JP2013053115A - METHOD OF PRODUCING α CYCLIC DIPEPTIDE - Google Patents
METHOD OF PRODUCING α CYCLIC DIPEPTIDE Download PDFInfo
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Abstract
Description
本発明は、α環状ジペプチドの製造方法及びその用途に関する。 The present invention relates to a method for producing an α-cyclic dipeptide and its use.
２以上のアミノ酸が結合した環状ペプチドは、様々な生理活性を有することが近年明らかになりつつあり、研究が盛んになりつつある。例えば、カイコ蛹エキスの鎮痛活性を示す画分からは環状ジグルタミルペプチドが単離されている。また、特に低分子ペプチド（例えばジ又はトリペプチド等の比較的低分子量のペプチド）は細胞膜透過性を有しているため、早くから研究が盛んであり、各種低分子ペプチドが研究又は臨床現場に供給されてきた。 In recent years, it has become clear that cyclic peptides in which two or more amino acids are bound have various physiological activities, and research is being actively conducted. For example, a cyclic diglutamyl peptide has been isolated from the fraction showing the analgesic activity of the silkworm cocoon extract. In particular, since low molecular weight peptides (eg, relatively low molecular weight peptides such as di- or tripeptides) have cell membrane permeability, research has been active from an early stage, and various low molecular weight peptides have been supplied to research or clinical settings. It has been.
しかしながら、ペプチドを合成する場合には、原料がアミノ酸であることがほとんどであり、アミノ酸はカルボキシル基やアミノ基を有するため、通常段階的な保護基の導入を要する。このため、副生成物が多く、収率がよくない場合が多い。また、高純度のものを得るためには、個別のプロセスごとに有機溶媒を用いた複雑な精製を行わなくてはならないことがほとんどであり、製造コストがかさむうえ、環境にも負荷をかけるおそれがある。 However, when synthesizing a peptide, the raw material is mostly an amino acid, and the amino acid has a carboxyl group or an amino group, so that it is usually necessary to introduce a protective group stepwise. For this reason, there are many by-products and the yield is often not good. In addition, in order to obtain a high-purity product, it is often necessary to carry out complicated purification using an organic solvent for each individual process, which increases the manufacturing cost and may place a burden on the environment. There is.
このため、新規低分子ペプチドの探求のみならず、既知低分子ペプチドの簡便且つ効率のよい製造方法の探求も盛んに行われている。例えば特許文献１には、環状ジグルタミルペプチドの効率的な製造方法が記載されている。 For this reason, not only the search for a novel low molecular weight peptide but also the search for a simple and efficient production method of a known low molecular weight peptide has been actively conducted. For example, Patent Document 1 describes an efficient method for producing a cyclic diglutamyl peptide.
本発明は、簡便かつ効率よく環状ペプチドを製造できる方法の提供を課題とする。 An object of the present invention is to provide a method capable of producing a cyclic peptide simply and efficiently.
本発明者らは、驚くべき事に、特定のアミノ酸を溶解した酸性溶液を加熱することにより、極めて簡便且つ効率的にα環状ジペプチドを製造できることを見出し、さらに改良を重ねて本発明を完成させるに至った。なお、α環状ジペプチドとは、２つのアミノ酸において、一方をアミノ酸Ａ、もう一方をアミノ酸Ｂとした場合、アミノ酸Ａのαカルボキシル基がアミノ酸Ｂのアミノ基と脱水縮合し、アミノ酸Ａのアミノ基がアミノ酸Ｂのαカルボキシル基と脱水縮合した構造を有する、環状ジペプチドである。 The present inventors have surprisingly found that an α-cyclic dipeptide can be produced extremely simply and efficiently by heating an acidic solution in which a specific amino acid is dissolved, and further improvements are made to complete the present invention. It came to. The α cyclic dipeptide means that when two amino acids are amino acid A and the other is amino acid B, the α carboxyl group of amino acid A undergoes dehydration condensation with the amino group of amino acid B, and the amino group of amino acid A It is a cyclic dipeptide having a structure dehydrated and condensed with the α carboxyl group of amino acid B.
すなわち、本発明は例えば以下の項に記載のα環状ジペプチドの製造方法及び血中尿酸値低下剤を包含する。
項１．
一般式（１）：
That is, the present invention includes, for example, the α-cyclic dipeptide production method and blood uric acid level-lowering agent described in the following section.
Item 1.
General formula (1):
〔式中、Ｘはアルキレン基を示す。好ましくは炭素数２〜５のアルキレン基を示す。〕
で表されるアミノ酸又はその塩を溶解した酸性溶液を、加熱する工程を含む、α環状ジペプチドの製造方法。
項２．
加熱が１１０℃以上で行われる、項１に記載のα環状ジペプチドの製造方法。
項３．
一般式（１）で表されるアミノ酸又はその塩を溶解した酸性水溶液又は酸性含水アルコール溶液を、加熱する工程を含む、項１又は２に記載のα環状ジペプチドの製造方法。
項４．
一般式（１）：
[Wherein, X represents an alkylene group. An alkylene group having 2 to 5 carbon atoms is preferable. ]
The manufacturing method of alpha cyclic dipeptide including the process of heating the acidic solution which melt | dissolved the amino acid represented by these, or its salt.
General formula (1):
〔式中、Ｘはアルキレン基を示す。好ましくは炭素数２〜５のアルキレン基を示す。〕
で表されるアミノ酸が溶解した酸性溶液を、加熱して得られる組成物を含む、血中尿酸値低下剤。
項５．
一般式（１）：
[Wherein, X represents an alkylene group. An alkylene group having 2 to 5 carbon atoms is preferable. ]
A blood uric acid level-lowering agent comprising a composition obtained by heating an acidic solution in which an amino acid represented by
General formula (1):
〔式中、Ｘはアルキレン基を示す。好ましくは炭素数２〜５のアルキレン基を示す。〕
で表されるアミノ酸又はその塩、
及び／又は
一般式（２）：
[Wherein, X represents an alkylene group. An alkylene group having 2 to 5 carbon atoms is preferable. ]
An amino acid represented by
And / or general formula (2):
〔式中、Ｘはアルキレン基を示す。好ましくは炭素数２〜５のアルキレン基を示す。〕
で表されるα環状ジペプチド、
を含む、血中尿酸値低下剤。
[Wherein, X represents an alkylene group. An alkylene group having 2 to 5 carbon atoms is preferable. ]
An α-cyclic dipeptide represented by
A blood uric acid level-lowering agent containing.
