JP2004269374A - Hypoglycemic composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hypoglycemic composition having effects on inhibition of a rise in blood glucose level and reducing the blood glucose level from a hyperglycemic state and therapeutic and/or prophylactic effects of complications caused by the hyperglycemic state in a prediabetic or a diabetic state. <P>SOLUTION: The hypoglycemic composition comprises a sphingoglycolipid having an α-oxyfatty acid and a sphingosine skeleton or the α-oxyfatty acid and a phytosphingosine skeleton and extracted from an echinoderm as an active ingredient. Furthermore, the hypoglycemic composition which is a medicinal composition, a food and drink composition or a feed composition is obtained. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００２１】
【化２】

【００２２】
ここで、「有効成分」とは、本発明の血糖降下組成物における前記に定義した作用を有する成分を意味し、本発明の血糖降下組成物の主たる有効成分が前記スフィンゴ糖脂質でなければならない必要は無く、また、本発明の血糖降下組成物にはスフィンゴ糖脂質以外に血糖低下作用又は血糖上昇抑制効果を有する成分が含まれていても良い。
【００２３】
本発明の血糖降下組成物の有効成分は、棘皮動物であれば何れを抽出原料として用いて抽出精製しても製造することが可能であり、その有効成分には、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、あるいはこれらの粗精製物又は精製物のいずれも含まれる。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、本発明の血糖降下組成物の有効成分を抽出精製することが可能である。
【００２４】
本発明の血糖降下組成物の有効成分の製造方法に関する一例としては、稲垣らの方法（〔ヨーロピアン・ジャーナル・オブ・オルガニック・ケミストリー［Ｅｕｒ． Ｊ． Ｏｒｇ． Ｃｈｅｍ．］、１９９８年、ｐ．１２９−１３１〕、〔ケミカル・ファーマシューティカル・ブレチン［Ｃｈｅｍ． Ｐｈａｒｍ． Ｂｕｌｌ．］、第４９巻、第１２号、２００１年、ｐ．１５２１−１５２５〕）、に基づいた、図２に示すフローチャートの方法が例示され、具体的には、次に記載するとおりである。即ち、棘皮動物をホモジナイズし、これを、有機溶媒、特に好ましくはクロロホルム・メタノール又は塩化メチレン・メタノール混合比が１：１から１：１０、好ましくは１：２から１：３のいずれかの混合比である混合溶液で抽出処理する。この抽出液を濃縮した後、酢酸エチル・ブタノール混合比が５：１から１：１、好ましくは３：１から２：１のいずれかの混合比である混合液、及び水を添加して、分配操作を行った後、酢酸エチル・ブタノール層（α層）と水層（β層）を分取する。このうち、酢酸エチル・ブタノール層（α層）を濃縮した後、アセトンを添加し、アセトン可溶性画分（セラミド混合物）を得る。また、水層（β層）に水飽和ブタノール（ｎ−ブタノールを使用することが好ましい。）を添加して分配操作を行い、ブタノール層と水層（δ）に分画する。このうち、水層（δ）を濃縮後、逆相カラムを用いて以下の条件で分離を行う。即ち、まず、溶出液であるメタノール又はエタノールの濃度を６０％から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分離を行う。メタノール濃度が１００％の画分を回収し、濃縮してガングリオシド混合物を得る。
【００２５】
前記、中間精製物として調製したセラミド混合物は、有効成分であるセラミドを１〜１０％含有していることが好ましく、少なくとも前記範囲を含有していれば、そのまま、本発明の血糖降下組成物の有効成分として使用することができる。
また、前記中間精製物として調製したガングリオシド混合物は、有効成分であるガングリオシドを２０〜６０％含有していることが好ましく、少なくとも前記範囲のガングリオシドを含有していれば、そのまま、本発明の血糖降下組成物の有効成分として使用することができる。
【００２６】
前記、セラミド混合物、及びガングリオシド混合物は、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を行うことによって、セラミド混合物からセラミド精製標品、同様にガングリオシド混合物からガングリオシド精製標品をそれぞれ精製することが可能であり、これらについても、本発明の有効成分として使用することができる。セラミド精製標品及びガングリオシド精製標品の純度は、薄層クロマトグラフィー等により決定することができ、それぞれ９０％以上、特に９５％以上であることが好ましい。
【００２７】
本発明の血糖降下組成物の有効成分を抽出精製する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族炭化水素、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。本発明では、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。
【００２８】
また、本発明の血糖降下組成物の製造方法の中で使用される有効成分の分離法は、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ことが可能である。
本発明の血糖降下組成物の有効成分としては、前記製造方法によって棘皮動物から得られたものの他に、同様の構造を有する化合物を化学合成によって製造したものを使用することも可能である。
【００２９】
本発明の血糖降下組成物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、及び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、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く、スフィンゴ糖脂質以外の成分を含有していても良い。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できる。
本発明の組成物の形態は、特に限定されるものではなく、本発明の血糖降下組成物を、例えば、医薬組成物、飲食品組成物、飼料組成物等の形態として使用することが可能である。
【００３０】
これらのうち、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でき、製剤化したスフィンゴ糖脂質は、血糖降下組成物として使用できる。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、０．００１〜１．０質量％、好ましくは０．０１〜０．１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。
【００３１】
また、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。
【００３２】
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。
【００３３】
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、前記医薬組成物は、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、０．０１〜１０００ｍｇ、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。
【００３４】
次に、飲食品組成物は、例えば、本発明の血糖降下組成物又は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血糖降下組成物又は有効成分であるスフィンゴ糖脂質の含有量は、０．００１〜１０質量％の範囲から適宜選択することができる。
【００３５】
次に、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血糖降下組成物又は有効成分であるスフィンゴ糖脂質の含有量は、０．００１〜１０質量％の範囲から適宜選択することができる。
【００３６】
本発明の組成物は、単独で使用しても良いが、その他の血糖降下組成物等と併用して使用しても良い。それらの併用によって、高血糖状態での血糖値低下作用を高めることができる。また、本発明の組成物を併用する血糖降下組成物の組成物中に有効成分として含有させても良いし、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。
【００３７】
本発明の血糖降下組成物は、血糖値の診断基準（前記、非特許文献１６：橘敏也著、「生活習慣病とその治療薬」、株式会社じほう、２００１年、ｐ．４２−４３）に区分された境界領域（空腹時：１１０〜１２６ｍｇ／ｄｌ、又は７５ｇブドウ糖負荷試験［ＯＧＴＴ］２時間後：１４０〜２００ｍｇ／ｄｌ、のいずれかの状態）、もしくは糖尿病領域（空腹時：１２６ｍｇ／ｄｌ以上、又は７５ｇブドウ糖負荷試験［ＯＧＴＴ］２時間後：２００ｍｇ／ｄｌ以上のいずれかの状態）の状態から、正常領域（空腹時：１１０ｍｇ／ｄｌ以下、又は７５ｇブドウ糖負荷試験［ＯＧＴＴ］２時間後：１４０ｍｇ／ｄｌ以下のいずれかの状態）に血糖値を低下させる作用、及び、正常領域から境界領域、正常領域から糖尿病領域、又は境界領域から糖尿病領域のいずれかの場合の血糖値の上昇を抑制する作用によって、前糖尿病もしくは糖尿病状態での高血糖状態における治療や、高血糖状態から引き起こされる合併症の治療及び／又は予防に有用である。
【００３８】
一般的に、高血糖状態から血糖値を低下させる作用、及び血糖値の上昇を抑制する作用は、糖尿病モデルマウスを使用したｉｎ ｖｉｖｏ による効果として、該マウスに血糖降下剤等の投与を開始し、一定期間投与をした段階での血糖値及び糖化ヘモグロビン量を測定することによって評価することができる。糖化ヘモグロビンは、ヘモグロビンＡ１ａ、ヘモグロビンＡ１ｂ、ヘモグロビンＡ１ｃの３つの成分から構成され、長時間にわたって血液中に残留する傾向をもっているので、血液中の糖化蛋白質の優れた指標となる。中でも、ヘモグロビンＡ１ｃ（ＨｂＡ１ｃ）のレベルは、測定の前の６〜８週間の平均的血糖コントロールレベルの指標として血中グルコース治療の有効性を反映するので、糖尿病の診断に有効なマーカーであり、血糖値の測定と併用して糖尿病の診断及び進行度の測定に利用されている〔ディアベティック・メディシン（Ｄｉａｂｅｔｉｃ Ｍｅｄｉｃｉｎｅ ）、アメリカ、第１４巻、１９９７年、ｐ．５８４−５８８〕。これらのことから、本発明の血糖降下組成物の血糖低下作用については、その検討方法として、前記血糖値及び糖化ヘモグロビン量を測定することによって評価した。
