JP4585172B2 - Bone formation promoter - Google Patents

Description

【００２０】
【化２】

【００２１】
【化３】

【００２２】
ここで、「有効成分」とは、本発明の骨形成促進剤において、哺乳動物等の骨芽細胞を増殖する効果を有する成分を意味し、前記スフィンゴ糖脂質が本発明の骨形成促進剤の主成分である必要は無い。また、本発明の骨形成促進剤にはスフィンゴ糖脂質以外に骨芽細胞増殖効果又は骨形成促進効果を有する成分が含まれていても良い。本発明の骨形成促進剤の有効成分は、棘皮動物から抽出精製して製造することが可能であり、該成分としては、例えば、棘皮動物を抽出原料として得られる抽出物、該抽出物の希釈液又は濃縮液、該抽出液を乾燥して得られる乾燥物、若しくはこれらの粗精製物又は精製物等が用いられる。棘皮動物としては、具体的に、海星〔ヒトデ〕綱（例えば、ホシヒトデ、アオヒトデ、ヤツデスナヒトデ、オニヒトデ、ムラサキヒトデ等）、蛇尾〔クモヒトデ〕綱（例えば、スナクモヒトデ、ニホンクモヒトデ等）、海胆〔ウニ〕綱（例えば、ムラサキウニ、バフンウニ等）、海鼠〔ナマコ〕綱（例えば、クロナマコ、シロナマコ等）、及び海百合〔ウミユリ〕綱（例えば、トリノアシ、ニッポンウミシダ、コアシウミシダ、オオウミシダ等）等が例示され、これらから前記有効成分を抽出精製することが可能である。
【００２３】
本発明の骨形成促進剤の有効成分の製造方法の一例としては、好適には、図２に示すフローチャートに基づいた方法が例示され、その具体内容は、次に記載するとおりである。即ち、棘皮動物をミキサーにてホモジナイズし、次いで、クロロホルムとメタノールの混合液で抽出を行い、ろ過した後に抽出液を濃縮する。これに水を加えて懸濁した後、酢酸エチルとｎ−ブタノールの混合液を添加して、分配操作を行い、酢酸エチル／ｎ−ブタノール層（α）と水層（β）に分取し、それぞれ別々に抽出操作を続ける。酢酸エチル／ｎ−ブタノール層（α）を、濃縮した後にアセトンを加えて混合し、アセトン可溶性画分とアセトン不溶性画分に分取する。次いで、各々の画分中に含まれる有機溶媒を揮発させてエキスＡ（アセトン可溶性画分）、エキスＢ（アセトン不溶性画分）を調製する。先に分取した水層（β）に水飽和ｎ−ブタノールを添加して分配操作を行い、ｎ−ブタノール層（γ）と水層（σ）に分取する。ｎ−ブタノール層（γ）の有機溶媒を揮発させてエキスＣとする。また、水層（σ）を濃縮した後に、６０％メタノールに溶解する。成分の分離には溶出液であるメタノール溶液の濃度を６０〜１００％まで段階的に上昇させて溶出させるステップワイズグラジエント法により逆相カラムクロマトグラフィーを行い、１００％メタノール溶出画分の有機溶媒を揮発させてエキスＤを調製する。調製した各エキスを、それぞれ引き続き順相カラムクロマトグラフィー等による分離工程に供することによって、エキスＡからセラミド、エキスＢからセレブロシド、エキスＣからラクトシルセラミド、及びエキスＤからスルファチド、ガングリオシド、又はスフィンゴリン糖脂質、をそれぞれ精製することができる。
【００２４】
本発明の骨形成促進剤の抽出精製の際に使用する溶媒は、特に限定されるものではないが、水、有機溶媒又はこれらの混合物を使用することが好ましい。抽出溶媒として使用し得る水には、水に各種処理（例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧の調整、緩衝化等）を施したもの、例えば、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。抽出溶媒として使用し得る有機溶媒としては、酢酸エチル、酢酸ブチルなどのエステル、メタノール、エタノール、ブタノール、イソプロピルアルコールなどのアルコール、ヘプタン、ヘキサン、イソオクタンなどの脂肪属炭化水素、アセトン、メチルエチルケトンなどのケトン、ベンゼン、トルエンなどの芳香族化合物、ジエチルエーテル、ｔ−ブチルエーテルなどのエーテル、塩化メチレン、クロロホルム、１，２−ジクロロエタンなどの低級脂肪族炭化水素化合物等が例示され、中でも、メタノール、ｎ−ブタノール、クロロホルムを使用することが好ましい。尚、製造に使用する原料の棘皮動物の種類や、製造する有効成分（スフィンゴ糖脂質）の種類に応じて、有機溶媒を適宜選択し、濃度を調整することができる。また、数種の有機溶媒を混合して使用することもでき、その混合比等は適宜調整することが可能である。
【００２５】
また、本発明の骨形成促進剤の有効成分の製造方法の中で使用される分離方法としては、好適には、吸着クロマトグラフィー、分配クロマトグラフィー、薄層クロマトグラフィー、高速液体クロマトグラフィー（ＨＰＬＣ）、ゲル充填のカラムクロマトグラフィー等を１種又は数種を組合わせて使用する方法が例示される。本発明の骨形成促進剤の有効成分は、前記製造方法によって棘皮動物から得られたものである。本発明の骨形成促進剤の有効成分であるスフィンゴ糖脂質の中で、α−オキシ脂肪酸及びスフィンゴシン骨格を有する化合物、α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有する化合物は、それぞれ単独で使用することも、あるいは複数種の化合物を選択して混合物として使用することも可能である。本発明の組成物の形態は特に限定されるものではなく、本発明の骨形成促進剤の形態としては、医薬組成物としての適宜の形態が挙げられる。本発明の組成物は、棘皮動物から抽出精製されるスフィンゴ糖脂質それ自体であっても良いし、スフィンゴ糖脂質以外の成分を含有しても良い。スフィンゴ糖脂質以外の成分は、組成物の形態に応じて適宜選択することができる。
【００２６】
医薬組成物は、例えば、スフィンゴ糖脂質を薬学的に許容され得る賦形剤、その他任意の添加剤を用いて製剤化することにより製造することができ、製剤化したスフィンゴ糖脂質は骨形成促進剤として使用することができる。製剤化する場合、製剤中のスフィンゴ糖脂質の含有量は、通常、０．０００００１〜０．１質量％、好ましくは０．０００１〜０．０１質量％である。製剤化にあたっては、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味矯臭剤、希釈剤、注射用溶剤等の添加剤を使用することが可能である。賦形剤としては、例えば、乳糖、ブドウ糖、マンニット、ソルビット等の糖誘導体；トウモロコシ澱粉、馬鈴薯澱粉、α−デンプン、デキストリン、カルボキシメチルデンプン等のデンプン誘導体；結晶セルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム等のセルロース誘導体；アラビアゴム；デキストラン；プルラン；軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウム等のケイ酸塩誘導体；リン酸ナトリウム、リン酸カルシウム等のリン酸塩誘導体；炭酸ナトリウム、炭酸カルシウム等の炭酸塩誘導体；硫酸ナトリウム、硫酸カルシウム等の硫酸塩誘導体が挙げられる。
【００２７】
結合剤としては、例えば、上記賦形剤の他、ゼラチン；ポリビニルピロリドン；マクロゴール等が挙げられる。崩壊剤としては、例えば、上記賦形剤の他、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドン等の化学修飾されたデンプン又はセルロース誘導体等が挙げられる。滑沢剤としては、例えば、タルク；ステアリン酸；ステアリン酸カルシウム、ステアリン酸マグネシウム等のステアリン酸金属塩；コロイドシリカ；ビーガム、ゲイロウ等のワックス類；ホウ酸；グリコール；フマル酸、アジピン酸等のカルボン酸類；安息香酸ナトリウム等のカルボン酸ナトリウム塩；硫酸ナトリウム等の硫酸塩；ロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等のラウリル硫酸塩；無水ケイ酸、ケイ酸水和物等のケイ酸類；デンプン誘導体等が挙げられる。安定剤としては、例えば、メチルパラベン、プロピルパラベン等のパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコール等のアルコール類；塩化ベンザルコニウム；無水酢酸；ソルビン酸等が挙げられる。矯味矯臭剤としては、例えば、甘味料、酸味料、香料等が挙げられる。注射用溶剤としては、例えば、水、エタノール、グリセリン等が挙げられる。
【００２８】
医薬組成物の投与経路としては、例えば、経口投与、経腸投与等の非経口投与が挙げられる。医薬組成物の投与剤形としては、例えば、注射剤、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、坐剤、軟膏、テープ剤等が挙げられる。また、医薬組成物は飲食品、又は飼料等に配合して投与することもできる。投与量及び投与回数は、目的とする作用効果、投与方法、治療期間、年齢、性別、体重等により異なるが、投与量は成人１日当たり、通常、０．００１〜１０００ｍｇ、好ましくは０．１〜１００ｍｇの範囲から適宜選択でき、投与回数は１日１回から数回の範囲から適宜選択できる。
【００２９】
ここで、参考例として示す飲食品組成物は、例えば、本発明の骨形成促進剤の有効成分であるスフィンゴ糖脂質に、デキストリン、デンプン等の糖類；ゼラチン、カゼイン、ホエー、大豆タンパク、トウモロコシタンパク等のタンパク質；アラニン、グルタミン、イソロイシン等のアミノ酸類；セルロース、グアガム、ジェランガム、アラビアガム等の多糖類；大豆油、中鎖脂肪酸トリグリセリド等の油脂類等を配合することにより製造できる。飲食品組成物の形態としては、清涼飲料、炭酸飲料、栄養飲料、果汁飲料、乳酸菌飲料等の飲料（これらの飲料の濃縮原液及び調整用粉末を含む）；アイスクリーム、シャーベット、氷菓等の冷菓；そば、うどん、はるさめ、餃子及びしゅうまいの皮、中華麺、即席麺等の麺類；飴、チューインガム、キャンディー、チョコレート、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、錠菓等の菓子類；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳、調製粉乳等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及び油脂加工食品；ソース、たれ等の調味料；スープ、シチュー、サラダ、惣菜、漬物；パン類；経腸栄養食；機能性食品等が例示される。尚、飲食品組成物を製造する際に添加する骨形成促進剤の有効成分であるスフィンゴ糖脂質の含有量は、０．０００１〜０．１質量％の範囲から適宜選択することができる。