本発明に係るα環状ジペプチドの製造方法によれば、非常に簡便にα環状ジペプチドが製造できる。また、原料の立体化学（Ｌ体かＤ体か）に依存したジペプチドが選択的に合成されるため、Ｌ−Ｄ体が生成される場合に比べて分離精製が非常に容易である。さらに、当該製造方法によれば、従来の方法に比べ収率が非常に高い。またさらに有機溶媒を用いずとも合成ができるため、環境への負荷も少ない上、精製せずとも生体へ適用することもできる。さらに、当該製造方法で得られる反応生成物は、血中尿酸値低下作用を示す。よって、当該反応生成物は、特に高尿酸血症や痛風等の予防又は治療に有用である。 According to the method for producing an α cyclic dipeptide according to the present invention, an α cyclic dipeptide can be produced very simply. In addition, since dipeptides depending on the stereochemistry (L-form or D-form) of the raw material are selectively synthesized, separation and purification are very easy as compared with the case where the LD form is produced. Furthermore, according to the manufacturing method, the yield is very high as compared with the conventional method. Furthermore, since the synthesis can be performed without using an organic solvent, the load on the environment is small, and it can be applied to a living body without purification. Furthermore, the reaction product obtained by the production method exhibits a blood uric acid level lowering action. Therefore, the reaction product is particularly useful for preventing or treating hyperuricemia and gout.
以下、本発明について、さらに詳細に説明する。 Hereinafter, the present invention will be described in more detail.
本発明のα環状ペプチドの製造方法に用いるアミノ酸は、下記一般式（１）で表されるアミノ酸（以下「アミノ酸（１）」とも表記する）である。また、当該アミノ酸の塩を用いることもできる。 The amino acid used in the method for producing an α-cyclic peptide of the present invention is an amino acid represented by the following general formula (1) (hereinafter also referred to as “amino acid (1)”). Moreover, the salt of the said amino acid can also be used.
式中、Ｘは、アルキレン基（−（ＣＨ２）ｎ−）を示す。当該アルキレン基は、炭素数（すなわちｎ数）１〜１０のものが好ましく、１〜８のものがより好ましく、１〜６のものがさらに好ましく、２、３、４、５又は６のものがよりさらに好ましい。中でも炭素数２〜５のアルキレン基が好ましく、−（ＣＨ２）３−又は−（ＣＨ２）４−が特に好ましい。なお、Ｘが−（ＣＨ２）３−のときアミノ酸（１）はオルニチンを、Ｘが−（ＣＨ２）４−のときアミノ酸（１）はリジンを、それぞれ示す。 In the formula, X represents an alkylene group (— (CH 2 ) n —). The alkylene group preferably has 1 to 10 carbon atoms (that is, n number), more preferably 1 to 8, more preferably 1 to 6, and more preferably 2, 3, 4, 5 or 6. Even more preferred. Among them, an alkylene group having 2 to 5 carbon atoms is preferable, and — (CH 2 ) 3 — or — (CH 2 ) 4 — is particularly preferable. When X is — (CH 2 ) 3 —, amino acid (1) represents ornithine, and when X is — (CH 2 ) 4 —, amino acid (1) represents lysine.
アミノ酸（１）には、その不斉炭素原子によるＤ体及びＬ体が存在し得るが、本発明に用いるアミノ酸（１）は、これらキラリティーは特に制限されず、適宜適当なものを選択して用い得る。すなわち、本発明に用いるアミノ酸（１）は、当該Ｌ体及びＤ体の何れであってもよいし、これらの混合物（例えばラセミ体）であってもよい。なお、本発明に係るα環状ジペプチドの製造方法により、製造されるジペプチドは、たとえＤ体及びＬ体の混合物を用いた場合であっても、Ｌ−Ｄ体はほとんど製造されないことから、それぞれＬ−Ｌ体およびＤ‐Ｄ体が原料におけるＤＬ混合比に応じて製造される。 The amino acid (1) can exist in the D-form and L-form due to its asymmetric carbon atom, but the chirality of the amino acid (1) used in the present invention is not particularly limited, and an appropriate one can be selected as appropriate. Can be used. That is, the amino acid (1) used in the present invention may be either the L-form or the D-form, or a mixture thereof (for example, a racemate). In addition, since the dipeptide manufactured by the manufacturing method of (alpha) cyclic dipeptide which concerns on this invention is a case where the mixture of D body and L body is used, since LD body is hardly manufactured, each L -L body and DD body are produced according to the DL mixing ratio in the raw material.
アミノ酸（１）の塩を用いる場合、本発明の効果を損なわない限り、塩としては特に制限されないが、無機塩であることが好ましい。例えばアミノ酸（１）の塩酸塩、硫酸塩、硫酸水素塩、リン酸塩等を用い得る。本発明に係るα環状ペプチドの製造方法では、アミノ酸（１）又はその塩を溶解した酸性溶液を用いるため、特に塩酸塩等であれば、溶媒に溶解させるだけで酸性溶液となり得るため、好ましい。 When the salt of amino acid (1) is used, the salt is not particularly limited as long as the effects of the present invention are not impaired, but an inorganic salt is preferable. For example, hydrochloride, sulfate, hydrogen sulfate, phosphate, etc. of amino acid (1) can be used. In the method for producing an α-cyclic peptide according to the present invention, an acidic solution in which amino acid (1) or a salt thereof is dissolved is used. In particular, hydrochloride or the like is preferable because it can be converted into an acidic solution only by dissolving in a solvent.
なお、アミノ酸（１）又はその塩は、公知の物質であるか、公知の方法により容易に製造することができ、容易に入手できる。市販品を購入して用いることもできる。 Amino acid (1) or a salt thereof is a known substance, or can be easily produced by a known method, and can be easily obtained. Commercial products can also be purchased and used.
