JP2004217544A - Protein nonenzymatic saccharification inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a protein nonenzymatic saccharification inhibitor which promotes inhibitory action on protein nonenzymatic saccharification and is effective for preventing/treating diabetes or diabetic complications. <P>SOLUTION: This protein nonenzymatic saccharification inhibitor comprises a substance as an active ingredient, which substance is derived from Hippophae rhamnoides and exhibits inhibitory activity against protein nonenzymatic saccharification. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００１７】
式ＩＩ又はＩＶ（新規化合物）：
【化２】

【００１８】
式ＩＩＩ（新規化合物）：
【化３】

【００１９】
また、本発明のタンパク質非酵素的糖化抑制剤は、沙棘葉部の抽出物・処理物を公知の医薬用担体と組合せて製剤化することができる。投与形態としては、特に制限はなく、必要に応じ適宜選択されるが、一般には錠剤、カプセル剤、顆粒剤、細粒剤、散剤、液剤、シロップ剤、懸濁剤、乳剤、エリキシル剤等の経口剤、又は注射剤、点滴剤、坐剤、吸入剤、経皮吸収剤、経粘膜吸収剤、貼付剤、軟膏剤等の非経口剤として使用される。
【００２０】
本発明のタンパク質非酵素的糖化抑制剤の投与量は、患者の年令、体重、疾患の程度、投与経路により異なるが、経口投与では、沙棘葉部の抽出物乾燥粉末として、通常１日１０〜３０００ｍｇであり、投与回数は、通常、経口投与では１日１〜３回である。
経口剤は、例えばデンプン、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等の賦形剤を用いて常法に従って製造される。
【００２１】
この種の製剤には、適宜前記賦形剤の他に、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を使用することができる。
【００２２】
結合剤の具体例としては、結晶セルロース、結晶セルロース・カルメロースナトリウム、メチルセルロース、ヒドロキシプロピルセルロース、低置換度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、ヒドロキシプロピルメチルセルロースフタレート、ヒドロキシプロピルメチルセルロースアセテートサクシネート、カルメロースナトリウム、エチルセルロース、カルボキシメチルエチルセルロース、ヒドロキシエチルセルロース、コムギデンプン、コメデンプン、トウモロコシデンプン、バレイショデンプン、デキストリン、アルファー化デンプン、部分アルファー化デンプン、ヒドロキシプロピルスターチ、プルラン、ポリビニルピロリドン、アミノアルキルメタクリレートコポリマーＥ、アミノアルキルメタクリレートコポリマーＲＳ、メタクリル酸コポリマーＬ、メタクリル酸コポリマー、ポリビニルアセタールジエチルアミノアセテート、ポリビニルアルコール、アラビアゴム、アラビアゴム末、寒天、ゼラチン、白色セラック、トラガント、精製白糖、マクロゴールが挙げられる。
【００２３】
崩壊剤の具体例としては、結晶セルロース、メチルセルロース、低置換度ヒドロキシプロピルセルロース、カルメロース、カルメロースカルシウム、カルメロースナトリウム、クロスカルメロースナトリウム、コムギデンプン、コメデンプン、トウモロコシデンプン、バレイショデンプン、部分アルファー化デンプン、ヒドロキシプロピルスターチ、カルボキシメチルスターチナトリウム、トラガントが挙げられる。
【００２４】
界面活性剤の具体例としては、大豆レシチン、ショ糖脂肪酸エステル、ステアリン酸ポリオキシル、ポリオキシエチレン硬化ヒマシ油、ポリオキシエチレンポリオキシプロピレングリコール、セスキオレイン酸ソルビタン、トリオレイン酸ソルビタン、モノステアリン酸ソルビタン、モノパルミチン酸ソルビタン、モノラウリン酸ソルビタン、ポリソルベート、モノステアリン酸グリセリン、ラウリル硫酸ナトリウム、ラウロマクロゴールが挙げられる。
【００２５】
滑沢剤の具体例としては、コムギデンプン、コメデンプン、トウモロコシデンプン、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、含水二酸化ケイ素、軽質無水ケイ酸、合成ケイ酸アルミニウム、乾燥水酸化アルミニウムゲル、タルク、メタケイ酸アルミン酸マグネシウム、リン酸水素カルシウム、無水リン酸水素カルシウム、ショ糖脂肪酸エステル、ロウ類、水素添加植物油、ポリエチレングリコールが挙げられる。
【００２６】
流動性促進剤の具体例としては、含水二酸化ケイ素、軽質無水ケイ酸、乾燥水酸化アルミニウムゲル、合成ケイ酸アルミニウム、ケイ酸マグネシウムが挙げられる。
【００２７】
また、本発明のタンパク質非酵素的糖化抑制剤は、液剤、シロップ剤、懸濁剤、乳剤、エリキシル剤として投与する場合には、矯味矯臭剤、着色剤を含有してもよい。
【００２８】