【００３９】
尚、本発明において、血糖値の測定については、固定化ブドウ糖酸化酵素膜・過酸化水素電極法（基礎と臨床、第３０巻、第５号、１９９６年５月、ｐ．１１２５−１１３２）に基づいた血糖測定装置（例えば、バイエル・三共社製：アントセンスＩＩ）等により測定した。また、糖化ヘモグロビン量の測定については、ラテックス凝集阻止反応に基づくＨｂＡ１ｃ の簡易測定法（医学と薬学、第３７巻、第４号、１９９７年４月、ｐ．１０２３−１０２８）による測定装置（例えば、バイエル・三共社製：ＤＣＡ２０００）等により測定した。
【００４０】
次に、本発明の血糖降下組成物の有効成分であるスフィンゴ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をミキサーに投入し、次いで、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リットルをミキサーに添加し、ホモジナイズして抽出を行い、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セラミド混合物約５．３ｇを得た。次いで、該セラミド混合物をクロロホルム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おいて既存のセラミドと同様の挙動を示す画分について回収し、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おいて単一物質の挙動を示すＬＭＣｅｒ−１（３ｍｇ）及びＬＭＣｅｒ−２（４ｍｇ）の２種類のセラミドをそれぞれ製造した。ＬＭＣｅｒ−１の構造式を化３、及びＬＭＣｅｒ−２の構造式を化４にそれぞれ示す。
（化３）
Ｈ−Ｃｅｒ１
（化４）
Ｈ−Ｃｅｒ２
【００４１】
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をミキサーに投入し、次いで、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リットルをミキサーに添加し、ホモジナイズして抽出を行い、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、分取した水層は、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。該水層は、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。分離は、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、ガングリオシド混合物約０．１８ｇを調製した。次いで、該ガングリオシド混合物をクロロホルム／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おいて単一物質の挙動を示すガングリオシドＬＭＧ−２（２．４ｍｇ）を製造した。ＬＭＧ−２の構造式を化５に示す。
【００４２】
（化５）
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）
【００４３】
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ガングリオシドの製造）
出発原料にアオヒトデ（学名：Ｌｉｎｃｋｉａ ｌａｅｖｉｇａｔａ ）１８ｋｇを用い、製造例２と同様の方法で、ガングリオシドＬＬＧ−３（５８ｍｇ）及びＬＬＧ−５（７０ｍｇ）をそれぞれ製造した。ＬＬＧ−３の構造式を化６、ＬＬＧ−５の構造式を化７に示す。
（化６）
８−Ｏ−ＣＨ_３ 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７）
８−Ｏ−ＣＨ_３ 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）
【００４４】
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）
出発原料にムラサキヒトデ（学名：Ａｓｔｅｒｉａｓ ａｍｕｒｅｎｓｉｓ ｖｅｒｓｉｃｏｌｏｒ ）６５ｋｇを用い、製造例２と同様の方法で、ガングリオシドＧＡＡ−６（２０ｍｇ）及びＧＡＡ−７（８６ｍｇ）を製造した。ＧＡＡ−６の構造式を化８、ＧＡＡ−７の構造式を化９に示す。
（化８）
８（１１）−Ｏ−ＣＨ_３ ＮｅｕＧｃα２→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）
（化９）
８−Ｏ−ＣＨ_３ ＮｅｕＧｃα２→６〔８−Ｏ−ＣＨ_３ 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）
【００４５】
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）
出発原料にオニヒトデ（Ａｃａｎｔｈａｓｔｅｒ ｐｌａｎｃｉ）１２ｋｇを用い、製造例２と同様の方法で、ガングリオシドＡＧ−１（３６．４ｍｇ）、ＡＧ−２（２９０ｍｇ）、 ＡＧ−３（８．９ｍｇ）を製造した。ＡＧ−１の構造式を化１０、ＡＧ−２の構造式を化１１、ＡＧ−３の構造式を化１２に示す。
（化１０）
Ｆｕｃｆβ１→４Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ｆｕｃｆ：フコフラノース）
（化１１）
Ｇａｌｆβ１→３Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ｇａｌｆ：ガラクトフラノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
（化１２）
Ｇａｌｆβ１→３Ｇａｌα１→３Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ｇａｌｆ：ガラクトフラノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
【００４６】
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）
出発原料にイトマキヒトデ（Ａｓｔｅｒｉｎａ ｐｅｃｔｉｎｉｆｅｒａ）４２ｋｇを用い、製造例２と同様の方法で、ガングリオシドＧＰ−１ａ（７２ｍｇ）、ＧＰ−１ｂ（１１９ｍｇ）、ＧＰ−２（４２１ｍｇ）を製造した。ＧＰ１ａの構造式を化１３、ＧＰ−１ｂの構造式を化１４、ＧＰ−２の構造式を化１５に示す。
（化１３）
Ａｒａｆα１→３Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ａｒａｆ：アラビノフラノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
（化１４）
Ａｒａｆα１→４〔Ａｒａｆα１→３〕Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ａｒａｆ：アラビノフラノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
（化１５）
Ａｒａｆα１→４Ｇａｌα１→６〔Ａｒａｆα１→４〕Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ａｒａｆ：アラビノフラノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
【００４７】
製造例７
（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）
出発原料にヒラモミジイガイ（Ａｓｔｒｏｐｅｃｔｅｎ ｌａｔｅｓｐｉｎｏｓｕｓ）３０ｋｇを用い、製造例２と同様の方法で、ガングリオシドＬＧ−１（１４６ｍｇ）、ＬＧ−２（５４５ｍｇ）を製造した。ＬＧ−１の構造式を化１６、ＬＧ−２の構造式を化１７に示す。
（化１６）
Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
（化１７）
Ａｒａβ１→４Ｇａｌα１→４ＮｅｕＡｃα２→３Ｇａｌβ１→４Ｇｌｃβ１→１Ｃｅｒ２
（Ａｒａ：アラビノース、ＮｅｕＡｃ：Ｎ−アセチルノイラミン酸）
尚、前記製造例１〜７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た。
【００４８】
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、棘皮動物から抽出分画したスフィンゴ糖脂質の中間精製物のマウスへの経口投与による血糖値低下作用を確認するために行った。
（１）試料の調製
前記製造例１及び２で調製したヤツデスナヒトデ由来のセラミド混合物（セラミド有効成分約１μｇ含有）及びガングリオシド混合物（ガングリオシド有効成分約８μｇ含有）を、それぞれ生理食塩水によって５００μｇ／ｍｌに調製した試料を、それぞれ試験試料１及び試験試料２とした。また、牛由来セラミド（和光純薬工業社製：カタログ番号０３１−１８４４１）、及びα−オキシ脂肪酸及びフィトスフィンゴシン骨格をもたない牛由来ガングリオシド混合液（和光純薬工業社製：カタログ番号０７４−０３６４３）を、生理食塩水によって２０μｇ／ｍｌに調製した試料を、それぞれ対照試料１及び対照試料２とした。更に、従来の血糖降下剤である２型経口糖尿病薬ジアベン（中外製薬社製：トルブタミド錠）を粉砕した後、２％カルボキシメチルセルロース（ナカライテスク社製）溶液にて、１ｍｇ／ｍｌに調製した試料を、対照試料３とした。尚、生理食塩水を陰性試料とした。
【００４９】
（２）試験方法
本試験に使用するマウスは、２型糖尿病モデルマウスである、６週齢、雄性ｄｂ／ｄｂマウス（日本クレア社より購入）を１群５匹に群分けして使用し、予め通常の血糖値を測定して、高血糖状態（３００ｍｇ／ｄｌ）であることを確認した。
該２型糖尿病モデルマウスに、試験試料１及び２、対照試料１〜３、並びに陰性試料を、それぞれ５００μｌ ずつ、１日１回、３６日間連続してゾンデを用いて強制経口投与した。投与を開始してから２７日目に、絶食時の血糖値及び糖化ヘモグロビン量（ＨｂＡ１Ｃ）を、それぞれアントセンスＩＩ及びＤＣＡ２０００（ともにバイエル三共社製）を用いて測定した。
【００５０】
（３）試験結果
本試験の結果は、表１に示すとおりである。表１は、２型糖尿病モデルマウスに試料の投与を開始してから２７日目の血糖値及び糖化ヘモグロビン量を測定した結果を示したものである。その結果、試料投与開始後２７日目において、陰性試料を投与したマウスでは血糖値が約１０％上昇し、対照試料１では約８％減少、対照試料２では約１９％減少、対照試料３では約２７％減少する結果が得られた。これに対し、試験試料１では血糖値が約６０％減少し、試験試料２では約４０％減少したことから、本発明の血糖降下組成物の有効成分は、従来のセラミドやガングリオシド、並びに２型糖尿病治療薬として用いられているジアベンのいずれよりも、優れた血糖値低下作用を有することが判明した。
また、糖化ヘモグロビン量についても、試験試料は、陰性試料及び対照試料より有意に低下していたことから、マウスへの試料の連続投与により血糖値の低下が維持されていたことが確認された。
【００５１】
【表１】

【００５２】
試験例２
本試験は、棘皮動物から精製したスフィンゴ糖脂質精製標品のマウスへの経口投与による血糖値低下作用を確認するために行った。
（１）試料の調製