本発明に係る、α環状ジペプチドの製造方法においては、上述したアミノ酸（１）又はその塩は、溶媒に溶解させて用いられる。ここで使用する溶媒としては、水性溶媒が好ましい。水性溶媒とは、水を主に含有する溶媒であり、より具体的には、水又は含水アルコールが例示できる。当該含水アルコールは、水の含有量（ｗ／ｗ％）が５０％以上であり、好ましくは６０％以上、より好ましくは７０％以上、さらに好ましくは８０％以上、よりさらに好ましくは９０％以上である。また、含水アルコールに含まれるアルコールとしては、炭素数１〜４のアルキルアルコールが好ましく、中でもエタノールが好ましい。なお、溶媒として特に好ましいのは水である。水を用いた場合、環境への負荷も少ない上、精製せずとも生体へ適用することもできる。 In the method for producing an α-cyclic dipeptide according to the present invention, the above-mentioned amino acid (1) or a salt thereof is used after being dissolved in a solvent. The solvent used here is preferably an aqueous solvent. The aqueous solvent is a solvent mainly containing water, and more specifically, water or hydrous alcohol can be exemplified. The water-containing alcohol has a water content (w / w%) of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and still more preferably 90% or more. is there. Moreover, as alcohol contained in a hydrous alcohol, a C1-C4 alkyl alcohol is preferable and especially ethanol is preferable. Particularly preferred as the solvent is water. When water is used, the load on the environment is small, and it can be applied to a living body without purification.
また、アミノ酸（１）又はその塩を溶媒に溶解した溶液（以下「アミノ酸（１）溶液」ともいう）は、酸性としたうえで用いられる。ここでの酸性とは、ｐＨが７未満であることをいい、好ましくは約６．５以下、より好ましくは０．３〜６．５程度である。なお、ｐＨは２５℃においてＫＳ７２３ｐＨメーター（３点校正）により測定した値である。 A solution in which amino acid (1) or a salt thereof is dissolved in a solvent (hereinafter also referred to as “amino acid (1) solution”) is used after being made acidic. Acidity here means that pH is less than 7, Preferably it is about 6.5 or less, More preferably, it is about 0.3-6.5. The pH is a value measured at 25 ° C. with a KS723 pH meter (3-point calibration).
アミノ酸（１）溶液のｐＨを酸性に調整するため、公知のｐＨ調整剤を用いることができる。例えば、塩酸、硫酸、硫酸水素塩（例えば硫酸水素カリウム、硫酸水素ナトリウム等）等を用いることができる。また、上述の通り、アミノ酸（１）の塩のうち、溶媒に溶解させるだけで溶液のｐＨが酸性となるものが存在するので、このようなアミノ酸（１）の塩を用いる場合は、必ずしもｐＨ調整剤を用いてｐＨを調整する必要はない。なお、アミノ酸（１）溶液のｐＨが中性又はアルカリ性（即ちｐＨが７以上）の場合は、目的とするα環状ジペプチドはほとんど製造できない。 In order to adjust the pH of the amino acid (1) solution to be acidic, a known pH adjuster can be used. For example, hydrochloric acid, sulfuric acid, hydrogen sulfate (for example, potassium hydrogen sulfate, sodium hydrogen sulfate, etc.) can be used. Further, as described above, among the salts of amino acid (1), there are those in which the pH of the solution becomes acidic only by dissolving in the solvent. Therefore, when such a salt of amino acid (1) is used, the pH is not necessarily limited. There is no need to adjust the pH with a regulator. In addition, when the pH of the amino acid (1) solution is neutral or alkaline (that is, pH is 7 or more), the target α-cyclic dipeptide can hardly be produced.
アミノ酸（１）溶液に含まれるアミノ酸（１）又はその塩の量は、本発明の効果が損なわれない限り特に制限されない。溶解可能な限界量まで溶解させてもよい。例えば、０．１〜１０ｗ／ｖ％程度の量が例示される。 The amount of amino acid (1) or a salt thereof contained in the amino acid (1) solution is not particularly limited as long as the effects of the present invention are not impaired. You may make it melt | dissolve to the limit amount which can be melt | dissolved. For example, an amount of about 0.1 to 10 w / v% is exemplified.
ｐＨが酸性であるアミノ酸（１）溶液を加熱することにより、２個のアミノ酸（１）のαカルボキシル基及びアミノ基が脱水縮合し、α環状ジペプチドが生成する。当該α環状ジペプチドの一般式を次の一般式（２）に示す。 By heating the amino acid (1) solution having an acidic pH, the α carboxyl group and amino group of the two amino acids (1) undergo dehydration condensation to form an α cyclic dipeptide. The general formula of the α cyclic dipeptide is shown in the following general formula (2).
式中、Ｘは、前記と同じである。 In the formula, X is the same as described above.
なお、以下一般式（２）で示されるα環状ジペプチドを「α環状ジペプチド（２）」ともいう。
また、当該反応において想定される反応式を以下に示す。
Hereinafter, the α-cyclic dipeptide represented by the general formula (2) is also referred to as “α-cyclic dipeptide (2)”.
Moreover, the reaction formula assumed in the said reaction is shown below.
式中、Ｘは前記と同じである。 In the formula, X is the same as described above.
加熱温度は、本願発明の効果が著しく損なわれなければ（特に、α環状ジペプチドが生成される限り）、特に制限されないが、好ましくは約１１０℃以上、より好ましくは約１１５℃以上である。加熱温度上限は本発明の効果が損なわれない限り特に制限はされないが、例えば約１５０℃以下が例示できる。なお、加熱方法も特に制限されず、例えば前記の温度まで加熱が可能な公知の方法又は装置を用いて行うことができる。例えば、酸性のアミノ酸（１）溶液を密封耐圧試験管にいれ、これをデジタル加熱ブロックにセットして所望の温度で加熱することができる。 The heating temperature is not particularly limited as long as the effects of the present invention are not significantly impaired (particularly as long as an α-cyclic dipeptide is produced), but is preferably about 110 ° C. or higher, more preferably about 115 ° C. or higher. The upper limit of the heating temperature is not particularly limited as long as the effect of the present invention is not impaired, and for example, about 150 ° C. or less can be exemplified. The heating method is not particularly limited, and for example, it can be performed using a known method or apparatus capable of heating to the above temperature. For example, an acidic amino acid (1) solution can be placed in a sealed pressure test tube, set in a digital heating block, and heated at a desired temperature.
