JP6860877B2 - Osteoblast differentiation promoter and bone formation promoter - Google Patents
Osteoblast differentiation promoter and bone formation promoter Download PDFInfo
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- JP6860877B2 JP6860877B2 JP2016193980A JP2016193980A JP6860877B2 JP 6860877 B2 JP6860877 B2 JP 6860877B2 JP 2016193980 A JP2016193980 A JP 2016193980A JP 2016193980 A JP2016193980 A JP 2016193980A JP 6860877 B2 JP6860877 B2 JP 6860877B2
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Description
本発明は、X−Hyp−Gly(式中Xは、Gly、HypおよびGlu以外のアミノ酸残基)で示されるトリペプチドまたはその医学的に許容可能な塩を含有する、骨芽細胞の分化促進剤および骨形成促進剤に関する。 The present invention promotes osteoblast differentiation containing a tripeptide represented by X-Hyp-Gly (where X is an amino acid residue other than Gly, Hyp and Glu) or a medically acceptable salt thereof. Regarding agents and bone formation promoters.
コラーゲンは、細胞内で−(Gly−X−Y)−(式中、XおよびYはそれぞれ同一でも異なってもよいアミノ酸残基を示す。)の繰り返し構造を有するポリペプチド鎖として合成され、Yに位置するProはほとんどがヒドロキシプロリン(Hyp)へと水酸化される。その後、3本のポリペプチド鎖が三重螺旋構造を形成して細胞外へと分泌され、さらにコラーゲン線維となって皮膚、腱、骨などの様々な結合組織に沈着し、主要な細胞外マトリックスタンパク質として機能する。これらの結合組織から抽出したコラーゲンやゼラチン(熱変性コラーゲン)にクロストリジウム属やグリモンティア属由来のコラゲナーゼを作用させると、N末端がGlyの、Gly−X−Yで示されるトリペプチドを生成する。これら微生物由来コラゲナーゼは、YとGlyとの間を切断するエンドプロテアーゼであり、トリペプチドのN末端はGlyとなる。これに対し、C末端がGlyのコラーゲン/ゼラチン由来のトリペプチドも存在する(特許文献1)。コラーゲンまたはゼラチンに、ProまたはHypのC末端側に隣接するアミノ酸残基とその次のアミノ酸残基との間のペプチド結合を切断するショウガ根茎由来酵素を添加すると、X−Hyp−Glyで示されるペプチドを生成することができるという。 Collagen is synthesized intracellularly as a polypeptide chain having a repeating structure of-(Gly-XY)-(in the formula, X and Y each indicate an amino acid residue which may be the same or different), and Y Most of the Pro located in is hydroxylated to hydroxyproline (Hyp). After that, three polypeptide chains form a triple helix structure and are secreted extracellularly, and then become collagen fibers that are deposited in various connective tissues such as skin, tendons, and bones, and are major extracellular matrix proteins. Functions as. When collagenase derived from Clostridium or Glymontia is allowed to act on collagen or gelatin (heat-denatured collagen) extracted from these connective tissues, a tripeptide represented by Gly-XY with an N-terminal of Gly is produced. These microbial collagenases are endoproteases that cleave between Y and Gly, and the N-terminus of the tripeptide is Gly. On the other hand, there is also a collagen / gelatin-derived tripeptide having a Gly C-terminal (Patent Document 1). Addition of ginger rhizome-derived enzyme to collagen or gelatin that cleaves the peptide bond between the amino acid residue adjacent to the C-terminal side of Pro or Hyp and the next amino acid residue is indicated by X-Hyp-Gly. It is said that it can produce peptides.