本発明のタンパク質非酵素的糖化抑制剤は、食品、チューインガム、飲料等に添加して、いわゆる特定保健用食品（例えば、糖尿病・糖尿病合併症予防食品）等とすることもできる。
【００２９】
本発明のタンパク質非酵素的糖化抑制剤の製造原料である沙棘は現在までに民間薬、化粧品等に供されており、安全性は確立されている。
【００３０】
【実施例】
以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。
【００３１】
（実施例１）沙棘葉部抽出物のタンパク質非酵素的糖化抑制作用の測定
沙棘葉部抽出物の調製
中国甘粛省より入手した沙棘葉部２ｋｇを粉砕後、超音波条件下、エタノール２３Ｌを用いて３０分間抽出を行った。グラスフィルターにより抽出液をろ過した後、ろ液を減圧濃縮してエタノール抽出物１４３ｇを得た。
【００３２】
このエタノール抽出物１４３ｇを、ＨＰ−２０カラムクロマトグラフィーに付し、４０％メタノール、６０％メタノール、７０％メタノール、１００％メタノール、アセトンにて順次溶出し画分１〜７に分画した（図１）。
【００３３】
タンパク質非酵素的糖化抑制作用の検討
実施例１で得られた画分１〜７について、ウシ血清アルブミン（ＢＳＡ）（和光純薬社製０１３−０７４９２）の非酵素的糖化後期反応生成物（Ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ ｐｒｏｄｕｃｔｓ：ＡＧＥｓ）形成に対する阻害率を測定し、沙棘葉部抽出物のタンパク質非酵素的糖化反応に対する阻害を検討した。
【００３４】
陽性コントロールとしてはクエルセチン（ＳＩＧＭＡ社製Ｑ１２５）を用いて実験を行った。
【００３５】
まず、ＢＳＡをリン酸緩衝液（ＳＩＧＭＡ社製Ｄ−１４０８）にて５０ｍｇ／ｍｌの濃度に調製し，これをＡ液とした。次にＤ（−）リボース（ＳＩＧＭＡ社製Ｒ７５００）をリン酸緩衝液にて０．５Ｍに調製し，これをＢ液とした。このＡ液とＢ液を等量ずつ混和した液（Ｃ液）を調製しておいた。次に９６穴プレートに１０ｍＭ、１ｍＭ、０．１ｍＭに調製した各サンプル１ｍＬを注入し，それぞれに上記Ｃ液１５０ｍＬを添加した。添加後、直ちに蛍光Ｐｌａｔｅ Ｒｅａｄｅｒ ＥＺＳ−ＦＬ（旭テクノグラス社製）を用いて、各混合液の吸光度を励起波長３５５ｎｍ、測定波長４５０ｎｍにて測定し、測定値を０時間反応液の吸光度とした。
【００３６】
次に、この９６穴プレートをモイストチャンバー内にて３７℃で４日間インキュベートした。インキュベート後に生成された蛍光物質の吸光度を励起波長３５５ｎｍ、測定波長４５０ｎｍにて測定し、測定値をサンプルの反応液の吸光度とした。なお、各サンプルは、終濃度６６μＭ、６．６μＭ、０．６μＭとなるようにジメチルスルホキシド（ＤＭＳＯ）に溶解した。
【００３７】
各サンプルの測定値より、下記式を用いて各サンプルのＡＧＥｓ形成に対する阻害率を算出した。
阻害率（％）＝［１−（サンプルの反応液の吸光度−０時間反応液の吸光度）／（コントロール−ブランク）］×１００
式中、コントロールはサンプルを含まないＤＭＳＯのインキュベート後の吸光度であり、ブランクはサンプルを含まないＤＭＳＯの０時間反応時の吸光度である。
【００３８】
各サンプルの阻害率を図２に示す。画分１〜７のいずれも、ＡＧＥｓ形成に対する阻害を示したことから、沙棘葉部の抽出物がタンパク質非酵素的糖化抑制作用を有していることがわかった。
【００３９】
（実施例２）化合物Ｉ〜ＶＩＩＩの精製
以下のカラムを用いて、実施例１で得られた画分１〜７をさらに逆相高速液体クロマトグラフィー（ｒｐＨＰＬＣ）に付した。
Ａ：ＳＨＩＳＥＩＤＯ ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８ ＵＧ ２０Φ ｘ ２５０ ｍｍ
Ｂ：ＳＨＩＳＥＩＤＯ ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８ ＭＧ ２０Φ ｘ ２５０ ｍｍ
Ｃ：ＳＨＩＳＥＩＤＯ ＣＡＰＣＥＬＬ ＰＡＫ Ｐｈ １０Φ ｘ ２５０ ｍｍ
Ｄ：Ｄｅｖｅｒｏｓｉｌ Ｒｐａｑｕｅｏｕｓ １０Φ ｘ ２５０ ｍｍ
Ｅ：Ｋａｓｅｉｓｏｒｂ ＬＣ ＯＤＳ−Ｐｈ １０Φ ｘ ２５０ ｍｍ
Ｆ：ＳＨＩＳＥＩＤＯ ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８ ＭＧ １０Φ ｘ ２５０ ｍｍ
【００４０】
画分４（３．８ｇ）をｒｐＨＰＬＣ（カラムＢ、５０％メタノール）により画分８〜１１に分画し、画分８（５４８ｍｇ）をさらに、ｒｐＨＰＬＣ（カラムＣ、２２％アセトニトリル）により画分１２〜１６に分画した。画分１３（６２ｍｇ）をｒｐＨＰＬＣ（カラムＤ、２０％アセトニトリル）により精製し、化合物Ｉ（１９ｍｇ）を得た。画分１５（８７ｍｇ）をｒｐＨＰＬＣ（カラムＣ、２１％アセトニトリル）により精製し、化合物ＩＩ（５２ｍｇ）を得た。また、画分１６（５５ｍｇ）をｒｐＨＰＬＣ（カラムＥ、４４％メタノール）により精製し、化合物ＩＩＩ（１２ｍｇ）を得た。さらに、画分１７（８５ｍｇ）をｒｐＨＰＬＣ（カラムＥ、１９％アセトニトリル）により精製し、化合物ＩＶ（１２ｍｇ）を得た。画分９（８３８ｍｇ）をｒｐＨＰＬＣ（カラムＣ、２５％アセトニトリル）により精製し、化合物Ｖ（５９ｍｇ）を得た。画分１０（２４７ｍｇ）をｒｐＨＰＬＣ（カラムＦ、２９％アセトニトリル）及びｒｐＨＰＬＣ（カラムＥ、２３％アセトニトリル）により精製し、化合物ＶＩ（４６ｍｇ）及び化合物ＶＩＩ（２１ｍｇ）を得た。
【００４１】