また、加熱時間も特に制限されず、適宜設定することができる。例えば、２４〜１４４時間（好ましくは４８〜１０８時間）程度の時間が例示される。 Also, the heating time is not particularly limited and can be set as appropriate. For example, a time of about 24 to 144 hours (preferably 48 to 108 hours) is exemplified.
加熱後、反応溶液中にはα環状ジペプチドが生成されている。当該反応溶液をそのまま用いることもできるし、例えば減圧乾固又は凍結乾燥させ、粉体とした上で保存及び使用することもできる。また、特に反応液中にＫＨＳＯ４を加えた場合は、減圧乾固後、メタノールを加え懸濁して、これをろ過することにより、残渣（残留ＫＨＳＯ４）と目的物を分離することが出来る。また、シリカゲルクロマトグラフィー等の公知の方法を用いて、さらにα環状ジペプチド（２）を精製することもできる。 After the heating, α-cyclic dipeptide is produced in the reaction solution. The reaction solution can be used as it is, or can be stored and used after being dried in a vacuum or freeze-dried to form a powder. In particular, when KHSO 4 is added to the reaction solution, the residue (residual KHSO 4 ) and the target product can be separated by drying and drying under reduced pressure, suspending methanol and filtering it. Further, the α-cyclic dipeptide (2) can be further purified using a known method such as silica gel chromatography.
このようにして調製される反応生成物（主成分はアミノ酸（１）及び／又はα環状ジペプチド（２））は、例えば試薬や医薬候補化合物として利用することができる。 The reaction product thus prepared (the main component is amino acid (1) and / or α-cyclic dipeptide (2)) can be used as, for example, a reagent or a drug candidate compound.
本発明は、このようにして得られる当該反応生成物の用途にも関する。当該反応生成物の主成分はアミノ酸（１）及び／又はα環状ジペプチド（２）であるので、本発明はアミノ酸（１）及び／又はα環状ジペプチド（２）の用途にも関するといえる。具体的には、本発明は、当該反応生成物を含む血中尿酸値低下剤を包含する。また、本発明は、アミノ酸（１）及び／又はα環状ジペプチド（２）を含む血中尿酸値低下剤を包含する。 The present invention also relates to the use of the reaction product thus obtained. Since the main component of the reaction product is amino acid (1) and / or α-cyclic dipeptide (2), it can be said that the present invention also relates to the use of amino acid (1) and / or α-cyclic dipeptide (2). Specifically, the present invention includes a blood uric acid level-lowering agent containing the reaction product. The present invention also includes a blood uric acid level-lowering agent comprising amino acid (1) and / or α-cyclic dipeptide (2).
以下、これらの血中尿酸値低下剤をまとめて「本発明の尿酸値低下剤」ということがある。本発明の尿酸値低下剤は、医薬分野及び食品分野で用いられ得る。また、“当該反応生成物”、又は“アミノ酸（１）及び／又はα環状ジペプチド（２）”を、医薬分野で用いられる場合「有効成分」ということが、食品分野で用いられる場合「関与成分」ということが、ある。 Hereinafter, these blood uric acid level lowering agents may be collectively referred to as “the uric acid level lowering agent of the present invention”. The uric acid level-lowering agent of the present invention can be used in the pharmaceutical field and food field. In addition, when “the reaction product” or “amino acid (1) and / or α-cyclic dipeptide (2)” is used in the pharmaceutical field, it is referred to as “active ingredient”. "There is.
本発明の尿酸値低下剤を医薬分野にて用いる場合、当該剤（以下「本発明に係る医薬剤」と記載することがある）は、有効成分そのものであってもよいし、これと他の薬理活性成分、薬学的に許容される基剤、担体、添加剤（例えば溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤等）等が必要に応じて配合され、錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤等の医薬製剤に調製されたものでもよい。本発明に係る医薬剤における有効成分含有量は、例えば０．１〜１００重量％であり得る。このような本発明に係る医薬剤は、特に経口投与により、血漿尿酸値を低下させることができる。このため、本発明に係る医薬剤は、特に高尿酸血症及び痛風の予防又は治療に好適に用いることができる。よって、当該医薬剤は、特に高尿酸血症患者、痛風患者に用いるのに好適である。なお、本発明に係る医薬剤の有効成分の一日摂取量は、患者の年齢や性別、病状の重篤度等に応じて、適宜設定することができる。 When the uric acid level-lowering agent of the present invention is used in the pharmaceutical field, the agent (hereinafter sometimes referred to as “the pharmaceutical agent according to the present invention”) may be the active ingredient itself, or other Pharmacologically active ingredients, pharmaceutically acceptable bases, carriers, additives (eg solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, etc.) are required And may be prepared into pharmaceutical preparations such as tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like. The active ingredient content in the pharmaceutical agent according to the present invention may be, for example, 0.1 to 100% by weight. Such a pharmaceutical agent according to the present invention can lower the plasma uric acid level, particularly by oral administration. Therefore, the pharmaceutical agent according to the present invention can be suitably used particularly for the prevention or treatment of hyperuricemia and gout. Therefore, the pharmaceutical agent is particularly suitable for use in hyperuricemia patients and gout patients. In addition, the daily intake of the active ingredient of the pharmaceutical agent according to the present invention can be appropriately set according to the age and sex of the patient, the severity of the medical condition, and the like.