X−Hyp−Glyで示されるトリペプチドとして、Ala−Hyp−Gly、Leu−Hyp−Gly、Phe−Hyp−Glyなどがある。これらトリペプチドを含むコラーゲン加水分解物を経口摂取すると、血中のX−Hyp−Gly濃度が対照のコラーゲン加水分解物を経口摂取した場合と比較して有意に上昇するとの報告がある(非特許文献1)。近年、コラーゲン加水分解物を経口摂取することにより、Pro−HypやX−Hyp−Gly型トリペプチドなどのコラーゲン由来オリゴペプチドが血中で非常に高濃度で検出されることが報告され、それらペプチドの効果効能に注目が集まっている。X−Hyp−Gly型トリペプチドの生理作用としては、Ala−Hyp−Gly、Pro−Hyp−Glyのコラーゲン合成促進効果(特許文献2)、Glu−Hyp−Gly、Ser−Hyp−Gly、Ala−Hyp−Gly、Leu−Hyp−Gly、Pro−Hyp−Glyのエラスチン産生促進効果(特許文献3)、Glu−Hyp−Gly、Leu−Hyp−Gly、Ala−Hyp−GlyのジペプチジルペプチダーゼIV阻害活性(特許文献4)、Pro−Hyp−Gly、Glu−Hyp−Gly、Ala−Hyp−Gly、Ser−Hyp−Glyのアンジオテンシン変換酵素阻害効果(非特許文献2)などが報告されている。 Examples of the tripeptide represented by X-Hyp-Gly include Ala-Hyp-Gly, Leu-Hyp-Gly, and Ph-Hyp-Gly. It has been reported that when a collagen hydrolyzate containing these tripeptides is orally ingested, the blood X-Hyp-Gly concentration is significantly increased as compared with the case where a control collagen hydrolyzate is orally ingested (non-patent). Document 1). In recent years, it has been reported that collagen-derived oligopeptides such as Pro-Hyp and X-Hyp-Gly type tripeptides are detected in blood at extremely high concentrations by oral ingestion of collagen hydrolysates. Attention is focused on the effects of collagen. The physiological actions of the X-Hyp-Gly type tripeptide include the collagen synthesis promoting effect of Ala-Hyp-Gly and Pro-Hyp-Gly (Patent Document 2), Glu-Hyp-Gly, Ser-Hyp-Gly, and Ala-. Elastin production promoting effect of Hyper-Gly, Leu-Hyp-Gly, Pro-Hyp-Gly (Patent Document 3), Gly-Hyp-Gly, Leu-Hyp-Gly, Ala-Hyp-Gly dipeptidyl peptidase IV inhibitory activity (Patent Document 4), Pro-Hyp-Gly, Glu-Hyp-Gly, Ala-Hyp-Gly, Ser-Hyp-Gly angiotensin-converting enzyme inhibitory effect (Non-Patent Document 2) and the like have been reported.
また、コラーゲン加水分解物摂取による骨代謝改善(特許文献5)、コラーゲン加水分解物による骨分化促進効果(非特許文献3、非特許文献4)などの報告もある。また、コラーゲン加水分解組成物に代えて、Pro−Hypによる骨分化促進効果も報告されている(非特許文献5)。 There are also reports of improvement of bone metabolism by ingestion of collagen hydrolyzate (Patent Document 5) and bone differentiation promoting effect of collagen hydrolyzate (Non-Patent Document 3 and Non-Patent Document 4). Further, it has been reported that Pro-Hyp has an effect of promoting bone differentiation instead of the collagen hydrolyzed composition (Non-Patent Document 5).
更に、Glu−Hyp−Glyを用いた、前駆軟骨細胞または骨芽細胞の増殖促進剤もある(特許文献6)。ペプチド固相合成法で調製したGlu−Hyp−Glyをマウス正常骨芽細胞MC3T3−E1に10nmol/mlで添加してアルカリホスファターゼ活性(以下、ALP活性と称する。)を測定したところ、対照に対して1.8倍という高い骨芽細胞の分化促進作用が示され、および対照に対して1.7倍という高い骨芽細胞の増殖促進率が示されたという。 Further, there is also a proliferative chondrocyte or osteoblast proliferation promoter using Glu-Hyp-Gly (Patent Document 6). Glu-Hyp-Gly prepared by the peptide solid phase synthesis method was added to normal mouse osteoblast MC3T3-E1 at 10 nmol / ml, and alkaline phosphatase activity (hereinafter referred to as ALP activity) was measured. It is said that a high osteoblast differentiation promoting effect of 1.8 times was shown, and an osteoblast proliferation promoting rate of 1.7 times higher than that of the control was shown.
非特許文献5では、Pro−HypによりMC3T3−E1細胞の分化が促進され、特許文献6ではGlu−Hyp−Glyにより、MC3T3−E1細胞のALP活性が増加したというが、より分化能や骨形成促進作用に優れ、また工業的に多量に生産可能なペプチドの開発が望まれる。 In Non-Patent Document 5, Pro-Hyp promoted the differentiation of MC3T3-E1 cells, and in Patent Document 6, Glu-Hyp-Gly increased the ALP activity of MC3T3-E1 cells. It is desired to develop a peptide which has an excellent promoting action and can be industrially produced in a large amount.
上記現状に鑑み、本発明は、X−Hyp−Gly(式中Xは、Pro、Gly、HypおよびGlu以外のアミノ酸残基)で示されるトリペプチドを含む、骨芽細胞の分化促進剤を提供することを目的とする。 In view of the above situation, the present invention provides an osteoblast differentiation-promoting agent containing a tripeptide represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Pro, Gly, Hyp and Glu). The purpose is to do.
また本発明は、X−Hyp−Gly(式中Xは、Gly、HypおよびGlu以外のアミノ酸残基)で示されるトリペプチドを含む、骨形成促進剤を提供することを目的とする。 Another object of the present invention is to provide a bone formation-promoting agent containing a tripeptide represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Gly, Hyp and Glu).