画分５（２４．４ｇ）をシリカゲルカラムクロマトグラフィーに付し、クロロホルム：メタノール＝１００：１、２０：１、９：１、クロロホルム：メタノール：水＝８：２：０．１、６：４：０．５、メタノールを用いて順次溶出し、画分１７〜２５に分画した。画分１８（６．１ｇ）をｒｐＨＰＬＣ（カラムＡ、９５％メタノール）により精製し、化合物ＶＩＩＩ（９３７ｍｇ）を得た（以上、図１参照）。
【００４２】
以上の精製により、上述の式Ｉ、Ｖ、ＶＩ、ＶＩＩ及びＶＩＩＩで表される既知の化合物と、式ＩＩ、ＩＩＩ又はＩＶで表される新規化合物が得られた。
【００４３】
新規化合物ＩＩ〜ＩＶのそれぞれの物性値を以下に示す。
化合物 ＩＩ
非晶質粉末
ｎｅｇａｔｉｖｅ ＨＲ−ＦＡＢＭＳ ｍ／ｚ； ９６１．２６１３［Ｍ−Ｈ］⁻ （９６１．２６１２ Ｃａｌｃｄ． ｆｏｒ Ｃ_４４Ｈ_４９Ｏ_２４）
［α］_Ｄ ６５．０°（ｃ＝１．０， ＭｅＯＨ）
ＵＶ λ_ｍａｘ （ＭｅＯＨ） ２４５ｎｍ （ε １５７００）， ２６８ｎｍ （ε １２７００）， ３３２ｎｍ （ε １７９００）
^１Ｈ−ＮＭＲ （５００ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １．６３ （３Ｈ， ｄ， Ｊ ＝ ６．１ Ｈｚ， Ｈ−６’’’’’）， ３．７８ （６Ｈ， ｓ， −ＯＣＨ_３ ｘ ２）， ４．９７−５．０６ （２Ｈ， ｍ， Ｈ−６’’’）， ５．６４ （１Ｈ， ｄ， Ｊ ＝ ７．６ Ｈｚ， Ｈ−１’’’）， ６．２３ （１Ｈ， ｂｒ ｓ， Ｈ−１’’’’’）， ６．４８ （１Ｈ， ｄ， Ｊ ＝ １５．５ Ｈｚ， Ｈ−８’’’’）， ６．５６ （１Ｈ， ｄ， Ｊ ＝ ７．６ Ｈｚ， Ｈ−１’’）， ６．６９ （１Ｈ， ｄ， Ｊ ＝ ２．１ Ｈｚ， Ｈ−６）， ６．８２ （３Ｈ， ｍ， Ｈ−８， ２’’’’， ６’’’’）， ７．３６ （２Ｈ， ｄ， Ｊ ＝ ９．０ Ｈｚ， Ｈ−３’， ５’）， ７．７９ （１Ｈ， ｄ， Ｊ ＝ １５．５ Ｈｚ， Ｈ−７’’’’）， ８．４８ （２Ｈ， ｄ， Ｊ ＝ ９．０ Ｈｚ， Ｈ−２’， ６’）， １３．３ （１Ｈ， ｂｒ ｓ， ５−ＯＨ）
^１３Ｃ−ＮＭＲ （１２５ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １８．７ （Ｃ−６’’’’’）， ５６．３ （−ＯＣＨ_３ ｘ ２）， ６２．３ （Ｃ−６’’）， ６４．３ （Ｃ−６’’’）， ７１．２ （Ｃ−４’’， ４’’’）， ７１．４ （Ｃ−５’’’’’）， ７１．６ （Ｃ−２’’’’’）， ７２．４ （Ｃ−３’’’’’）， ７３．６ （Ｃ−４’’’’）， ７５．８ （Ｃ−５’’’）， ７６．２ （Ｃ−２’’’）， ７８．２ （Ｃ−３’’’）， ７８．３ （Ｃ−３’’）， ７８．９ （Ｃ−５’’）， ８４．１ （Ｃ−２’’）， ９４．５ （Ｃ−８）， １００．０ （Ｃ−１’’）， １００．１ （Ｃ−１’’’’’）， １００．３ （Ｃ−６）， １０６．１ （Ｃ−１’’’）， １０６．４ （Ｃ−１０）， １０６．６ （Ｃ−６’’’’）， １１５．３ （Ｃ−８’’’’）， １１６．３ （Ｃ−２’， ５’）， １２２．０ （Ｃ−１’）， １２５．１ （Ｃ−１’’’’）， １３２．１ （Ｃ−２’， ６’）， １３４．８ （Ｃ−３）， １４０．４ （Ｃ−４’’’’）， １４５．９ （Ｃ−７’’’’）， １４９．９ （Ｃ−２’’’’， ５’’’’）， １５６．７ （Ｃ−９）， １５７．０ （Ｃ−２）， １６１．８ （Ｃ−４’）， １６２．４ （Ｃ−５）， １６２．６ （Ｃ−７）， １６７．５ （Ｃ−９’’’’）， １７８．９ （Ｃ−４）
【００４４】
化合物 ＩＩＩ
非晶質粉末
ｎｅｇａｔｉｖｅ ＨＲ−ＦＡＢＭＳ ｍ／ｚ； ９３１．２５０５［Ｍ−Ｈ］⁻ （９６１．２５０７ Ｃａｌｃｄ． ｆｏｒ Ｃ_４３Ｈ_４７Ｏ_２３）
［α］_Ｄ ８７．９°（ｃ＝１．０， ＭｅＯＨ）
ＵＶ λ_ｍａｘ （ＭｅＯＨ） ２４６ｎｍ （ε １６９００）， ２６８ｎｍ （ε １８１００）， ３２７ｎｍ （ε ２１９００）
^１Ｈ−ＮＭＲ （５００ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １．６３ （３Ｈ， ｄ， Ｊ ＝ ６．１ Ｈｚ， Ｈ−６’’’’’）， ３．７４ （３Ｈ， ｓ， −ＯＣＨ_３）， ５．００ （２Ｈ， ｍ， Ｈ−６’’’）， ５．６４ （１Ｈ， ｄ， Ｊ ＝ ７．５ Ｈｚ， Ｈ−１’’’）， ６．２１ （１Ｈ， ｄ， Ｊ ＝ １．２ Ｈｚ， Ｈ−１’’’’’）， ６．４６ （１Ｈ， ｄ， Ｊ ＝ １５．５ Ｈｚ， Ｈ−８’’’’）， ６．５６ （１Ｈ， ｄ， Ｊ ＝ ７．５ Ｈｚ， Ｈ−１’’）， ６．６９ （１Ｈ， ｄ， Ｊ ＝ ２．１ Ｈｚ， Ｈ−６）， ６．８４ （１Ｈ， ｄ， Ｊ ＝ ２．１ Ｈｚ， Ｈ−８）， ７．００−７．１２ （３Ｈ， ｍ， Ｈ−２’’’’， ５’’’’， ６’’’’）， ７．３７ （２Ｈ， ｄ， Ｊ ＝ ８．８ Ｈｚ， Ｈ−３’， ５’）， ７．７９ （１Ｈ， ｄ， Ｊ ＝ １５．５ Ｈｚ， Ｈ−７’’’’）， ８．４９ （２Ｈ， ｄ， Ｊ ＝ ８．９ Ｈｚ， Ｈ−２’， ６’）， １３．３ （１Ｈ， ｂｒ ｓ， ５−ＯＨ）