【００３０】
また、参考例として示す飼料組成物は、例えば、スフィンゴ糖脂質に、トウモロコシ、小麦、大麦、ライ麦等の穀類；ふすま、麦糠、米糠、脱脂米糠等の糠類；コーングルテンミール、コーンジャムミール等の製造粕類；脱脂粉乳、ホエー、魚粉、骨粉等の動物性飼料類；ビール酵母等の酵母類；リン酸カルシウム、炭酸カルシウム等の鉱物質飼料；油脂類；アミノ酸類；糖類等を配合することにより製造することができる。飼料組成物の形態としては、愛玩動物用飼料（ペットフード等）、家畜飼料、養魚飼料等が例示される。尚、飼料組成物を製造する際に添加する骨形成促進剤の有効成分であるスフィンゴ糖脂質の含有量は、０．０００１〜０．１質量％の範囲から適宜選択することができる。本発明の医薬組成物は、単独で使用しても良いが、その他の骨形成促進剤や骨疾患予防・治療剤等と併用しても良い。併用によって、骨形成や骨疾患の予防・治療効果を高めることができる。また、併用する骨形成促進剤や骨疾患予防・治療剤等は、本発明の組成物中に有効成分として含有させても良いし、本発明の組成物中には含有させずに別個の薬剤として組み合わせて製品として良い。
【００３１】
次に、本発明の骨形成促進剤の有効成分であるスフィンゴ糖脂質の製造例及び参考例として示すスフィンゴ糖脂質（スフィンゴリン糖脂質）の製造例を説明する。尚、各スフィンゴ糖脂質の構造は、マススペクトル法（ＭＳ）、核磁気共鳴スペクトル法（ＮＭＲ）等を利用して決定した。
製造例１
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するセラミドの製造）
ヤツデスナヒトデ（学名：Ｌｕｉｄｉａ ｍａｃｕｌａｔａ）１ｋｇをミキサーにてホモジナイズし、クロロホルム／メタノール（クロロホルム：メタノール＝１：２）の混合液２リットルで抽出を行い、ろ過した後に抽出液を濃縮した。該濃縮画分（４１．４ｇ）に水を２５０ｍｌ加えて懸濁した後、酢酸エチル／ｎ−ブタノール（酢酸エチル：ｎ−ブタノール＝３：１）の混合液２５０ｍｌを添加して、分配操作を行い、酢酸エチル／ｎ−ブタノール層と水層を分取した。このうち酢酸エチル／ｎ−ブタノール層を濃縮し、この濃縮画分（９．９ｇ）にアセトンを加えて混合し、アセトン可溶性画分を回収した後に有機溶媒を揮発させてエキスＡ約５．３ｇを得た。次いで、該エキスＡをクロロホルム５ｍｌに溶解して順相カラム（Ｓｉｌｉｃａ ｇｅｌ ６０：メルク社製）に添加し、溶出溶媒にクロロホルム／メタノール混合液（クロロホルム：メタノール＝１００：０、９８：２、９５：５、９：１、８５：１５、７：３、及び６：４の各混合比）を使用し、メタノール濃度を段階的に上昇させるステップワイズグラジエント法により溶出した。順相薄層クロマトグラフィーにて既存のセラミドと同様の挙動を示す画分を回収し、該画分について、更に、順相カラムに添加してクロロホルムメタノール混合液（クロロホルム：メタノール＝９７：３→９５：５）で溶出させ、順相薄層クロマトグラフィーにて単一物質の挙動を示すＬＭＣｅｒ−１（３ｍｇ）及びＬＭＣｅｒ−２（４ｍｇ）の２種類のセラミドを各々製造した。ＬＭＣｅｒ−１の構造式を化４、及びＬＭＣｅｒ−２の構造式を化５に各々示す。
【００３２】
化４
Ｈ−Ｃｅｒ１
化５
Ｈ−Ｃｅｒ２
【００３３】
製造例２
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するセレブロシドの製造）
ヤツデスナヒトデ１ｋｇをミキサーにてホモジナイズし、クロロホルム／メタノール（クロロホルム：メタノール＝１：２）の混合液２リットルで抽出を行い、ろ過した後に抽出液を濃縮した。この濃縮画分に水を２５０ｍｌ加えて懸濁した後、酢酸エチル／ｎ−ブタノール（酢酸エチル：ｎ−ブタノール＝３：１）の混合液２５０ｍｌを添加して、分配操作を行い、酢酸エチル／ｎ−ブタノール層と水層を分取した。酢酸エチル／ｎ−ブタノール層は、濃縮した後にアセトンを加えて混合して、アセトン不溶性画分を回収し、更に、有機溶媒を揮発させてエキスＢ約４．５ｇを得た。次いで、該エキスＢをクロロホルム／メタノール／水（混合比＝９：１：０．０５）混合液５ｍｌに溶解し、順相カラム（Silica gel 60 ：メルク社製）に添加して、同混合液で溶出した。順相薄層クロマトグラフィーにて既存のセレブロシドと同様の挙動を示す画分について回収し、該画分を逆相カラム（Cosmosil １４０ Ｃ１８−ＰＲＥＰ：ナカライ社製）に添加して、１００％メタノールにて溶出した。次いで、順相薄層クロマトグラフィーにて既存のセレブロシドと同様の挙動を示す画分を、ゲル濾過カラム（Sephadex ＬＨ−２０：ファルマシア社製）に添加して、クロロホルム／メタノール（クロロホルム：メタノール＝１：１）混合液を溶出液としてカラムクロマトグラフィーを行い、順相薄層クロマトグラフィーにて単一物質の挙動を示すＬＭＣ−１（５ｍｇ）及びＬＭＣ−２（６ｍｇ）の２種類のセレブロシドを各々製造した。ＬＭＣ−１の構造式を化６、及びＬＭＣ−２の構造式を化７に各々示す。
【００３４】
化６
Ｇｌｃβ１→１Ｃｅｒ１
（Ｇｌｃ：グルコース）
化７
Ｇｌｃβ１→１Ｃｅｒ２
（Ｇｌｃ：グルコース）
【００３５】
製造例３
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するラクトシルセラミドの製造）
ヤツデスナヒトデ１ｋｇをミキサーにてホモジナイズし、クロロホルム／メタノール（クロロホルム：メタノール＝１：２）の混合液２リットルで抽出を行い、ろ過した後に抽出液を濃縮した。これに水を２５０ｍｌ加えて懸濁した後、酢酸エチル／ｎ−ブタノール（酢酸エチル：ｎ−ブタノール＝３：１）の混合液２５０ｍｌを添加して、分配操作を行い、酢酸エチル／ｎ−ブタノール層と水層として分取した。ここで、分取した水層に更に水飽和ｎ−ブタノールを添加して分配操作を行い、ｎ−ブタノール層と水層として分取し、このうちｎ−ブタノール層について有機溶媒を揮発させてエキスＣとした。エキスＣの乾燥重量は約３．２ｇであった。次いで、該エキスＣをクロロホルム／メタノール（クロロホルム：メタノール＝１：１）混合液５ｍｌに溶解し、逆相カラム（Cosmosil 1４０Ｃ１８−ＰＲＥＰ：ナカライ社製）に添加し、同混合液にて溶出した。次いで、順相薄層クロマトグラフィーにて既存のラクトシルセラミドと同様の挙動を示す画分を回収した。該画分を順相カラム（Silica gel ６０：メルク社製）に添加し、混合比がクロロホルム：メタノール：水＝９：１．５：０．５、８：２：０．２、及び６：４：１の各クロロホルム／メタノール／水混合液を使用して、メタノール濃度を段階的に上昇させるステップワイズグラジエント法により溶出した。溶出画分のうち、順相薄層クロマトグラフィーにて単一物質の挙動を示すＬＭＣＤＨ−１（１．７ｍｇ）及びＬＭＣＤＨ−２（１．４ｍｇ）の２種類のラクトシルセラミドを各々製造した。ＬＭＣＤＨ−１の構造式を化８、及びＬＭＣＤＨ−２の構造式を化９に各々示す。
【００３６】
化８
Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ１
（Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
化９
Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
【００３７】
製造例４
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するガングリオシドの製造）
ヤツデスナヒトデ１ｋｇをミキサーにてホモジナイズし、クロロホルム／メタノール（クロロホルム：メタノール＝１：２）の混合液２リットルで抽出を行い、ろ過した後に抽出液を濃縮した。これに水を２５０ｍｌ加えて懸濁した後、酢酸エチル／ｎ−ブタノール（酢酸エチル：ｎ−ブタノール＝３：１）の混合液２５０ｍｌを添加して、分配操作を行い、酢酸エチル／ｎ−ブタノール層と水層として分取した。ここで、分取した水層に更に水飽和ｎ−ブタノールを添加して分配操作を行い、ｎ−ブタノール層と水層として分取して、そのうちの水層を回収した。該水層を濃縮した後に、逆相カラム（Cosmosil １４０ Ｃ１８−ＰＲＥＰ：ナカライ社製）を用いて分離を行った。成分の分離には溶出液であるメタノール溶液の濃度を６０〜１００％まで段階的に上昇させて溶出させるステップワイズグラジエント法により行った。メタノールの濃度が１００％に達した時点の溶出画分の有機溶媒を揮発させてエキスＤ約０．１８ｇを調製した。次いで、該エキスＤをクロロホルム／メタノール／水（クロロホルム：メタノール：水＝６：４：１）混合液に溶解し、順相カラム（Silica gel ６０：メルク社製）に添加して同混合液で溶出した。次いで、ゲル濾過カラム（Sephadex ＬＨ−２０：ファルマシア社製）に添加して脱塩し、順相薄層クロマトグラフィーにて単一物質の挙動を示すスルファチドＬＭＧ−１（０．６７ｍｇ）及びガングリオシドＬＭＧ−２（２．４ｍｇ）を各々製造した。ＬＭＧ−１の構造式を化１０、ＬＭＧ−２の構造式を化１１に各々示す。
【００３８】
化１０
３−Ｏ−ＨＳＯ₃ Ｇａｌβ１→４Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
化１１
ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸、Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
【００３９】
製造例５
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するガングリオシドの製造）
出発原料として、アオヒトデ（学名：Linckia laevigata ）１８ｋｇを用い、製造例４と同様の方法でガングリオシドＬＬＧ−３（５８ｍｇ）及びＬＬＧ−５（７０ｍｇ）を各々製造した。ＬＬＧ−３の構造式を化１２、ＬＬＧ−５の構造式を化１３に示す。
【００４０】
化１２
８−Ｏ−ＣＨ₃ ＮｅｕＡｃα２→１１ＮｅｕＧｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸、ＮｅｕＧｃ：Ｎ−グリコリルノイラミン酸、Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