本発明の尿酸値低下剤を食品添加剤として用いる場合、当該剤（以下「本発明に係る食品添加剤」と記載することがある）は、関与成分そのものであってもよいし、これと食品衛生学上許容される基剤、担体、添加剤や、その他食品添加剤として利用され得る成分・材料が適宜配合されたものでもよい。本発明に係る食品添加剤における関与成分含有量は、例えば０．１〜１００重量％であり得る。また、このような食品添加剤の形態としては、例えば液状、粉末状、フレーク状、顆粒状、ペースト状のものが挙げられるがこれらに限定されない。具体的には、調味料（醤油、ソース、ケチャップ、ドレッシング等）、フレーク（ふりかけ）、焼き肉のたれ、スパイス、ルーペースト（カレールーペースト等）等が例示できる。このような食品添加剤は、常法に従って適宜調製することができる。 When the uric acid level-lowering agent of the present invention is used as a food additive, the agent (hereinafter sometimes referred to as “the food additive according to the present invention”) may be the involved component itself or this and the food. Hygienically acceptable bases, carriers, additives, and other components and materials that can be used as food additives may be appropriately blended. The content of the components involved in the food additive according to the present invention may be, for example, 0.1 to 100% by weight. Examples of such food additives include, but are not limited to, liquids, powders, flakes, granules, and pastes. Specific examples include seasonings (soy sauce, sauce, ketchup, dressing, etc.), flakes (sprinkles), grilled meat sauce, spices, roux paste (carrille paste, etc.) and the like. Such food additives can be appropriately prepared according to conventional methods.
このような本発明に係る食品添加剤は、該食品添加剤が添加された食品を食べることにより摂取される。なお、当該添加は食品調理中又は製造中に行ってもよいし、調理済みの食品を食べる直前又は食べながら行ってもよい。当該食品添加剤はこのようにして経口摂取することにより、血中尿酸値低下効果を発揮し、痛風等の症状の改善効果を奏する。当該食品添加物は、高尿酸血症患者、痛風患者の他、尿酸値が高めの人にも有用である。なお、本発明に係る食品添加剤の関与成分の一日摂取量は適宜設定することができる。 Such a food additive according to the present invention is ingested by eating a food to which the food additive is added. In addition, the said addition may be performed during food preparation or manufacture, and may be performed immediately before eating cooked food or while eating. When the food additive is taken orally in this way, it exhibits an effect of lowering the blood uric acid level and an effect of improving symptoms such as gout. The food additive is useful not only for hyperuricemia patients and gout patients but also for people with high uric acid levels. In addition, the daily intake of the component in which the food additive which concerns on this invention is concerned can be set suitably.
本発明の尿酸値低下剤を血中尿酸値低下用の飲食品として用いる場合、当該剤（以下「本発明に係る飲食品」と記載することがある）は、関与成分と、食品衛生学上許容される基剤、担体、添加剤や、その他食品として利用され得る成分・材料等が適宜配合されたものである。関与成分が配合されてなる飲食品ということもできる。例えば、関与成分を含む、血中尿酸値低下用の加工食品、飲料、健康食品（栄養機能食品、特定保健用食品等）、サプリメント、病者用食品（病院食、病人食又は介護食等）等が例示できる。また、関与成分を、飲料類（ジュース等）、菓子類（例えばガム、チョコレート、キャンディー、ビスケット、クッキー、おかき、煎餅、プリン、杏仁豆腐等）、パン類、スープ類（粉末スープ等を含む）、加工食品等の各種飲食品に含有させたものであってもよい。このような、関与成分を含んでなる飲食品である抗アトピー性皮膚炎剤は、血中尿酸値を低下させるために好ましく用いることができる。 When the uric acid level-lowering agent of the present invention is used as a food / beverage product for lowering blood uric acid level, the agent (hereinafter sometimes referred to as “the food / beverage product according to the present invention”), Acceptable bases, carriers, additives, and other ingredients and materials that can be used as foods are appropriately blended. It can also be said to be a food or drink comprising a component involved. For example, processed foods, beverages, health foods (nutrient functional foods, foods for specified health use, etc.), supplements, foods for sick people (hospital foods, sick foods, nursing foods, etc.) for reducing blood uric acid levels, including components involved Etc. can be illustrated. In addition, the ingredients involved include beverages (juice etc.), confectionery (eg gum, chocolate, candy, biscuits, cookies, rice cakes, rice crackers, pudding, apricot tofu etc.), breads, soups (including powdered soups etc.) It may be contained in various foods and beverages such as processed foods. Such an anti-atopic dermatitis agent that is a food or drink comprising the component concerned can be preferably used in order to reduce the blood uric acid level.
健康食品（栄養機能食品、特定保健用食品等）、サプリメントとして、当該飲食品からなる尿酸値低下剤を調製する場合は、継続的な摂取が行いやすいように、例えば顆粒、カプセル、錠剤（チュアブル剤等を含む）、飲料（ドリンク剤）等の形態で調製することが好ましく、なかでもカプセル、タブレット、錠剤、ドリンク剤の形態が摂取の簡便さの点からは好ましいが、特にこれらに限定されるものではない。顆粒、カプセル、錠剤、ドリンク剤等の形態の当該飲食品からなる尿酸値低下剤は、薬学的及び／又は食品衛生学的に許容される担体等を用いて、常法に従って適宜調製することができる。また、他の形態に調製する場合であっても、従来の方法に従えばよい。 When preparing a uric acid level-lowering agent consisting of the food or drink as a health food (nutrient functional food, food for specified health use, etc.) or supplement, for example, granules, capsules, tablets (chewable) It is preferable to prepare in the form of beverages (drinks) and the like. Among them, capsules, tablets, tablets and drinks are preferable from the viewpoint of ease of ingestion, but are not particularly limited to these. It is not something. The uric acid level-lowering agent comprising the food or drink in the form of granules, capsules, tablets, drinks, etc. can be appropriately prepared according to a conventional method using a pharmaceutically and / or food hygienically acceptable carrier. it can. Moreover, even if it is a case where it prepares in another form, what is necessary is just to follow the conventional method.
本発明に係る飲食品の摂取量、摂取対象等は、例えば上述した本発明に係る食品添加剤のそれらと同様であることが好ましい。 It is preferable that the amount of food and drink ingested according to the present invention, the ingestion target, and the like are the same as those of the food additive according to the present invention described above, for example.
なお、病院食とは病院に入院した際に供される食事であり、病人食は病人用の食事であり、介護食とは被介護者用の食事である。本発明に係る飲食品は、特に高尿酸血症又は痛風（及び何らかの合併症を患っている）患者であって、入院、自宅療養等されている者、あるいは介護を受けられている者用の病院食、病人食又は介護食として好ましく用いることができる。 The hospital food is a meal provided when hospitalized, the sick food is a meal for the sick, and the care food is a meal for the care recipient. The food and drink according to the present invention is especially for patients with hyperuricemia or gout (and suffering from some complications) who are hospitalized, treated at home, or who are receiving nursing care. It can be preferably used as hospital food, sick food or nursing food.
