106988A - Bile acid binder, serum cholesterol reducing agent, and method for reducing serum cholesterol - Google Patents
Bile acid binder, serum cholesterol reducing agent, and method for reducing serum cholesterol Download PDFInfo
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- JP2012106988A JP2012106988A JP2011221902A JP2011221902A JP2012106988A JP 2012106988 A JP2012106988 A JP 2012106988A JP 2011221902 A JP2011221902 A JP 2011221902A JP 2011221902 A JP2011221902 A JP 2011221902A JP 2012106988 A JP2012106988 A JP 2012106988A
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- bile acid
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- cholesterol
- serum cholesterol
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- 239000003613 bile acid Substances 0.000 title claims abstract description 87
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
Description
本発明は、胆汁酸結合能を有するローヤルゼリー由来のタンパク質を有効成分とする胆汁酸結合剤及び血清コレステロール量低減剤、並びに、当該タンパク質を用いた血清コレステロール量を低下させる方法に関する。 The present invention relates to a bile acid binding agent and a serum cholesterol level reducing agent containing a protein derived from royal jelly having bile acid binding ability as an active ingredient, and a method for reducing the serum cholesterol level using the protein.
世界保健機関（ＷＨＯ）の統計では、世界の死因の第１位は、動脈硬化症等の心臓血管疾患である。血液中のＬＤＬ−コレステロール濃度が過剰に高いことが動脈硬化症の主因と考えられており、コレステロール代謝を改善するためのより効果的な医薬品や機能性食品の開発が強く求められている。 According to World Health Organization (WHO) statistics, the number one cause of death worldwide is cardiovascular disease such as arteriosclerosis. An excessively high concentration of LDL-cholesterol in the blood is considered to be the main cause of arteriosclerosis, and there is a strong demand for the development of more effective pharmaceuticals and functional foods for improving cholesterol metabolism.
近年、ローヤルゼリーの摂取により、高コレステロール血症が改善されるという報告がなされている（例えば、非特許文献１〜４参照。）。また、ローヤルゼリーには、一般成分として粗タンパク質が１２〜１５％含まれているほかに、特殊成分として１０−ヒドロキシデセン酸とロイヤリシンが含まれているが、この１０−ヒドロキシデセン酸は、高脂血症ラットの血液中のトリグリセリド、総コレステロールを減少させるという報告がなされている（例えば、非特許文献５参照。）。 In recent years, it has been reported that the intake of royal jelly improves hypercholesterolemia (see, for example, Non-Patent Documents 1 to 4). Royal jelly contains 12-15% of crude protein as a general component and 10-hydroxydecenoic acid and roylicin as special components. This 10-hydroxydecenoic acid is a high fat. It has been reported that triglycerides and total cholesterol in the blood of septic rats are reduced (see, for example, Non-Patent Document 5).
一方で、コール酸を結合させた担体を充填させたカラムを胆汁酸結合アフィニティーカラム（胆汁酸結合能を有する成分に対するアフィニティーカラム）として用い、血清アルブミンを単離する方法（例えば、非特許文献６参照。）や、胆汁酸結合アフィニティーカラムを用いて、大豆タンパク質の中から胆汁酸結合能を有するタンパク質を単離・同定する方法（例えば、非特許文献７及び８参照。）等が開示されている。 On the other hand, a method of isolating serum albumin using a column packed with a carrier to which cholic acid is bound as a bile acid binding affinity column (affinity column for a component having bile acid binding ability) (for example, Non-Patent Document 6). And a method for isolating and identifying a protein having bile acid binding ability from soybean protein using a bile acid binding affinity column (for example, see Non-Patent Documents 7 and 8). Yes.
また、ローヤルゼリー（以下、ＲＪ）は、働き蜂が下咽頭腺と大腮腺から分泌する乳白色ゼリー状の栄養物質である。ローヤルゼリーは女王蜂幼虫の唯一の食糧であり、様々なビタミン類、ミネラル、糖質、アミノ酸、タンパク質成分を含み、非常に栄養価が高い。また、科学的に立証されてはいないものの、昔から、疲労回復作用、抗アレルギー作用、抗癌作用、免疫増強作用等の多くの薬理作用を有すると考えられており、栄養補助食品や医薬品原料として広く用いられている。このため、ＲＪ由来の成分を有効成分とすることにより、安全な新しい高コレステロール血症予防・改善のための食品や医薬品を開発し得ることが期待される。 Royal jelly (hereinafter referred to as RJ) is a milky white jelly-like nutritional substance that the worker bee secretes from the hypopharyngeal gland and the greater vagina. Royal jelly is the only food of the queen bee larvae and contains various vitamins, minerals, sugars, amino acids and protein components, and is very nutritious. Although not scientifically proven, it has long been considered to have many pharmacological actions such as fatigue recovery, antiallergic action, anticancer action, and immune enhancement action. Is widely used. For this reason, it is expected that safe foods and pharmaceuticals for preventing and improving hypercholesterolemia can be developed by using RJ-derived components as active ingredients.
ＲＪ中には様々な成分が含まれているため、新規の医薬品や機能性食品を開発するためには、ＲＪ中の有効成分の特定が不可欠である。しかしながら、非特許文献１〜４では、ＲＪ自身には、高コレステロール血症改善効果があることは明らかにされているものの、その有効成分については全く解明されておらず、その作用機序も未解明である。 Since various components are contained in RJ, it is indispensable to identify active components in RJ in order to develop new pharmaceuticals and functional foods. However, in Non-Patent Documents 1 to 4, although it has been clarified that RJ itself has an effect of improving hypercholesterolemia, its active ingredient has not been elucidated at all and its mechanism of action has not been revealed. It is elucidation.
本発明は、ＲＪに含まれている胆汁酸結合能や高コレステロール血症改善能を備える特定の成分を有効成分とする胆汁酸結合剤、及び当該特定成分を用いて血清コレステロール量を低下させる方法を提供することを目的とする。 The present invention relates to a bile acid binding agent containing a specific component having an ability to improve bile acid binding and hypercholesterolemia contained in RJ as an active ingredient, and a method for reducing the amount of serum cholesterol using the specific component The purpose is to provide.
本発明者らは、上記課題を解決すべく鋭意研究した結果、胆汁酸結合アフィニティーカラムを用いてＲＪから胆汁酸結合能を有する複数の成分を特定し、これらの成分の胆汁酸結合能や血清コレステロールに対する影響を調べることにより、本発明を完成させた。 As a result of diligent research to solve the above problems, the present inventors have identified a plurality of components having bile acid binding ability from RJ using a bile acid binding affinity column, and the bile acid binding ability and serum of these components. The present invention was completed by examining the effect on cholesterol.
すなわち、本発明は、
（１） ＭＲＪＰ１、ＭＲＪＰ２、及びＭＲＪＰ３からなる群より選択される１種以上を有効成分とすることを特徴とする、胆汁酸結合剤、
（２） ＭＲＪＰ１を有効成分とすることを特徴とする、血清コレステロール量低下剤、
（３） ＭＲＪＰ１を有効成分とすることを特徴とする、コレステロールミセルの溶解抑制剤、
（４） ＭＲＪＰ１を経口摂取することを特徴とする、血清コレステロール量を低下させる方法を提供するものである。
That is, the present invention
(1) A bile acid binder characterized by comprising one or more selected from the group consisting of MRJP1, MRJP2, and MRJP3 as an active ingredient,
(2) A serum cholesterol level-lowering agent characterized by comprising MRJP1 as an active ingredient,
(3) A cholesterol micelle dissolution inhibitor characterized by comprising MRJP1 as an active ingredient,
(4) The present invention provides a method for reducing the amount of serum cholesterol, which comprises orally ingesting MRJP1.
本発明の胆汁酸結合剤や血清コレステロール量を低下させる方法においては、ＲＪ由来の胆汁酸結合剤を経口服用することにより、血清コレステロール量を安全に低下させることができる。したがって、本発明の胆汁酸結合剤等は、特に高コレステロール血症や動脈硬化症等の、血清中のコレステロール量の増大に起因する疾患に対する治療や予防効果が期待される。 In the method of reducing the amount of bile acid binder and serum cholesterol of the present invention, the amount of serum cholesterol can be safely reduced by taking RJ-derived bile acid binder orally. Therefore, the bile acid binder of the present invention is expected to have a therapeutic or preventive effect on diseases caused by an increase in the amount of cholesterol in serum, such as hypercholesterolemia and arteriosclerosis.