^１３Ｃ−ＮＭＲ （１２５ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １８．７ （Ｃ−６’’’’’）， ５５．８ （−ＯＣＨ_３）， ６２．３ （Ｃ−６’’）， ６４．３ （Ｃ−６’’’）， ７１．２ （Ｃ−４’’， ４’’’）， ７１．４ （Ｃ−５’’’’’）， ７１．６ （Ｃ−２’’’’’）， ７２．４ （Ｃ−３’’’’’）， ７３．６ （Ｃ−４’’’’’）， ７５．８ （Ｃ−５’’’）， ７６．２ （Ｃ−２’’’）， ７８．２ （Ｃ−３’’’）， ７８．３ （Ｃ−３’’）， ７８．９ （Ｃ−５’’）， ８４．１ （Ｃ−２’’）， ９４．６ （Ｃ−８）， １００．０ （Ｃ−１’’， １’’’’’）， １００．３ （Ｃ−５， ６）， １０６．２ （Ｃ−１’’’）， １０６．９ （Ｃ−１０）， １１１．２ （Ｃ−２’’’’）， １１５．０ （Ｃ−８’’’’）， １１６．４ （Ｃ−３’， ５’）， １１６．６ （Ｃ−５’’’’）， １２２．０ （Ｃ−１’）， １２３．７ （Ｃ−６’’’’）， １２６．４ （Ｃ−１’’’’）， １３２．１ （Ｃ−２’， ６’）， １３４．８ （Ｃ−３）， １４５．５ （Ｃ−７’’’’）， １４８．８ （Ｃ−３’’’’）， １５０．９ （Ｃ−４’’’’）， １５６．８ （Ｃ−９）， １５７．０ （Ｃ−２）， （Ｃ−５）， １６１．８ （Ｃ−４’）， １６２．６ （Ｃ−７）， １６７．６ （Ｃ−９’’’’）， １７８．９ （Ｃ−４）
【００４５】
化合物 ＩＶ
非晶質粉末
ｎｅｇａｔｉｖｅ ＨＲ−ＦＡＢＭＳ ｍ／ｚ； ９７７．２５７０［Ｍ−Ｈ］⁻ （９６１．２５６１ Ｃａｌｃｄ． ｆｏｒ Ｃ_４４Ｈ_４９Ｏ_２５）
［α］_Ｄ １０９．６°（ｃ＝０．５， Ｈ_２Ｏ）
ＵＶ λ_ｍａｘ （ＭｅＯＨ） ２０４ｎｍ （ε ５２７００）， ２６２ｎｍ （ε ２５２００）， ３４３ｎｍ （ε １９２００）
^１Ｈ−ＮＭＲ （５００ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １．６３ （３Ｈ， ｄ， Ｊ ＝ ６．１ Ｈｚ， Ｈ−６’’’’’）， ３．７８ （３Ｈ， ｓ， −ＯＣＨ_３）， ５．００−５．１３ （２Ｈ， ｍ， Ｈ−６’’’）， ５．６６ （１Ｈ， ｄ， Ｊ ＝ ７．６ Ｈｚ， Ｈ−１’’’）， ６．１９ （１Ｈ， ｂｒ ｓ， Ｈ−１’’’’’）， ６．４８ （１Ｈ， ｄ， Ｊ ＝ １５．９ Ｈｚ， Ｈ−８’’’’）， ６．６３（１Ｈ， ｄ， Ｊ ＝ ７．６ Ｈｚ， Ｈ−１’’）， ６．６７ （１Ｈ， ｄ， Ｊ ＝ ２．１ Ｈｚ， Ｈ−６）， ６．６５ （１Ｈ， ｄ，Ｊ ＝ ２．１ Ｈｚ， Ｈ−８）， ６．８４ （２Ｈ， ｓ， Ｈ−２’’’’， ６’’’’）， ７．４４ （１Ｈ， ｄ， Ｊ ＝ ８．５ Ｈｚ， Ｈ−５’）， ７．７７ （１Ｈ， ｄ， Ｊ ＝ １５．９ Ｈｚ， Ｈ−７’’’’）， ８．２４ （２Ｈ， ｍ， Ｈ−２’， ６’）， １３．３ （１Ｈ， ｂｒ ｓ， ５−ＯＨ）
^１３Ｃ−ＮＭＲ （１２５ ＭＨｚ， δ ｉｎ Ｐｙ−ｄ_５）； １８．７ （Ｃ−６’’’’’）， ５６．３ （−ＯＣＨ_３ ｘ ２）， ６２．３ （Ｃ−６’’）， ６４．３ （Ｃ−６’’’）， ７１．２ （Ｃ−４’’， ４’’’）， ７１．４ （Ｃ−５’’’’’）， ７１．６ （Ｃ−２’’’’’）， ７２．４ （Ｃ−３’’’’’）， ７３．７ （Ｃ−４’’’’’）， ７５．８ （Ｃ−５’’’）， ７６．３ （Ｃ−２’’’）， ７８．２ （Ｃ−３’’）， ７８．３ （Ｃ−３’’’）， ７８．８ （Ｃ−５’’）， ８４．７ （Ｃ−２’’）， ９４．３ （Ｃ−８）， ９９．９ （Ｃ−１’’）， １００．０ （Ｃ−１’’’’’）， １００．２ （Ｃ−６）， １０６．３ （Ｃ−１’’’）， １０６．６ （Ｃ−２’’’’， ６’’’’）， １０６．９ （Ｃ−１０）， １１５．３ （Ｃ−８’’’’）， １１６．５ （Ｃ−５’）， １１７．５ （Ｃ−２’）， １２３．０（Ｃ−１’）， １２５．２ （Ｃ−１’’’’）， １３５．０ （Ｃ−３）， １３５．５ （Ｃ−６’）， １４０．４ （Ｃ−４’’’’）， １４５．８ （Ｃ−７’’’’）， １４７．０ （Ｃ−３’）， １４９．１ （Ｃ−３’’’’， ５’’’’）， １５６．７ （Ｃ−９）， １５７．０ （Ｃ−２）， １５０．６ （Ｃ−４’）， １６２．３ （Ｃ−５）， １６２．５ （Ｃ−７）， １６７．５ （Ｃ−９’’’’）， １７８．９ （Ｃ−４）
【００４６】
（実施例３）化合物Ｉ〜ＶＩＩのタンパク質非酵素的糖化抑制作用の検討
化合物Ｉ〜ＶＩＩについて、実施例１と同様の方法により、ＡＧＥｓ形成に対する阻害率を測定し、化合物Ｉ〜ＶＩＩのタンパク質非酵素的糖化反応に対する阻害を検討した。
【００４７】
陽性コントロールとしてはクエルセチン（ＳＩＧＭＡ社製Ｑ１２５）及びケンフェロール（東京化成社）を用いて実験を行い、実施例１で用いた式により各サンプルのＡＧＥｓ形成に対する阻害率を算出した。
【００４８】
各サンプルの阻害率を図３に示す。新規化合物ＩＩ〜ＩＶを含む化合物Ｉ〜ＶＩＩのいずれも、ＡＧＥｓ形成に対する阻害を示した。特に新規化合物ＩＩは、サンプル濃度６６μＭ及び６．６μＭにおいては、検討した化合物中最も高い阻害率を示した。この結果より、化合物Ｉ〜ＶＩＩがタンパク質非酵素的糖化抑制作用を有していることがわかった。
【００４９】
【発明の効果】
本発明のタンパク質非酵素的糖化抑制剤は、タンパク質非酵素的糖化抑制作用を促進するため、糖尿病又は糖尿病合併症を予防・治療するのに有効である。
【図面の簡単な説明】
【図１】図１は、沙棘抽出物の画分を示す。
【図２】図２は、ＡＧＥｓ形成に対する画分１〜７の阻害率を示す。
【図３】図３は、ＡＧＥｓ形成に対する化合物Ｉ〜ＶＩＩの阻害率を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a protein non-enzymatic saccharification inhibitor containing an active ingredient derived from prickle.