本発明は、高尿酸血症又は痛風（及び何らかの合併症を患っている）患者又は哺乳動物に対し、本発明の尿酸値低下剤を経口投与又は摂取することを特徴とする高尿酸血症又は痛風の改善方法及び治療方法をも提供する。当該方法は、具体的には、前述の本発明の尿酸値低下剤を投与又は摂取することで実施される。なお、当該方法における、経口投与又は摂取量等の各条件は前述の通りである。 The present invention relates to hyperuricemia or gout characterized by orally administering or ingesting a uric acid level-lowering agent of the present invention to a patient or mammal having hyperuricemia or gout (and suffering from some complication). Methods for improving and treating gout are also provided. Specifically, this method is carried out by administering or ingesting the aforementioned uric acid level-lowering agent of the present invention. In this method, each condition such as oral administration or intake is as described above.
以下、本発明を具体的に説明するが、本発明は下記の例に限定されるものではない。
実施例１．α環状ジペプチドの製造
以下のようにしてオルニチンからα環状オルニチニックジペプチド（以下「ＡＣＯＰ」ともいう）を製造した。なお、L(+)-オルニチン(塩酸塩)、D(-)-オルニチン(塩酸塩)、硫酸水素カリウム、炭酸水素カリウムは、いずれも和光純薬（株）の特級試薬を購入して用いた。塩酸はナカライテスク製標準試薬（6 mol/L）を用時調製して用いた。
Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples.
Example 1. Production of α-cyclic dipeptide An α-cyclic ornithine dipeptide (hereinafter also referred to as “ACOP”) was produced from ornithine as follows. L (+)-ornithine (hydrochloride), D (-)-ornithine (hydrochloride), potassium hydrogen sulfate, and potassium hydrogen carbonate were purchased from Wako Pure Chemical Co., Ltd. . Hydrochloric acid was prepared by using a standard reagent (6 mol / L) manufactured by Nacalai Tesque.
密封耐圧試験管（１０ｍＬ サイズ）に約１％（ｗ／ｖ）となるように原料（Ｌ−オルニチン、Ｄ−オルニチン、又はＬ−オルニチン及びＤ−オルニチンの等量混合物）５０ｍｇを量り採り、これに５ｍＬの精製水を加えて溶解させた。さらに適量のＫＨＳＯ４又はＨＣｌを加え、あるいは加えず、専用のスクリューキャップで密封した（ＫＨＳＯ４を添加した場合の結果を表１に示す。ＫＨＳＯ４の添加量を表１に記載する）。これを、アルミブロック式のデジタル加熱ブロック（東京理化、ＭＧ−２０００）を用い、９６時間１２０℃で加熱してＡＣＯＰを製造した。この後、反応液をそのまま減圧乾固（又は凍結乾燥）し、反応生成物を得た。 Weigh 50 mg of raw material (L-ornithine, D-ornithine, or an equal mixture of L-ornithine and D-ornithine) so that it becomes approximately 1% (w / v) in a sealed pressure test tube (10 mL size). 5 mL of purified water was added and dissolved. Further adding an appropriate amount of KHSO 4 or HCl, or not added, (describing amount of .KHSO 4 shown in Table 1 the results of the case of adding KHSO 4 in Table 1) was sealed with special screw cap. This was heated at 120 ° C. for 96 hours using an aluminum block digital heating block (Tokyo Rika, MG-2000) to produce ACOP. Thereafter, the reaction solution was directly dried under reduced pressure (or lyophilized) to obtain a reaction product.
製造されたＡＣＯＰを含む反応生成物を１Ｈ−ＮＭＲ（４００ＭＨｚ、：４５℃の重水（Ｄ２Ｏ）中で測定）で解析し、原料とＡＣＯＰのシグナル面積の総和に占めるＡＣＯＰシグナル面積値の相対比率（％）を、目的物の収率とした。結果を表１に示す。 The produced reaction product containing ACOP was analyzed by 1 H-NMR (400 MHz, measured in heavy water (D 2 O) at 45 ° C.), and the ACOP signal area value in the total of the signal area of the raw material and ACOP was calculated. The relative ratio (%) was defined as the yield of the target product. The results are shown in Table 1.
なお、ＮＭＲ解析においては、オルニチン様構造の対称体ダイマーであるＡＣＯＰは、２つのオルニチン様構造が与える個々のシグナルそれぞれが、全く同位置に同じ形で現れるため、“ＡＣＯＰシグナル”は、オルニチンのそれと比較すると相対的には２倍の面積シグナルを示すことになる。例えば、表１において、ＫＨＳＯ４の添加量０％でＬ体オルニチンを４時間反応させた場合、ＡＣＯＰ生成率は４２．８％であるが、これはＡＣＯＰシグナル：オルニチンシグナルの面積比が２：２．７という結果が得られたところから、｛２／（２＋２．７）｝×１００≒４２．８と算出した値であり、この場合、ＡＣＯＰとオルニチンのモル比は１：２．７ということになる（“ＡＣＯＰシグナル”は、相対的には２倍の面積シグナルを示すため）。そして、当該モル比並びにＡＣＯＰ及びオルニチン塩酸塩の分子量（それぞれ２２８．１５及び１６８．６２）から、この反応生成物に含まれるＡＣＯＰとオルニチン塩酸塩の重量比は約１：２であることも算出できる。 In the NMR analysis, ACOP, which is a symmetric dimer having an ornithine-like structure, appears in the same form at the same position as each signal given by the two ornithine-like structures. In comparison with that, the area signal is relatively doubled. For example, in Table 1, when L form ornithine was reacted for 4 hours with 0% addition of KHSO 4 , the ACOP production rate was 42.8%, which means that the area ratio of ACOP signal: ornithine signal was 2: Since the result of 2.7 was obtained, it was a value calculated as {2 / (2 + 2.7)} × 100≈42.8. In this case, the molar ratio of ACOP to ornithine is 1: 2.7. ("ACOP signal" is a relatively double area signal). From the molar ratio and the molecular weight of ACOP and ornithine hydrochloride (228.15 and 168.62 respectively), it is also calculated that the weight ratio of ACOP and ornithine hydrochloride contained in this reaction product is about 1: 2. it can.