本発明の胆汁酸結合剤は、ＭＲＪＰ１（Ｍａｊｏｒ ｒｏｙａｌ ｊｅｌｌｙ ｐｒｏｔｅｉｎ １）、ＭＲＪＰ２、及びＭＲＪＰ３からなる群より選択される１種以上を有効成分とすることを特徴とする。ＭＲＪＰ１、ＭＲＪＰ２、及びＭＲＪＰ３は、後記実施例において示すように、胆汁酸結合アフィニティーカラムを用いて、ＲＪから初めて単離・同定された胆汁酸結合タンパク質である。胆汁酸結合アフィニティークロマトグラフィーを使用した胆汁酸結合タンパク質の単離・同定法は、本発明者らにより初めて、ＲＪに対して適用された。 The bile acid binding agent of the present invention is characterized in that one or more selected from the group consisting of MRJP1 (Major royal jelly protein 1), MRJP2, and MRJP3 is an active ingredient. MRJP1, MRJP2, and MRJP3 are bile acid binding proteins that have been isolated and identified for the first time from RJ using a bile acid binding affinity column, as shown in Examples below. The method for isolating and identifying bile acid-binding proteins using bile acid-binding affinity chromatography was first applied to RJ by the present inventors.
ＭＲＪＰ１はＲＪに含まれているタンパク質全体の約３９．４％を占めるタンパク質であり、ＭＲＪＰ２はＲＪに含まれているタンパク質全体の約２１．５％を占めるタンパク質であり、ＭＲＪＰ３はＲＪに含まれているタンパク質全体の約２１．２％を占めるタンパク質である（泉宏樹、米倉政実著。「ローヤルゼリータンパク質のプロテオーム解析」、社団法人全国ローヤルゼリー公正取引協議会、平成２０年度報告書（２００８年）、第２１ページ。）。ＭＲＪＰ１は、４３２アミノ酸からなるタンパク質であり、一般的にはＳＤＳ−ＰＡＧＥでは約５５ｋＤａの分子量として知られている（J.Schmitozova et. al, Cell.Mol.Life Sci., vol.54, p1020-1030 (1998)）。また、ＭＲＪＰ１は、アメリカ合衆国のＮＣＢＩ（国立生物工学情報センター）の配列データベースに、アクセッション番号ＮＰ＿００１０１１５７９として登録されている。ＭＲＪＰ２は、４５２アミノ酸からなるタンパク質であり、一般的にはＳＤＳ−ＰＡＧＥでは約４９ｋＤａの分子量として知られている（J.Schmitozova et. al, Cell.Mol.Life Sci., vol.54, p1020-1030 (1998)）。また、ＭＲＪＰ２のＮＣＢＩのアクセッション番号はＮＰ＿００１０１１５８０である。ＭＲＪＰ３は、５４４アミノ酸からなるタンパク質であり、一般的にＳＤＳ−ＰＡＧＥでは６０〜７０ｋＤａの分子量である（J.Schmitozova et. al, Cell.Mol.Life Sci., vol.54, p1020-1030 (1998)）。また、ＭＲＪＰ３のＮＣＢＩのアクセッション番号はＮＰ＿００１０１１６０１である。
以下、これらのタンパク質を、「本発明のＲＪ由来胆汁酸結合タンパク質」と総称することがある。
MRJP1 is a protein that accounts for approximately 39.4% of the total protein contained in RJ, MRJP2 is a protein that accounts for approximately 21.5% of the total protein included in RJ, and MRJP3 is included in RJ. (Prof. Hiroki Izumi, Masami Yonekura, “Proteome analysis of royal jelly protein”, Japan Royal Jelly Fair Trade Council, 2008 report (2008), Page 21.). MRJP1 is a protein consisting of 432 amino acids, and is generally known as a molecular weight of about 55 kDa by SDS-PAGE (J. Schmitozova et. Al, Cell. Mol. Life Sci., Vol. 54, p1020-). 1030 (1998)). MRJP1 is registered as an accession number NP_001011579 in the sequence database of NCBI (National Center for Biotechnology Information) in the United States. MRJP2 is a protein consisting of 452 amino acids, and is generally known as a molecular weight of about 49 kDa by SDS-PAGE (J. Schmitozova et. Al, Cell. Mol. Life Sci., Vol. 54, p1020-). 1030 (1998)). The accession number of NCBI of MRJP2 is NP_001011580. MRJP3 is a protein consisting of 544 amino acids, and generally has a molecular weight of 60-70 kDa in SDS-PAGE (J. Schmitozova et. Al, Cell. Mol. Life Sci., Vol. 54, p1020-1030 (1998). )). The accession number of NCBI of MRJP3 is NP_001011601.
Hereinafter, these proteins may be collectively referred to as “the RJ-derived bile acid binding protein of the present invention”.
さらに、本発明においては、ＭＲＪＰ１、ＭＲＪＰ２、又はＭＲＪＰ３が有する胆汁酸結合能を損なわない限り、ＭＲＪＰ１、ＭＲＪＰ２、又はＭＲＪＰ３中の１〜数個のアミノ酸を欠失、置換若しくは付加させてもよい。このようなアミノ酸を付加等させたタンパク質も、本発明の胆汁酸結合剤の有効成分とすることができる。例えば、ＭＲＪＰ１、ＭＲＪＰ２、又はＭＲＪＰ３のＮ末端やＣ末端にそれぞれ、１〜１０個、好ましくは１〜５個のアミノ酸を付加することができる。本発明のＲＪ由来胆汁酸結合タンパク質のＮ末端やＣ末端に付加させるアミノ酸配列としては、例えば、Ｈｉｓタグ等の通常、リコンビナントタンパク質の生成に用いられるタグ配列等が挙げられる。 Furthermore, in the present invention, one to several amino acids in MRJP1, MRJP2, or MRJP3 may be deleted, substituted, or added as long as the bile acid binding ability of MRJP1, MRJP2, or MRJP3 is not impaired. A protein to which such an amino acid is added can also be used as an active ingredient of the bile acid binder of the present invention. For example, 1 to 10, preferably 1 to 5 amino acids can be added to the N-terminus or C-terminus of MRJP1, MRJP2, or MRJP3, respectively. Examples of the amino acid sequence to be added to the N-terminus or C-terminus of the RJ-derived bile acid binding protein of the present invention include tag sequences usually used for the production of recombinant proteins such as His tags.
本発明のＲＪ由来胆汁酸結合タンパク質のうち、ＭＲＪＰ１は、胆汁酸結合能に加えて、コレステロールミセルの溶解低下作用と、血清コレステロール量低下作用とを有する。すなわち、本発明のＲＪ由来胆汁酸結合タンパク質は、コレステロールミセルの溶解抑制剤及び血清コレステロール量低下剤の有効成分として好適である。なお、ＭＲＪＰ１の１〜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、かつコレステロールミセルの溶解抑制能や血清コレステロール量低下能を有するタンパク質も、ＭＲＪＰ１と同様にコレステロールミセルの溶解抑制剤及び血清コレステロール量低下剤の有効成分として好適である。 Among the RJ-derived bile acid binding proteins of the present invention, MRJP1 has a cholesterol micelle dissolution lowering action and a serum cholesterol content lowering action in addition to the bile acid binding ability. That is, the RJ-derived bile acid binding protein of the present invention is suitable as an active ingredient of a cholesterol micelle dissolution inhibitor and a serum cholesterol level lowering agent. In addition, a protein having an amino acid sequence in which one to several amino acids of MRJP1 are deleted, substituted, or added, and having the ability to suppress dissolution of cholesterol micelles or the ability to lower serum cholesterol levels is also dissolved in cholesterol micelles, similar to MRJP1. It is suitable as an active ingredient of an inhibitor and a serum cholesterol level lowering agent.