化１３
８−Ｏ−ＣＨ₃ ＮｅｕＧｃα２→１１ＮｅｕＧｃα２→１１ＮｅｕＧｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（ＮｅｕＧｃ：Ｎ−グリコリルノイラミン酸、Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
【００４１】
製造例６
（α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するガングリオシドの製造）
出発原料として、ムラサキヒトデ（学名：Asterias amurensis versicolor ）６５ｋｇを用い、製造例４と同様の方法でガングリオシドＧＡＡ−７（８６ｍｇ）を製造した。ＧＡＡ−７の構造式を化１４に示す。
【００４２】
化１４
８−Ｏ−ＣＨ₃ ＮｅｕＧｃα２→６〔８−Ｏ−ＣＨ₃ ＮｅｕＧｃα２→３〕ＧａｌＮＡｃβ１→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（ＮｅｕＧｃ：Ｎ−グリコリルノイラミン酸、ＧａｌＮＡｃ：Ｎ−アセチルガラクトサミン、Ｇａｌ：ガラクトース、Ｇｌｃ：グルコース）
【００４３】
製造例７
（スフィンゴリン糖脂質の製造）
出発原料として、ニッポンウミシダ（学名：Comanthus japonica）７．６ｋｇを用い、製造例４と同様の方法でニッポンウミシダ由来のエキスＤを製造した。該エキスＤをクロロホルム／メタノール／水混合液（クロロホルム：メタノール：水＝８：２：０．２）２ｍｌに溶解し、順相カラム（Silica gel ６０：メルク社製）に添加して、混合比がクロロホルム：メタノール：水＝８：２：０．２及び６：４：１の各クロロホルム／メタノール／水混合液を使用して、メタノール濃度を上昇させるステップワイズグラジエント法により溶出した。薄層クロマトグラフィー上でスフィンゴリン糖脂質ＣＪＰ−１（９２ｍｇ）、ＣＪＰ−２（１７０．６ｍｇ）及びＣＪＰ−３（５６８．５ｍｇ）を製造した。ＣＪＰ−１の構造式を化１５、ＣＪＰ−２の構造式を化１６、ＣＪＰ−３の構造式を化１７に示す。
【００４４】
化１５
Ｉｎｓ１→Ｐｈｏｓｐｈｏ−Ｃｅｒ
（Ｉｎｓ ：イノシトール）
化１６
９−ＯＣＨ₃ ＮｅｕＧｃα２→３Ｉｎｓ１→Ｐｈｏｓｐｈｏ−Ｃｅｒ
（ＮｅｕＧｃ：Ｎ−グリコリルノイラミン酸、Ｉｎｓ：イノシトール）
化１７
９−ＯＣＨ₃ ＮｅｕＧｃα２→１１〔９−ＯＣＨ₃ 〕ＮｅｕＧｃα２→３Ｉｎｓ１→Ｐｈｏｓｐｈｏ−Ｃｅｒ
（ＮｅｕＧｃ：Ｎ−グリコリルノイラミン酸、Ｉｎｓ：イノシトール）
【００４５】
尚、前記製造例１〜６において、本発明者等が棘皮動物から抽出精製したα−オキシ脂肪酸、及びスフィンゴシン骨格又はフィトスフィンゴシン骨格を有するスフィンゴ糖脂質は、哺乳類や植物から精製されるものとは異なり、棘皮動物特有の構造を有することが確認され、分子内の脂肪酸のα位がオキシ化されている点で、前記特許文献３に記載のスフィンゴシン骨格を有する化合物とは構造上異なる化合物であることが分かった。また、前記製造例７で製造したスフィンゴリン糖脂質は、ウミユリ類やウミシダ類に特有のスフィンゴリン糖脂質の構造を有していた。
【００４６】
次に、試験例を示して本発明を詳細に説明する。
試験例１
本試験は、棘皮動物から抽出精製したスフィンゴ糖脂質の骨芽細胞増殖促進活性を測定するために行った。
（１）試料の調製
前記製造例１〜７で製造した各スフィンゴ糖脂質（ＬＭＣｅｒ−１、ＬＭＣｅｒ−２、ＬＭＣ−１、ＬＭＣ−２、ＬＭＣＤＨ−１、ＬＭＣＤＨ−２、ＬＬＧ−３、ＬＬＧ−５、ＧＡＡ−７、ＣＪＰ−１、ＣＪＰ−２、ＣＪＰ−３）をそれぞれ試験試料として使用した。また、α−オキシ脂肪酸及びフィトスフィンゴシン骨格を持たない従来のスフィンゴ糖脂質の一種であるＣ２セラミド（N-Acetyl-D-sphingosine semisynthetic：シグマ社製、カタログＮｏ．Ａ７１９１）を対照試料として使用した。
【００４７】
（２）試験方法
マウス骨芽細胞株ＭＣ３Ｔ３−Ｅ１（ＲＣＢ１１２６：理研セルバンク）を、各々１０％牛胎児血清を含むα−ＭＥＭ培地（ギブコＢＲＬ社製）中で、１ウェル当たり５×１０⁴ 個となるように９６ウェルマイクロプレートに分注し、３７℃、５％炭酸ガス存在下で１８時間培養した。培養後に培地を除去し、改めて２％牛胎児血清を含むα−ＭＥＭ培地を各ウェルに１００μｌずつ添加した。次いで、各ウェルに各試験試料及び対照試料を最終濃度が０．２μｇ／ｍｌとなるように添加して更に２日間培養した。培養終了時間の６時間前に各ウェルに５０ｎＣｉ量となるようにトリチウムチミジンを添加した。培養終了後、各ウェルのＤＮＡ合成量を液体シンチレーションカウンター（ＬＫＢ１２１９ ＲＡＣＫ−ＢＥＴＡ：ＬＫＢ社製）を使用して測定した。
【００４８】
（３）試験結果
本試験の結果を図３に示す。図３は、棘皮動物から抽出精製したスフィンゴ糖脂質による骨芽細胞の増殖促進活性を示す。その結果、各試験試料に使用した棘皮動物由来のスフィンゴ糖脂質は、対照試料のＣ２セラミドに対して高い骨芽細胞増殖促進効果を有することが明らかとなった。その中でもフィトスフィンゴシン骨格を有するスフィンゴ糖脂質（ＬＭＣｅｒ−２、ＬＭＣ−２、ＬＭＣＤＨ−２、ＬＬＧ−３、ＬＬＧ−５、ＧＡＡ−７）はスフィンゴシン骨格を有するそれに比して、総じて活性が強いことが判明した。従って、棘皮動物に特異的な構造（例えば、α−オキシ脂肪酸、フィトスフィンゴシン骨格を有する等）を有するスフィンゴ糖脂質は従来のスフィンゴ糖脂質に比して、より強い骨芽細胞増殖促進効果を有することが明らかとなった。
【００４９】
試験例２
本試験は、棘皮動物から抽出精製したスフィンゴ糖脂質を投与したマウスのｅｘ ｖｉｖｏにおける骨芽細胞増殖促進活性を測定するために行った。
（１）試料の調製
前記製造例で製造したスフィンゴ糖脂質のうち、ＬＭＣｅｒ−２、ＬＭＣＤＨ−２、ＣＪＰ−３、ＬＧＧ−３をそれぞれ試験試料として使用した。また、陰性試料として生理食塩水を使用した。
【００５０】
（２）試験方法
本試験においてマウスは、４週齢、雌性ｄｄｙマウス（日本エスエルシー社より購入）を使用した。前記マウスの卵巣を摘出した後、７週間低カルシウム食を給餌して作成した、骨粗鬆症モデルマウスを１群６匹に群分けした。生理食塩水にて１０μｇ／ｍｌに調製した各試験試料溶液を、該骨粗鬆症モデルマウスに各々２００μｌずつ、尾静脈より１日１回１４日間静脈内投与した。投与完了後、大腿骨及び脛骨の骨髄細胞をＲＰＭＩ１６４０培地（ギブコＢＲＬ社製）中に採取した。採取した骨芽細胞を１ウェル当たり１×１０⁶ 個となるように６ウェルマイクロプレートに分注し、１０％牛胎児血清を含むＲＰＭＩ１６４０培地にて７日間培養した。培養終了後、アルカリフォスファターゼ染色（DIAGNOSTICS,Ｎｏ．８５：シグマ社製）を行い、陽性のコロニーを骨芽細胞コロニーとしてカウントした。
【００５１】
（３）試験結果
本試験の結果を図４に示す。図４は、棘皮動物から抽出精製したスフィンゴ糖脂質による骨粗鬆症モデルマウス骨髄細胞中の骨芽細胞の増殖促進活性を示す。その結果、何れの試験試料においても生体内の骨芽細胞を増殖させる作用を有することが判明した。
【００５２】
試験例３
本試験は、棘皮動物から粗精製した中間精製物であるスフィンゴ糖脂質エキスを投与したマウスのｅｘ ｖｉｖｏにおける骨芽細胞増殖促進活性を測定するために行った。
（１） 試料の調製
前記製造例１で粗精製したエキスＡ（セラミド組成物）、製造例２で粗精製したエキスＢ（セレブロシド組成物）、製造例３で粗精製したエキスＣ（ラクトシルセラミド組成物）、製造例４で粗精製したエキスＤ（ガングリオシド組成物）を各々試験試料とし、生理食塩水で５００μｇ／ｍｌとなるように溶解して試験に供した。この時、エキスＡはセラミドを１μｇ含有し、エキスＢはセレブロシドを１．２μｇ含有し、エキスＣはラクトシルセラミドを０．５μｇ含有し、エキスＤはガングリオシドを８μｇ含有している。また、哺乳類由来のセラミド（和光純薬工業社製、カタログＮｏ．０３１−１８４４１）を生理食塩水で２０μｇ／ｍｌに調製した溶液を対照試料Ａとし、セレブロシド（和光純薬工業社製、カタログＮｏ．０７６−０３９６３）を生理食塩水で２０μｇ／ｍｌに調製した溶液を対照試料Ｂとし、ガングリオシド混合物（和光純薬工業社製、カタログＮｏ．０７４−０３６４３）を生理食塩水で２０μｇ／ｍｌに調製した溶液を対照試料Ｄとし、更に、生理食塩水を陰性試料として各々試験に供した。
【００５３】
（２）試験方法
本試験において、マウスは、６週齢、雄性ｄｂ／ｄｂマウス（日本クレア社より購入）を使用した。前記マウスを１群５匹に群分けし、エキスＡ、エキスＢ、エキスＣ、エキスＤ、対照試料Ａ、対照試料Ｂ、対照試料Ｄ、及び陰性試料をマウスに各々５００μｌずつ、１日１回ゾンデを用いて３６日間連続して強制経口投与した。投与完了後、大腿骨及び脛骨の骨髄細胞をＲＰＭＩ１６４０培地（ギブコＢＲＬ社製）中に採取した。採取した骨芽細胞を１ウェル当たり１×１０⁶個となるように６ウェルマイクロプレートに分注し、１０％牛胎児血清を含むＲＰＭＩ１６４０培地にて７日間培養した。培養終了後、アルカリフォスファターゼ染色（ＤＩＡＧＮＯＳＴＩＣＳ，Ｎｏ．８５：シグマ社製）を行い、陽性のコロニーを骨芽細胞コロニーとしてカウントし、１群５匹の平均値を算出した。
【００５４】
（３）試験結果
本試験の結果を表１に示す。表１は、棘皮動物から粗精製した中間精製物であるスフィンゴ糖脂質エキスによる骨芽細胞の増殖促進活性を示す。その結果、エキスＡ、エキスＢ、エキスＣ、エキスＤ、対照試料Ａ、対照試料Ｂ、及び対照試料Ｄは、いずれも陰性試料に対して骨芽細胞コロニー数が増加し、増殖促進活性が高いことが判明した。しかしながら、本発明のスフィンゴ糖脂質エキス（エキスＡ、エキスＢ、エキスＣ、エキスＤ）と、市販のセラミド（対照試料Ａ）、セレブロシド（対照試料Ｂ）、ガングリオシド混合物（対照試料Ｄ）について、陰性試料に対する骨芽細胞コロニー数の増加の割合を比較したところ、対照試料Ａ：３．２に対しエキスＡ（セラミド組成物）：５．０、対照試料Ｂ：１．１に対しエキスＢ（セレブロシド組成物）：２．４、対照試料Ｄ：１．３に対しエキスＤ（ガングリオシド組成物）：７．６であったことから、棘皮動物由来のスフィンゴ糖脂質エキスは、従来の哺乳動物由来のスフィンゴ糖脂質に比して優れた骨芽細胞増殖促進効果を有することが判明した。
【００５５】
【表１】