前記製造例１におけるセラミド混合物を順相カラムクロマトグラフィーにて純度を高めたセラミド精製標品（ＬＭＣｅｒ−２）、及び前記製造例３におけるガングリオシド混合物を順相カラムクロマトグラフィーにて純度を高めたガングリオシド精製標品（ＬＬＧ−３）を、それぞれ生理食塩水によって２μｇ／ｍｌに調製した試料を、それぞれ試験試料３及び試験試料４とした。また、生理食塩水を陰性試料とした。
【００５３】
（２）試験方法
試験例１で使用したマウスと同系の２型糖尿病モデルマウスに、試験試料３、試験試料４又は陰性試料を、それぞれ２００μｌずつ、１日１回、４０日間連続して尾静脈より静脈内投与した。投与を開始してから３０日目に、絶食時の血糖値及び糖化ヘモグロビン量（ＨｂＡ１Ｃ）を、それぞれアントセンスＩＩ及びＤＣＡ２０００（ともにバイエル三共社製）を用いて測定した。
【００５４】
（３）試験結果
本試験の結果は、表２に示されるとおりである。表２は、２型糖尿病モデルマウスに試料の投与を開始してから３０日目の血糖値及び糖化ヘモグロビン量を測定した結果を示したものである。表２から明らかなとおり、陰性試料を投与したマウスでは、投与開始後３０日目の血糖値が２倍に上昇（１０４％増加）したのに対し、試験試料３（セラミド：ＬＭＣｅｒ−２）及び試験試料４（ガングリオシド：ＬＧＧ−３）を投与したマウスでは、投与開始後３０日目の血糖値が、それぞれ約１．５倍の上昇（約５０％増加）に留まり、セラミドやガングリオシドによって血糖値の上昇が抑制されることが判明した。また、同様にセラミドやガングリオシドによって糖化ヘモグロビン量も有意に減少し、血糖値の低下が維持されていることが判明した。
【００５５】
【表２】

【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、本発明の範囲は、以下の実施例によって何ら限定されるものではない。
実施例１
次の組成からなる組成物を調製し、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】
実施例２
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１で製造したセラミド（ＬＭＣｅｒ−２）６０ｇを添加し、均一に混合して、スフィンゴ糖脂質を含有する経腸栄養食粉末約５６ｋｇを製造した。
【００５８】
実施例３
前記製造例４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、及びグリセリン脂肪酸エステル製剤（理研ビタミン社製）３４ｇの各粉末を配合して均一に混合し、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】
実施例４
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。これを約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それぞれ１００ｇずつ添加した。これを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５で製造したガングリオシド（ＡＧ−３）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を有するスフィンゴ糖脂質、又は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血糖降下組成物に係るものであり、本発明により、次のような効果が奏される。
（１）本発明の血糖降下組成物は、血糖降下作用を有し、血糖値の上昇を抑制し、高血糖状態の血糖値を低下させる効果を有する。
（２）この組成物は、前糖尿病又は糖尿病状態での高血糖状態から引き起こされる合併症の治療及び／又は予防効果を有する。
（３）血糖降下作用を有する新規医薬組物、飲食品組成物又は飼料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を、飲食品組成物及び飼料組成物等としての新しい有効利用法を提供することが可能である。
【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である。
【図２】図２は、棘皮動物からスフィンゴ糖脂質であるセラミド及びガングリオシドを製造する方法を示すフローチャートである。[0001]
BACKGROUND OF THE INVENTION
The present invention is caused by a hypoglycemic composition having a hypoglycemic effect, that is, an effect of suppressing an increase in blood glucose level, an effect of lowering the blood glucose level from a hyperglycemic state, and a hyperglycemic state in a pre-diabetes or diabetic state The present invention relates to a hypoglycemic composition having an effect in the treatment and / or prevention of complications, and more specifically, an α-oxy fatty acid and a sphingosine skeleton purified from an organism belonging to the echinoderm, and an α-oxy fatty acid and a phyto The present invention relates to a hypoglycemic composition containing a sphingoglycolipid having a sphingosine skeleton as an active ingredient.
[0002]
[Prior art]
In general, starfish etc. belonging to Echinodermata feed on coral etc., and there is concern about destroying the ecosystem of fishing grounds. Is regarded as a serious environmental problem. In such echinoderms, an effective method of use has not yet been found, and research for exploring the value as a resource has been actively conducted in various fields.
[0003]
On the other hand, in recent years, various studies on sphingolipids have been conducted. Sphingolipids are widely present in nature as important membrane components of cells, and most of them are present as glycosphingolipids and sphingophospholipids. Sphingolipids are known to play an important role in life phenomena such as regulatory effects on cell proliferation and differentiation (for example, see Non-Patent Document 1) and apoptosis induction (for example, see Non-Patent Document 2). . These phenomena involve cytokines such as TNF (see, for example, Non-Patent Document 3), IL-1 (see, for example, Non-Patent Document 4), and Fas ligand (for example, see Non-Patent Document 5). It is clear that In addition, sphingolipids regulate the activation of EGF receptors (see, for example, Non-Patent Document 6), intracellular fluidization of calcium ions (see, for example, Non-Patent Document 7), and inhibition / activation of protein kinase C activity. It is also known to be involved in
[0004]
Among the sphingolipids, the glycosphingolipid is a ceramide composed of a long-chain base and a fatty acid as an aglycone, and a cerebroside in which a monosaccharide such as galactose or glucose is bound to the aglycon part, a lactosylceramide in which lactose is bound, Examples include sulfatide in which a sugar (monosaccharide or oligosaccharide) and a sulfate group are bonded, and ganglioside in which an oligosaccharide and sialic acid are bonded (for example, see Non-Patent Document 8). Among them, gangliosides containing sialic acid are mutually recognized between cells (for example, see Non-patent Document 9), adhesion (for example, see Non-Patent Document 10), proliferation (for example, see Non-Patent Document 11), or differentiation induction. It is said that it is involved in various important phenomena in the living body such as (see, for example, Non-Patent Document 12). This compound is also used for treatment of ischemic injury and brain injury caused by Parkinson's disease (for example, see Non-Patent Document 13).