本発明者等は、X−Hyp−Gly(式中Xは、Pro、Gly、HypおよびGlu以外のアミノ酸残基)で示されるトリペプチドについて詳細に検討したところ、これらが骨芽細胞の分化促進作用を有することを見出し、本発明を完成させた。 The present inventors have examined in detail the tripeptides represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Pro, Gly, Hyp and Glu), and found that these promote osteoblast differentiation. We have found that it has an action and completed the present invention.
すなわち本発明は、Ala−Hyp−Gly、Phe−Hyp−Gly、Leu−Hyp−Gly、Ser−Hyp−GlyおよびThr−Hyp−Glyからなる群から選択される1以上のペプチド、またはその医学的に許容可能な塩を含有する、骨芽細胞の分化促進剤を提供するものである。 That is, the present invention is one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly , or medically thereof. It provides an agent for promoting the differentiation of osteoblasts, which contains an acceptable salt in Glycine.
また本発明は、前記トリペプチドが、Ala−Hyp−GlyまたはLeu−Hyp−Glyのいずれかであるペプチド、またはその医学的に許容可能な塩を含有する、前記骨芽細胞の分化促進剤を提供するものである。 The present invention also presents the osteoblast differentiation-promoting agent, wherein the tripeptide contains a peptide of either Ala-Hyp-Gly or Leu-Hyp-Gly, or a medically acceptable salt thereof. It is to provide.
更に、本発明は、前記骨芽細胞の分化促進剤を含む、骨形成促進剤を提供するものである。 Furthermore, the present invention provides a bone formation-promoting agent containing the osteoblast differentiation-promoting agent.
本発明によれば、Ala−Hyp−Gly、Phe−Hyp−Gly、Leu−Hyp−Gly、Ser−Hyp−GlyおよびThr−Hyp−Glyからなる群から選択される1以上のペプチドを含む骨分化促進剤等が提供される。 According to the present invention, bone differentiation containing one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly. Accelerators and the like are provided.
本発明は、Ala−Hyp−Gly、Phe−Hyp−Gly、Leu−Hyp−Gly、Ser−Hyp−GlyおよびThr−Hyp−Glyからなる群から選択される1以上のペプチド、またはその医学的に許容可能な塩を含有する、骨芽細胞の分化促進剤である。以下、本発明を詳細に説明する。
The present invention relates to one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly , or medically thereof. An osteoblast differentiation-promoting agent containing an acceptable salt. Hereinafter, the present invention will be described in detail.
(1)X−Hyp−Gly
本発明で使用するトリペプチドは、X−Hyp−Glyで示され、Xは、Pro、Gly、HypおよびGlu以外のアミノ酸残基である。好ましくは、Ala−Hyp−Gly、Phe−Hyp−Gly、Leu−Hyp−Gly、Ser−Hyp−Gly、Thr−Hyp−Glyである。上記式で示されるトリペプチドを生成するには、特許文献4を参照して、コラーゲンやゼラチンの−(Gly−X−Y)−で示されるペプチド鎖に、1次酵素としてコラゲナーゼや黄色コウジカビ由来プロテアーゼを作用させ、次いで2次酵素としてN末端から2番目にプロリンまたはヒドロキシプロリン以外のアミノ酸が存在する場合にN末端からアミノ酸を遊離させるペプチダーゼを作用させてもよい。また、ProまたはHypのC末端側に隣接するアミノ酸残基とその次のアミノ酸残基との間のペプチド結合を切断するショウガ根茎由来酵素を添加し、その単独の酵素反応によってより簡便かつ多量にX−Hyp−Glyで示されるペプチドを生成することができる。X−Hyp−Glyで示されるペプチドは、これらのペプチド組成物から液体クロマトグラフィーその他の方法で精製して調製することができる。
(1) X-Hyp-Gly
The tripeptides used in the present invention are represented by X-Hyp-Gly, where X is an amino acid residue other than Pro, Gly, Hyp and Glu. Preferably, it is Ala-Hyp-Gly, Phe-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly, Thr-Hyp-Gly. In order to produce the tripeptide represented by the above formula, refer to Patent Document 4, and the peptide chain represented by-(Gly-XY)-of collagen or gelatin is derived from collagenase or yellow red mold as a primary enzyme. A protease may be allowed to act, and then a peptide that releases the amino acid from the N-terminal when an amino acid other than proline or hydroxyproline is present second from the N-terminal as a secondary enzyme may be allowed to act. In addition, a ginger rhizome-derived enzyme that cleaves the peptide bond between the amino acid residue adjacent to the C-terminal side of Pro or Hyp and the next amino acid residue is added, and the enzyme reaction alone makes it easier and more abundant. The peptide represented by X-Hyp-Gly can be produced. The peptides represented by X-Hyp-Gly can be prepared by purifying from these peptide compositions by liquid chromatography or other methods.