【００５６】
【実施例】
次に、実施例を示して本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。
実施例１
次の組成の注射剤を常法により製造した。
製造例１で製造したセラミド（ＬＭＣｅｒ−２） ０．０１（％）
アクチノマイシンＤ（シグマ社製） ０．００５
塩化ナトリウム（和光純薬社製） ０．９
マンニトール（関東化学社製） １．０
注射用蒸留水（大塚製薬社製） ９８．０８５
【００５７】
参考例１
ホエー蛋白酵素分解物（森永乳業社製）１０．８ｋｇ、デキストリン（昭和産業社製）３６ｋｇ、及び少量の水溶性ビタミンとミネラルを水２００ｋｇに溶解し、水相をタンク内に調製した。これとは別に、大豆サラダ油（太陽油脂社製）３ｋｇ、パーム油（太陽油脂社製）８．５ｋｇ、サフラワー油（太陽油脂社製）２．５ｋｇ、レシチン（味の素社製）０．２ｋｇ、脂肪酸モノグリセリド（花王社製）０．２ｋｇ、及び少量の脂溶性ビタミンを混合溶解し、油相を調製した。タンク内の水相に油相を添加し、攪拌して混合した後、７０℃に加温し、更に、ホモゲナイザーにより１４．７ＭＰａの圧力で均質化した。次いで、９０℃で１０分間殺菌した後、濃縮し、噴霧乾燥して、中間製品粉末約５９ｋｇを調製した。この中間製品粉末５０ｋｇに、蔗糖（ホクレン社製）６．８ｋｇ、アミノ酸混合粉末（味の素社製）１６７ｇ、及び前記製造例７で製造したスフィンゴリン糖脂質（ＣＪＰ−３
）６０ｇを添加し、均一に混合して、スフィンゴリン糖脂質を含有する骨形成促進効果を有する経腸栄養食粉末約５６ｋｇを製造した。
【００５８】
参考例２
前記製造例６で製造したガングリオシド（ＧＡＡ−７）１００ｇ、ラクチュロース粉末（森永乳業社製）１１０ｇ、マルツデキストリン（松谷化学工業社製）６５５ｇ、脱脂粉乳（森永乳業社製）９５ｇ、ステビア甘味料（三栄源エフ・エフ・アイ社製）１ｇ、ヨーグルト・フレーバー（三栄源エフ・エフ・アイ社製）５ｇ、グリセリン脂肪酸エステル製剤（理研ビタミン社製）３４ｇの各粉末を添加して均一に混合し、打錠機（畑鉄鋼所社製）を使用して、錠剤１錠当り０．５ｇとし、１２錠／分の打錠速度、９．８ＫＰａの圧力で前記混合粉末を連続的に打錠し、ガングリオシド（ＧＡＡ−７）含有タブレット１８００錠（約９００ｇ）を製造した。
【００５９】
参考例３
乳脂肪含量３．５％、無脂乳固形分含量９．２％の生乳１０ｋｇを均質化し、９０〜９２℃で１０分間加熱殺菌した。約４２℃に冷却し、スターターとして市販のストレプトコッカス・サーモフィルス（Streptococcus thermophilus）及びラクトバシラス・ブルガリカス（Lactobacillus delbrueckii
subsp. bulgaricus ）の牛乳カルチャーを各々１００ｇずつ添加した。発酵タンク内で充分に攪拌し、４２〜４５℃で４時間静置して発酵させた。この発酵乳を攪拌しながら５〜８℃に冷却し、次いで、ホモゲナイザーで均質化することによって均質化発酵乳を調製した。次に、１７％のグラニュウ糖（東洋精糖社製）液５ｋｇにペクチン（三栄源ＦＦＩ社製）６０ｇ及び前記製造例２で製造したセレブロシド（ＬＭＣ−２）３０ｇを添加して、９０〜９２℃で１０分間殺菌した後、５〜８℃に冷却して糖液を調製した。これらの方法で調製した均質化発酵乳、糖液のそれぞれ全量をタンク内で十分に混合し、紙容器に１２０ｍｌずつ充填した後、密封してドリンクヨーグルトを製造した。
【００６０】
【発明の効果】
以上詳記したとおり、本発明は、棘皮動物から抽出されるα−オキシ脂肪酸及びスフィンゴシン骨格、α−オキシ脂肪酸及びフィトスフィンゴシン骨格を有するスフィンゴ糖脂質を有効成分として含有する医薬用の骨形成促進剤に係るものであり、本発明により、以下のような効果が奏される。
（１）哺乳動物等の骨芽細胞の増殖を促進する新規骨形成促進剤を提供することができる。
（２）本発明の骨形成促進剤は、骨粗鬆症やその他の骨代謝疾患の治療及び／又は予防に効果を有する。
（３）骨粗鬆症等の予防・治療用医薬組成物を提供することができる。
（４）水産業の現場において大量に捕獲される棘皮動物の有効な利用法を提供することができる。
【図面の簡単な説明】
【図１】図１は、スフィンゴ糖脂質の基本構造を示す。
【図２】図２は、棘皮動物から、α−オキシ脂肪酸、及びスフィンゴシン骨格又はフィトスフィンゴシン骨格を有するスフィンゴ糖脂質、並びにスフィンゴリン糖脂質を製造する方法を示すフローチャートである。
【図３】図３は、棘皮動物から抽出精製したスフィンゴ糖脂質による骨芽細胞の増殖促進活性を示す。
【図４】図４は、棘皮動物から抽出精製したスフィンゴ糖脂質による骨粗鬆症モデルマウス骨髄細胞中の骨芽細胞の増殖促進活性を示す。[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to an osteogenesis promoter that promotes the proliferation of osteoblasts and has an effect on treatment and / or prevention aimed at strengthening bone, and more specifically, is purified from an organism belonging to the echinoderm. Α-oxy fatty acid and sphingosine skeleton, and sphingoglycolipid having α-oxy fatty acid and phytosphingosine skeletonQualityThe present invention relates to a pharmaceutical osteogenesis promoter contained as an active ingredient. In the present invention, the osteogenesis promoter means an osteogenesis promoter as a pharmaceutical composition. The present invention provides a novel osteogenesis promoter, and makes it possible to provide a food / beverage composition or a feed composition containing the above components.
[0002]
[Prior art]
With aging, bone diseases such as osteoporosis, fractures, and back pain are increasing. Osteoblasts and osteoclasts are present in the bone tissue, and are responsible for bone formation and bone resorption, respectively, and so on. Known diseases caused by abnormal bone metabolism include osteoporosis, rheumatoid arthritis, Paget's disease of bone, and osteoarthritis. The problem of increasing bedridden elderly due to these bone diseases is As the society continues, it has become an issue that must be solved. Bone loss caused by abnormal bone metabolism is improved by promoting bone formation or suppressing bone resorption. The promotion of osteogenesis is the promotion of osteoblast proliferation / differentiation. In addition, suppression of bone resorption can be achieved by suppression of proliferation / differentiation of osteoclast precursor cells, function suppression of mature osteoclasts, and the like. The development of proteins and other compounds having such activities as promoting bone formation or inhibiting bone resorption is currently being promoted in various fields.
[0003]
Osteoblasts are mononuclear cells derived from mesenchymal stem cells and are in a resting state that covers pre-osteoblasts, osteoblasts, and bone cells embedded in the bone matrix and bone surface Differentiate into cells. Proliferated and differentiated osteoblasts produce bone matrix (ostoids) and fill the bone resorption pits. Furthermore, under the control of osteoblasts, osteoids mature, mineralize into bone. Pre-osteoblasts and osteoblasts not only produce such bone matrix, but also insulin-like growth factors (IGF-1, IGF-2), β2-microglobulin, osteoinductive protein (BMP), transforming proliferation. Factor-β (TGF-β), interleukin-1 (IL-1), tumor necrosis factor (TNF-α), prostaglandin E₂(PGE₂ ) And other local factors, and the autocrine / paracrine mechanism is thought to control bone formation. Furthermore, osteoblasts are composed of parathyroid hormone (PTH), epidermal growth factor (EGF), active vitamin [1α, 25 (OH)₂ D_Three ], Estrogen, TNF-α, PGE₂Since it has receptors such as IL-1, it is considered that the control of proliferation / differentiation of osteoblasts itself and the control of differentiation / activation of osteoclasts are performed through these receptors.
[0004]
Currently, bisphosphonate compounds, calcitonin preparations, active vitamin D are used to treat bone diseases._Three Formulation, Estradiol-containing hormone preparation, Vitamin K₂Such drugs are generally used. Aiming at an excellent therapeutic drug with few side effects, active vitamin D_Three Derivatives, estradiol derivatives, third-generation bisphosphonate compounds and the like have been developed. However, treatment methods for bone diseases using the aforementioned drugs are not always satisfactory, and there are some that are limited in terms of side effects. Furthermore, since multi-drug combination therapy has become the mainstream for the treatment of bone metabolic diseases, development of a new drug having an action mechanism different from that of conventional drugs is expected.
[0005]
Sphingolipids exist widely in nature as important membrane components of cells, and most of them exist as glycosphingolipids and sphingophospholipids. Sphingolipids are known to play important roles in life phenomena such as regulatory effects on cell proliferation and differentiation (Non-patent Document 1) and apoptosis induction (Non-patent Document 2). It has been clarified that TNF (Non-patent Document 3), IL-1 (Non-patent Document 4) and Fas ligand (Non-patent Document 5), which are cytokines, are involved. Sphingolipids are involved in regulation of EGF receptor activation (Non-patent Document 6), intracellular fluidization of calcium ions (Non-patent Document 7), protein kinase C activity inhibition and activation, and the like. It is also known.
For glycosphingolipids, ceramide consisting of a long-chain base and a fatty acid is used as an aglycon, and cerebroside to which a monosaccharide such as galactose and glucose is bound, lactosylceramide to which lactose is bound, sugar (monosaccharide or oligosaccharide). Sugar) and a sulfate group to which sulfatide is bonded, and ganglioside to which oligosaccharide and sialic acid are bonded are included (Non-patent Document 8). Among them, gangliosides containing sialic acid are used for mutual recognition (non-patent document 9), adhesion (non-patent document 10), proliferation (non-patent document 11), differentiation induction (non-patent document 12), and the like. It is said to be involved in various important phenomena in the body. This compound is also used for treatment of ischemic injury and brain injury caused by Parkinson's disease (Non-patent Document 13).
[0006]
The main skeleton of glycosphingolipids derived from organisms belonging to the marine invertebrates Echinodermata (for example, starfishes, spider starfishes, sea urchins, sea cucumbers, sea lilies, etc.) are mostly α-oxy fatty acids and sphingosine type Mammal origin that has a ceramide part (aglycone part) composed of long-chain bases or phytosphingosine-type long-chain bases, and is composed of fatty acids that are not oxidized at the α-position (unsubstituted fatty acids) and sphingosine-type long-chain bases It is structurally different from the glycosphingolipid (Non-Patent Document 8, Non-Patent Document 14). The phytosphingosine skeleton has a structure in which the double bond portion at the 4th and 5th positions in the sphingosine skeleton is saturated with a hydroxyl group at the 4th position and hydrogen at the 5th position. It is expected to have.
[0007]
Among the glycosphingolipids derived from echinoderms, glycolipids obtained from sea lilies (particularly, sea urchins) are sphingophosphoglycosides containing inositol phosphoceramide in the basic structure. This inositol phosphoceramide has been found in plants and anterior mouth animals, but has been found only in those derived from echinoderms from hind mouth animals. Unlike the conventional echinoderm glycosphingolipids, the ceramide part is composed of unsubstituted fatty acids and sphingosine-type long-chain bases, so the sphingoglycolipids of sea urchins are significantly different from those of other echinoderms. It is positioned as. As a technique using ceramide having a phytosphingosine skeleton, a cosmetic composition (Patent Document 1), an external medicine for skin diseases (Patent Document 2), and the like are disclosed. In addition, a compound having a sphingosine skeleton such as ceramide, sphingomyelin, sphingoglycolipid, ganglioside and the like derived from bovine brain and milk, and further, one or more substances selected from calcium, vitamin D and vitamin K An added preventive and ameliorating agent for bone and joint diseases is disclosed (Patent Document 3).
[0008]
[Patent Document 1]
JP-A-8-502961
[Patent Document 2]
JP 2001-39859 A
[Patent Document 3]
JP 2001-158736 A
[Non-Patent Document 1]
FEBS Lett., 524, 103-106, 2002
[Non-Patent Document 2]
FEBS Lett., 526, 15-20, 2002
[Non-Patent Document 3]
EMBO J. Journal, Vol. 19, No. 13, pp. 3304-3313, 2000
[Non-Patent Document 4]
Journal of Biological Chemistry (J. Biol. Chem.), 275, 45, 35617-35623, 2000
[Non-Patent Document 5]
Journal of Biological Chemistry (J. Biol. Chem.), 276, 26, 23954-23961, 2001
[Non-Patent Document 6]
Journal of Biological Chemistry (J. Biol. Chem.), Vol. 277, No. 12, pp. 10108-10113, 2002
[Non-Patent Document 7]
Proc. Natl. Acad. Sci. USA, Vol. 98, No. 1, pp. 307-312, 2001
[Non-Patent Document 8]
Nobuhiro Fushiya and Hiroshi Hamada, “Structural analysis of natural organic compounds”, Springer Fairlark Tokyo, pages 81-140, 1994
[Non-patent document 9]
Journal of Biological Chemistry (J. Biol. Chem.), 264, 20159-20162, 1989
[Non-Patent Document 10]
Journal of Biological Chemistry (J. Biol. Chem.), 266, 17552-17558, 1991
[Non-Patent Document 11]
Brain Res., 284, 215-221, 1983
[Non-Patent Document 12]
Proceding National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Volume 76, pages 3367-3371, 1979
[Non-Patent Document 13]
Neurology, Volume 50, pp. 1630-1636, 1998
[Non-Patent Document 14]
Pharmacia, Vol. 38, No. 9, pp. 851-855, 2002
[0009]
[Problems to be solved by the invention]
Various drugs are used to treat osteoporosis, one of the bone metabolic diseases, but it is necessary to grasp the patient's bone metabolic status when selecting a therapeutic drug, and bone turnover is highly metabolized. The applicable drug is determined depending on whether it is a rotational type or a low-turnover type. In the high turnover type, a bone resorption inhibitor that inhibits the effect of osteoclastic bone resorption is used, and in the low turnover type, an osteogenesis promoter that increases osteoblasts is used (Toshio Matsumoto, “Molecules” Bone Metabolism and Osteoporosis ”, Medical View, 293-295, 1996: These may be abbreviated as Prior Art 1). The compound having a sphingosine skeleton described in Patent Document 3 is a compound inherent to mammals, and inhibits bone resorption by osteoclasts, thereby exhibiting an effect of promoting the increase in bone density. Met.
[0010]
However, according to other prior art documents, osteoclasts and osteoblasts secrete local factors with each other and affect their differentiation and activation. Describes that it is considered highly likely to cause inactivation of osteoblasts and slow down bone turnover (Japanese Patent Laid-Open No. 10-29950, column 2, line 18). As is clear from this, it has been awaited in the technical field to develop a novel osteogenesis promoter useful for osteoporosis and other bone metabolic diseases.
[0011]
  The present inventors are considering the effective use of starfish belonging to the Echinodermata, which is feared to destroy the ecosystem of the fishing ground, using sharks as a diet, and being captured and discarded in large quantities. The α-oxy fatty acid and sphingoglycolipid having sphingosine skeleton or phytosphingosine skeleton extracted and purified from these, and sphingoline glycolipid have been found to have an effect of promoting osteoblast proliferation, and the present invention was completed. It came to do.
  The present invention provides a novel useful for the treatment and / or prevention of osteoporosis and other bone metabolic diseases.MedicinalThe object is to provide an osteogenesis promoter.
  On the other hand, the present invention is an echinoderm that is regarded as a serious environmental problem in the field of fisheries and has not yet been found to be effective even though it is captured in large quantities in the coastal areas of Japan. Use and have an effect of promoting bone formationAbove ingredientsIn addition to solving environmental problems by producing food and beverage compositions and feed compositions with added food, it is also possible to provide technology that contributes to the development of the fishery industryPossibleIt is what.
[0012]
[Means for Solving the Problems]
  The first aspect of the present invention for solving the above problems is as follows.A compound purified from an organism belonging to the echinoderm,Has α-oxy fatty acid and sphingosine skeletonRepresented byCeramide,And represented byA bone formation promoter for pharmaceutical use, comprising as an active ingredient a mixture of one or more selected from cerebrosideThe However, R ₂ -Cer1 represents the structural formula represented by Chemical Formula 1.The present invention also provides a desirable embodiment in which the osteogenesis promoter is a prophylactic / therapeutic agent for osteoporosis, rheumatoid arthritis, Paget's disease of bone, or osteoarthritis.
[0013]
  The second aspect of the present invention for solving the above problems isA compound purified from an organism belonging to the echinoderm,Has α-oxy fatty acid and phytosphingosine skeletonRepresented byCeramide,Represented byCerebroside,Represented bySulfatide, andRepresented by Chemical Formulas 11-14A pharmaceutical osteogenesis promoter comprising one or a mixture selected from the group of gangliosides as an active ingredientThe However, R ₂ -Cer2 represents the structural formula represented by Chemical Formula 2.The present invention also provides a desirable embodiment in which the osteogenesis promoter is a prophylactic / therapeutic agent for osteoporosis, rheumatoid arthritis, Paget's disease of bone, or osteoarthritis.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in more detail. In the present specification, percentages are expressed by mass unless otherwise specified.
The osteogenesis promoter of the present invention can be used for treatment and / or prevention aimed at bone strengthening such as treatment of bone metabolic diseases by promoting the proliferation of osteoblasts. It is considered useful as a therapeutic agent for rheumatoid arthritis, Paget's disease of bone, and osteoarthritis. Therefore, the osteoblast proliferation-promoting effect of the osteogenesis promoter of the present invention is an action located in the opposite relationship in bone metabolism to the effect of suppressing bone resorption by osteoclasts described in Patent Document 3. This is an effect that is not expected from the technique disclosed in Patent Document 3.
[0016]
  The active ingredient of the osteogenesis promoter of the present invention is purified from an organism belonging to the echinoderm.TheHere, the organisms belonging to the Echinodermata are classified, specifically, the star class (eg, starfish, blue starfish, starfish starfish, starfish, purple starfish), snake starfish (eg, starfish) , Sea spider starfish, Japanese spider starfish, etc.), sea gall (e.g., sea urchin, bafun sea urchin, etc.), ridge (e.g., sea cucumber, e.g., sea cucumber), (Nippon sea fern, core sea fern, great sea fern etc.).
[0017]
In general, glycosphingolipids, as shown in FIG. 1, are ceramides consisting of a long-chain base having a sphingosine skeleton or a phytosphingosine skeleton and a fatty acid as aglycone, and in this aglycon part, neutral galactose, glucose or the like Cerebrosides with sugars, lactosylceramide with lactose, sulfatides with monosaccharides or oligosaccharides and sulfate groups, and neutral sugars and amino sugars (may not be included) and sial Those with acid binding are classified as gangliosides, etc. Incidentally, in glycosphingolipids derived from organisms belonging to the marine invertebrate Echinodermata, there are compounds in which the α-position of the fatty acid moiety shown in FIG. 1 is oxidized.
[0018]
  As the glycosphingolipid as an active ingredient of the osteogenesis promoter of the present invention, specifically, ceramide, cerebroside, lactosylceramide, sulfatide, which has a sphingosine skeleton in which the α-position of fatty acid in the molecule is oxidized, And ganglioside, etc., or the α-position of fatty acid in the molecule is oxidized and a phytosphingosine skeleton (that is, a hydroxyl group at the 4-position and a hydrogen-position at the 5-position of the 4-bond and 5-position double bonds in the sphingosine skeleton, respectively) And ceramide, cerebroside, lactosylceramide, sulfatide, ganglioside, etc. having a saturated structure). Basic structure of ceramide having α-oxy fatty acid and sphingosine skeleton (R₂-Cer1) ## STR1 ## Basic structure of ceramide having α-oxy fatty acid and phytosphingosine skeleton (R₂-Cer2) is shown in Chemical Formula 2, respectively. Also,Reference exampleAs an active ingredient of the osteogenesis promoter, sphingophosphoglycolipid, which is a glycosphingolipid containing inositol phosphoceramide in the basic structure, includes sphingophosphoglycolipid, specifically, inositol phosphoceramide or this. An example is sialosyl inositol phosphoceramide in which sialic acid or the like is bound. Basic structure of inositol phosphoceramide (R₃-Phospho-Cer) is shown in Chemical Formula 3. In the present specification, except for the case where special classification or explanation is made, the above-mentioned glycosphingolipid having an α-oxy fatty acid and a sphingosine skeleton, a glycosphingolipid having an α-oxy fatty acid and a phytosphingosine skeleton, and Glycosphingolipids are collectively referred to as glycosphingolipids.
[0019]
[Chemical 1]