[0005]
Regarding the chemical structures of these glycosphingolipids, glycosphingolipids derived from organisms belonging to the marine invertebrate Echinodermata (eg, starfishes, spider starfishes, sea urchins, sea cucumbers, sea lilies, etc.) Major skeleton has α-oxy fatty acid and ceramide part (aglycone part) composed of sphingosine long-chain base or phytosphingosine-type long chain base. Substituted fatty acids) and glycosphingolipids derived from mammals composed of sphingosine-type long-chain bases are structurally different (see, for example, Non-Patent Document 8 and Non-Patent Document 14).
[0006]
The phytosphingosine skeleton has a structure in which the 4-bond and 5-position double bonds in the sphingosine skeleton are saturated with a hydroxyl group at the 4-position and hydrogen at the 5-position, respectively. Each glycosphingolipid having a skeleton is expected to have different actions.
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. The ceramide part is also composed of unsubstituted fatty acids and sphingosine-type long-chain bases, unlike conventional echinoderm sphingoglycolipids, so the sphingoglycolipids of sea urchins are quite different from those of other echinoderms. It is positioned as an object.
[0007]
By the way, diabetes is a general term for a group of metabolic diseases in which various diseases are accompanied by the persistence of hyperglycemia. Diabetes mellitus is said to develop in combination with a decrease in insulin secretion and a decrease in insulin sensitivity, and severe symptoms such as kidney damage, neuropathy, and retinopathy appear.
Diabetes is classified into type 1 diabetes [Insulin Dependent Diabetes Mellitus (IDDM)] and type 2 diabetes [Non-Insulin Dependent Diabetes Mellitus (NIDDM)]. Type 1 diabetes is a type that is caused by an absolute deficiency of insulin due to necrosis or dysfunction of pancreatic β cells by an autoimmune mechanism. On the other hand, type 2 diabetes is said to be one of lifestyle-related diseases. For example, hyperglycemia caused by insulin secretion deficiency caused by various factors such as overeating, stress, lack of exercise, and relative insulin deficiency due to insulin resistance. It is considered to be a type indicating
[0008]
The fear of diabetes is not only hyperglycemia but also its various and serious complications, and the main cause of death is complications. Diabetes are not only hyperglycemic, but their complications lead to a healthy life, and if left untreated, they can lead to physical disability and death due to a combination of many organ disorders.
Microangiopathy is the most specific complication of diabetes, and its retinopathy, neuropathy and nephropathy appear from the earliest and most frequently. In recent years, with the aging of diabetic patients, an increase in arteriosclerosis has been conspicuous, and has become a major position among diabetic complications. Thus, since complications in diabetes lead to a significant decrease in QOL, the main purpose of diabetes treatment is to prevent such complications (for example, Non-Patent Document 15 and Non-Patent Document 16). reference).
[0009]
In current treatments for diabetes, synthetic human insulin preparations and the like should generally be used for hyperglycemia associated with weight loss, etc., including type 1 diabetes, and oral hypoglycemic drugs should not be used. It is said that there is no. On the other hand, in the treatment of type 2 diabetes, diet therapy, exercise therapy, oral hypoglycemic drug administration, and insulin treatment can be mentioned, and different methods are used to treat diabetes depending on either type 1 or type 2. (For example, refer nonpatent literature 16).
Type 2 diabetes is said to account for about 90% of diabetic patients, and dietary therapy and exercise therapy are incorporated for mild symptoms among these types. In particular, suppression of postprandial hyperglycemia is said to be effective in suppressing the onset of type 2 diabetes, an α-glucosidase inhibitor as a sugar absorption inhibitor, a sulfonylurea drug as an insulin secretion promoter, and insulin resistance. Thiazolysin derivatives and the like are used as pharmaceuticals as property improving agents.
[0010]
Among the foods recommended for people with high blood sugar levels, for example, indigestible dextrin, guava polyphenol, wheat albumin, or L- Foods such as tea and soup containing arabinose are commercially available. However, in any case, although the safety is high, since it is a food, its effect does not reach the expected level.
Conventionally, in prior art documents, for example, an antihyperglycemic agent containing lactic acid bacteria as an active ingredient (for example, see Patent Document 1), mushroom-derived diabetes prevention / amelioration as having an effect of suppressing an increase in blood glucose level An agent and a functional food containing the same (for example, see Patent Document 2), a blood sugar level increase inhibitor (for example, see Patent Document 3) containing a hydrolyzate peptide of collagen and gelatin as active ingredients are disclosed.
[0011]
In addition, in the prior art documents, as a technique related to diabetes mellitus including glycosphingolipid and the like, for example, pharmaceutical preparations for diabetes and related complications caused by sulfated glycolipids (sulfatides) and antibodies thereof, and those diseases are related. Disclosed are methods for detecting islet cell antibodies (ICA) (for example, see Patent Document 4), galactosylceramide, glucosylceramide, lactosylceramide and pharmaceutical preparations for diabetes and related complications caused by the antibodies (for example, see Patent Document 5). Has been. Furthermore, the prior art document discloses an NKT cell activator containing α-glycosylceramide and an autoimmune disease therapeutic agent (see, for example, Patent Document 6).
[0012]
[Patent Document 1]
Japanese Patent Laid-Open No. 10-7777
[Patent Document 2]
JP 2002-187851 A
[Patent Document 3]
JP 2002-326951 A
[Patent Document 4]
Japanese Patent Publication No. 6-506943
[Patent Document 5]
JP 2001-527046 A
[Patent Document 6]
International Publication No. 98/044928 Pamphlet
[Non-Patent Document 1]
FEBS Lett., Netherlands, Vol. 524, 2002, p. 103-106
[Non-Patent Document 2]
FEBS Lett., Netherlands, Vol. 526, 2002, p. 15-20
[Non-Patent Document 3]
EM BJ Journal (EMBO J.), UK, Vol. 19, No. 13, 2000, p. 3304-3313
[Non-Patent Document 4]
Journal of Biological Chemistry (J. Biol. Chem.), USA, 275, 45, 2000, p. 35617-35623
[Non-Patent Document 5]
Journal of Biological Chemistry (J. Biol. Chem.), USA, 276, 26, 2001, p. 23954-23961
[Non-Patent Document 6]
Journal of Biological Chemistry (J. Biol. Chem.), USA, 277, 12, 2002, p. 10108-10113
[Non-Patent Document 7]
Proceding National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), USA, Vol. 98, No. 1, 2001, p. 307-312
[Non-Patent Document 8]
Nobuhiro Fushiya and Hiroshi Hamada, “Structural analysis of natural organic compounds”, Springer Fairlark Tokyo, 1994, p. 81-140
[Non-patent document 9]
Journal of Biological Chemistry (J. Biol. Chem.), USA, 264, 1989, p. 2014-19-20162
[Non-Patent Document 10]
Journal of Biological Chemistry (J. Biol. Chem.), USA, 266, 1991, p. 17552-17558
[Non-Patent Document 11]
Developmental Brain Research, Netherlands, 284, 1983, p. 215-221
[Non-Patent Document 12]
Proceding National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), USA, Vol. 76, 1979, p. 3367-3371
[Non-Patent Document 13]
Neurology, USA, 50, 1998, p. 1630-1636
[Non-Patent Document 14]
Pharmacia, Vol. 38, No. 9, 2002, p. 851-855
[Non-Patent Document 15]
Toshiya Tachibana, “Lifestyle-related diseases and their remedies”, Jiho Co., Ltd., 2001, p. 39-68
[Non-Patent Document 16]
Supervised by Yoshio Yazaki, “Advances in Molecular Diabetes”, Kanbara Publishing Co., Ltd., 2002, p. 134-138
[0013]
Among the prior art documents showing the above prior art, Patent Document 4 or Patent Document 5 discloses an invention substantially relating to a method for detecting an islet cell antibody derived from a type 1 diabetes patient, comprising sulfatide and galactosylceramide. There is no teaching of data showing the therapeutic effect (especially hypoglycemic action) on type 1 diabetic patients with glycosphingolipids such as glucosylceramide and lactosylceramide. Patent Document 6 discloses the effect of α-glycosylceramide to suppress the spontaneous development of diabetes in NOD mice, which are model mice for type 1 diabetes. In this invention, the effect of α-glycosylceramide is disclosed. Is an effect on autoimmune diseases. Specifically, this document suppresses the specific destruction of pancreatic islets of Langerhans by promoting activation of NKT cells and maintains insulin production, resulting in an increase in blood glucose level. This document shows the effect of suppressing the onset of diabetes by being suppressed, and this document does not describe any effect of directly acting on a high blood sugar level to lower the blood sugar level.