また、「固相合成法」や「液相合成法」などの公知のペプチド合成法によって製造することもできる。固相合成法としては、Fmoc法、Boc法の何れであってもよい。表面をアミノ基で修飾した樹脂ビーズを固相として用い、縮合剤としてジイソプロピルカルボジイミド(DIC)を用いる。トリペプチドのC−末端にあたるGlyのN−末端をFmoc基で保護し、上記樹脂ビーズのアミノ基とペプチド結合させる。固相を溶媒で洗浄し、その後、固相に結合しているGlyのFmoc基の脱保護を行う。溶媒で樹脂ビーズを洗浄後、Fmoc−Hyp−OH、DICおよびHOBtをそれぞれ反応溶液に加える。反応後、樹脂ビーズを溶媒で洗浄し、N−末端のFmoc基を脱保護し、溶媒で樹脂ビーズを洗浄する。その後、Fmoc−X−OH、DICおよびHOBtをそれぞれ加え、固相上でペプチドを合成する。合成後に、N−末端のFmoc基を除去し、減圧乾燥した後に得られた乾燥樹脂ビーズをTFAで処理し、樹脂ビーズからX−Hyp−Gly粗生成物をTFAに溶出させる。得られた粗生成物を精製水に溶解し、液体クロマトグラフィー等で精製し、X−Hyp−Glyを単離する。 It can also be produced by a known peptide synthesis method such as "solid phase synthesis method" or "liquid phase synthesis method". The solid-phase synthesis method may be either the Fmoc method or the Boc method. Resin beads whose surface is modified with an amino group are used as a solid phase, and diisopropylcarbodiimide (DIC) is used as a condensing agent. The N-terminal of Gly, which corresponds to the C-terminal of the tripeptide, is protected by an Fmoc group and peptide-bonded to the amino group of the resin beads. The solid phase is washed with a solvent, and then the Fmoc group of Gly bonded to the solid phase is deprotected. After washing the resin beads with a solvent, Fmoc-Hyp-OH, DIC and HOBt are added to the reaction solution, respectively. After the reaction, the resin beads are washed with a solvent, the N-terminal Fmoc group is deprotected, and the resin beads are washed with a solvent. Then, Fmoc-X-OH, DIC and HOBt are added, respectively, to synthesize a peptide on a solid phase. After the synthesis, the N-terminal Fmoc group is removed, the dried resin beads obtained after drying under reduced pressure are treated with TFA, and the X-Hyp-Gly crude product is eluted from the resin beads into TFA. The obtained crude product is dissolved in purified water and purified by liquid chromatography or the like to isolate X-Hyp-Gly.
上記X−Hyp−Glyで示されるトリペプチドは、塩を形成するものであってもよい。塩は、医学的に許容可能な塩であることが好ましい。「医学的に許容可能な塩」とは、薬理学的に許容可能であり、投与された対象に対して略無毒である塩形態をいう。例えば、無毒の有機酸塩または無機酸塩がある。無機酸としては、塩化水素酸、硫酸、またはリン酸などがある。有機酸としては、カルボン酸、ホスホン酸、スルホン酸があり、たとえば酢酸、プロピオン酸、グリコール酸、乳酸、ヒドロキシブチル酸、リンゴ酸、マレイン酸、マロン酸、サリチル酸、フマル酸、琥珀酸、アジピン酸、酒石酸、クエン酸、グルタル酸、2−または3−グリセロリン酸、ならびに当業者には周知の他の鉱物の酸がある。また、無機塩基や有機塩基との塩であってもよい。無機塩基としては、アルカリまたはアルカリ土類金属(たとえば、ナトリウム、カリウム、リチウム、カルシウム、またはマグネシウム)水酸化物、アンモニア、アンモニウム水酸化物などがある。また、有機塩基としては、アルキルアミン、ヒドロキシアルキルアミン、N−メチルグルカミン、ベンジルアミン、ピペリジン、ピロリジンなどがある。 The tripeptide represented by X-Hyp-Gly may form a salt. The salt is preferably a medically acceptable salt. "Medically acceptable salt" refers to a salt form that is pharmacologically acceptable and is substantially non-toxic to the subject to whom it is administered. For example, there are non-toxic organic or inorganic acid salts. Examples of the inorganic acid include hydrochloride, sulfuric acid, and phosphoric acid. Organic acids include carboxylic acids, phosphonic acids and sulfonic acids, such as acetic acid, propionic acid, glycolic acid, lactic acid, hydroxybutyl acid, malic acid, maleic acid, malonic acid, salicylic acid, fumaric acid, amber acid and adipic acid. , Tartrate acid, citric acid, glutaric acid, 2- or 3-glycerophosphate, and other mineral acids known to those of skill in the art. Further, it may be a salt with an inorganic base or an organic base. Inorganic bases include alkali or alkaline earth metal (eg, sodium, potassium, lithium, calcium, or magnesium) hydroxides, ammonia, ammonium hydroxides, and the like. Further, examples of the organic base include alkylamine, hydroxyalkylamine, N-methylglucamine, benzylamine, piperidine, pyrrolidine and the like.