[0020]
[Chemical 2]

[0021]
[Chemical Formula 3]

[0022]
Here, the “active ingredient” means an ingredient having an effect of proliferating osteoblasts such as mammals in the osteogenesis promoter of the present invention, wherein the glycosphingolipid is the osteogenesis promoter of the present invention. It need not be the main component. Further, the osteogenesis promoter of the present invention may contain an ingredient having an osteoblast proliferation effect or an osteogenesis promoting effect in addition to the glycosphingolipid. The active ingredient of the osteogenesis promoter of the present invention can be produced by extraction and purification from echinoderms. Examples of the ingredient include an extract obtained from echinoderms as a raw material for extraction, and dilution of the extract. A liquid or a concentrated liquid, a dried product obtained by drying the extract, or a roughly purified product or a purified product thereof is used. Specific examples of echinoderms include marine starfish (for example, starfish, blue starfish, starfish starfish, starfish, purple starfish), serpentine starfish (eg, starfish, Japanese spider starfish), sea gall [ Sea urchins (for example, sea urchins, sea urchins, etc.), sea bream (for sea cucumbers) (for example, black sea cucumbers, blue sea cucumbers, etc.) From these, the active ingredient can be extracted and purified.
[0023]
As an example of the method for producing the active ingredient of the osteogenesis promoter of the present invention, a method based on the flowchart shown in FIG. 2 is preferably exemplified, and the specific contents thereof are as described below. That is, echinoderms are homogenized with a mixer, then extracted with a mixed solution of chloroform and methanol, filtered, and then the extract is concentrated. After adding water and suspending it, a mixed solution of ethyl acetate and n-butanol was added to perform a partitioning operation, and fractionated into an ethyl acetate / n-butanol layer (α) and an aqueous layer (β). Continue the extraction operation separately. The ethyl acetate / n-butanol layer (α) is concentrated, then acetone is added and mixed, and fractionated into an acetone-soluble fraction and an acetone-insoluble fraction. Next, the organic solvent contained in each fraction is volatilized to prepare extract A (acetone soluble fraction) and extract B (acetone insoluble fraction). Water-saturated n-butanol is added to the previously separated aqueous layer (β) to perform a partitioning operation, and the n-butanol layer (γ) and the aqueous layer (σ) are separated. The organic solvent in the n-butanol layer (γ) is volatilized to obtain extract C. Moreover, after concentrating a water layer ((sigma)), it melt | dissolves in 60% methanol. For separation of components, reverse phase column chromatography is performed by a stepwise gradient method in which the concentration of the methanol solution, which is the eluent, is increased stepwise to 60 to 100% and eluted, and the organic solvent of the 100% methanol elution fraction is removed. Volatilize to prepare extract D. By subjecting each prepared extract to a separation step by normal phase column chromatography or the like, extract A to ceramide, extract B to cerebroside, extract C to lactosylceramide, and extract D to sulfatide, ganglioside, or sphingoline Each glycolipid can be purified.
[0024]
The solvent used in the extraction and purification of the osteogenesis promoter of the present invention is not particularly limited, but it is preferable to use water, an organic solvent, or a mixture thereof. Water that can be used as an extraction solvent is water that has been subjected to various treatments (eg, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc.), such as purified water, hot water, Ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline and the like are also included. Organic solvents that can be used as the extraction solvent include esters such as ethyl acetate and butyl acetate, alcohols such as methanol, ethanol, butanol, and isopropyl alcohol, aliphatic hydrocarbons such as heptane, hexane, and isooctane, and ketones such as acetone and methyl ethyl ketone. , Aromatic compounds such as benzene and toluene, ethers such as diethyl ether and t-butyl ether, lower aliphatic hydrocarbon compounds such as methylene chloride, chloroform and 1,2-dichloroethane, etc., among which methanol, n-butanol It is preferable to use chloroform. The concentration can be adjusted by appropriately selecting an organic solvent according to the kind of raw echinoderms used for production and the kind of active ingredient to be produced (sphingoglycolipid). In addition, several organic solvents can be mixed and used, and the mixing ratio and the like can be appropriately adjusted.
[0025]
  The separation method used in the method for producing the active ingredient of the osteogenesis promoter of the present invention is preferably adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography (HPLC). Examples include a method of using one or a combination of gel-filled column chromatography and the like. The active ingredient of the osteogenesis promoter of the present invention is obtained from an echinoderm by the above production method.Becauseis there. Among the glycosphingolipids that are the active ingredients of the bone formation promoter of the present invention, a compound having an α-oxy fatty acid and a sphingosine skeleton, a compound having an α-oxy fatty acid and a phytosphingosine skeletonThingThese can be used alone, or a plurality of compounds can be selected and used as a mixture. The form of the composition of the present invention is not particularly limited, and examples of the form of the osteogenesis promoter of the present invention include an appropriate form as a pharmaceutical composition. The composition of the present invention may be a glycosphingolipid itself extracted and purified from echinoderms, or may contain components other than the glycosphingolipid. Components other than the glycosphingolipid can be appropriately selected according to the form of the composition.
[0026]
The pharmaceutical composition can be produced, for example, by formulating a glycosphingolipid using a pharmaceutically acceptable excipient or any other additive, and the formulated glycosphingolipid promotes bone formation. It can be used as an agent. In the case of formulation, the content of glycosphingolipid in the formulation is usually 0.000001 to 0.1% by mass, preferably 0.0001 to 0.01% by mass. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and solvents for injection can be used. Examples of excipients include sugar derivatives such as lactose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropylcellulose, hydroxypropyl Cellulose derivatives such as methylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium aluminosilicate; phosphorus such as sodium phosphate, calcium phosphate Acid salt derivatives; carbonate derivatives such as sodium carbonate and calcium carbonate; and sulfate derivatives such as sodium sulfate and calcium sulfate.
[0027]
Examples of the binder include gelatin, polyvinyl pyrrolidone, macrogol and the like in addition to the above excipients. Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone. Examples of the lubricant include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as bee gum and geirow; boric acid; glycol; carvone such as fumaric acid and adipic acid. Acids; Sodium carboxylic acid salts such as sodium benzoate; Sulfates such as sodium sulfate; Leucine; Lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; Silicic acids such as silicic anhydride and silicic acid hydrate; Starch derivatives Etc. Examples of the stabilizer include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like. Examples of the flavoring agent include sweeteners, acidulants, and fragrances. Examples of the solvent for injection include water, ethanol, glycerin and the like.
[0028]
Examples of the administration route of the pharmaceutical composition include parenteral administration such as oral administration and enteral administration. Examples of the dosage form of the pharmaceutical composition include injections, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, ointments, tapes and the like. In addition, the pharmaceutical composition can be administered by being blended with food or drink or feed. The dose and frequency of administration vary depending on the intended effect, administration method, treatment period, age, sex, body weight, etc., but the dose is usually 0.001 to 1000 mg, preferably 0.1 to 0.1 mg per adult day. It can be appropriately selected from the range of 100 mg, and the number of administrations can be appropriately selected from the range of once to several times a day.
[0029]
  Here is shown as a reference exampleThe food / beverage composition composition includes, for example, glycosphingolipid, which is an active ingredient of the osteogenesis promoter of the present invention, saccharides such as dextrin and starch; proteins such as gelatin, casein, whey, soy protein, and corn protein; alanine, glutamine Amino acids such as isoleucine; polysaccharides such as cellulose, guar gum, gellan gum, and gum arabic; and fats and oils such as soybean oil and medium chain fatty acid triglycerides. As a form of the food / beverage product composition, soft drinks, carbonated drinks, nutrition drinks, fruit juice drinks, lactic acid bacteria drinks and other drinks (including concentrated concentrates and powders for adjustment of these drinks); ice cream, sherbet, ice confectionery, etc. Noodles such as buckwheat, udon, harusame, dumplings and cucumber peels, Chinese noodles, instant noodles, etc .; confectionery such as candy, chewing gum, candy, chocolate, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, tablet confectionery; Fish and fishery processed foods such as kamaboko, ham and sausage; Dairy products such as processed milk, fermented milk, and formula milk; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils; Seasonings such as sauces, soups, soups, stews, salads, prepared dishes, pickles; breads; enteral nutrition; functions Food and the like. In addition, content of the sphingoglycolipid which is an active ingredient of the bone formation promoter added when manufacturing a food-drinks composition can be suitably selected from the range of 0.0001-0.1 mass%.
[0030]
  Also shown as a reference exampleThe feed composition includes, for example, glycosphingolipids, cereals such as corn, wheat, barley and rye; bran such as bran, wheat straw, rice bran and defatted rice bran; Animal feeds such as skim milk powder, whey, fish meal, bone meal, etc .; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; amino acids; . Examples of the form of the feed composition include pet food (pet food, etc.), livestock feed, fish feed and the like. In addition, content of the sphingoglycolipid which is an active ingredient of the bone formation promoter added when manufacturing a feed composition can be suitably selected from the range of 0.0001-0.1 mass%. The pharmaceutical composition of the present invention may be used alone, but may be used in combination with other osteogenesis promoters, bone disease prevention / treatment agents, and the like. The combined use can enhance the effect of preventing and treating bone formation and bone disease. Further, the osteogenesis promoter and bone disease preventive / therapeutic agent to be used in combination may be contained as active ingredients in the composition of the present invention, or separate drugs without being contained in the composition of the present invention. Combined as a good product.
[0031]
  Next, production example of glycosphingolipid which is an active ingredient of the osteogenesis promoter of the present inventionExamples of glycosphingolipids (sphingolin glycolipids) shown as reference examplesWill be explained. In addition, the structure of each glycosphingolipid was determined using a mass spectrum method (MS), a nuclear magnetic resonance spectrum method (NMR), etc.
Production Example 1
(Production of ceramide having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish (scientific name: Luidia maculata) was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and the extract was concentrated. The concentrated fraction (41.4 g) was suspended by adding 250 ml of water, and then 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform the partitioning operation. The ethyl acetate / n-butanol layer and the aqueous layer were separated. Of these, the ethyl acetate / n-butanol layer was concentrated, acetone was added to the concentrated fraction (9.9 g) and mixed, and the acetone-soluble fraction was recovered, and then the organic solvent was volatilized to extract about 5.3 g of Extract A. Got. Next, the extract A was dissolved in 5 ml of chloroform and added to a normal phase column (Silica gel 60: manufactured by Merck & Co., Inc.). A chloroform / methanol mixture (chloroform: methanol = 100: 0, 98: 2, 95) was used as an elution solvent. : 5, 9: 1, 85:15, 7: 3, and 6: 4), and the elution was performed by a stepwise gradient method in which the methanol concentration was increased stepwise. A fraction showing the same behavior as that of the existing ceramide was collected by normal phase thin layer chromatography, and the fraction was further added to a normal phase column and mixed with chloroform / methanol (chloroform: methanol = 97: 3 → 95: 5), and two types of ceramides, LMCer-1 (3 mg) and LMCer-2 (4 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, were produced. The structural formula of LMCer-1 is shown in Chemical Formula 4, and the structural formula of LMCer-2 is shown in Chemical Formula 5, respectively.
[0032]
4
H-Cer1
5
H-Cer2
[0033]
Production Example 2
(Production of cerebroside having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this concentrated fraction and suspending it, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to carry out a partitioning operation. The n-butanol layer and the aqueous layer were separated. The ethyl acetate / n-butanol layer was concentrated and then added with acetone and mixed to collect an acetone-insoluble fraction, and the organic solvent was evaporated to obtain about 4.5 g of extract B. Next, the extract B is dissolved in 5 ml of a mixed solution of chloroform / methanol / water (mixing ratio = 9: 1: 0.05) and added to a normal phase column (Silica gel 60: manufactured by Merck). Eluted with. The fraction showing the same behavior as that of the existing cerebroside was collected by normal phase thin layer chromatography, and the fraction was added to a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai Co., Ltd.) And eluted. Next, a fraction showing the same behavior as that of existing cerebroside in normal phase thin layer chromatography was added to a gel filtration column (Sephadex LH-20: manufactured by Pharmacia), and chloroform / methanol (chloroform: methanol = 1). 1) Column chromatography using the mixed solution as an eluent, and two types of cerebrosides, LMC-1 (5 mg) and LMC-2 (6 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, Manufactured. The structural formula of LMC-1 is shown in Chemical Formula 6, and the structural formula of LMC-2 is shown in Chemical Formula 7, respectively.