Furthermore, the treatment with sulfonylurea, which is an oral hypoglycemic agent currently used in clinical practice of diabetes, is concerned about side effects such as hypoglycemia shock due to a rapid decrease in blood glucose level. Therefore, in the technical field, development of a novel hypoglycemic composition capable of controlling blood glucose level more safely and reliably has been eagerly awaited, particularly in the medical field in the treatment of diabetes.
[0014]
[Problems to be solved by the invention]
In such a situation, in view of the above prior art, the present inventors are concerned about destroying the ecosystem of the fishing ground, using sharks as a food, and are captured and discarded in large quantities. In various studies on effective utilization methods such as starfish belonging to the phylum, α-oxy fatty acid and sphingoglycolipid having sphingosine skeleton or phytosphingosine skeleton, which are extracted and purified from these, lower blood sugar level from hyperglycemic state As a result, the present invention was completed.
That is, the present invention has an effect of suppressing an increase in blood sugar level, an effect of lowering blood sugar level from a hyperglycemic state, and a therapeutic and / or preventive effect on complications caused by a hyperglycemic state in a pre-diabetes or diabetic state, An object of the present invention is to provide a hypoglycemic composition having
In addition, the present invention is an echinoderm, which is regarded as a serious environmental problem in the field of fisheries, and has not yet found an effective use method despite being captured in large quantities in the coastal area of Japan. By producing and providing a pharmaceutical composition that can be used effectively as a hypoglycemic agent, a food / beverage product composition and a feed composition imparted with a blood glucose level-lowering effect, and thereby solving environmental problems It aims to provide technology that contributes to the development of the fishery industry.
[0015]
[Means for Solving the Problems]
A first invention of the present invention for solving the above-mentioned problems is a hypoglycemic composition having a hypoglycemic action, characterized by comprising a compound having an α-oxy fatty acid and a sphingosine skeleton as active ingredients, and the present invention. The preferred embodiment is that the compound is ceramide and / or ganglioside and contains a compound purified from an organism belonging to the echinoderm as an active ingredient.
The second invention of the present invention for solving the above-mentioned problems is a hypoglycemic composition having a hypoglycemic action, characterized by comprising a compound having an α-oxy fatty acid and a phytosphingosine skeleton as active ingredients. It is desirable that the compound is a ceramide and / or ganglioside, and contains a compound purified from an organism belonging to the echinoderm as an active ingredient.
A third invention of the present invention that solves the above problem is the hypoglycemic composition according to the first or second invention, wherein the composition is a pharmaceutical composition, a food or drink composition, or a feed composition.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in more detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In the present specification, the percentage is expressed by mass unless otherwise specified.
In the present invention, the hypoglycemic composition is a composition having an action of controlling (for example, suppressing) an increase in blood glucose level, and more specifically, at the onset of diabetes or the blood glucose level is higher than a normal level. An action that reduces the blood glucose level in a state (glucose in the blood is in a hyperglycemic state, for example, 110 mg / dl or more on an empty stomach) to a normal level or almost normal level (below 110 mg / dl) It may be described as a hypoglycemic action.), Or a drug having an action of suppressing an increase in blood sugar level caused by disorder of hormone balance or the like.
[0017]
The active ingredient of the hypoglycemic composition of the present invention is preferably purified from an organism belonging to the echinoderm. In the present invention, the organisms belonging to the Echinodermata are specifically classified as class starfish (starfish, starfish, blue starfish, starfish, starfish, purple starfish, etc.), snake (starfish), For example, snail starfish, Japanese spider starfish, etc., sea urchins (eg, sea urchins, bafun sea urchins, etc.), sea sharks (eg, sea cucumbers, white sea cucumbers), and sea lily (eg, Torinoasis) , Japanese sea urchins, core sea urns, sea urchins, etc.).
[0018]
Generally, as shown in FIG. 1, a glycosphingolipid is a ceramide composed of a long-chain base having a sphingosine skeleton or a phytosphingosine skeleton and a fatty acid, and the aglycon part contains neutral sugars and amino sugars (included). In some cases, it is a combination of sialic acid and is classified as a ganglioside. 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.
[0019]
As the glycosphingolipid which is an active ingredient of the hypoglycemic composition of the present invention, specifically, ceramide and ganglioside each having a sphingosine skeleton in which α-position of fatty acid in the molecule is oxidized, or intramolecular Each of the ceramides having a phytosphingosine skeleton in which the α-position of the fatty acid is oxidized (a structure in which double bonds at positions 4 and 5 in the sphingosine skeleton are hydroxyl at the 4-position and hydrogen at the 5-position, respectively) And gangliosides. The basic structure of ceramide having an α-oxy fatty acid and a sphingosine skeleton (R ₂ -Cer1) ## STR1 ## Basic structure of ceramide having α-oxy fatty acid and phytosphingosine skeleton (R ₂ -Cer2) is shown in Chemical Formula 2, respectively. In the present specification, the sphingoglycolipid having the α-oxy fatty acid and the sphingosine skeleton, and the sphingoglycolipid having the α-oxy fatty acid and the phytosphingosine skeleton, unless otherwise specified or explained. Collectively referred to as glycosphingolipid.
[0020]
[Chemical 1]

[0021]
[Chemical 2]

[0022]
Here, the “active ingredient” means an ingredient having the action as defined above in the hypoglycemic composition of the present invention, and the main active ingredient of the hypoglycemic composition of the present invention must be the glycosphingolipid. It is not necessary, and the blood glucose lowering composition of the present invention may contain a component having a blood glucose lowering action or a blood sugar rise suppressing effect in addition to the glycosphingolipid.
[0023]
The active ingredient of the hypoglycemic composition of the present invention can be produced by extraction and purification using any of the echinoderms as an extraction raw material, and the effective ingredient is obtained from the echinoderm as an extraction raw material. Extract, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product or a purified product thereof. 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 of the hypoglycemic composition of the present invention can be extracted and purified.