(2)骨芽細胞の分化促進剤
上記X−Hyp−Glyで示されるトリペプチドやその医学的に許容可能な塩は、骨芽細胞の分化促進剤として使用することができる。
骨組織が形成されることを骨化といい、膜内骨化と軟骨内骨化とに大別される。膜内骨化は頭蓋骨等で観察され、間葉系細胞が直接、骨芽細胞に分化して骨基質を産生する骨形成様式をいう。一方、軟骨内骨化とは、未分化間葉系細胞が軟骨細胞に分化して軟骨原基を形成し、軟骨細胞が肥大軟骨細胞に分化し、周囲の軟骨膜に含まれる細胞が骨芽細胞に分化し、これらによって石灰化した軟骨組織を骨組織に置換する骨形成様式をいう。何れの骨形成過程でも骨芽細胞が関与する点で共通する。骨芽細胞の分化が促進されると骨形成が促進される。
(2) Osteoblast Differentiation Promoting Agent The tripeptide represented by X-Hyp-Gly and a medically acceptable salt thereof can be used as an osteoblast differentiation promoting agent.
The formation of bone tissue is called ossification and is roughly divided into intramembranous ossification and endochondral ossification. Intramembranous ossification is observed in the skull and the like, and refers to a bone formation mode in which mesenchymal cells directly differentiate into osteoblasts to produce bone matrix. On the other hand, intrachondral ossification means that undifferentiated mesenchymal cells differentiate into cartilage cells to form cartilage primordia, cartilage cells differentiate into hypertrophic cartilage cells, and cells contained in the surrounding cartilage membrane are osteoblasts. A mode of bone formation that differentiates into cells and replaces the cartilage tissue calcified by these with bone tissue. It is common in that osteoblasts are involved in all bone formation processes. Bone formation is promoted when osteoblast differentiation is promoted.
マウス頭蓋冠由来MC3T3−E1細胞は、胎児マウスの頭蓋骨より樹立された前駆骨芽細胞株である。骨芽細胞へと分化誘導されるとアルカリホスファターゼ(ALP)を発現するため、骨芽細胞の分化の指標としてALP活性が用いられている。よって、MC3T3−E1細胞培養時にサンプルを添加し、未処置対照に対するサンプル添加時のALP活性を求めると、骨芽細胞への分化に対する作用を調べることができる。骨芽細胞の分化誘導は、生体を構成する種々の骨形成にポジティブに寄与して骨形成が促進されると考えられる。ただし、ALP活性の上昇と細胞増殖とは同義ではない。一般の細胞分化の過程では増殖が止まってから分化が起こり、MC3T3−E1細胞も同様である。後記する実施例に示すように、前記X−Hyp−Glyは、骨芽細胞の分化を促進し、ゆえに骨芽細胞の分化促進剤として使用することができる。 The mouse calvaria-derived MC3T3-E1 cell is a precursor osteoblast line established from the skull of a fetal mouse. Alkaline phosphatase (ALP) is expressed when differentiation is induced into osteoblasts, so ALP activity is used as an index of osteoblast differentiation. Therefore, if a sample is added during MC3T3-E1 cell culture and the ALP activity at the time of sample addition to an untreated control is determined, the effect on osteoblast differentiation can be investigated. It is considered that the induction of osteoblast differentiation positively contributes to various bone formations constituting the living body and promotes bone formation. However, increased ALP activity and cell proliferation are not synonymous. In the general process of cell differentiation, differentiation occurs after proliferation has stopped, and the same applies to MC3T3-E1 cells. As shown in Examples described later, the X-Hyp-Gly promotes osteoblast differentiation and can therefore be used as an osteoblast differentiation promoter.
(3)骨形成促進剤
前記X−Hyp−Glyやその医学的に許容可能な塩は、骨形成促進剤として使用することができる。上記したように、前記X−Hyp−Glyで示されるトリペプチドやその医学的に許容可能な塩は、骨芽細胞の分化促進作用があり、この分化促進作用によって骨形成を促進することができる。
(3) Bone Formation Promoting Agent The X-Hyp-Gly or a medically acceptable salt thereof can be used as a bone formation promoting agent. As described above, the tripeptide represented by X-Hyp-Gly and its medically acceptable salt have an osteoblast differentiation promoting action, and this differentiation promoting action can promote bone formation. ..