食事由来のコレステロールは、レシチン、脂肪酸、胆汁酸等とミセルを形成し、水溶性となり、非撹拌水層（ＵＷＬ）を通過し、小腸から吸収される。このような小腸におけるコレステロール吸収メカニズムから、本発明のＲＪ由来胆汁酸結合タンパク質は、小腸内において、胆汁酸と結合することにより、コレステロールのミセル形成を抑制する結果、コレステロールの吸収を抑制すると推察される。 Dietary cholesterol forms micelles with lecithin, fatty acids, bile acids, etc., becomes water-soluble, passes through the unstirred water layer (UWL), and is absorbed from the small intestine. From such a cholesterol absorption mechanism in the small intestine, it is speculated that the RJ-derived bile acid binding protein of the present invention inhibits cholesterol absorption by binding to bile acids in the small intestine, thereby suppressing cholesterol micelle formation. The
本発明の胆汁酸結合剤、コレステロールミセルの溶解抑制剤、及び血清コレステロール量低下剤（以下、胆汁酸結合剤等）の有効成分であるＲＪ由来胆汁酸結合タンパク質は、ＲＪから精製されたものであってもよく、遺伝子組換え技術を用いた公知の発現系により合成されたリコンビナントタンパク質であってもよく、ペプチド合成により得られた合成品であってもよい。例えば、ＲＪタンパク質溶液を胆汁酸結合アフィニティークロマトグラフィーによって分画し、ＲＪ由来胆汁酸結合タンパク質を含有する画分を回収する。この回収された画分を、本発明の胆汁酸結合剤等の有効成分とすることができる。また、ＲＪタンパク質溶液をゲル濾過クロマトグラフィーによって分画し、ＭＲＪＰ１、ＭＲＪＰ２、又はＭＲＪＰ３を含有する画分を回収する。これらの回収された画分を、本発明の胆汁酸結合剤等の有効成分とすることもできる。 The RJ-derived bile acid binding protein, which is an active ingredient of the bile acid binding agent, cholesterol micelle dissolution inhibitor, and serum cholesterol level lowering agent (hereinafter referred to as bile acid binding agent) of the present invention, is purified from RJ. It may be a recombinant protein synthesized by a known expression system using a gene recombination technique, or a synthetic product obtained by peptide synthesis. For example, the RJ protein solution is fractionated by bile acid binding affinity chromatography, and the fraction containing RJ-derived bile acid binding protein is collected. This collected fraction can be used as an active ingredient such as the bile acid binder of the present invention. Further, the RJ protein solution is fractionated by gel filtration chromatography, and the fraction containing MRJP1, MRJP2, or MRJP3 is collected. These recovered fractions can be used as active ingredients such as the bile acid binder of the present invention.
本発明の胆汁酸結合剤等は、医薬品やサプリメント等の飲食品として単独で摂取されてもよく、他の飲食用組成物や医薬用組成物と同様に、飲食品や医薬品への添加剤として用いることもできる。 The bile acid binder of the present invention may be taken alone as a food or drink such as pharmaceuticals or supplements, and as other food and drink compositions or pharmaceutical compositions, as an additive to food or drink or medicines. It can also be used.
本発明の胆汁酸結合剤等の剤型は、特に限定されるものではない。例えば、ソフトカプセル剤、ハードカプセル剤、錠剤、顆粒剤、散剤、液剤、軟膏剤、クリーム剤、ゲル剤等であってもよい。培養細胞等へ用いられる場合には、乾燥粉末や、水や緩衝液等の適当な溶液に溶解させた液剤であることが好ましい。一方、生物個体へ用いられる場合には、経口投与に適した剤型であることが好ましく、腸溶剤であることがより好ましい。 The dosage form such as the bile acid binder of the present invention is not particularly limited. For example, soft capsules, hard capsules, tablets, granules, powders, solutions, ointments, creams, gels and the like may be used. When used for cultured cells or the like, it is preferably a solution prepared by dissolving in a dry powder or an appropriate solution such as water or a buffer solution. On the other hand, when used for living organisms, it is preferably a dosage form suitable for oral administration, and more preferably an enteric solvent.
本発明の胆汁酸結合剤等に含まれるＲＪ由来胆汁酸結合タンパク質は、１種類のみであってもよく、２種類以上を組み合わせてもよい。また、これらの胆汁酸結合剤等に含まれるＲＪ由来胆汁酸結合タンパク質の量は、該ＲＪ由来胆汁酸結合タンパク質の胆汁酸結合能、コレステロールミセル溶解抑制能、又は血清コレステロール低下能が発揮され得る量であれば、特に限定されるものではなく、ＲＪ由来胆汁酸結合タンパク質の種類や、剤型等を考慮して、適宜決定することができる。 Only one type of RJ-derived bile acid binding protein contained in the bile acid binder or the like of the present invention may be used, or two or more types may be combined. The amount of RJ-derived bile acid-binding protein contained in these bile acid-binding agents can exhibit the bile acid-binding ability, cholesterol micelle dissolution-inhibiting ability, or serum cholesterol-lowering ability of the RJ-derived bile acid-binding protein. The amount is not particularly limited, and can be appropriately determined in consideration of the type, dosage form, and the like of the RJ-derived bile acid binding protein.
本発明の胆汁酸結合剤等は、本発明のＲＪ由来胆汁酸結合タンパク質のみからなるものであってもよく、その他の成分を含有するものであってもよい。その他の成分としては、本発明のＲＪ由来胆汁酸結合タンパク質の活性を損なわない限り、どのような成分であってもよく、例えば、本発明のＲＪ由来胆汁酸結合タンパク質以外のＲＪ由来の成分を含有していてもよく、胆汁酸結合能や血清コレステロール低下能を有する機能性ペプチドを含有していてもよい。その他にも、本発明の胆汁酸結合剤等は、例えば、賦型剤、結合剤、界面活性剤、酸化防止剤、ｐＨ調整剤、崩壊剤、滑沢剤、防腐剤、殺菌剤、着色剤、矯味矯臭剤等の、医薬品や飲食品に添加される成分を含有することができる。 The bile acid binding agent and the like of the present invention may be composed only of the RJ-derived bile acid binding protein of the present invention or may contain other components. The other components may be any components as long as the activity of the RJ-derived bile acid binding protein of the present invention is not impaired. For example, RJ-derived components other than the RJ-derived bile acid binding protein of the present invention may be used. It may contain, and may contain the functional peptide which has a bile acid binding ability and a serum cholesterol lowering ability. In addition, the bile acid binder of the present invention includes, for example, an excipient, a binder, a surfactant, an antioxidant, a pH adjuster, a disintegrant, a lubricant, an antiseptic, a disinfectant, and a colorant. Ingredients added to pharmaceuticals and foods and drinks such as flavoring agents can be contained.
本発明の胆汁酸結合剤等の製造方法は、含有される本発明のＲＪ由来胆汁酸結合タンパク質の活性を損なわない方法であれば特に限定されるものではなく、通常、機能性タンパク質を含有する飲食用組成物又は医薬用組成物を製造する場合に使用される方法を用いて製造することができる。 The method for producing the bile acid binder and the like of the present invention is not particularly limited as long as it does not impair the activity of the RJ-derived bile acid binding protein of the present invention, and usually contains a functional protein. It can manufacture using the method used when manufacturing a food-drinking composition or a pharmaceutical composition.
本発明の胆汁酸結合剤等の投与量は、ＲＪ由来胆汁酸結合タンパク質の胆汁酸結合等の活性が発揮され得る量であれば、特に限定されるものではなく、ＲＪ由来胆汁酸結合タンパク質の種類、対象とする細胞の種類や状態、剤形、投与方法等を考慮して適宜決定することができる。例えば、培養細胞等の生物個体外の細胞へ用いる場合には、本発明の胆汁酸結合剤又はコレステロールミセル溶解抑制剤を、培養液中のＲＪ由来胆汁酸結合タンパク質の含有量が１０ｍｇ〜１００mｇ／ｍＬとなるように培養液へ添加することができる。 The dose of the bile acid binding agent and the like of the present invention is not particularly limited as long as the activity such as bile acid binding of the RJ-derived bile acid binding protein can be exerted. It can be appropriately determined in consideration of the type, the type and state of the target cell, the dosage form, the administration method, and the like. For example, when used for cells outside an organism such as cultured cells, the bile acid binding agent or cholesterol micelle dissolution inhibitor of the present invention has a content of RJ-derived bile acid binding protein in the culture solution of 10 mg to 100 mg / mg. It can add to a culture solution so that it may become mL.
生物個体に摂取させる場合には、本発明の胆汁酸結合剤等の摂取量は、該ＲＪ由来胆汁酸結合タンパク質の胆汁酸結合等の活性が発揮され得る量であれば、特に限定されるものではなく、摂取する人や動物の体重、年齢、性別、剤型等により適宜決定することができる。例えば、ＲＪ由来胆汁酸結合タンパク質の一日当たりの摂取量が３００ｍｇ〜１ｇ／ｋｇとなるように、１度に又は数回に分けて摂取させることが好ましい。 When ingested by a living individual, the amount of the bile acid binding agent or the like of the present invention is particularly limited as long as the amount of the RJ-derived bile acid binding protein can exert an activity such as bile acid binding. Instead, it can be determined appropriately depending on the weight, age, sex, dosage form, etc. of the person or animal to be ingested. For example, it is preferable to take the RJ-derived bile acid binding protein at a time or in several divided doses so that the daily intake is 300 mg to 1 g / kg.