[0024]
As an example of the method for producing the active ingredient of the hypoglycemic composition of the present invention, the method of Inagaki et al. ([European Journal of Organic Chemistry [Eur. J. Org. Chem.], 1998, p. 129-131], [Chemical Pharmaceutical Bulletin [Chem. Pharm. Bull.], Vol. 49, No. 12, 2001, p. 1521-1525]). This method is exemplified and specifically described as follows. That is, echinoderms are homogenized and mixed with an organic solvent, particularly preferably any mixture of chloroform / methanol or methylene chloride / methanol in a mixing ratio of 1: 1 to 1:10, preferably 1: 2 to 1: 3. Extraction treatment is performed with a mixed solution having a ratio. After concentrating the extract, an ethyl acetate / butanol mixture ratio of 5: 1 to 1: 1, preferably 3: 1 to 2: 1 is added, and water is added. After the partitioning operation, the ethyl acetate / butanol layer (α layer) and the aqueous layer (β layer) are separated. Among these, after concentrating the ethyl acetate / butanol layer (α layer), acetone is added to obtain an acetone-soluble fraction (ceramide mixture). In addition, water-saturated butanol (preferably using n-butanol) is added to the aqueous layer (β layer) to perform a partitioning operation, and the aqueous layer (β layer) is divided into a butanol layer and an aqueous layer (δ). Among these, after concentrating the aqueous layer (δ), separation is performed under the following conditions using a reverse phase column. That is, first, separation is performed by a stepwise gradient method in which the concentration of methanol or ethanol as an eluent is increased stepwise from 60% to 100% and eluted. The fraction with a methanol concentration of 100% is collected and concentrated to obtain a ganglioside mixture.
[0025]
The ceramide mixture prepared as an intermediate purified product preferably contains 1 to 10% of ceramide as an active ingredient. If at least the above range is contained, the ceramide mixture of the present invention is used as it is. It can be used as an active ingredient.
Further, the ganglioside mixture prepared as the intermediate purified product preferably contains 20 to 60% of ganglioside as an active ingredient. If at least the ganglioside in the above range is contained, the blood glucose lowering level of the present invention is maintained as it is. It can be used as an active ingredient of a composition.
[0026]
The ceramide mixture and the ganglioside mixture can be purified from the ceramide mixture by the normal phase column chromatography etc., respectively, and the ganglioside purified sample can be purified from the ganglioside mixture as well. They can also be used as the active ingredient of the present invention. The purity of the purified ceramide preparation and the purified ganglioside preparation can be determined by thin layer chromatography or the like, and each is preferably 90% or more, particularly preferably 95% or more.
[0027]
Although the solvent used when extracting and purifying the active ingredient of the hypoglycemic composition of the present invention is not particularly limited, 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. In the present invention, an organic solvent can be selected and the concentration can be adjusted according to the type of echinoderm as a raw material used for production and the type of active ingredient (sphingoglycolipid) to be produced. In addition, several organic solvents can be mixed and used, and the mixing ratio and the like can be appropriately adjusted.
[0028]
The separation method of the active ingredient used in the method for producing a hypoglycemic composition of the present invention includes adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography (HPLC), and a gel packed column. Chromatography or the like can be used alone or in combination.
As the active ingredient of the hypoglycemic composition of the present invention, in addition to those obtained from echinoderms by the above production method, those produced by chemical synthesis of compounds having the same structure can be used.
[0029]
Among the glycosphingolipids that are active ingredients of the hypoglycemic composition of the present invention, the compound having an α-oxy fatty acid and a sphingosine skeleton, and the compound having an α-oxy fatty acid and a phytosphingosine skeleton should be used alone. It is also possible to select a plurality of compounds and use them as a mixture.
The composition of the present invention may be a glycosphingolipid extracted and purified from echinoderms itself, or may contain components other than the glycosphingolipid. Components other than the glycosphingolipid can be appropriately selected depending on the form of the composition.
The form of the composition of the present invention is not particularly limited, and the hypoglycemic composition of the present invention can be used as, for example, a form of a pharmaceutical composition, a food and drink composition, a feed composition, and the like. is there.
[0030]
Among these, the pharmaceutical composition can be produced by, for example, formulating a glycosphingolipid using a pharmaceutically acceptable excipient, or any other additive, and the formulated glycosphingolipid is, It can be used as a hypoglycemic composition. When formulating, the content of glycosphingolipid in the formulation is usually 0.001 to 1.0% by mass, preferably 0.01 to 0.1% by mass. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and solvents for injection can be used.
[0031]
Examples of the excipient 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, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate; sodium phosphate, calcium phosphate, etc. And phosphate derivatives such as sodium carbonate and calcium carbonate; and sulfate derivatives such as sodium sulfate and calcium sulfate.
[0032]
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. Lubricants include, for example, 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; carboxyl 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.
[0033]
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. Moreover, the said pharmaceutical composition can also be mix | blended and administered to food-drinks 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 0.01 to 1000 mg per day for an adult, preferably 0.1 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.
[0034]
Next, the food / beverage composition includes, for example, a glycosphingolipid that is a hypoglycemic composition of the present invention or an active ingredient, sugars such as dextrin and starch; proteins such as gelatin, casein, whey, soy protein, and corn protein; Amino acids such as alanine, glutamine, and 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 can be used. 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 sauce, soup, soup, stew, salad, prepared dishes, pickles; breads; enteral nutrition; function Food and the like. In addition, content of the hypoglycemic composition or glycosphingolipid which is an active ingredient added when manufacturing a food-drinks composition can be suitably selected from the range of 0.001-10 mass%.
[0035]
Next, the feed composition is produced, for example, from glycosphingolipids, cereals such as corn, wheat, barley, rye; bran such as bran, wheat straw, rice bran, defatted rice bran; corn gluten meal, corn jam meal, etc. Manufactured by blending animal feeds such as skim milk powder, whey, fish meal and bone meal; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; amino acids; be able to. 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 hypoglycemic composition or the sphingoglycolipid which is an active ingredient added when manufacturing a feed composition can be suitably selected from the range of 0.001-10 mass%.
[0036]
The composition of the present invention may be used alone or in combination with other hypoglycemic compositions. Their combined use can enhance the blood glucose level lowering action in a hyperglycemic state. Moreover, you may make it contain as an active ingredient in the composition of the hypoglycemic composition which uses the composition of this invention together, and it is good to combine it as a separate chemical | medical agent, without making it contain in a composition.
[0037]
The hypoglycemic composition of the present invention is based on a diagnostic standard for blood sugar level (Non-Patent Document 16: Toshiya Tachibana, “Lifestyle-related diseases and therapeutic agents thereof”, Jiho Co., Ltd., 2001, p. 42-43). Segmented border area (fasting: 110-126 mg / dl, or 75 g glucose tolerance test [OGTT] 2 hours: 140-200 mg / dl), or diabetic area (fasting: 126 mg / dl) From the state of the above or 75 g glucose tolerance test [OGTT] 2 hours: any state of 200 mg / dl or more), normal region (fasting: 110 mg / dl or less, or 75 g glucose tolerance test [OGTT] 2 hours) : Any state of 140 mg / dl or less) to lower the blood glucose level, and from the normal region to the border region, from the normal region to the diabetes region, or from the border region Useful for the treatment of pre-diabetic or diabetic hyperglycemia and for the treatment and / or prevention of complications caused by hyperglycemia by suppressing blood glucose levels in any of the urine disease areas is there.
[0038]
In general, the action of lowering the blood sugar level from the hyperglycemic state and the action of suppressing the rise of the blood sugar level are the effects of in vivo using a diabetes model mouse, and administration of a hypoglycemic agent or the like to the mouse is started. It can be evaluated by measuring the blood glucose level and the amount of glycated hemoglobin at the stage of administration for a certain period. Glycated hemoglobin is composed of three components, hemoglobin A1a, hemoglobin A1b, and hemoglobin A1c, and has a tendency to remain in the blood for a long time. Therefore, it is an excellent indicator of glycated protein in blood. Among them, the level of hemoglobin A1c (HbA1c) reflects the effectiveness of blood glucose treatment as an indicator of the average glycemic control level for 6 to 8 weeks before measurement, and is therefore an effective marker for the diagnosis of diabetes. It is used for diagnosis of diabetes and measurement of progress in combination with measurement of blood glucose level [Diabetic Medicine, USA, Vol. 14, 1997, p. 584-588]. From these facts, the hypoglycemic effect of the hypoglycemic composition of the present invention was evaluated by measuring the blood glucose level and the amount of glycated hemoglobin as the examination method.