[0002]
[Prior art]
Hyperglycemia due to diabetes may cause various complications such as neuropathy, cataract, renal disorder, retinopathy, arteriosclerosis, atherosclerosis, and diabetic gangrene. Complications are mainly due to vascular disorders caused by non-enzymatic glycation, where proteins in the patient's blood bind sugar, and cell destruction caused by the accumulation of sorbitol, which occurs when sugar is metabolized. In particular, vascular disorders often affect the life of a patient or greatly impair the quality of life of the patient over a long period of time. For this reason, prevention / treatment of vascular disorders is an important issue in the treatment of diabetes or diabetic complications, and the relationship between non-enzymatic glycated proteins and diseases has been studied in detail (for example, see Non-Patent Documents 1 to 7). ).
[0003]
On the other hand, Hippophae rhamnoides is a plant of the family Gumicaceae distributed mainly in the Yellow River basin of China, and in the arid region of Russia and the Middle East, and its fruits and leaves have long been used for skin ulcer disease, stomach, liver and respiratory organs. It is used as a folk medicine for chronic systemic diseases, and in Japan, patents have been given to bath compositions and cosmetics that use an extract of saury as a raw material (for example, Patent Documents 1 and 2).
[0004]
Although many flavonoid glycosides have been obtained as the main components of the saury, the relationship between the flavonoid glycosides and the pharmacological activity has not yet been elucidated.
[0005]
[Non-patent document 1]
"Biochemical and Biophysical Research Communications", Vol. 67, 1975, p. 103
[Non-patent document 2]
"Journal of Biological Chemistry", Vol. 254, 1979, p. 702
[Non-Patent Document 3]
"Proceedings of the National Academy of Sciences," USA, Vol. 78, 1982, p. 2393
[Non-patent document 4]
"Diabetes", Vol. 31, 1982, p. 283
[Non-Patent Document 5]
"Proceedings of the National Academy of Sciences", U.S.A., 75, 1978, p. 2918
[Non-Patent Document 6]
"Journal of Clinical Pathology", Vol. 37, 1984, p. 841
[Non-Patent Document 7]
"Annals of Clinical Biochemistry", Vol. 21, 1984, p. 2
[Patent Document 1]
Japanese Patent No. 2811711 [Patent Document 2]
Japanese Patent No. 2515525 [0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a protein non-enzymatic saccharification inhibitor derived from spinach.
[0007]
[Means for Solving the Problems]
The present invention includes the following inventions.
(1) A protein non-enzymatic saccharification inhibitor comprising, as an active ingredient, a substance having a protein non-enzymatic saccharification inhibitory activity derived from saury.
(2) A protein non-enzymatic saccharification inhibitor comprising, as an active ingredient, a spiny leaf extract or a processed product thereof.
(3) The protein non-enzymatic glycation inhibitor according to the above (1) or (2), which is used for treating diabetes or diabetic complications.
(4) The protein non-enzymatic saccharification inhibitor according to any of (1) to (3) above, which is added to a food, chewing gum or beverage.
(5) The protein non-enzymatic saccharification inhibitor according to any of the above (1) to (4), comprising a flavonoid glycoside.
(6) A prophylactic / therapeutic agent for diabetes or diabetic complications, which comprises, as an active ingredient, a substance having a protein non-enzymatic saccharification inhibitory activity derived from spines.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
TECHNICAL FIELD The present invention relates to a protein non-enzymatic saccharification inhibitor containing, as an active ingredient, a substance having a protein non-enzymatic saccharification inhibitory activity derived from spinach.
[0009]
Sharp spines (Hippophae rhamnoides) are a member of the family Gummyceae distributed mainly in the Yellow River basin in China. As the spines, those conventionally used in folk medicine and the like can be used. In the present invention, it is particularly preferable to use the leaves of the spines.
[0010]
The protein non-enzymatic saccharification inhibitor of the present invention may contain a substance which is derived from spinach and exhibits protein non-enzymatic saccharification inhibitory activity. The prickle may be used after being pulverized by crushing, pulverization, or the like, but is preferably used as an extract or a processed product thereof.
[0011]
Examples of the extraction solvent include water; lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and isobutanol; ethers such as ethyl ether, dioxane; ketones such as acetone; A mixed solvent of water and ethanol is preferred.
Usually, 5 to 25 L of extraction solvent is used per 1 kg of spinach.
[0012]
The extraction temperature is not particularly limited and is within the range of the melting point of the solvent or the boiling point of the solvent, and supercritical extraction may be performed. The supercritical extraction is preferably performed by using carbon dioxide as a main solvent and adding an alcohol such as methanol or ethanol as an entrainer. The extraction is usually performed under normal pressure, but may be performed under increased or reduced pressure. The extraction time varies depending on the extraction temperature and the like, and is usually 5 minutes to 1 day.