本発明の胆汁酸結合剤等は、経口摂取することにより、生体内において、胆汁酸と結合し、さらに、食事として摂取されたコレステロールのミセルの溶解性を低下させる結果、血清コレステロール量を低減させる。このため、本発明の胆汁酸結合剤等は、高コレステロール血症や動脈硬化症等の、血清中のコレステロール量が過剰となることに起因する疾患の治療や予防に好適である。 The bile acid binder of the present invention, when taken orally, binds to bile acids in vivo and further reduces the solubility of cholesterol micelles taken as a meal, resulting in a reduction in serum cholesterol levels. . For this reason, the bile acid binder of the present invention is suitable for the treatment and prevention of diseases such as hypercholesterolemia and arteriosclerosis caused by an excessive amount of cholesterol in the serum.
本発明の血清コレステロール量を低下させる方法（以下、血清コレステロール量低下方法）は、ＭＲＪＰ１、ＭＲＪＰ２、及びＭＲＪＰ３からなる群より選択される１種以上を経口摂取することを特徴とする。ＲＪ由来胆汁酸結合タンパク質を経口摂取させる生物は、小腸や胆管等の組織を有する動物であれば特に限定されるものではなく、ヒトであってもよく、マウス、ラット、イヌ、ネコ、サル、ウシ等のヒト以外の動物であってもよい。 The method for reducing the amount of serum cholesterol of the present invention (hereinafter referred to as the method for reducing the amount of serum cholesterol) is characterized by orally ingesting one or more selected from the group consisting of MRJP1, MRJP2, and MRJP3. The organism that orally ingests the RJ-derived bile acid binding protein is not particularly limited as long as it is an animal having a tissue such as the small intestine or the bile duct, and may be a human, mouse, rat, dog, cat, monkey, It may be a non-human animal such as a cow.
次に実施例等を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example etc. are shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
［参考例１］
＜胆汁酸結合アフィニティーカラムの作製＞
胆汁酸結合アフィニティーカラムは、カルボジイミドをスペーサーとして、コール酸をカラム担体であるＥＡＨ−Ｓｅｐｈａｒｏｓｅ ４Ｂに結合させることにより作製した。胆汁酸結合アフィニティーカラムの調製方法は、Ｐａｔｔｉｎｓｏｎらの方法（非特許文献６）に準じて行った。
具体的には、まず、ＥＡＨ−Ｓｅｐｈａｒｏｓｅ ４Ｂ担体５０ｍＬ容量に対し、６２５ｍＬの胆汁酸溶液（スペーサーとして３３．４ｍＭ １−ｅｔｈｙｌ−３−（３−ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ）ｃａｒｂｏｄｉｉｍｉｄｅ ＨＣｌと３．７ｍＭのコール酸を含む５０％エタノール溶液）をｐＨ６．４で１６時間反応させ、担体にコール酸を結合させた。その後、カラムに非吸着の過剰なコール酸溶液を０．５Ｍ ＮａＣｌを含む５０％エタノールで洗浄したものを充填することにより、胆汁酸結合アフィニティーカラム（カラムのベッド容量：５０ｍＬ）を作製した。なお、カラムは使用前に０．０２％ＮａＮ３溶液にて平衡化した。
[Reference Example 1]
<Preparation of bile acid binding affinity column>
The bile acid binding affinity column was prepared by binding cholic acid to EAH-Sepharose 4B, which is a column carrier, using carbodiimide as a spacer. The bile acid binding affinity column was prepared according to the method of Pattinson et al. (Non-patent Document 6).
Specifically, first, for 50 mL volume of EAH-Sepharose 4B carrier, 625 mL of bile acid solution (33.4 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl and 3.7 mM cholic acid as a spacer is included. 50% ethanol solution) was reacted at pH 6.4 for 16 hours to bind cholic acid to the carrier. Thereafter, a bile acid-binding affinity column (column bed volume: 50 mL) was prepared by filling the column with a non-adsorbed excess cholic acid solution washed with 50% ethanol containing 0.5 M NaCl. The column was equilibrated with a 0.02% NaN 3 solution before use.
＜胆汁酸結合アフィニティーカラムによる分画＞
次いで、作製されたカラムが、実際に胆汁酸結合能を有する化合物に対するアフィニティーを備えているかどうかを、ＢＳＡとＯｖａｌｂｕｍｉｎをアプライすることにより、確認した。カラムからの溶出条件は、マキノらの方法（非特許文献７）に準じて行った。
まず、作製された胆汁酸結合アフィニティーカラムに、１．５ｍｇ／ｍＬのＢＳＡ溶液又はＯｖａｌｂｕｍｉｎ溶液をアプライした。次に、当該カラムに第１次洗浄液（０．５Ｍ ＮａＣｌ、１０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）を通し、当該カラム中の担体に吸着していなかった物質を洗浄除去した。次いで、当該カラムに、溶出液（０．５％Ｓｏｄｉｕｍ ｄｅｏｘｙｃｈｏｌａｔｅ、１０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）を通し、コール酸と結合したタンパク質、すなわち胆汁酸結合能を有するタンパク質を溶出した。最後に、当該カラムに、第２次洗浄液（８Ｍ ｕｒｅａ、１０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）を通し、当該カラムに非特異的に結合した物質を溶出した。
<Fractionation by bile acid binding affinity column>
Subsequently, it was confirmed by applying BSA and Ovalbumin whether or not the prepared column actually had an affinity for a compound having a bile acid binding ability. Elution conditions from the column were performed according to the method of Makino et al. (Non-patent Document 7).
First, a 1.5 mg / mL BSA solution or Ovalbumin solution was applied to the prepared bile acid binding affinity column. Next, a primary washing solution (0.5 M NaCl, 10 mM Tris-HCl, pH 8.0) was passed through the column to wash away substances that were not adsorbed on the carrier in the column. Next, an eluate (0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 8.0) was passed through the column to elute proteins bound to cholic acid, that is, proteins having bile acid binding ability. Finally, a secondary washing solution (8 Murea, 10 mM Tris-HCl, pH 8.0) was passed through the column to elute substances that non-specifically bound to the column.
各画分の移動度と２８０ｎｍの吸光度との関係を図１に示す。図１中、「（１）」が第１次洗浄液の画分であり、「（２）」が溶出液の画分であり、「（３）」が第２次洗浄液の画分である。この結果、Ｏｖａｌｂｕｍｉｎは第１次洗浄液の画分に溶出されており、ＢＳＡは溶出液の画分に溶出されていた。これらの結果から、作製された胆汁酸結合アフィニティーカラムは、非特許文献７に記載されている胆汁酸結合アフィニティーカラム同様に、Ｏｖａｌｂｕｍｉｎは吸着せず、ＢＳＡを吸着することが確認された。 The relationship between the mobility of each fraction and the absorbance at 280 nm is shown in FIG. In FIG. 1, “(1)” is a fraction of the primary washing solution, “(2)” is a fraction of the eluate, and “(3)” is a fraction of the secondary washing solution. As a result, Ovalbumin was eluted in the fraction of the first washing solution, and BSA was eluted in the fraction of the eluate. From these results, it was confirmed that the prepared bile acid-binding affinity column adsorbs BSA without adsorbing Ovalbumin, like the bile acid-binding affinity column described in Non-Patent Document 7.
［実施例１］
＜胆汁酸結合アフィニティーカラムによるＲＪタンパク質の分画＞
参考例１で作製された胆汁酸結合アフィニティーカラムを用いて、ＲＪタンパク質の分画を行い、胆汁酸結合能を有するタンパク質を単離・精製した。この胆汁酸結合アフィニティーカラムを用いたＲＪ由来胆汁酸結合タンパク質の単離精製は、これまで報告が無く、非常に効率的な精製法である。
まず、ＲＪタンパク質溶液を調製した。具体的には、ＲＪ（中国産）に純水を加えて懸濁したものを、孔径１０ｋＤａの透析膜を用いて、純水にて７２時間透析した。透析膜内液を回収し、凍結乾燥した後、再びＮａＮ３含有トリス緩衝液（０．２％ＮａＮ３、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０））に懸濁し、１１７ｍｇ／２５ｍＬのＲＪタンパク質溶液（１０ｋＤａ ｃｕｔ ｏｆｆ ＲＪ）を調製した。
[Example 1]
<Fractionation of RJ protein by bile acid binding affinity column>
Using the bile acid binding affinity column produced in Reference Example 1, RJ protein fractionation was performed, and a protein having bile acid binding ability was isolated and purified. The isolation and purification of RJ-derived bile acid-binding protein using this bile acid-binding affinity column has not been reported so far and is a very efficient purification method.