[0039]
In the present invention, the blood glucose level is measured by the immobilized glucose oxidase membrane / hydrogen peroxide electrode method (Basic and Clinical, Vol. 30, No. 5, May 1996, p. 1125-1132). Based on a blood glucose measurement device (for example, Bayer Sankyo Co., Ltd .: Antsense II) and the like. As for the measurement of the amount of glycated hemoglobin, a measuring device (for example, a simple measurement method of HbA1c based on latex aggregation inhibition reaction (medicine and pharmacy, Vol. 37, No. 4, April 1997, p. 1023-1028) , Bayer Sankyo Co., Ltd .: DCA2000) and the like.
[0040]
Next, production examples of glycosphingolipids which are active ingredients of the hypoglycemic composition of the present invention will be described. 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 a starfish starfish (scientific name: Luidia maculata) was put into a mixer, and then 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2) was added to the mixer, followed by homogenization, extraction, and filtration. Later 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. Among 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 collected, and then the organic solvent was volatilized and the ceramide mixture was about 5. 3 g was obtained. Next, the ceramide mixture was dissolved in 5 ml of chloroform and added to a normal phase column (Silica gel 60: manufactured by Merck). 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. The fraction showing the same behavior as that of the existing ceramide in normal phase thin layer chromatography was recovered, and the fraction was further added to a normal phase column to be mixed with a chloroform methanol mixture (chloroform: methanol = 97: 3 → 95). : 2 types of ceramides, LMCer-1 (3 mg) and LMCer-2 (4 mg), which were eluted in 5) and showed the behavior of a single substance in normal phase thin layer chromatography, respectively. The structural formula of LMCer-1 is shown in Chemical Formula 3, and the structural formula of LMCer-2 is shown in Chemical Formula 4, respectively.
(Chemical formula 3)
H-Cer1
(Chemical formula 4)
H-Cer2
[0041]
Production Example 2
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was put into a mixer, then 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2) was added to the mixer, homogenized to perform extraction, filtered and the extract was 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, the separated water layer was further distributed by adding water-saturated n-butanol, and separated as an n-butanol layer and an aqueous layer, and the aqueous layer was recovered. The aqueous layer was concentrated and then separated using a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai). Separation was performed by a stepwise gradient method in which the concentration of a methanol solution as an eluent was increased stepwise to 60 to 100% and eluted. The organic solvent of the elution fraction when the concentration of methanol reached 100% was volatilized to prepare about 0.18 g of a ganglioside mixture. Next, the ganglioside mixture was dissolved in a chloroform / methanol / water (chloroform: methanol: water = 6: 4: 1) mixed solution, added to a normal phase column (Silica gel 60: manufactured by Merck), and added with the same mixed solution. Eluted. Subsequently, it was added to a gel filtration column (Sephadex LH-20: manufactured by Pharmacia) for desalting to produce ganglioside LMG-2 (2.4 mg) showing the behavior of a single substance in normal phase thin layer chromatography. The structural formula of LMG-2 is shown in Chemical Formula 5.
[0042]
(Chemical formula 5)
NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, Gal: galactose, Glc: glucose)
[0043]
Production Example 3
(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 2 using 18 kg of Ao starfish (scientific name: Linckia laevigata) as a starting material. The structural formula of LLG-3 is shown in Chemical formula 6, and the structural formula of LLG-5 is shown in Chemical formula 7.
(Chemical formula 6)
8-O-CH ₃ NeuAcα2 → 11NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
(Chemical formula 7)
8-O-CH ₃ NeuGcα2 → 11NeuGcα2 → 11NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
[0044]
Production Example 4
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside GAA-6 (20 mg) and GAA-7 (86 mg) were produced in the same manner as in Production Example 2 using 65 kg of Murasaki starfish (scientific name: Asterias aurensis versicolor) as a starting material. The structural formula of GAA-6 is shown in Chemical formula 8, and the structural formula of GAA-7 is shown in Chemical formula 9.
(Chemical formula 8)
8 (11) -O-CH ₃ NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
(Chemical 9)
8-O-CH ₃ NeuGcα2 → 6 [8-O-CH ₃ NeuGcα2 → 3] GalNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, GalNAc: N-acetylgalactosamine, Gal: galactose, Glc: glucose)
[0045]
Production Example 5
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside AG-1 (36.4 mg), AG-2 (290 mg), and AG-3 (8.9 mg) were produced in the same manner as in Production Example 2, using 12 kg of starfish (Acanthator plant) as a starting material. The structural formula of AG-1 is shown in Chemical formula 10, the structural formula of AG-2 is shown in Chemical formula 11, and the structural formula of AG-3 is shown in Chemical formula 12.
(Chemical Formula 10)
Fucfβ1 → 4Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Fucf: Fucofuranose)
(Chemical Formula 11)
Galβ1 → 3Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Galf: galactofuranose, NeuAc: N-acetylneuraminic acid)
(Chemical Formula 12)
Galβ1 → 3Galα1 → 3Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Galf: galactofuranose, NeuAc: N-acetylneuraminic acid)
[0046]
Production Example 6
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside GP-1a (72 mg), GP-1b (119 mg) and GP-2 (421 mg) were produced in the same manner as in Production Example 2 using 42 kg of Asterina pectinifera as a starting material. The structural formula of GP1a is shown in Chemical formula 13, the structural formula of GP-1b is shown in Chemical formula 14, and the structural formula of GP-2 is shown in Chemical formula 15.
(Chemical Formula 13)
Arafα1 → 3Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Araf: Arabinofuranose, NeuAc: N-acetylneuraminic acid)
(Chemical formula 14)
Arafα1 → 4 [Arafα1 → 3] Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Araf: Arabinofuranose, NeuAc: N-acetylneuraminic acid)
(Chemical 15)
Arafα1 → 4Galα1 → 6 [Arafα1 → 4] Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Araf: Arabinofuranose, NeuAc: N-acetylneuraminic acid)
[0047]
Production Example 7
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside LG-1 (146 mg) and LG-2 (545 mg) were produced in the same manner as in Production Example 2, using 30 kg of Astrospecten latespinosus as the starting material. The structural formula of LG-1 is shown in Chemical formula 16, and the structural formula of LG-2 is shown in Chemical formula 17.
(Chemical formula 16)
Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid)
(Chemical Formula 17)
Araβ1 → 4Galα1 → 4NeAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(Ara: arabinose, NeuAc: N-acetylneuraminic acid)
In the production examples 1 to 7, 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. Unlikely, it was confirmed to have a structure specific to echinoderms.
[0048]
Next, a test example is shown and this invention is demonstrated in detail.
Test example 1
This test was conducted to confirm the blood glucose level-lowering effect of orally administered to the mouse an intermediate purified product of glycosphingolipid extracted and fractionated from echinoderms.