また、表１において、ＫＨＳＯ４の添加量が０％の反応溶液のｐＨは５．８〜６．０であり、ＫＨＳＯ４の添加量が０．１６％の反応溶液のｐＨは約０．７であり、ＫＨＳＯ４の添加量が０．３４％以上のものの反応溶液のｐＨは０．７より小さい。また、原料としてＬ−オルニチンを用いた場合はＬ−Ｌ体が得られ、Ｄ−オルニチンを用いた場合はＤ−Ｄ体が得られた。原料としてＬ−オルニチン及びＤ−オルニチンの等量混合物を用いた場合であっても、Ｌ−Ｌ体及びＤ−Ｄ体のみが得られ、Ｌ−Ｄ体は得られなかった。ＡＣＯＰのＬ−Ｌ体の構造式を下に示す。 In Table 1, the pH of the reaction solution with 0% KHSO 4 added is 5.8 to 6.0, and the pH of the reaction solution with 0.16% KHSO 4 added is about 0.7. The pH of the reaction solution having an addition amount of KHSO 4 of 0.34% or more is less than 0.7. Further, when L-ornithine was used as a raw material, an LL isomer was obtained, and when D-ornithine was used, a DD isomer was obtained. Even when an equal amount mixture of L-ornithine and D-ornithine was used as a raw material, only the LL form and DD form were obtained, and the LD form was not obtained. The structural formula of ACOP LL form is shown below.
なお、特に反応液中にＫＨＳＯ４を加えた場合は、減圧乾固後、メタノールを加え懸濁して、これをろ過することにより、残渣（残留ＫＨＳＯ４）と目的物を分離することが出来る。また、シリカゲルクロマトグラフィー等の公知の方法を用いて、さらにＡＣＯＰを精製することもできる。 In particular, when KHSO 4 is added to the reaction solution, the residue (residual KHSO 4 ) and the target product can be separated by drying and drying under reduced pressure, suspending methanol and filtering it. Further, ACOP can be further purified using a known method such as silica gel chromatography.
本発明に係るα環状ジペプチドの製造方法によれば、非常に簡便にα環状ジペプチドが製造できることが表１より明らかである。また、原料の立体化学（Ｌ体かＤ体か）に依存したジペプチドが選択的に合成されるため、Ｌ−Ｄ体が精製される場合に比べて分離精製が非常に容易である。さらに、当該製造方法によれば、従来の方法に比べ収率が非常に高い。またさらに有機溶媒を用いずとも合成ができるため、環境への負荷も少ない上、精製せずとも生体へ適用することもできる。 It is clear from Table 1 that an α-cyclic dipeptide can be manufactured very simply according to the method for manufacturing an α-cyclic dipeptide according to the present invention. In addition, since dipeptides depending on the stereochemistry (L-form or D-form) of the raw material are selectively synthesized, separation and purification are very easy as compared with the case where the LD form is purified. Furthermore, according to the manufacturing method, the yield is very high as compared with the conventional method. Furthermore, since the synthesis can be performed without using an organic solvent, the load on the environment is small, and it can be applied to a living body without purification.
実施例２．反応生成物が有する効果の検討
実施例１において、原料としてＬ−オルニチンを用いて製造された反応生成物（減圧乾固物）を血漿中尿酸濃度が高濃度となったモデルマウスへ投与し、その効果を検討した。具体的には次のようにして行った。
Example 2 Examination of effect of reaction product In Example 1, a reaction product (vacuum dried product) produced using L-ornithine as a raw material was administered to a model mouse having a high plasma uric acid concentration, The effect was examined. Specifically, it was performed as follows.
オキソン酸カリウム 0.125 g を0.5 %CMC 溶液 1 mL に溶解し、オキソン酸0.5 ％カルボキシメチルセルロース（CMC）懸濁液を調製した。そして、8週齢の雄性ICR マウスに250 mg/kg の当該オキソン酸0.5 ％CMC懸濁液を腹腔内投与することにより、高尿酸血症モデルマウスを作製した。当該腹腔内投与から1 時間後に、当該高尿酸血症モデルマウスに対して2 g/kg の試料をゾンデで強制経口投与した。なお、当該試料は反応生成物（表１において、ＫＨＳＯ４の添加量０％でＬ体オルニチンを４時間反応させて得た反応組成物）６０mgを生理食塩水300 μLに溶解させたものである。また、対照実験として、高尿酸血症モデルマウスに、生理食塩水３００μＬをゾンデで強制経口投与した。 0.125 g of potassium oxonate was dissolved in 1 mL of 0.5% CMC solution to prepare a 0.5% carboxymethylcellulose (CMC) suspension of oxonate. Then, hyperuricemia model mice were prepared by intraperitoneally administering 250 mg / kg of the oxonic acid 0.5% CMC suspension to 8-week-old male ICR mice. One hour after the intraperitoneal administration, a 2 g / kg sample was forcibly administered orally with a sonde to the hyperuricemia model mouse. The sample was prepared by dissolving 60 mg of a reaction product (reaction composition obtained by reacting L-ornithine for 4 hours with 0% addition of KHSO 4 in Table 1) in 300 μL of physiological saline. . As a control experiment, 300 μL of physiological saline was forcibly orally administered to a hyperuricemia model mouse with a sonde.