First, an RJ protein solution was prepared. Specifically, a suspension obtained by adding pure water to RJ (made in China) was dialyzed against pure water for 72 hours using a dialysis membrane having a pore size of 10 kDa. The dialysis membrane solution was collected and lyophilized, and then suspended again in NaN 3 -containing Tris buffer (0.2% NaN 3 , 10 mM Tris-HCl (pH 8.0)) and 117 mg / 25 mL of RJ protein solution ( 10 kDa cut off RJ).
次いで、参考例１で作製された胆汁酸結合アフィニティーカラムに、ＲＪタンパク質溶液をアプライした後、参考例１と同様にして、第１次洗浄液、溶出液、第２次洗浄液を順次当該カラムにアプライした。各画分の移動度と２８０ｎｍの吸光度との関係を図２に示す。図２中、「（１）」、「（２）」、及び「（３）」は図１と同じである。
さらに、溶出液により溶出された画分（図２中の「Ａ」、以下、「画分Ａ」）を回収した。
Next, after applying the RJ protein solution to the bile acid-binding affinity column prepared in Reference Example 1, the first washing solution, the eluate, and the second washing solution were sequentially applied to the column in the same manner as in Reference Example 1. did. The relationship between the mobility of each fraction and the absorbance at 280 nm is shown in FIG. In FIG. 2, “(1)”, “(2)”, and “(3)” are the same as those in FIG.
Further, the fraction eluted by the eluate (“A” in FIG. 2, hereinafter “fraction A”) was collected.
＜胆汁酸結合アフィニティーカラムにより単離・精製されたＲＪ由来胆汁酸結合タンパク質の同定＞
胆汁酸結合アフィニティーカラムにより単離・精製されたＲＪ由来胆汁酸結合タンパク質の同定を行った。
まず、画分Ａを孔径１０ｋＤａの透析膜を用いて、純水にて４℃、７２時間透析・脱塩した。透析膜内液を回収し、凍結乾燥することにより、脱塩画分Ａを調製した。
次いで、脱塩画分Ａを１０％のＳＤＳ−ＰＡＧＥに供し、含まれているタンパク質を分離した。ＳＤＳ−ＰＡＧＥの結果を図３に示す。図３中、「ｓｔｄ」は分子量マーカーを、「Ａ」は脱塩画分Ａを意味し、これらをそれぞれアプライしたものである。この結果、「Ａ」レーンには約４９〜７５ｋＤａの範囲内に３本のバンド（図３中、（１）〜（３））が検出された。この結果から、脱塩画分Ａ溶液には、胆汁酸と結合する３種類のＲＪ由来タンパク質が含まれていたことが明らかである。
<Identification of RJ-derived bile acid binding protein isolated and purified by bile acid binding affinity column>
RJ-derived bile acid binding protein isolated and purified by a bile acid binding affinity column was identified.
First, the fraction A was dialyzed and desalted with pure water at 4 ° C. for 72 hours using a dialysis membrane having a pore size of 10 kDa. The desalted fraction A was prepared by collecting the dialyzed membrane solution and freeze-drying it.
Subsequently, the desalted fraction A was subjected to 10% SDS-PAGE to separate contained proteins. The result of SDS-PAGE is shown in FIG. In FIG. 3, “std” means a molecular weight marker, and “A” means a desalted fraction A, which are applied respectively. As a result, in the “A” lane, three bands ((1) to (3) in FIG. 3) were detected within a range of about 49 to 75 kDa. From this result, it is clear that the desalted fraction A solution contained three types of RJ-derived proteins that bind to bile acids.
各バンドをＳＤＳ−ＰＡＧＥのゲルから切り出し、還元アルキル化した後、トリプシン処理によりＩｎ−ｇｅｌ ｄｉｇｅｓｔｉｏｎを行った。その後、ゲルより抽出したペプチド断片を脱塩精製して、ＭＡＬＤＩ−ＴＯＦ／ＭＳにて質量分析を行った。この結果、図３中、バンド（１）はＭＲＪＰ３、バンド（２）はＭＲＪＰ１、バンド（３）はＭＲＪＰ２であると同定された。ＳＤＳ−ＰＡＧＥの結果、得られたＣＢＢ染色像を画像解析（Ｉｍａｇｅ Ｊ分析）したところ、これら３種のタンパク質の割合は、ＭＲＪＰ１が７６％、ＭＲＪＰ２が２２％、ＭＲＪＰ３が２％であった。 Each band was cut out from the SDS-PAGE gel, subjected to reductive alkylation, and then subjected to In-gel digestion by trypsin treatment. Thereafter, the peptide fragment extracted from the gel was desalted and purified, and mass spectrometry was performed with MALDI-TOF / MS. As a result, in FIG. 3, band (1) was identified as MRJP3, band (2) as MRJP1, and band (3) as MRJP2. As a result of SDS-PAGE, the obtained CBB-stained image was subjected to image analysis (Image J analysis). The ratio of these three proteins was 76% for MRJP1, 22% for MRJP2, and 2% for MRJP3.
［実施例２］
ＲＪから単離精製されたＭＲＪＰ１及びＭＲＪＰ２の胆汁酸結合能を測定した。
[Example 2]
The bile acid binding ability of MRJP1 and MRJP2 isolated and purified from RJ was measured.
＜ＲＪからのＭＲＪＰ１の単離精製＞
まず、ＲＪからゲル濾過クロマトグラフィーを用いてＭＲＪＰ１を精製した。具体的には、まず、実施例１と同様にして調製した２３．５ｍｇ／５ｍＬのＲＪタンパク質溶液（
１０ｋＤａ ｃｕｔ ｏｆｆ ＲＪ）を５ｍＬ、ＨｉＬｏａｄ ｓｕｐｅｒｄｅｘ ２００ｐ．ｇ（ＧＥ ｈｅａｌｔｈｃａｒｅ社製）にアプライした。その後、溶出液（２０ｍＭ Ｎａ２ＨＰＯ４・２０ｍＭ ＮａＨ２ＰＯ４、１５０ｍＭ ＮａＣｌ、ｐＨ７．５）を溶出速度１．５ｍＬ／ｍｉｎで当該カラムに通し、５ｍＬずつを１画分として回収した。各画分の移動度と２８０ｎｍの吸光度との関係を図４に示す。また、ＲＪタンパク質溶液と同時に、当該カラムに３６ｍｇ／ｍｌのＧｅｌ Ｆｉｌｔｒａｔｉｏｎ Ｓｔａｎｄａｒｄ （ＢＩＯ−ＲＡＤ社製）をアプライし、移動度（溶出開始からの総溶出量）と、当該画分に含まれる分子の大きさとの関係を調べ、検量線を作成した。作成された検量線を図５に示す。
本発明者らは、前記の胆汁酸結合アフィニティーカラムから溶出した複合体の、ゲル解析において量的に一番多かったＭＲＪＰ１画分に着目した。図４に示すゲル濾過クロマトグラフィーのうち、画分Ｂ（Frac.B：図４中、点線で囲まれた画分）は、図５に示すスタンダードの移動度と分子量から得た検量線から、分子量約２９０ｋＤａ付近であることがわかった。この画分を１５％のＳＤＳ−ＰＡＧＥに供したところ、図６に示すように、約５５ｋＤａの位置に単一なバンドとして検出された。また、当該画分に含まれている分子に対してＭＡＬＤＩ−ＴＯＦ／ＭＳにて質量分析を行ったところ、ＭＲＪＰ１を含む画分であると判明した。これらの結果から、ＭＲＪＰ１は約５５ｋＤａであり、ＲＪ中では、５〜６量体として存在していることが確認された。以下、当該画分をＭＲＪＰ１画分という。
なお、当該ＭＲＪＰ１画分には、非特許文献５において、高脂血症ラットの血液中のトリグリセリド、総コレステロールを減少させる作用が報告されている１０−デセン酸は含まれていないことが、定量的に明らかにされた。
<Isolation and purification of MRJP1 from RJ>
First, MRJP1 was purified from RJ using gel filtration chromatography. Specifically, first, a 23.5 mg / 5 mL RJ protein solution prepared in the same manner as in Example 1 (
10 kDa cut off RJ), HiLoad superdex 200 p. g (manufactured by GE healthcare) was applied. Thereafter, the eluate (20 mM Na 2 HPO 4 .20 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.5) was passed through the column at an elution rate of 1.5 mL / min, and 5 mL each was collected as one fraction. The relationship between the mobility of each fraction and the absorbance at 280 nm is shown in FIG. Simultaneously with the RJ protein solution, 36 mg / ml Gel Filtration Standard (manufactured by BIO-RAD) was applied to the column, the mobility (total elution amount from the start of elution) and the molecules contained in the fraction. The relationship with the size was examined, and a calibration curve was created. The created calibration curve is shown in FIG.