[0034]
6
Glcβ1 → 1Cer1
(Glc: glucose)
7
Glcβ1 → 1Cer2
(Glc: glucose)
[0035]
Production Example 3
(Production of lactosylceramide having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this and suspending, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform a partitioning operation, and ethyl acetate / n-butanol was added. Separated as a layer and an aqueous layer. Here, a water-saturated n-butanol is further added to the separated aqueous layer to perform a partitioning operation, and the n-butanol layer and the aqueous layer are separated, and the organic solvent is volatilized in the n-butanol layer and extracted. C. The dry weight of Extract C was about 3.2 g. Next, the extract C was dissolved in 5 ml of a chloroform / methanol (chloroform: methanol = 1: 1) mixed solution, added to a reverse phase column (Cosmosil 140C18-PREP: manufactured by Nacalai Co., Ltd.), and eluted with the same mixed solution. Subsequently, the fraction which showed the same behavior as the existing lactosylceramide was collect | recovered by normal phase thin layer chromatography. The fraction was added to a normal phase column (Silica gel 60: manufactured by Merck), and the mixing ratio was chloroform: methanol: water = 9: 1.5: 0.5, 8: 2: 0.2, and 6: Elution was performed by a stepwise gradient method using a 4: 1 each chloroform / methanol / water mixture and gradually increasing the methanol concentration. Among the eluted fractions, two types of lactosylceramide, LMCDH-1 (1.7 mg) and LMCDH-2 (1.4 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, were produced. The structural formula of LMCDH-1 is shown in Chemical Formula 8, and the structural formula of LMCDH-2 is shown in Chemical Formula 9, respectively.
[0036]
8
Galβ1 → 4Glcβ1 → 1Cer1
(Gal: galactose, Glc: glucose)
9
Galβ1 → 4Glcβ1 → 1Cer2
(Gal: galactose, Glc: glucose)
[0037]
Production Example 4
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this and suspending, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform a partitioning operation, and ethyl acetate / n-butanol was added. Separated as a layer and an aqueous layer. Here, water-saturated n-butanol was further added to the separated water layer to perform a partitioning operation, and the n-butanol layer and the water layer were separated, and the water layer was recovered. After concentrating the aqueous layer, separation was performed using a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai Co., Ltd.). Separation of the components was performed by a stepwise gradient method in which the concentration of the methanol solution as an eluent was gradually increased to 60 to 100% and eluted. About 0.18 g of extract D was prepared by volatilizing the organic solvent of the elution fraction when the concentration of methanol reached 100%. Next, the extract D is dissolved in a mixed solution of chloroform / methanol / water (chloroform: methanol: water = 6: 4: 1) and added to a normal phase column (Silica gel 60: manufactured by Merck). Eluted. Subsequently, sulfatide LMG-1 (0.67 mg) and ganglioside LMG, which are added to a gel filtration column (Sephadex LH-20: manufactured by Pharmacia), desalted, and exhibit the behavior of a single substance by normal phase thin layer chromatography. -2 (2.4 mg) was prepared respectively. The structural formula of LMG-1 is shown in Chemical Formula 10, and the structural formula of LMG-2 is shown in Chemical Formula 11, respectively.
[0038]
10
3-O-HSO_Three Galβ1 → 4Galβ1 → 4Glcβ1 → 1Cer2
(Gal: galactose, Glc: glucose)
11
NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, Gal: galactose, Glc: glucose)
[0039]
Production Example 5
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside LLG-3 (58 mg) and LLG-5 (70 mg) were produced in the same manner as in Production Example 4 using 18 kg of blue starfish (scientific name: Linckia laevigata) as a starting material. The structural formula of LLG-3 is shown in Chemical formula 12, and the structural formula of LLG-5 is shown in Chemical formula 13.
[0040]
12
8-O-CH_Three NeuAcα2 → 11NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
13
8-O-CH_Three NeuGcα2 → 11NeuGcα2 → 11NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
[0041]
Production Example 6
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside GAA-7 (86 mg) was produced in the same manner as in Production Example 4 using 65 kg of purple starfish (scientific name: Asterias amurensis versicolor) as a starting material. The structural formula of GAA-7 is shown in Chemical formula 14.
[0042]
14
8-O-CH_Three NeuGcα2 → 6 [8-O-CH_Three NeuGcα2 → 3] GalNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, GalNAc: N-acetylgalactosamine, Gal: galactose, Glc: glucose)
[0043]
Production Example 7
(Manufacturing of sphingoline glycolipid)
Using 7.6 kg of Japanese sea urchin (scientific name: Comanthus japonica) as a starting material, an extract D derived from Nippon sea urchin was produced in the same manner as in Production Example 4. The extract D is dissolved in 2 ml of a chloroform / methanol / water mixture (chloroform: methanol: water = 8: 2: 0.2) and added to a normal phase column (Silica gel 60: manufactured by Merck). Were eluted by a stepwise gradient method using increasing chloroform concentration using chloroform: methanol: water = 8: 2: 0.2 and 6: 4: 1 chloroform / methanol / water mixtures. Sphingoline glycolipids CJP-1 (92 mg), CJP-2 (170.6 mg) and CJP-3 (568.5 mg) were prepared on thin layer chromatography. The structural formula of CJP-1 is shown in chemical formula 15, the structural formula of CJP-2 is chemical formula 16, and the structural formula of CJP-3 is shown in chemical formula 17.
[0044]
15
Ins1 → Phospho-Cer
(Ins: Inositol)
16
9-OCH_Three NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
17
9-OCH_Three NeuGcα2 → 11 [9-OCH_Three ] NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
[0045]
In the production examples 1 to 6, the α-oxy fatty acid and the sphingoglycolipid having a sphingosine skeleton or phytosphingosine skeleton extracted and purified from the echinoderms by the present inventors are purified from mammals and plants. Unlike the compound having a sphingosine skeleton described in Patent Document 3, the compound is confirmed to have a structure specific to echinoderms and the α-position of the fatty acid in the molecule is oxidized. I understood that. Moreover, the sphingoline glycolipid produced in Production Example 7 had a sphingophosphoglycolipid structure unique to sea lilies and sea urchins.
[0046]
Next, a test example is shown and this invention is demonstrated in detail.
Test example 1
This test was conducted to measure the osteoblast proliferation promoting activity of glycosphingolipid extracted and purified from echinoderms.
(1) Sample preparation
Each glycosphingolipid produced in Production Examples 1 to 7 (LMer-1, LMer-2, LMC-1, LMC-2, LMCDH-1, LMCDH-2, LLG-3, LLG-5, GAA-7, CJP-1, CJP-2, CJP-3) were used as test samples, respectively. Further, C2 ceramide (N-Acetyl-D-sphingosine semisynthetic: Sigma, catalog No. A7191), which is a kind of conventional glycosphingolipid having no α-oxy fatty acid and phytosphingosine skeleton, was used as a control sample.
[0047]
(2) Test method
The mouse osteoblast cell line MC3T3-E1 (RCB1126: RIKEN Cell Bank) is 5 × 10 5 per well in α-MEM medium (Gibco BRL) containing 10% fetal calf serum.^Four The cells were dispensed into 96-well microplates and cultured at 37 ° C. in the presence of 5% carbon dioxide gas for 18 hours. After culturing, the medium was removed, and 100 μl of α-MEM medium containing 2% fetal calf serum was added to each well. Then, each test sample and control sample were added to each well so that the final concentration was 0.2 μg / ml, and further cultured for 2 days. Tritium thymidine was added to each well at an amount of 50 nCi 6 hours before the end of the culture. After completion of the culture, the amount of DNA synthesized in each well was measured using a liquid scintillation counter (LKB1219 RACK-BETA: manufactured by LKB).
[0048]
(3) Test results
The results of this test are shown in FIG. FIG. 3 shows the activity of promoting osteoblast proliferation by glycosphingolipid extracted and purified from echinoderms. As a result, it was revealed that the glycosphingolipid derived from echinoderms used in each test sample has a high osteoblast proliferation promoting effect with respect to C2 ceramide as a control sample. Among them, glycosphingolipids (LMer-2, LMC-2, LMCDH-2, LLG-3, LLG-5, GAA-7) having a phytosphingosine skeleton are generally more active than those having a sphingosine skeleton. There was found. Therefore, glycosphingolipids having a structure specific to echinoderms (for example, having an α-oxy fatty acid, a phytosphingosine skeleton, etc.) have a stronger osteoblast proliferation promoting effect than conventional glycosphingolipids. It became clear.
[0049]
Test example 2
This test was performed to measure the ex vivo osteoblast proliferation-promoting activity of mice administered with glycosphingolipid extracted and purified from echinoderms.
(1) Sample preparation
Among the glycosphingolipids produced in the above production examples, LMcer-2, LMCDH-2, CJP-3, and LGG-3 were used as test samples, respectively. Moreover, physiological saline was used as a negative sample.
[0050]
(2) Test method
In this test, a 4-week-old female ddy mouse (purchased from Japan SLC) was used. After the ovaries of the mice were removed, osteoporosis model mice prepared by feeding a low calcium diet for 7 weeks were divided into 6 groups per group. Each test sample solution prepared to 10 μg / ml with physiological saline was intravenously administered to the osteoporosis model mouse, 200 μl each, from the tail vein once a day for 14 days. After the administration was completed, bone marrow cells of femur and tibia were collected in RPMI 1640 medium (Gibco BRL). Collected osteoblasts at 1 x 10 per well⁶ The cells were dispensed into 6-well microplates and cultured in RPMI 1640 medium containing 10% fetal bovine serum for 7 days. After completion of the culture, alkaline phosphatase staining (DIAGNOSTICS, No. 85: manufactured by Sigma) was performed, and positive colonies were counted as osteoblast colonies.
[0051]
(3) Test results
The results of this test are shown in FIG. FIG. 4 shows the activity of promoting osteoblast proliferation in osteoporosis model mouse bone marrow cells by glycosphingolipid extracted and purified from echinoderms. As a result, it was found that any of the test samples had an action of proliferating osteoblasts in vivo.
[0052]
Test example 3
This test was performed to measure the ex vivo osteoblast proliferation promoting activity of mice administered with a sphingoglycolipid extract, an intermediate purified product from echinoderms.
(1) Sample preparation
Extract A (ceramide composition) roughly purified in Preparation Example 1, Extract B (Cerebroside composition) roughly purified in Preparation Example 2, Extract C (lactosylceramide composition) roughly purified in Preparation Example 3, and Preparation Example Extract D (ganglioside composition) roughly purified in 4 was used as a test sample, dissolved in physiological saline to 500 μg / ml, and used for the test. At this time, extract A contains 1 μg of ceramide, extract B contains 1.2 μg of cerebroside, extract C contains 0.5 μg of lactosylceramide, and extract D contains 8 μg of ganglioside. Further, a solution prepared by preparing a ceramide derived from a mammal (manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 031-18441) to 20 μg / ml with physiological saline was used as a control sample A, and cerebroside (manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 0.076-03963) with physiological saline to 20 μg / ml was used as control sample B, and a ganglioside mixture (manufactured by Wako Pure Chemical Industries, Catalog No. 074-03643) was prepared with physiological saline to 20 μg / ml. The obtained solution was used as a control sample D, and physiological saline was used as a negative sample for each test.
[0053]
(2) Test method
In this test, a 6-week-old male db / db mouse (purchased from CLEA Japan) was used. The mice were grouped into 5 mice per group, and 500 μl each of extract A, extract B, extract C, extract D, control sample A, control sample B, control sample D, and negative sample were once a day. Forcible oral administration was continued for 36 days using a sonde. After the administration was completed, bone marrow cells of femur and tibia were collected in RPMI 1640 medium (Gibco BRL). Collected osteoblasts at 1 x 10 per well⁶The cells were dispensed into 6-well microplates and cultured in RPMI 1640 medium containing 10% fetal bovine serum for 7 days. After completion of the culture, alkaline phosphatase staining (DIAGNOSTICS, No. 85: manufactured by Sigma) was performed, positive colonies were counted as osteoblast colonies, and the average value of 5 mice per group was calculated.
[0054]
(3) Test results
The results of this test are shown in Table 1. Table 1 shows osteoblast proliferation promoting activity by glycosphingolipid extract, which is an intermediate purified product from echinoderms. As a result, the extract A, the extract B, the extract C, the extract D, the control sample A, the control sample B, and the control sample D all have an increased osteoblast colony count and a high growth promoting activity with respect to the negative sample. It has been found. However, it is negative for glycosphingolipid extract of the present invention (extract A, extract B, extract C, extract D), commercially available ceramide (control sample A), cerebroside (control sample B), and ganglioside mixture (control sample D). When the ratio of increase in the number of osteoblast colonies relative to the sample was compared, the extract A (ceramide composition): 5.0 compared to the control sample A: 3.2, and the extract B (cerebroside) compared to the control sample B: 1.1. Composition): 2.4, Control sample D: Extract D (ganglioside composition): 7.6 compared to 1.3, and therefore, the echinoderm-derived glycosphingolipid extract is derived from a conventional mammal. It was found to have an excellent osteoblast proliferation promoting effect as compared with glycosphingolipids.
[0055]
[Table 1]