(4)剤型等
骨芽細胞の分化促進剤や骨形成促進剤は、経口的、または非経口的に投与することができる。経口投与の場合は、前記X−Hyp−Glyで示されるトリペプチドやその医学的に許容可能な塩をそのまま散剤、顆粒剤、丸剤としてもよく、表面に糖衣その他で加工したコーティング剤や、カプセルに充填したカプセル剤、適当な溶媒に溶解した経口液剤や懸濁剤、乳化剤によって乳化してなる乳剤などの何れであってもよい。非経口投与の場合は、ローション剤、軟膏剤、貼付剤(パップ剤)、注射剤、坐剤、点鼻剤等がある。
(4) Dosage form, etc. Osteoblast differentiation promoters and bone formation promoters can be administered orally or parenterally. In the case of oral administration, the tripeptide represented by X-Hyp-Gly or a medically acceptable salt thereof may be used as it is as a powder, a granule, a pill, a coating agent whose surface is processed with a sugar coating or the like, or a coating agent. It may be a capsule filled in a capsule, an oral solution or suspension dissolved in an appropriate solvent, an emulsion obtained by emulsifying with an emulsifier, or the like. In the case of parenteral administration, there are lotions, ointments, patches (pups), injections, suppositories, nasal drops and the like.
上記骨芽細胞の分化促進剤や骨形成促進剤には、剤型に応じて賦形剤、崩壊剤、滑沢剤、保存料、抗酸化剤、界面活性剤、pH調整剤、保湿剤、増粘剤、無機充填剤、結合剤、希釈剤、着色料、香料、紫外線吸収剤などを添加してもよい。なお、X−Hyp−Glyとしては、コラーゲン含有食品を分解して精製したものに限定されず、精製を行わずX−Hyp−Glyを含有するタンパク質加水分解物を使用してもよい。 The osteoblast differentiation promoters and bone formation promoters include excipients, disintegrants, lubricants, preservatives, antioxidants, surfactants, pH regulators, moisturizers, etc., depending on the dosage form. Thickeners, inorganic fillers, binders, diluents, colorants, fragrances, UV absorbers and the like may be added. The X-Hyp-Gly is not limited to the one obtained by decomposing and purifying a collagen-containing food, and a protein hydrolyzate containing X-Hyp-Gly may be used without purification.
骨芽細胞の分化促進剤や骨形成促進剤として使用する際の上記ペプチドの投与量は、対象の状態や体重、剤型、投与経路等によって異なるが、成人1日当たり、経口投与の場合は、1〜5000mg、好ましくは2〜2000mg、より好ましくは5〜500mgである。患部に直接投与する場合は、0.1〜500mg、好ましくは0.2〜200mg、より好ましくは0.5〜50mgである。製剤は、1日1〜数回に分けて投与してもよく、または1〜数日に1回投与してもよい。 The dose of the above peptide when used as an osteoblast differentiation promoter or bone formation promoter varies depending on the condition, body weight, dosage form, administration route, etc. of the subject, but in the case of oral administration per day for adults, It is 1 to 5000 mg, preferably 2 to 2000 mg, and more preferably 5 to 500 mg. When directly administered to the affected area, it is 0.1 to 500 mg, preferably 0.2 to 200 mg, and more preferably 0.5 to 50 mg. The preparation may be administered in 1 to several divided doses per day, or may be administered once in 1 to several days.
上記骨芽細胞の分化促進剤や骨形成促進剤は、飲食物に添加してもよい。このような飲食物として、野菜やフルーツ、乳酸菌などを含むジュースその他の飲料、ゼリー、ヨーグルト、プリン、アイスクリームなどの半流動性食品などがあり、また他の食材に混練して固形食品に調製してもよい。 The above-mentioned osteoblast differentiation-promoting agent and bone formation-promoting agent may be added to foods and drinks. Such foods and drinks include juices and other beverages containing vegetables, fruits, lactic acid bacteria, and semi-fluid foods such as jelly, yogurt, pudding, and ice cream, and are kneaded with other ingredients to prepare solid foods. You may.
次に実施例を挙げて本発明を具体的に説明するが、これらの実施例は何ら本発明を制限するものではない。 Next, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention in any way.
(製造例1)
ショウガ根茎の外皮を除去して細切後−30℃で凍結保存し、この凍結ショウガに対し5倍量(重量/容量)の冷アセトンを添加してホモジナイザーで十分破砕した。この破砕液をろ過して残渣を分取し、再度5倍量の冷アセトンで洗浄・ろ過してから室温で風乾して、ショウガ酵素粉末を得た。
ウシ皮膚由来I型コラーゲン溶液を60℃で30分間加熱して調製したウシゼラチン溶液に、塩酸を加えてpH4.0に調整し、2mMとなるようにジチオトレイトールを添加して5%ゼラチン溶液を調製した。これに、1/10量の前記ショウガ酵素粉末を添加し、振盪しながら50℃で16時間反応させた。反応終了後、ショウガ酵素粉末をろ過により除き、ろ液をさらに限外ろ過(Vivaspin 20−10K、GEヘルスケア社製)した。回収したろ液を凍結乾燥し、ペプチド組成物を得た。このペプチド組成物の重量平均分子量は590であった。ついで、下記LC/MS測定条件で分析し、主要なX−Hyp−Gly型トリペプチドの生成量を定量した。ゼラチン1gあたりの主要なトリペプチドの生成量を表1に示す。
(Manufacturing example 1)
The exodermis of ginger rhizome was removed, shredded and stored frozen at −30 ° C., and 5 times the amount (weight / volume) of cold acetone was added to the frozen ginger and sufficiently crushed with a homogenizer. The crushed solution was filtered to separate the residue, which was washed and filtered again with 5 times the amount of cold acetone, and then air-dried at room temperature to obtain ginger enzyme powder.