The present inventors paid attention to the MRJP1 fraction, which was quantitatively the most in the gel analysis of the complex eluted from the bile acid binding affinity column. Among the gel filtration chromatography shown in FIG. 4, fraction B (Frac.B: the fraction surrounded by a dotted line in FIG. 4) is obtained from a calibration curve obtained from the mobility and molecular weight of the standard shown in FIG. It was found that the molecular weight was around 290 kDa. When this fraction was subjected to 15% SDS-PAGE, it was detected as a single band at a position of about 55 kDa, as shown in FIG. Moreover, when mass analysis was performed by MALDI-TOF / MS with respect to the molecule | numerator contained in the said fraction, it became clear that it was a fraction containing MRJP1. From these results, MRJP1 was about 55 kDa, and it was confirmed that it exists as a 5-6 mer in RJ. Hereinafter, this fraction is referred to as MRJP1 fraction.
In addition, it is quantitatively determined that the MRJP1 fraction does not contain 10-decenoic acid, which is reported in Non-Patent Document 5, which has been reported to reduce triglycerides and total cholesterol in the blood of hyperlipidemic rats. Was revealed.
＜ＲＪからのＭＲＪＰ２の単離精製＞
本発明者らはさらに、前記の胆汁酸結合アフィニティーカラムから溶出した複合体の、ゲル解析において量的に一番多かったＭＲＪＰ１画分に次いで多かったＭＲＪＰ２に着目した。図４に示すゲル濾過クロマトグラフィーのうち、画分Ｅ（Frac.E：図４中、一点鎖線で囲まれた画分）に含まれているタンパク質を調べた。当該画分Ｅは、図５に示すスタンダードの移動度と分子量から得た検量線から、分子量約５１ｋＤａ付近であることがわかった。この画分Ｅを１５％のＳＤＳ−ＰＡＧＥに供したところ、図７に示すように、４６〜６６ｋＤａの位置に２本のバンドとして検出された。
また、この画分Ｅを陰イオンクロマトグラフィーに供し、単一のタンパク質に精製した。具体的には、画分Ｅを結合用溶液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）で１２５ｍｇ／５ｍＬに希釈した上で、５ｍＬをＨｉＰｒｅｐ Ｑ ＦＦ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）にアプライした。その後、溶出液（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、０．５Ｍ ＮａＣｌ、ｐＨ８．０）を用いて、移動相中の塩化ナトリウム濃度を図８中の点線で示すような０〜０．５Ｍにグラジエントをかけることにより、タンパク質を溶出させた。なお、移動相の移動速度は１．５ｍＬ／ｍｉｎとした。
各画分の移動度と２８０ｎｍの吸光度との関係を図８に示す。図８中に示す得られたピークのうちの１〜４を、それぞれ１５％のＳＤＳ−ＰＡＧＥに供したところ、図９に示すように、ピーク１から２までは単一なバンドとして検出された。そこで、ピーク１及び２を合わせて回収し、当該画分に含まれている分子に対してＭＡＬＤＩ−ＴＯＦ／ＭＳにて質量分析を行ったところ、ＭＲＪＰ２を含む画分であると判明した。以下、当該画分をＭＲＪＰ２画分という。
<Isolation and purification of MRJP2 from RJ>
The present inventors further focused attention on MRJP2, which was the largest after the MRJP1 fraction quantitatively most in the gel analysis of the complex eluted from the bile acid-binding affinity column. In the gel filtration chromatography shown in FIG. 4, the protein contained in the fraction E (Frac. E: the fraction surrounded by the one-dot chain line in FIG. 4) was examined. The fraction E was found to have a molecular weight of about 51 kDa from the calibration curve obtained from the mobility and molecular weight of the standard shown in FIG. When this fraction E was subjected to 15% SDS-PAGE, it was detected as two bands at a position of 46 to 66 kDa as shown in FIG.
In addition, this fraction E was subjected to anion chromatography and purified to a single protein. Specifically, fraction E was diluted to 125 mg / 5 mL with a binding solution (20 mM Tris-HCl, pH 8.0), and 5 mL was applied to HiPrep Q FF (manufactured by GE Healthcare). Then, using an eluent (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0), apply a gradient from 0 to 0.5 M as indicated by the dotted line in FIG. 8 for the sodium chloride concentration in the mobile phase. To elute the protein. The moving speed of the mobile phase was 1.5 mL / min.
The relationship between the mobility of each fraction and the absorbance at 280 nm is shown in FIG. When 1-4 of the obtained peaks shown in FIG. 8 were each subjected to 15% SDS-PAGE, peaks 1 and 2 were detected as a single band as shown in FIG. . Thus, peaks 1 and 2 were collected together and subjected to mass spectrometry using MALDI-TOF / MS for the molecules contained in the fraction, and found to be a fraction containing MRJP2. Hereinafter, this fraction is referred to as MRJP2 fraction.
＜透析法による胆汁酸結合試験＞
上記で精製されたＭＲＪＰ１及びＭＲＪＰ２の胆汁酸結合能の評価を、透析法により行った。ポジティブコントロールとして抗高コレステロール剤であるコレスチラミンを、ネガティブコントロールとしてカゼインをそれぞれ用いた。
具体的には、タウロコール酸含有リン酸緩衝液（５０ｍＭ タウロコール酸、１００ｍＭリン酸緩衝液、ｐＨ７．４）中に、コレスチラミン、カゼイン、ＭＲＪＰ１画分、ＭＲＪＰ２画分、又は生ＲＪを凍結乾燥させたものを、それぞれ１００ｍｇ／ｍＬとなるように添加したものを反応溶液とした。これらの反応溶液を３７℃で２時間インキュベートした後、室温で７２時間透析した。透析膜外液に含まれている総胆汁酸量を測定することにより、透析膜内液に含まれている（すなわち、各分子と結合した）胆汁酸の割合を算出した。
算出された各分子と結合した胆汁酸の割合を図１０に示す。図１０中、「Ｉｎｔａｃｔ ＲＪ」は、生ＲＪを凍結乾燥させたものを添加した反応溶液の結果である。この結果、胆汁酸結合能はネガティブコントロール群のカゼイン（３３％）と比較し、ＭＲＪＰ１は４６％、ＭＲＪＰ２は３７％であった。すなわち、ＭＲＪＰ１のほうがＭＲＪＰ２よりも高い胆汁酸結合能を示した。
<Bile acid binding test by dialysis method>
The bile acid binding ability of MRJP1 and MRJP2 purified as described above was evaluated by a dialysis method. Cholestyramine, an anti-high cholesterol agent, was used as a positive control, and casein was used as a negative control.
Specifically, cholestyramine, casein, MRJP1 fraction, MRJP2 fraction, or raw RJ is lyophilized in a taurocholic acid-containing phosphate buffer (50 mM taurocholic acid, 100 mM phosphate buffer, pH 7.4). The reaction solution was prepared by adding 100 mg / mL of each solution. These reaction solutions were incubated at 37 ° C. for 2 hours and then dialyzed at room temperature for 72 hours. By measuring the total amount of bile acids contained in the dialysis membrane liquid, the ratio of bile acids contained in the dialysis membrane solution (that is, bound to each molecule) was calculated.
FIG. 10 shows the calculated ratio of bile acids bound to each molecule. In FIG. 10, “Intact RJ” is the result of the reaction solution to which the raw RJ lyophilized was added. As a result, bile acid binding ability was 46% for MRJP1 and 37% for MRJP2 compared to casein (33%) in the negative control group. That is, MRJP1 showed higher bile acid binding ability than MRJP2.