[0056]
【Example】
EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.
Example 1
An injection having the following composition was produced by a conventional method.
Ceramide (LMCer-2) produced in Production Example 1 0.01 (%)
Actinomycin D (manufactured by Sigma) 0.005
Sodium chloride (Wako Pure Chemical Industries, Ltd.) 0.9
Mannitol (manufactured by Kanto Chemical Co., Inc.) 1.0
Distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) 98.085
[0057]
referenceExample1
  10.8 kg of whey protein enzyme degradation product (manufactured by Morinaga Milk Industry Co., Ltd.), 36 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), and a small amount of water-soluble vitamins and minerals were dissolved in 200 kg of water, and an aqueous phase was prepared in the tank. Separately, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 3 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 8.5 kg, safflower oil (manufactured by Taiyo Yufu Co., Ltd.) 2.5 kg, lecithin (manufactured by Ajinomoto Co., Inc.) 0.2 kg, An oil phase was prepared by mixing and dissolving 0.2 kg of a fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of a fat-soluble vitamin. The oil phase was added to the aqueous phase in the tank, stirred and mixed, then heated to 70 ° C., and further homogenized with a homogenizer at a pressure of 14.7 MPa. Next, the mixture was sterilized at 90 ° C. for 10 minutes, concentrated and spray-dried to prepare about 59 kg of intermediate product powder. 50 kg of this intermediate product powder, 6.8 kg of sucrose (manufactured by Hokuren), 167 g of amino acid mixed powder (manufactured by Ajinomoto Co., Inc.), and sphingophosphoglycolipid (CJP-3) produced in Production Example 7 above
) 60 g was added and mixed uniformly to produce about 56 kg of enteral nutrition food powder containing sphingoline glycolipid and having osteogenesis promoting effect.
[0058]
referenceExample2
  100 g of ganglioside (GAA-7) produced in Production Example 6, 110 g of lactulose powder (manufactured by Morinaga Milk Industry), 655 g of maltextrin (manufactured by Matsutani Chemical Industry), 95 g of skim milk powder (manufactured by Morinaga Milk Industry), stevia sweetener ( Each powder of 1 g of Saneigen F.F.I.), 5 g of yogurt flavor (manufactured of Saneigen F.F.I.), and 34 g of glycerin fatty acid ester preparation (manufactured by Riken Vitamin Co., Ltd.) was added and mixed uniformly. Using a tableting machine (manufactured by Hata Steel Co., Ltd.), the mixed powder was continuously tableted at a tableting speed of 12 tablets / minute and a pressure of 9.8 KPa at a tablet weight of 0.5 g. 1800 tablets (about 900 g) containing ganglioside (GAA-7) were produced.
[0059]
referenceExample3
  10 kg of raw milk having a milk fat content of 3.5% and a non-fat milk solid content of 9.2% was homogenized and sterilized by heating at 90 to 92 ° C. for 10 minutes. After cooling to about 42 ° C., commercially available Streptococcus thermophilus and Lactobacillus delbrueckii as starters
Subsp. bulgaricus) milk culture was added 100 g each. The mixture was sufficiently stirred in the fermentation tank and allowed to stand at 42 to 45 ° C. for 4 hours for fermentation. The fermented milk was cooled to 5-8 ° C. with stirring, and then homogenized with a homogenizer to prepare homogenized fermented milk. Next, 60 g of pectin (manufactured by Saneigen FFI Co.) and 30 g of cerebroside (LMC-2) produced in Production Example 2 were added to 5 kg of 17% granulated sugar (manufactured by Toyo Seika Co., Ltd.), and 90-92 ° C. Was sterilized for 10 minutes and cooled to 5-8 ° C. to prepare a sugar solution. All the homogenized fermented milk and sugar solution prepared by these methods were thoroughly mixed in a tank, filled in 120 ml each in a paper container, and sealed to produce a drink yogurt.
[0060]
【The invention's effect】
  As described above in detail, the present invention provides an α-oxy fatty acid and a sphingosine skeleton extracted from echinoderms, a sphingoglycolipid having an α-oxy fatty acid and a phytosphingosine skeleton.QualityThe present invention relates to a pharmaceutical osteogenesis promoter contained as an active ingredient, and the following effects are exhibited by the present invention.
(1) A novel osteogenesis promoter that promotes the proliferation of osteoblasts such as mammals can be provided.
(2) The osteogenesis promoter of the present invention is effective for the treatment and / or prevention of osteoporosis and other bone metabolic diseases.
(3) A pharmaceutical composition for prevention / treatment of osteoporosis and the like can be provided.
(4) It is possible to provide an effective utilization method of echinoderms captured in large quantities in the field of fisheries.
[Brief description of the drawings]
FIG. 1 shows the basic structure of glycosphingolipids.
FIG. 2 is a flowchart showing a method for producing an α-oxy fatty acid, a sphingoglycolipid having a sphingosine skeleton or a phytosphingosine skeleton, and a sphingoline glycolipid from echinoderms.
FIG. 3 shows osteoblast proliferation promoting activity by glycosphingolipid extracted and purified from echinoderms.
FIG. 4 shows the activity of promoting the proliferation of osteoblasts in osteoporosis model mouse bone marrow cells by glycosphingolipid extracted and purified from echinoderms.