[0013]
The target extract can be obtained by filtering the extract obtained as described above with a cloth, a stainless steel filter, filter paper, or the like to remove impurities and the like. Further, the extract after filtration may be subjected to a treatment such as a spray-dry treatment, a freeze-dry treatment, and a supercritical treatment.
[0014]
The extract thus obtained can be used as it is as an active ingredient of the protein non-enzymatic saccharification inhibitor of the present invention. Further, the extract may be treated by various purification means such as ion exchange chromatography, gel filtration chromatography, and dialysis, and may be used as a treated substance having further enhanced activity.
[0015]
The present inventors further purify the spiny extract to obtain the triterpene ursolic acid (VIII), as well as the known compounds of formulas I, V, VI and VII shown below and formulas II, III Or isolating and determining the structure of a flavonoid glycoside containing three kinds of novel compounds represented by IV, and obtaining a compound represented by the formulas I to VII including the novel compound represented by the formula II, III or VI. It has been shown to have non-enzymatic saccharification inhibitory activity.
[0016]
Formulas I, V, VI, VII and VIII (known compounds):
Embedded image

[0017]
Formula II or IV (new compounds):
Embedded image

[0018]
Formula III (new compound):
Embedded image

[0019]
In addition, the protein non-enzymatic saccharification inhibitor of the present invention can be formulated by combining an extract / treated product of the spiny leaf portion with a known pharmaceutical carrier. The administration form is not particularly limited and may be appropriately selected as necessary. In general, tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, elixirs and the like It is used as an oral preparation or a parenteral preparation such as an injection, a drip, a suppository, an inhalant, a transdermal absorbent, a transmucosal absorbent, a patch, an ointment and the like.
[0020]
The dose of the protein non-enzymatic glycation inhibitor of the present invention varies depending on the age, body weight, degree of disease, and administration route of the patient. The dose is usually 1 to 3 times a day for oral administration.
Oral preparations are produced in a conventional manner using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like.
[0021]
In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavor, and the like can be appropriately used in addition to the excipient.
[0022]
Specific examples of the binder include crystalline cellulose, crystalline cellulose and carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, and carmellose sodium , Ethyl cellulose, carboxymethyl ethyl cellulose, hydroxyethyl cellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl Metak Rate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.
[0023]
Specific examples of disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth.
[0024]
Specific examples of the surfactant include soy lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glycerin monostearate, sodium lauryl sulfate, lauromacrogol.
[0025]
Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dried aluminum hydroxide gel, talc, Examples include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid esters, waxes, hydrogenated vegetable oils, and polyethylene glycol.
[0026]
Specific examples of the fluidity promoter include hydrous silicon dioxide, light silicic anhydride, dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.
[0027]
When administered as a solution, syrup, suspension, emulsion or elixir, the protein non-enzymatic saccharification inhibitor of the present invention may contain a flavoring agent and a coloring agent.
[0028]
The protein non-enzymatic saccharification inhibitor of the present invention can be added to foods, chewing gums, beverages, and the like to produce so-called foods for specified health use (eg, foods for preventing diabetes and diabetic complications).
[0029]
Sharp, which is a raw material for producing the protein non-enzymatic saccharification inhibitor of the present invention, has been used in folk medicines, cosmetics, and the like, and its safety has been established.
[0030]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.
[0031]
(Example 1) Measurement of protein non-enzymatic saccharification inhibitory action of spiny leaf extract
Preparation of Sharp Leaves Extract 2 kg of Sharp Leaves obtained from Gansu Province, China, were pulverized and then extracted for 30 minutes using 23 L of ethanol under ultrasonic conditions. After the extract was filtered through a glass filter, the filtrate was concentrated under reduced pressure to obtain 143 g of an ethanol extract.
[0032]
143 g of this ethanol extract was subjected to HP-20 column chromatography, and eluted sequentially with 40% methanol, 60% methanol, 70% methanol, 100% methanol and acetone, and fractionated into fractions 1 to 7 (FIG. 1).
[0033]
Examination of protein non-enzymatic saccharification inhibitory action Regarding fractions 1 to 7 obtained in Example 1, non-enzymatic saccharification late reaction of bovine serum albumin (BSA) (013-07492 manufactured by Wako Pure Chemical Industries, Ltd.) The rate of inhibition of the formation of advanced glycation end products (AGEs) was measured, and the inhibition of the non-enzymatic saccharification reaction of the spinach leaf extract was examined.
[0034]
An experiment was performed using quercetin (Q125 manufactured by SIGMA) as a positive control.
[0035]
First, BSA was adjusted to a concentration of 50 mg / ml with a phosphate buffer (D-1408 manufactured by SIGMA), and this was used as solution A. Next, D (-) ribose (R7500 manufactured by SIGMA) was adjusted to 0.5 M with a phosphate buffer, and this was used as solution B. A solution (solution C) was prepared by mixing equal amounts of this solution A and solution B. Next, 1 mL of each sample adjusted to 10 mM, 1 mM, and 0.1 mM was injected into a 96-well plate, and 150 mL of the above solution C was added to each. Immediately after the addition, the absorbance of each mixed solution was measured at an excitation wavelength of 355 nm and a measurement wavelength of 450 nm using a fluorescent plate reader EZS-FL (manufactured by Asahi Techno Glass Co.), and the measured value was defined as the absorbance of the reaction solution for 0 hour. .
[0036]
Next, the 96-well plate was incubated at 37 ° C. for 4 days in a moist chamber. The absorbance of the fluorescent substance generated after the incubation was measured at an excitation wavelength of 355 nm and a measurement wavelength of 450 nm, and the measured value was taken as the absorbance of the sample reaction solution. Each sample was dissolved in dimethyl sulfoxide (DMSO) so that the final concentration was 66 μM, 6.6 μM, and 0.6 μM.
[0037]
From the measured value of each sample, the inhibition rate of each sample against AGEs formation was calculated using the following equation.
Inhibition rate (%) = [1− (absorbance of reaction solution of sample−absorbance of reaction solution of 0 hour) / (control−blank)] × 100
In the formula, the control is the absorbance of DMSO containing no sample after incubation, and the blank is the absorbance of DMSO containing no sample at the time of 0 hour reaction.
[0038]
FIG. 2 shows the inhibition rate of each sample. All of fractions 1 to 7 showed inhibition on AGEs formation, indicating that the spiny leaf extract had a protein non-enzymatic glycation inhibitory action.