試料を経口投与してから１、２又は３時間後（すなわち、オキソン酸を投与してから２、３又は４時間後）にマウスの尾静脈から採血し（ｎ＝１０）、得られた血漿を用いて次のようにして血漿尿酸値を定量した。血漿 １０μＬ、ＭｅＯＨ １００μＬ、ＣＨＣｌ３ １００μＬ、Ｈ２Ｏ ９０μＬ、を順次コスモスピンフィルターＨへ加え、よく混和した。（６サンプルずつ処理を行った）。これを遠心機で４０分間遠心ろ過し、除蛋白を行った。ろ液を遠心式加熱デシケーターＭｉｃｒｏ ｖａｃ ＭＶ−１００を用いて、９０分間減圧乾燥させた。チューブ中の乾燥残渣にＨ２Ｏ １５μＬを加えた。そして、チューブの蓋を閉めてソニケーターで混和し、遠心機で壁面の水滴を遠沈し、均一化させた後３μＬを採り、下記条件でＨＰＬＣにより分析して、血中尿酸値の定量を行った。ＨＰＬＣ分析は同一血漿について３回繰り返した。３回の平均値をＨＰＬＣ分析結果とした。 Blood was collected from the tail vein of mice (n = 10) at 1, 2 or 3 hours after administration of the sample orally (that is, 2, 3 or 4 hours after administration of oxonic acid). Was used to quantify plasma uric acid levels as follows. 10 μL of plasma, 100 μL of MeOH, 100 μL of CHCl 3 and 90 μL of H 2 O were sequentially added to the Cosmo spin filter H and mixed well. (6 samples were processed). This was subjected to centrifugal filtration with a centrifuge for 40 minutes for deproteinization. The filtrate was dried under reduced pressure for 90 minutes using a centrifugal heating desiccator Micro vac MV-100. 15 μL of H 2 O was added to the dry residue in the tube. Then, close the tube lid, mix with a sonicator, centrifuge the water droplets on the wall with a centrifuge, homogenize, take 3 μL, analyze by HPLC under the following conditions, and quantify the blood uric acid level It was. HPLC analysis was repeated three times for the same plasma. The average of three times was used as the HPLC analysis result.
＜HPLC 分析条件＞
Column:COSMOSIL 4.6 ×250 mm 5C18-AR-II(type : water)
inject：3 μL Column temp.：30 ℃
solvent：30 m mol/L リン酸水溶液 flow rate：1.0 ｍL/min
detector: UV（280 nm） RANGE：0.02 ATTEN：1
<HPLC analysis conditions>
Column: COSMOSIL 4.6 × 250 mm 5C18-AR-II (type: water)
inject: 3 μL Column temp .: 30 ℃
solvent: 30 ml / L phosphoric acid aqueous solution flow rate: 1.0 ml / min
detector: UV (280 nm) RANGE: 0.02 ATTEN: 1
そして、当該分析結果をもとにt 検定 (Bonferroni の修正t 検定)を行った。
結果を図１に示す。図中、正常群は何ら処理をしていないマウスの群を示し、対照群は高尿酸血症モデルマウスに生理食塩水を経口投与させた群を示し、ＤＡＣＯＰ投与群はＡＣＯＰを含む反応生成物を経口投与させた群を示す。図１から、Ｌ−オルニチンを用いて製造された反応生成物（特にＬ−Ｌ体のＡＣＯＰ）は、特に高尿酸血症において血中尿酸値を低下させる作用を有することが明らかになった。
Based on the analysis results, a t-test (Bonferroni's modified t-test) was performed.
The results are shown in FIG. In the figure, the normal group shows a group of mice not treated at all, the control group shows a group in which physiological saline is orally administered to hyperuricemia model mice, and the DACOP administration group is a reaction product containing ACOP The group which orally administered was shown. FIG. 1 reveals that the reaction product produced using L-ornithine (especially LL ACOP) has the effect of lowering the blood uric acid level particularly in hyperuricemia.
Claims (5)
で表されるアミノ酸又はその塩を溶解した酸性溶液を、加熱する工程を含む、α環状ジペプチドの製造方法。 General formula (1):
The manufacturing method of alpha cyclic dipeptide including the process of heating the acidic solution which melt | dissolved the amino acid represented by these, or its salt.
で表されるアミノ酸が溶解した酸性溶液を、加熱して得られる組成物を含む、血中尿酸値低下剤。 General formula (1):
A blood uric acid level-lowering agent comprising a composition obtained by heating an acidic solution in which an amino acid represented by
で表されるアミノ酸又はその塩、
及び／又は
一般式（２）：
で表されるα環状ジペプチド、
を含む、血中尿酸値低下剤。 General formula (1):
An amino acid represented by
And / or general formula (2):
An α-cyclic dipeptide represented by
A blood uric acid level-lowering agent containing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015194035A1 (en) * | 2014-06-20 | 2015-12-23 | サントリーホールディングス株式会社 | Cyclic dipeptide-containing composition |
| WO2015194034A1 (en) * | 2014-06-20 | 2015-12-23 | サントリーホールディングス株式会社 | Uric acid level lowering agent |
| CN105254576A (en) * | 2014-02-26 | 2016-01-20 | 中国科学院长春应用化学研究所 | Method for preparing glutamic piperazinedione |
| WO2017038799A1 (en) * | 2015-09-04 | 2017-03-09 | サントリーホールディングス株式会社 | Urease activity inhibitor |
-
2011
- 2011-09-06 JP JP2011193879A patent/JP2013053115A/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254576A (en) * | 2014-02-26 | 2016-01-20 | 中国科学院长春应用化学研究所 | Method for preparing glutamic piperazinedione |
| WO2015194035A1 (en) * | 2014-06-20 | 2015-12-23 | サントリーホールディングス株式会社 | Cyclic dipeptide-containing composition |
| WO2015194034A1 (en) * | 2014-06-20 | 2015-12-23 | サントリーホールディングス株式会社 | Uric acid level lowering agent |
| WO2015194205A1 (en) * | 2014-06-20 | 2015-12-23 | サントリーホールディングス株式会社 | Cyclic dipeptide-containing composition |
| CN105407907A (en) * | 2014-06-20 | 2016-03-16 | 三得利控股株式会社 | Uric acid level lowering agent |
| JPWO2015194205A1 (en) * | 2014-06-20 | 2017-04-20 | サントリーホールディングス株式会社 | Cyclic dipeptide-containing composition |
| US9650415B2 (en) | 2014-06-20 | 2017-05-16 | Suntory Holdings Limited | Uric acid-lowering agent |
| WO2017038799A1 (en) * | 2015-09-04 | 2017-03-09 | サントリーホールディングス株式会社 | Urease activity inhibitor |
| JPWO2017038799A1 (en) * | 2015-09-04 | 2018-07-12 | サントリーホールディングス株式会社 | Urease activity inhibitor |
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