Claims (3)

1. A bone formation promoter for use in medicine, which is a compound purified from an organism belonging to the Echinodermata and is selected from the following ceramides having the α-oxy fatty acid and the sphingosine skeleton, An osteogenesis promoter comprising a seed or a mixture of a plurality of kinds as an active ingredient.
4
H-Cer1

6
Glcβ1 → 1Cer1
(Glc: glucose)
_However, R 2 -Cer1 represents a structure represented by the following formula 1.

1

A bone formation promoter for pharmaceutical use, which is a compound purified from an organism belonging to the echinodermia , having an α-oxy fatty acid and a phytosphingosine skeleton, the following ceramide, chem 7 cerebrosid, chem 10 An osteogenesis promoter characterized by comprising, as an active ingredient, one or a mixture of sulfatide and a ganglioside of Chemical Formulas 11-14.
5
H-Cer2

7
Glcβ1 → 1Cer2
(Glc: glucose)

10
3-O-HSO ₃ Galβ1 → 4Galβ1 → 4Glcβ1 → 1Cer2
(Gal: galactose, Glc: glucose)

11
NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, Gal: galactose, Glc:
glucose)

12
8-O-CH ₃ NeuAcα2 → 11 NeuGcα2 → 3Galβ1 → 4Gl
cβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)

13
8-O-CH ₃ NeuGcα2 → 11NeuGcα2 → 11NeuGcα2 →
3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc
:glucose)

14
8-O-CH ₃ NeuGcα2 → 6 [8-O-CH ₃ NeuGcα2 → 3] G
alNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, GalNAc: N-acetylgalactosamine, Gal: galactose, Glc: glucose)
However, R < ₂ > -Cer2 shows the structural formula represented by the following Chemical formula 2.

2

The osteogenesis promoter according to claim 1 or 2 , which is a pharmaceutical composition for prevention / treatment of osteoporosis, rheumatoid arthritis, Paget's disease of bone, or osteoarthritis.

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