[0039]
(Example 2) Purification of compounds I to VIII Using the following columns, fractions 1 to 7 obtained in Example 1 were further subjected to reversed-phase high performance liquid chromatography (rpHPLC).
A: SHISEIDO CAPCELL PAK C18 UG 20Φ x 250 mm
B: SHISEIDO CAPCELL PAK C18 MG 20Φ x 250 mm
C: SHISEIDO CAPCELL PAK Ph 10Φ x 250 mm
D: Deverosil Rpaqueous 10Φ x 250 mm
E: Kasesorb LC ODS-Ph 10Φ x 250 mm
F: SHISEIDO CAPCELL PAK C18 MG 10Φ x 250 mm
[0040]
Fraction 4 (3.8 g) was fractionated by rpHPLC (column B, 50% methanol) into fractions 8-11, and fraction 8 (548 mg) was further fractionated by rpHPLC (column C, 22% acetonitrile). Fractionated into 12-16. Fraction 13 (62 mg) was purified by rpHPLC (column D, 20% acetonitrile) to give compound I (19 mg). Fraction 15 (87 mg) was purified by rpHPLC (column C, 21% acetonitrile) to give compound II (52 mg). Further, fraction 16 (55 mg) was purified by rpHPLC (column E, 44% methanol) to obtain compound III (12 mg). Further, fraction 17 (85 mg) was purified by rpHPLC (column E, 19% acetonitrile) to obtain compound IV (12 mg). Fraction 9 (838 mg) was purified by rpHPLC (column C, 25% acetonitrile) to give compound V (59 mg). Fraction 10 (247 mg) was purified by rpHPLC (column F, 29% acetonitrile) and rpHPLC (column E, 23% acetonitrile) to give compound VI (46 mg) and compound VII (21 mg).
[0041]
Fraction 5 (24.4 g) was subjected to silica gel column chromatography, chloroform: methanol = 100: 1, 20: 1, 9: 1, chloroform: methanol: water = 8: 2: 0.1, 6: 4. : 0.5, eluted sequentially with methanol and fractionated into fractions 17 to 25. Fraction 18 (6.1 g) was purified by rpHPLC (column A, 95% methanol) to obtain compound VIII (937 mg) (see FIG. 1).
[0042]
By the above purification, known compounds represented by the above formulas I, V, VI, VII and VIII and novel compounds represented by the formulas II, III or IV were obtained.
[0043]
The physical properties of the new compounds II to IV are shown below.
Compound II
Amorphous powder ^{negative HR-FABMS m / z;} 961.2613 [M-H] - (. 961.2612 Calcd for C 44 H 49 O 24)
[Α] _D 65.0 ° (c = 1.0, MeOH)
UV λ _max (MeOH) 245 nm (ε 15700), 268 nm (ε 12700), 332 nm (ε 17900)
^{1 H-NMR (500 MHz,} δ in Py-d 5); 1.63 (3H, d, J = 6.1 Hz, H-6 '''''), 3.78 (6H, s, - OCH ₃ x 2), 4.97-5.06 (2H, m, H-6 ′ ″), 5.64 (1H, d, J = 7.6 Hz, H−1 ′ ″), 6 .23 (1H, brs, H-1 ′ ″ ″), 6.48 (1H, d, J = 15.5 Hz, H−8 ″ ″), 6.56 (1H, d, J = 7.6 Hz, H-1 ″), 6.69 (1H, d, J = 2.1 Hz, H-6), 6.82 (3H, m, H-8, 2 ′ ″) ', 6''''), 7.36 (2H, d, J = 9.0 Hz, H-3', 5 '), 7.79 (1H, d, J = 15.5 Hz, H- 7 ″ ″), 8.48 (2H, d, J = 9.0 Hz, H−2 ′, 6 ′), 13.3 (1H, brs, 5-OH)
¹³ C-NMR (125 MHz, δ in Py-d ₅ ); 18.7 (C-6 ′ ″ ″), 56.3 (—OCH ₃ x2), 62.3 (C-6 ″). ), 64.3 (C-6 '''), 71.2 (C-4'',4'''), 71.4 (C-5 '''), 71.6 (C- 2 ''"), 72.4 (C-3""), 73.6 (C-4""), 75.8 (C-5"), 76.2. (C-2 '''), 78.2 (C-3'''), 78.3 (C-3 ''), 78.9 (C-5 ''), 84.1 (C-2 ''), 94.5 (C-8), 100.0 (C-1 ''), 100.1 (C-1 '''''), 100.3 (C-6), 106.1 (C-1 ′ ″), 106.4 (C-10), 106.6 (C-6 ″ ″), 115.3 (C-8 ″ ″), 116.3 (C− 2 ', 5'), 122.0 (C 1 '), 125.1 (C-1 ""), 132.1 (C-2', 6 '), 134.8 (C-3), 140.4 (C-4 ""). ), 145.9 (C-7 ""), 149.9 (C-2 "", 5 ""), 156.7 (C-9), 157.0 (C-2 ), 161.8 (C-4 '), 162.4 (C-5), 162.6 (C-7), 167.5 (C-9 ""), 178.9 (C-4 )
[0044]
Compound III
Amorphous powder negative HR-FABMS m / z; 931.2505 [MH] ⁻ (961.2507 Calcd. For C ₄₃ H ₄₇ O ₂₃ )
[Α] _D 87.9 ° (c = 1.0, MeOH)
UV λ _max (MeOH) 246 nm (ε 16900), 268 nm (ε 18100), 327 nm (ε 21900)
^{1 H-NMR (500 MHz,} δ in Py-d 5); 1.63 (3H, d, J = 6.1 Hz, H-6 '''''), 3.74 (3H, s, - _{OCH 3), 5.00 (2H,} m, H-6 '''), 5.64 (1H, d, J = 7.5 Hz, H-1'''), 6.21 (1H, d , J = 1.2 Hz, H-1 ′ ″ ″), 6.46 (1H, d, J = 15.5 Hz, H−8 ″ ″), 6.56 (1H, d, J = 7.5 Hz, H-1 ″), 6.69 (1H, d, J = 2.1 Hz, H-6), 6.84 (1H, d, J = 2.1 Hz, H -8), 7.00-7.12 (3H, m, H-2 "", 5 "", 6 ""), 7.37 (2H, d, J = 8.8). Hz, H-3 ', 5'), 7.79 (1H, d, J = 15.5 Hz, H-7 ''''), 8.49 (2H, d, J = 8.9 Hz, H-2', 6 '), 13.3 (1H, brs, 5-OH)