A bovine skin-derived type I collagen solution was prepared by heating at 60 ° C. for 30 minutes, and hydrochloric acid was added to adjust the pH to 4.0, and dithiotreitol was added to make a 5% gelatin solution. Was prepared. To this, 1/10 amount of the ginger enzyme powder was added, and the mixture was reacted at 50 ° C. for 16 hours with shaking. After completion of the reaction, the ginger enzyme powder was removed by filtration, and the filtrate was further ultrafiltered (Vivaspin 20-10K, manufactured by GE Healthcare). The recovered filtrate was freeze-dried to obtain a peptide composition. The weight average molecular weight of this peptide composition was 590. Then, the analysis was performed under the following LC / MS measurement conditions, and the amount of major X-Hyp-Gly type tripeptide produced was quantified. Table 1 shows the amount of major tripeptide produced per 1 g of gelatin.
(結果)
ショウガ根茎由来酵素を用いることでゼラチンから様々なX−Hyp−Gly型トリペプチドが多量に生成された。中でもAla−Hyp−Glyの生成量は7.49mg/g、Leu−Hyp−Glyの生成量は7.99mg/gと、他のX−Hyp−Gly型トリペプチドと比較し、特に高いペプチド生成が示された。
(result)
A large amount of various X-Hyp-Gly type tripeptides were produced from gelatin by using an enzyme derived from ginger rhizome. Among them, the amount of Ala-Hyp-Gly produced was 7.49 mg / g, and the amount of Leu-Hyp-Gly produced was 7.99 mg / g, which was particularly high compared to other X-Hyp-Gly type tripeptides. It has been shown.
(1)LC/MS測定条件
高速液体クロマトグラフ:1200Series(Agilent Technologies)、
質量分析装置:3200QTRAP(AB Sciex)、
分析カラム:Ascentis Express F5 5μm, 4.6mmi.d.×250mm(SUPELCO)、
カラム温度:40℃、
移動相:A液;10mM酢酸アンモニウム、B液;100%アセトニトリル、
グラジエント条件:
0〜7.5分:A液100%、
7.5〜20分:A液100〜25%;B液0〜75%、
20〜25分:A液25%;B液75%、
25〜30分:A液100%、
流速:0.4mL/min、
(2)質量分析条件:
イオン化:ESI、ポジティブ、
分析モード:Multiple Reaction Monitoring(MRM)モード、
イオンスプレー電圧:3kV、
イオンソース温度:700℃
(1) LC / MS measurement conditions High-performance liquid chromatograph: 1200Series (Agilent Technologies),
Mass spectrometer: 3200QTRAP (AB Sciex),
Analytical column: Ascentis Express F5 5 μm, 4.6 mm i.d. × 250 mm (SUPELCO),
Column temperature: 40 ° C,
Mobile phase: Solution A; 10 mM ammonium acetate, Solution B; 100% acetonitrile,
Gradient condition:
0-7.5 minutes: Solution A 100%,
7.5 to 20 minutes: Solution A 100 to 25%; Solution B 0 to 75%,
20 to 25 minutes: Solution A 25%; Solution B 75%,
25 to 30 minutes: Solution A 100%,
Flow velocity: 0.4 mL / min,
(2) Mass spectrometry conditions:
Ionization: ESI, positive,
Analysis mode: Multiple Reaction Monitoring (MRM) mode,
Ion spray voltage: 3kV,
Ion source temperature: 700 ° C
(実施例1)
マウス頭蓋冠由来骨芽細胞株MC3T3−E1細胞を用いて、表1に記載される各X−Hyp−Gly型トリペプチドの骨芽細胞分化促進効果について評価を行った。なお、表1に記載されるトリペプチドは、Anygen社のカスタム合成サービスにより化学合成したものを使用した。
MC3T3−E1細胞は、10%ウシ胎児血清含有アルファMEM中に5×104細胞/ウェルの条件で24−ウェル細胞培養プレート中に準備した。翌日、細胞がコンフルエントになっていることを確認後、培地を分化培地(10%透析済みウシ胎児血清(Thermo Scientific社製)、100μg/mLアスコルビン酸、10mMグリセロール2−リン酸二ナトリウム含有アルファMEM)で置換し、表1に示すトリペプチドを終濃度200nmol/mLになるように添加した。対照には、トリペプチドに代えて蒸留水を同量添加した。2日おきに分化培地を交換し、表1に示すトリペプチドを終濃度200nmol/mLになるように添加した。6日間培養後、リン酸緩衝生理食塩水(PBS)で洗浄し、3.7%ホルムアルデヒドにて固定した。
(Example 1)
Using mouse calvaria-derived osteoblast line MC3T3-E1 cells, the osteoblast differentiation promoting effect of each X-Hyp-Gly type tripeptide shown in Table 1 was evaluated. As the tripeptides shown in Table 1, those chemically synthesized by a custom synthesis service of Anygen Co., Ltd. were used.