(1) Sample preparation
Samples prepared by preparing the ceramide mixture (containing about 1 μg of ceramide active ingredient) and the ganglioside mixture (containing about 8 μg of ganglioside active ingredient) prepared in Preparation Examples 1 and 2 to 500 μg / ml with physiological saline, respectively. Test sample 1 and test sample 2, respectively. Further, cattle-derived ceramide (manufactured by Wako Pure Chemical Industries, Ltd .: catalog number 031-18441) and a bovine-derived ganglioside mixture having no α-oxy fatty acid and phytosphingosine skeleton (manufactured by Wako Pure Chemical Industries, Ltd .: catalog number 074-) Samples prepared by adjusting saline to 30 μg / ml were designated as control sample 1 and control sample 2, respectively. Furthermore, after pulverizing type 2 oral antidiabetic drug diaben (manufactured by Chugai Pharmaceutical Co., Ltd .: tolbutamide tablet) which is a conventional hypoglycemic agent, a sample prepared to 1 mg / ml with a 2% carboxymethyl cellulose (manufactured by Nacalai Tesque) solution Was designated as Control Sample 3. Saline was used as a negative sample.
[0049]
(2) Test method
The mice used in this test are type 2 diabetes model mice, 6-week-old male db / db mice (purchased from CLEA Japan, Inc.) divided into 5 groups per group. Was measured, and it was confirmed that the blood glucose level was high (300 mg / dl).
To the type 2 diabetes model mouse, test samples 1 and 2, control samples 1 to 3, and a negative sample were orally administered by gavage using a sonde once a day for 36 days, 500 μl each. On the 27th day after the start of administration, fasting blood glucose level and glycated hemoglobin amount (HbA1C) were measured using Antsense II and DCA2000 (both manufactured by Bayer Sankyo Co., Ltd.), respectively.
[0050]
(3) Test results
The results of this test are as shown in Table 1. Table 1 shows the results of measuring the blood glucose level and the amount of glycated hemoglobin on the 27th day after the administration of the sample to the type 2 diabetes model mouse was started. As a result, on the 27th day after the start of sample administration, the blood glucose level increased about 10% in the mouse administered with the negative sample, decreased about 8% in the control sample 1, decreased about 19% in the control sample 2, and decreased in the control sample 3. A result of about 27% reduction was obtained. On the other hand, since the blood glucose level decreased about 60% in test sample 1 and about 40% in test sample 2, the active ingredients of the hypoglycemic composition of the present invention are conventional ceramides, gangliosides, and type 2 It has been found that it has a superior blood glucose level lowering effect than any of the diabens used as a therapeutic agent for diabetes.
Moreover, since the amount of glycated hemoglobin was significantly lower in the test sample than in the negative sample and the control sample, it was confirmed that the decrease in blood glucose level was maintained by continuous administration of the sample to mice.
[0051]
[Table 1]

[0052]
Test example 2
This test was performed to confirm the blood glucose level-lowering effect by oral administration to mice of a purified glycosphingolipid preparation purified from echinoderms.
(1) Sample preparation
The purified ceramide (LMCer-2) in which the ceramide mixture in Production Example 1 was purified by normal phase column chromatography, and the ganglioside in which the ganglioside mixture in Production Example 3 was purified by normal phase column chromatography. Samples prepared by purifying the purified sample (LLG-3) to 2 μg / ml with physiological saline were used as test sample 3 and test sample 4, respectively. Saline was used as a negative sample.
[0053]
(2) Test method
200 μl each of test sample 3, test sample 4 or negative sample was administered intravenously from the tail vein for 40 consecutive days to type 2 diabetes model mice of the same strain as the mouse used in Test Example 1. . On the 30th day after the start of administration, the blood glucose level at the time of fasting and the amount of glycated hemoglobin (HbA1C) were measured using Antsense II and DCA2000 (both manufactured by Bayer Sankyo).
[0054]
(3) Test results
The results of this test are as shown in Table 2. Table 2 shows the results of measuring the blood glucose level and the amount of glycated hemoglobin 30 days after the administration of the sample to the type 2 diabetes model mouse was started. As is apparent from Table 2, in the mice administered with the negative sample, the blood glucose level on day 30 after the start of administration was doubled (increased by 104%), whereas test sample 3 (ceramide: LM Mer-2) and In mice administered with test sample 4 (ganglioside: LGG-3), the blood glucose level on the 30th day after the start of administration remained approximately 1.5 times as high (approximately 50% increase), and the blood glucose level was increased by ceramide or ganglioside. It has been found that the rise of is suppressed. Similarly, it was found that the amount of glycated hemoglobin was significantly reduced by ceramide and ganglioside, and the decrease in blood glucose level was maintained.
[0055]
[Table 2]

[0056]
【Example】
Next, the present invention will be specifically described with reference to examples. However, the scope of the present invention is not limited by the following examples.
Example 1
A composition comprising the following composition was prepared, and an injection 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]
Example 2
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. To the intermediate product powder 50 kg, sucrose (made by Hokuren) 6.8 kg, amino acid mixed powder (made by Ajinomoto Co.) 167 g, and ceramide (LMer-2) 60 g produced in Production Example 1 were added and mixed uniformly. Thus, about 56 kg of enteral nutrition food powder containing glycosphingolipid was produced.
[0058]
Example 3
100 g of ganglioside (GAA-7) produced in Production Example 4, 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 ( Uniform blending of 1 g of Saneigen F.F.I.), 5 g of yogurt flavor (San-Eigen F.F.I.), and 34 g of glycerin fatty acid ester preparation (Riken Vitamin). Using a tableting machine (manufactured by Hata Steel Co., Ltd.), the mixture powder was continuously tableted at a tableting speed of 12 tablets / minute and a pressure of 9.8 KPa at 0.5 g per tablet. 1800 tablets (about 900 g) containing ganglioside (GAA-7) were produced.
[0059]
Example 4
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 heat-sterilized at 90 to 92 ° C. for 10 minutes. This was cooled to about 42 ° C., and 100 g each of milk cultures of commercially available Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus was added as a starter. This was sufficiently stirred in a 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 ganglioside (AG-3) produced in Production Example 5 were added to 5 kg of 17% granulated sugar (manufactured by Toyo Seika Co., Ltd.), and 90 to 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 is a blood sugar containing an α-oxy fatty acid and a sphingoglycolipid having a sphingosine skeleton extracted from echinoderms or a glycosphingolipid having an α-oxy fatty acid and a phytosphingosine skeleton as active ingredients. The present invention relates to a descending composition, and the following effects are exhibited by the present invention.
(1) The blood glucose lowering composition of the present invention has a blood glucose lowering effect, and has an effect of suppressing an increase in blood sugar level and lowering a blood sugar level in a hyperglycemic state.
(2) This composition has the effect of treating and / or preventing complications caused by a pre-diabetic or hyperglycemic state in a diabetic state.
(3) A novel pharmaceutical composition, food / beverage product composition or feed composition having a hypoglycemic effect can be provided.
(4) It is possible to provide a new effective utilization method of echinoderms captured in large quantities in the field of fisheries industry as food and beverage composition and feed composition.
[Brief description of the drawings]
FIG. 1 is a diagram showing the basic structure of a glycosphingolipid.
FIG. 2 is a flowchart showing a method for producing ceramide and ganglioside, which are glycosphingolipids, from echinoderms.

Claims (6)

A hypoglycemic composition having a hypoglycemic action, comprising an α-oxy fatty acid and a compound having a sphingosine skeleton as active ingredients. The hypoglycemic composition according to claim 1, wherein the compound having an α-oxy fatty acid and a sphingosine skeleton is ceramide and / or ganglioside. An antihyperglycemic composition having a hypoglycemic action, comprising an α-oxy fatty acid and a compound having a phytosphingosine skeleton as active ingredients. The hypoglycemic composition according to claim 3, wherein the compound having an α-oxy fatty acid and a phytosphingosine skeleton is ceramide and / or ganglioside. The hypoglycemic composition according to any one of claims 1 to 4, comprising as an active ingredient a compound purified from an organism belonging to the echinoderm. The blood glucose lowering composition according to any one of claims 1 to 5, wherein the composition is a pharmaceutical composition, a food / beverage product composition, or a feed composition.

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