¹³ C-NMR (125 MHz, δ in Py-d ₅ ); 18.7 (C-6 ′ ″ ″), 55.8 (—OCH ₃ ), 62.3 (C-6 ″), 64.3 (C-6 '''), 71.2 (C-4'',4'''), 71.4 (C-5 '''''), 71.6 (C-2'''''), 72.4 (C-3 '''), 73.6 (C-4'''''), 75.8 (C-5 '''), 76.2 ( C-2 "), 78.2 (C-3"), 78.3 (C-3 "), 78.9 (C-5"), 84.1 (C-2 '). '), 94.6 (C-8), 100.0 (C-1'',1'''''), 100.3 (C-5, 6), 106.2 (C-1 ''). '), 106.9 (C-10), 111.2 (C-2 ""), 115.0 (C-8 ""), 116.4 (C-3', 5 '). , 116.6 (C-5 ″ ″), 122. 0 (C-1 ′), 123.7 (C-6 ″ ″), 126.4 (C-1 ″ ″), 132.1 (C-2 ′, 6 ′), 134.8. (C-3), 145.5 (C-7 ""), 148.8 (C-3 ""), 150.9 (C-4 ""), 156.8 (C -9), 157.0 (C-2), (C-5), 161.8 (C-4 '), 162.6 (C-7), 167.6 (C-9'''') , 178.9 (C-4)
[0045]
Compound IV
Amorphous powder ^{negative HR-FABMS m / z;} 977.2570 [M-H] - (. 961.2561 Calcd for C 44 H 49 O 25)
[Α] _D 109.6 ° (c = 0.5, H ₂ O)
UV λ _max (MeOH) 204 nm (ε 52700), 262 nm (ε 25200), 343 nm (ε 19200)
^{1 H-NMR (500 MHz,} δ in Py-d 5); 1.63 (3H, d, J = 6.1 Hz, H-6 '''''), 3.78 (3H, s, - _{OCH 3), 5.00-5.13 (2H,} m, H-6 '''), 5.66 (1H, d, J = 7.6 Hz, H-1'''), 6.19 (1H, brs, H-1 ′ ″ ″), 6.48 (1H, d, J = 15.9 Hz, H−8 ″ ″), 6.63 (1H, d, J = 7.6 Hz, H-1 ″), 6.67 (1H, d, J = 2.1 Hz, H-6), 6.65 (1H, d, J = 2.1 Hz, H-8) ), 6.84 (2H, s, H-2 "", 6 ""), 7.44 (1H, d, J = 8.5 Hz, H-5 '), 7.77 ( 1H, d, J = 15.9 Hz, H-7 ""), 8.24 (2H, m, H-2 ′, 6 ′), 13.3 (1H, brs, 5-OH)
¹³ C-NMR (125 MHz, δ in Py-d ₅ ); 18.7 (C-6 ′ ″ ″), 56.3 (—OCH ₃ x2), 62.3 (C-6 ″). ), 64.3 (C-6 '''), 71.2 (C-4'',4'''), 71.4 (C-5 '''), 71.6 (C- 2 ''"), 72.4 (C-3""), 73.7 (C-4""), 75.8 (C-5"), 76. 3 (C-2 '''), 78.2 (C-3''), 78.3 (C-3'''), 78.8 (C-5 ''), 84.7 (C- 2 ''), 94.3 (C-8), 99.9 (C-1 ''), 100.0 (C-1 '''''), 100.2 (C-6), 106. 3 (C-1 ""), 106.6 (C-2 "", 6 ""), 106.9 (C-10), 115.3 (C-8 ""). , 116.5 (C-5 '), 117.5 (C 2 ′), 123.0 (C-1 ′), 125.2 (C-1 ″ ″), 135.0 (C-3), 135.5 (C-6 ′), 140.4 ( C-4 ""), 145.8 (C-7 ""), 147.0 (C-3 '), 149.1 (C-3 "", 5 "") , 156.7 (C-9), 157.0 (C-2), 150.6 (C-4 '), 162.3 (C-5), 162.5 (C-7), 167.5 (C-9 ''''), 178.9 (C-4)
[0046]
Example 3 Investigation of Inhibitory Effect of Compounds I to VII on Protein Nonenzymatic Saccharification Inhibition rate on AGEs formation was measured for Compounds I to VII in the same manner as in Example 1, and the protein I The inhibition on the enzymatic saccharification reaction was examined.
[0047]
Experiments were performed using quercetin (Q125, manufactured by SIGMA) and kaempferol (Tokyo Kasei) as positive controls, and the inhibition rate on AGEs formation of each sample was calculated by the formula used in Example 1.
[0048]
FIG. 3 shows the inhibition rate of each sample. All of the compounds I-VII, including the novel compounds II-IV, showed inhibition on AGEs formation. In particular, the novel compound II showed the highest inhibition rate among the compounds examined at the sample concentrations of 66 μM and 6.6 μM. From these results, it was found that Compounds I to VII had a protein non-enzymatic saccharification inhibitory action.
[0049]
【The invention's effect】
The protein non-enzymatic saccharification inhibitor of the present invention promotes the protein non-enzymatic saccharification inhibitory action, and is therefore effective for preventing and treating diabetes or diabetic complications.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the fraction of prickly pear extract.
FIG. 2 shows the percentage of inhibition of fractions 1 to 7 on AGEs formation.
FIG. 3 shows the percentage of inhibition of compounds I-VII on AGEs formation.

Claims (6)

A protein non-enzymatic saccharification inhibitor comprising, as an active ingredient, a substance having a protein non-enzymatic saccharification inhibitory activity, which is derived from Saji thorn. A protein non-enzymatic saccharification inhibitor comprising, as an active ingredient, a spiny leaf extract or a processed product thereof. The protein non-enzymatic glycation inhibitor according to claim 1 or 2, which is used for treating diabetes or diabetic complications. The protein non-enzymatic saccharification inhibitor according to any one of claims 1 to 3, which is added to a food, a chewing gum or a beverage. The protein non-enzymatic saccharification inhibitor according to any one of claims 1 to 4, comprising a flavonoid glycoside. A preventive / therapeutic agent for diabetes or diabetic complications comprising, as an active ingredient, a substance having a protein non-enzymatic saccharification inhibitory activity, which is derived from the spinach.

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