MC3T3-E1 cells were prepared in 24-well cell culture plates under the condition of 5 × 10 4 cells / well in alpha MEM containing 10% fetal bovine serum. The next day, after confirming that the cells were confluent, the medium was differentiated medium (10% dialed fetal bovine serum (manufactured by Thermo Scientific), 100 μg / mL ascorbic acid, alpha MEM containing 10 mM glycerol 2-sodium phosphate. ), And the tripeptide shown in Table 1 was added to a final concentration of 200 nmol / mL. As a control, the same amount of distilled water was added instead of the tripeptide. The differentiation medium was changed every two days, and the tripeptides shown in Table 1 were added to a final concentration of 200 nmol / mL. After culturing for 6 days, the cells were washed with phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde.
PBS、Tween20を含むトリス緩衝生理食塩水、Tween20を含むTNM溶液(0.1M Tris−HCl、pH9.5、0.1M NaCl、0.05M MgCl2)で洗浄後、NBT/BCIP(18.75mg/mL p−ニトロブルーテトラゾリウムクロリド、9.4mg/mL 5−ブロモ−4−クロロ−3−インドリルリン酸p−トルイジン塩を67%メチルスルホキシド(v/v)に溶解した液)をTNM溶液で50倍希釈し、各ウェルに300μLずつ添加してALP染色を行った。Image Jにてウェル中でALP陽性を示す面積を数値化した。 After washing with PBS, Tris buffered saline containing Tween 20, and TNM solution containing Tween 20 (0.1M Tris-HCl, pH 9.5, 0.1M NaCl, 0.05M MgCl 2 ), NBT / BCIP (18.75 mg) / ML p-nitroblue tetrazolium chloride, 9.4 mg / mL 5-bromo-4-chloro-3-indrill phosphate p-toluidine salt (solution of 67% methyl phospoxide (v / v)) in TNM solution It was diluted 50 times with, and 300 μL was added to each well for ALP staining. The area showing ALP positive in the well was quantified by Image J.
N=3で独立して2回くりかえし、計6サンプルの平均値および標準誤差を、対照を1とした相対評価で示した。t検定にて、対照に対してp<0.05で統計的有意差ありと設定した。結果を表2に示す。 It was repeated twice independently at N = 3, and the mean value and standard error of a total of 6 samples were shown by relative evaluation with 1 as the control. In the t-test, it was set that there was a statistically significant difference with respect to the control at p <0.05. The results are shown in Table 2.
(比較例1)
トリペプチドに代えてPro−Hyp(Bachem社製)を使用した以外は実施例2と同様に操作し、ALP活性を算出した。結果を表2に示す。
(Comparative Example 1)
The ALP activity was calculated in the same manner as in Example 2 except that Pro-Hyp (manufactured by Bachem) was used instead of the tripeptide. The results are shown in Table 2.
(結果)
MC3T3−E1細胞に各X−Hyp−Gly型トリペプチドおよびPro−Hypを添加すると、それぞれALP活性が上昇した。さらに、ALP活性の上昇はPro−Hypでは対照に対して1.4倍であったのに対し、各トリペプチド全てでそれ以上のALP活性の上昇を示した。MC3T3−E1細胞は、骨芽細胞へと分化誘導されるとALPを発現するため、各トリペプチドは、骨芽細胞の分化誘導作用が高いと考えられる。中でもAla−Hyp−GlyおよびLeu−Hyp−Glyは特に高い骨芽細胞の分化誘導作用を持つことが示された。
(result)
Addition of each X-Hyp-Gly type tripeptide and Pro-Hyp to MC3T3-E1 cells increased ALP activity, respectively. Furthermore, the increase in ALP activity was 1.4-fold in Pro-Hyp compared to the control, whereas all the tripeptides showed a further increase in ALP activity. Since MC3T3-E1 cells express ALP when they are induced to differentiate into osteoblasts, it is considered that each tripeptide has a high effect of inducing differentiation of osteoblasts. Among them, Ala-Hyp-Gly and Leu-Hyp-Gly were shown to have a particularly high osteoblast differentiation-inducing effect.
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