［実施例３］
＜コレステロールミセル溶解性試験＞
ＲＪから単離精製されたＭＲＪＰ１及びＭＲＪＰ２のコレステロールミセル溶解抑制能を測定した。ＭＲＪＰ１及びＭＲＪＰ２は、実施例２において調製したＭＲＪＰ１画分及びＭＲＪＰ２画分をそれぞれ用いた。また、ネガティブコントロールとしてカゼインを用いた。
[Example 3]
<Cholesterol micelle solubility test>
The ability of MRJP1 and MRJP2 isolated and purified from RJ to inhibit cholesterol micelle dissolution was measured. As MRJP1 and MRJP2, the MRJP1 fraction and MRJP2 fraction prepared in Example 2 were used, respectively. Casein was used as a negative control.
まず、最終濃度が３．７ｋＢｑ／ｍＬ（２．１Ｇｂｑ／ｍｍｏｌ，ＮＥＮ）となるように［４−１４Ｃ］コレステロール／クロロホルム（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｌｉｆｅ Ｓｃｉｅｎｃｅ社製）を、２μＭとなるようにコレステロール（片山化学工業社製）／クロロホルムを、２０μＭとなるようにＯｌｅｉｃ ａｃｉｄ（ＳＩＧＭＡ社製）／クロロホルムを、５μＭとなるようにＭｏｎｏ ｏｌｅｏｙｌ−ｒａｃ−Ｇｌｙｃｅｒｏｌ（ＳＩＧＭＡ社製）／クロロホルムを、０．６ｍＭとなるようにＬ−α−ｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ（ＳＩＧＭＡ社製）／クロロホルムを２０ｍＬ容量のガラスバイアルに分取し、よく混合した後、窒素ガスを吹き付け乾固させた。その後、得られた乾固物を、６．６ｍＭのＴａｕｒｏｃｈｏｌｉｃ ａｃｉｄ（ＳＩＧＭＡ社製）及び１３２ｍＭ ＮａＣｌを含む１５ｍＭリン酸緩衝液（ｐＨ７．４）に溶解させた。ボルテックミキサーで２分間攪拌した後、超音波処理（２５Ｗ、ｏｕｔｐｕｔ６、３分間）し、その後３７℃で２４時間振とうしながらインキュベートすることにより、［１４Ｃ］−コレステロールミセル溶液を調製した。 First, [4- 14 C] cholesterol / chloroform (manufactured by Perkin Elmer Life Science) so that the final concentration becomes 3.7 kBq / mL (2.1 Gbq / mmol, NEN) is 2 μM cholesterol (Katayama). Chemical Industries) / chloroform, Oleic acid (manufactured by SIGMA) / chloroform to 20 μM, Monooleyl-rac-Glycerol (manufactured by SIGMA) / chloroform to 0.6 mM to 5 μM As described above, L-α-phosphatidylcholine (manufactured by SIGMA) / chloroform was fractionated into a 20 mL glass vial and mixed well, and then nitrogen gas was blown to dryness. Thereafter, the dried product obtained was dissolved in a 6.6 mM Taurocholic acid (manufactured by SIGMA) and a 15 mM phosphate buffer (pH 7.4) containing 132 mM NaCl. [ 14 C] -cholesterol micelle solution was prepared by stirring with a vortex mixer for 2 minutes, followed by sonication (25 W, output 6, 3 minutes), followed by incubation with shaking at 37 ° C. for 24 hours.
コレステロールミセル溶解性試験は、具体的には、［１４Ｃ］−コレステロールミセル溶液に、最終濃度が１０ｍｇ／ｍＬとなるようにＣａｓｅｉｎ、ＭＲＪＰ１、又はＭＲＪＰ２を添加したものを反応溶液とした。各反応溶液をボルテックミキサーで２分間攪拌した後、超音波処理（２５Ｗ、ｏｕｔｐｕｔ ６、３分間）し、３７℃で１時間インキュベートした後、３８０μＬを３７℃、１００，０００×ｇで1時間遠心分離を行い、上清を回収した。回収された上清５０μＬに、乳化シンチレーターを１０ｍＬ加えて、液体シンチレーションカウンターにより、上清中の［１４Ｃ］−コレステロールを測定した。上清中には、水溶液中に安定して溶解しているコレステロールミセルが存在する。このため、反応溶液に予め添加した［１４Ｃ］−コレステロール量に対する上清中の［１４Ｃ］−コレステロール量の割合から、水溶液中に安定して溶解しているコレステロールミセルの割合が算出できる。 Specifically, the cholesterol micelle solubility test was obtained by adding Casein, MRJP1, or MRJP2 to the [ 14 C] -cholesterol micelle solution so that the final concentration was 10 mg / mL. Each reaction solution was stirred with a vortex mixer for 2 minutes, sonicated (25 W, output 6, 3 minutes), incubated at 37 ° C. for 1 hour, and then centrifuged at 380 μL at 37 ° C. at 100,000 × g for 1 hour. Separation was performed and the supernatant was collected. 10 mL of an emulsion scintillator was added to 50 μL of the collected supernatant, and [ 14 C] -cholesterol in the supernatant was measured with a liquid scintillation counter. In the supernatant, there are cholesterol micelles that are stably dissolved in the aqueous solution. For this reason, the ratio of the cholesterol micelle stably dissolved in the aqueous solution can be calculated from the ratio of the [ 14 C] -cholesterol amount in the supernatant to the [ 14 C] -cholesterol amount added in advance to the reaction solution.
ミセルへのコレステロールの溶解割合を図１１に示す。各試験区は、Ｄｕｎｃａｎ’ｓ ｍｕｌｔｉｐｌｅ ｒａｎｇｅ ｔｅｓｔによりｐ＜０．０５で統計学的な有意差検定処理した。その結果、ネガティブコントロール群のカゼイン（７９％）と比較し、ＭＲＪＰ１は４６％であり、有意に低下していた。これに対してＭＲＪＰ２は９３％であり、有意な上昇が見られた。すなわち、ＭＲＪＰ１はコレステロールミセル溶解抑制能を有することが明らかである。 The dissolution rate of cholesterol in micelles is shown in FIG. Each test section was subjected to statistical significance test by p <0.05 by Duncan's multiple range test. As a result, MRJP1 was 46% compared with casein (79%) in the negative control group, which was significantly decreased. In contrast, MRJP2 was 93%, showing a significant increase. That is, it is clear that MRJP1 has cholesterol micelle dissolution inhibiting ability.
［実施例４］
＜ラットへの経口投与試験＞
高コレステロール血症のモデルである１％コレステロール摂取ラットに、ＲＪから単離精製されたＭＲＪＰ１を経口投与し、血清コレステロールに与える影響を調べた。ＭＲＪＰ１は、実施例２において調製したＭＲＪＰ１画分を用いた。また、ネガティブコントロールとしてカゼインを用いた。
図１２に示すように、実験期間の３日間、コレステロールを含む食餌を与えたラットに、１日１回、ＭＲＪＰ１又はカゼインを経口投与した。３回目の投与から２４時間経過後、４時間絶食させた後、心臓採血によりラットを屠殺した。採取された血液を用いて、血清コレステロールを測定した。さらに解剖し、肝臓重量を測定した。その他の具体的な実験条件は以下の通りである。
[Example 4]
<Oral administration test to rats>
MRJP1 isolated and purified from RJ was orally administered to rats fed with 1% cholesterol, which is a model of hypercholesterolemia, and the effect on serum cholesterol was examined. As MRJP1, the MRJP1 fraction prepared in Example 2 was used. Casein was used as a negative control.
As shown in FIG. 12, MRJP1 or casein was orally administered once a day to rats fed a diet containing cholesterol for 3 days during the experimental period. After 24 hours from the third administration, the animals were fasted for 4 hours and then sacrificed by cardiac blood sampling. Serum cholesterol was measured using the collected blood. Further dissection and liver weight were measured. Other specific experimental conditions are as follows.
実験群：４週齢のＷｉｓｔａｒ系雄ラット（７０ｇ）
実験群数：ｎ＝９
投与開始時間：ＡＭ８：００
試験飼育期間：３日間（予備飼育３日間）
絶食時間：４時間
実験サンプル：カゼインナトリウム（ＣＳ）又はＭＲＪＰ１（ＭＲ）
サンプル投与量：３００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙ、又は６００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙ
食餌組成：２０％カゼイン＋１％コレステロール
Experimental group: 4-week-old Wistar male rats (70 g)
Number of experimental groups: n = 9
Administration start time: AM 8:00
Test breeding period: 3 days (preliminary breeding 3 days)
Fasting time: 4 hours Experimental sample: Sodium caseinate (CS) or MRJP1 (MR)
Sample dose: 300 mg / kg (BW) / day or 600 mg / kg (BW) / day
Diet composition: 20% casein + 1% cholesterol
血清は、３０００ｒｐｍ、１５分間の遠心分離により調製した。血清コレステロールの定量は酵素法、具体的には市販のキット（コレステロールＥ−テストワコー；和光純薬工業社製））を用いて測定した。同様にして、ＨＤＬ−コレステロールはＨＤＬ−コレステロールＥ−テストワコー（和光純薬工業社製）を用いて測定した。ＬＤＬ＋ＶＬＤＬ−コレステロールは計算により求めた。 Serum was prepared by centrifugation at 3000 rpm for 15 minutes. Serum cholesterol was quantified using an enzymatic method, specifically, a commercially available kit (cholesterol E-Test Wako; manufactured by Wako Pure Chemical Industries, Ltd.). Similarly, HDL-cholesterol was measured using HDL-cholesterol E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). LDL + VLDL-cholesterol was obtained by calculation.
各種測定結果などを図１３〜１６に示す。なお、各数値は、１群９匹の平均±標準誤差とし、実験結果の統計的分析には、Ｄｕｎｃａｎ’ｓ ｍｕｌｔｉｐｌｅ ｒａｎｇｅ ｔｅｓｔとＳｔｕｄｅnｔ’ｓ ｔ−ｔｅｓｔを用いた。
各実験群の体重の変化を図１３に、１日当たりの食餌摂取量を図１４に、肝臓重量を図１５に、それぞれ示す。この結果、各群のうち、カゼインナトリウム（ＣＳ）投与群とＭＲＪＰ１（ＭＲ）投与群とでは、いずれも特に差は観察されなかった。
一方、図１６のように、血清総コレステロール量は、３００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙの投与群と６００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙの投与群のいずれにおいても、カゼインナトリウム（ＣＳ）投与群よりもＭＲＪＰ１（ＭＲ）投与群のほうが、血清総コレステロール量が低下する傾向が観察された。特に６００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙの投与群では、カゼインナトリウム（ＣＳ）投与群よりもＭＲＪＰ１（ＭＲ）投与群のほうが、血清総コレステロール量が有意に（Ｄｕｎｃａｎ’ｓ ｍｕｌｔｉｐｌｅ ｒａｎｇｅ ｔｅｓｔとＳｔｕｄｅｕｔ’ｓ ｔ−ｔｅｓｔの両方でｐ＜０．０５）低下していた。
これらの結果から、単離精製されたＭＲＪＰ１を経口投与することにより、血清総コレステロール量を低下させられることが明らかである。
Various measurement results and the like are shown in FIGS. In addition, each numerical value was made into the average +/- standard error of 9 animals per group, and Duncan's multiple range test and Student's t-test were used for the statistical analysis of an experimental result.
The change in body weight of each experimental group is shown in FIG. 13, the daily food intake is shown in FIG. 14, and the liver weight is shown in FIG. As a result, no particular difference was observed between the casein sodium (CS) administration group and the MRJP1 (MR) administration group.
On the other hand, as shown in FIG. 16, the serum total cholesterol amount was sodium caseinate in both the 300 mg / kg (BW) / day administration group and the 600 mg / kg (BW) / day administration group. In the MRJP1 (MR) administration group, a tendency to decrease the total serum cholesterol level was observed compared to the (CS) administration group. Particularly in the administration group of 600 mg / kg (BW) / day, the MRJP1 (MR) administration group showed significantly higher serum total cholesterol (Duncan's multiple range test) than the casein sodium (CS) administration group. Both of the Study's t-tests were reduced by p <0.05).
From these results, it is clear that the total serum cholesterol level can be reduced by oral administration of isolated and purified MRJP1.
［実施例５］
＜ラットへの経口投与試験＞
実験群数を１０匹（ｎ＝１０）、試験飼育期間を７日間（予備飼育３日間）、サンプル投与量を６００ｍｇ／ｋｇ（Ｂ．Ｗ．）／ｄａｙとした以外は、実施例４と同様にして、高コレステロール血症のモデルである１％コレステロール摂取ラットに、ＲＪから単離精製されたＭＲＪＰ１を７日間経口投与し、血清コレステロールに与える影響を調べた。
[Example 5]
<Oral administration test to rats>
Example 4 except that the number of experimental groups was 10 (n = 10), the test breeding period was 7 days (preliminary breeding 3 days), and the sample dose was 600 mg / kg (BW) / day. Thus, MRJP1 isolated and purified from RJ was orally administered to 1% cholesterol-ingested rats, a model of hypercholesterolemia, for 7 days, and the effect on serum cholesterol was examined.
各実験群の体重の変化を図１７に、１日当たりの食餌摂取量を図１８に、肝臓重量を図１９に、血清総コレステロール量を図２０に、それぞれ示す。なお、各数値は、１群１０匹の平均±標準誤差とし、実験結果の統計的分析には、Ｓｔｕｄｅｎｔ’ｓ ｔ−ｔｅｓｔを用いた。
この結果、体重の変化、１日当たりの食餌摂取量、及び肝臓重量は、カゼインナトリウム（ＣＳ）投与群とＭＲＪＰ１（ＭＲ）投与群とでは、いずれも有意差は観察されなかった。
一方、血清総コレステロール量は、カゼインナトリウム（ＣＳ）投与群よりもＭＲＪＰ１（ＭＲ）投与群のほうが、血清総コレステロール量が有意に（約２６％）低下した（Ｓｔｕｄｅｎｔ’ｓ ｔ−ｔｅｓｔでｐ＜０．０５）。特に、血清ＬＤＬコレステロール及び血清ＶＬＤＬコレステロールの総量が有意に低下した。
これらの結果から、単離精製されたＭＲＪＰ１を７日間経口投与することにより、血清総コレステロール量を低下させられることが明らかである。
FIG. 17 shows the change in body weight of each experimental group, FIG. 18 shows the daily food intake, FIG. 19 shows the liver weight, and FIG. 20 shows the total serum cholesterol level. In addition, each numerical value was made into the average +/- standard error of 10 animals per group, and Student's t-test was used for the statistical analysis of an experimental result.
As a result, no significant difference was observed in changes in body weight, daily food intake, and liver weight between the casein sodium (CS) administration group and the MRJP1 (MR) administration group.
On the other hand, the total serum cholesterol level was significantly (about 26%) lower in the MRJP1 (MR) administration group than in the casein sodium (CS) administration group (Student's t-test p < 0.05). In particular, the total amount of serum LDL cholesterol and serum VLDL cholesterol was significantly reduced.
From these results, it is clear that the total serum cholesterol level can be reduced by oral administration of isolated and purified MRJP1 for 7 days.
本発明の胆汁酸結合剤や血清コレステロール量低下方法は、ＲＪ由来の胆汁酸結合剤を経口服用することにより、血清コレステロール量を安全に低下させることができる。このため、本発明の胆汁酸結合剤等は、特に高コレステロール血症や動脈硬化症の治療や予防のための医薬や機能性食品等の有効成分として利用が可能である。 The bile acid binder and the method for lowering serum cholesterol level of the present invention can safely reduce the serum cholesterol level by orally taking an RJ-derived bile acid binder. For this reason, the bile acid binder etc. of this invention can be utilized as an active ingredient, such as a pharmaceutical for a treatment and prevention of hypercholesterolemia and arteriosclerosis especially, and a functional food.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001002577A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Lipid metabolism improver |
| JPWO2004019971A1 (en) * | 2002-08-29 | 2005-12-15 | 林原 健 | Antiallergic agent |
| JP2007137779A (en) * | 2005-11-15 | 2007-06-07 | Pola Chem Ind Inc | Pltp expression inhibitor and food containing the same |
| JP2007137778A (en) * | 2005-11-15 | 2007-06-07 | Pola Chem Ind Inc | Expression accelerator of low density lipoprotein receptor and food containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001002577A (en) * | 1999-06-22 | 2001-01-09 | Biox:Kk | Lipid metabolism improver |
| JPWO2004019971A1 (en) * | 2002-08-29 | 2005-12-15 | 林原 健 | Antiallergic agent |
| JP2007137779A (en) * | 2005-11-15 | 2007-06-07 | Pola Chem Ind Inc | Pltp expression inhibitor and food containing the same |
| JP2007137778A (en) * | 2005-11-15 | 2007-06-07 | Pola Chem Ind Inc | Expression accelerator of low density lipoprotein receptor and food containing the same |
Non-Patent Citations (1)
| Title |
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| JPN6015028194; Ｐｒｏｔｅｏｍｉｃｓ Ｖｏｌ．９, 2009, ｐ．２１３１-８ * |
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