JP4182251B2 - Propane-1,2,3-triol derivative, process for producing the same, and α-glucosidase inhibitor containing the same - Google Patents

Description

【０００６】
請求項２記載の本発明は、シソ科植物ヒソップ（Hyssopus officinalis）の地上部から含水メタノール溶液、含水エタノール溶液又は含水ブタノール溶液で抽出した後、酢酸エチルと水を用いて有機溶媒可溶部と水可溶部に分配することを特徴とする下記化学式 ( １ ) または ( ２ ) で表されるプロパン−１,２,３−トリオール誘導体の製造方法である。
【０００７】
【化７】
（2S,3S）1-O-β-D-6'-O-cinnamoylglucopyranosyl-3-(3’’,5’’-dimethoxy-4’’-hydroxyphenyl)propane-1,2,3-triol （１）

【０００８】
【化８】
（2S,3S）1-O-β-D-glucopyranosyl-3-(3’’,5’’-dimethoxy-4’’-hydroxyphenyl)propane-1,2,3-triol （２）

【０００９】
請求項３記載の本発明は、下記化学式 ( １ ) または ( ２ ) で表されるプロパン−１,２,３−トリオール誘導体の一方もしくは両方からなるα−グルコシダーゼ阻害剤である。
【００１０】
【化９】
（2S,3S）1-O-β-D-6'-O-cinnamoylglucopyranosyl-3-(3’’,5’’-dimethoxy-4’’-hydroxyphenyl)propane-1,2,3-triol （１）

【００１１】
【化１０】
（2S,3S）1-O-β-D-glucopyranosyl-3-(3’’,5’’-dimethoxy-4’’-hydroxyphenyl)propane-1,2,3-triol （２）

【００１２】
請求項４記載の本発明は、請求項１記載のプロパン−１ , ２ , ３−トリオール誘導体を添加したことを特徴とする食品もしくは飲料である。
【００１３】
【発明の実施の形態】
本発明に係る新規化合物のプロパン−１,２,３−トリオール誘導体は、上記の化学式(１)および(２)で表される。以下において、前者を化合物（Ａ）、後者を化合物（Ｂ）と称する。
【００１４】
本発明に係る化合物（Ａ）および化合物（Ｂ）は、ヒソップ地上部から得ることができる。本発明に使用することができるヒソップ（和名：ヤナギハッカ）の品種としては、ヒソップ，ホワイト・ヒソップ，ピンク・ヒソップなどを例示することができるが，これらの品種に限定されるものではない。また、地上部としては、葉，茎，芽等を例示できるが、有効成分について粉末化などの加工を施すには、葉部の使用が適している。
【００１５】
上記の植物から目的とする物質を抽出する方法としては、例えば以下に例示する方法が可能であるが、これに限定されるものではない。また、使用目的や使用濃度に応じて、各分画段階における粗抽出画分が利用可能である。
【００１６】
粗抽出工程として、ヒソップ乾燥葉などの原料を、アセトン，エタノール，ブタノール，アセトニトリル，テトラヒドロフランなどの有機溶媒の含水物を用いて抽出する。抽出する際の温度は常温でよく、抽出時間についてはヒソップ乾燥葉の場合、１週間乃至１０日ほどである。
【００１７】
抽出操作の終了後、濾紙やガラスフィルター等を用いる固−液分離手段で抽出物を濾液と残渣に分離する。次いで、濾液を減圧濃縮する。得られた濃縮液を有機溶媒可溶部と水可溶部に分配する。すなわち、酢酸エチルなどの有機溶媒と水を用いて有機溶媒可溶部と水可溶部に分配する。α−グルコシダーゼ阻害活性は両可溶部に観察され、有機溶媒可溶部から前記化合物（Ａ）が、水可溶部から化合物（Ｂ）が得られる。なお、この有機溶媒可溶部と水可溶部に分配する工程は、省略することができ、両化合物を含む混合物の形態で本発明に用いることができる。
【００１８】
上記化合物（Ａ）を分離する方法について説明すると、抽出第一段階として、有機溶媒可溶部を濃縮し、これを活性炭あるいはダイヤイオンHP-20（三菱化学(株)製）を担体に用いた吸着カラムクロマトグラフィーに供する。次いで、水，含水メタノール溶液，メタノールを用いて溶出し、阻害活性を有する含水メタノール溶液溶出区を濃縮する。
次に、これをシリカゲル(wakogelc-200、和光純薬製)担体カラムクロマトグラフィーに供して有機溶媒混合液、例えばCHCl₃並びにMeOH：CHCl₃の混合液を用いて溶出する。目的とする阻害活性の確認された溶出区を減圧し、濃縮する。
【００１９】
上記化合物（Ａ）の抽出第二段階として、この分画をゲル濾過クロマトグラフィー、例えばセファデックスLH-20 (Amersham Pharmacia Biotech社製)に供し、有機溶媒を用いて溶出し、阻害活性の高い溶出区を濃縮する。
次いで、化合物（Ａ）の抽出第三段階として、この分画を逆相中圧液体カラムクロマトグラフィーに供する。カラムとしては、例えば Lpbar LiChroprep RP-18 (Merck社製)等を使用することができる。移動層として含水有機溶媒、例えばメタノール：水：酢酸混液を用いて溶出し、阻害活性の高い溶出区を濃縮する。
さらに、化合物（Ａ）の抽出第四段階として、この分画を逆相高圧液体カラムクロマトグラフィーに供する。カラムとしては、例えば YMC-Pack ODS-AMカラム (YMC社製)等を使用することができる。移動層としては、含水有機溶媒、例えばメタノール：水：酢酸混液を用い、阻害活性の高い溶出区を得る。これを濃縮して上記化合物（Ａ）をシラップ状にて得る。
【００２０】
次に、前記化合物（Ｂ）を分離する方法について説明する。化合物（Ｂ）の抽出第一段階として、水可溶部を濃縮した後、エタノール等の有機溶媒を用いて適当な有機溶媒濃度になるように調製する。
得られた濃縮液を、低温、好ましくは−２５℃程度で１〜４日間、好ましくは２日間静置する。沈殿が生じた場合は、濾紙やガラスフィルター等を用いる固−液分離手段により当該沈殿を除去する。
【００２１】
上記化合物（Ｂ）の抽出第二段階として、濃縮液を活性炭あるいはダイヤイオンHP-20（三菱化学(株)製）を担体に用いた吸着カラムクロマトグラフィーに供する。吸着物質を含水有機溶剤、例えば含水メタノール溶液を用いて溶出し、目的とする阻害活性を有する溶出区を濃縮する。
次に、化合物（Ｂ）の抽出第三段階として、この分画をゲル濾過クロマトグラフィー、例えばセファデックスLH-20 (Amersham Pharmacia Biotech社製)に供して、酢酸エチル等の含水有機溶媒を用いて溶出し、阻害活性の高い分画を得る。
【００２２】
また、化合物（Ｂ）の抽出第四段階として、この分画を逆相系中圧液体カラムクロマトグラフィーに供する。カラムとしては、例えば Lpbar LiChroprep RP-18 (Merck社製)等を使用することができる。含水有機溶媒、例えばメタノール：水：酢酸混液等を用いて溶出して、抗酸化活性の高い分画を得る。
さらに、化合物（Ｂ）の抽出第五段階として、この分画を濃縮し、逆相高圧液体カラムクロマトグラフィーに供する。このとき用いるカラムとしては、例えばInertsil ODS カラム（GL Science社製）等を使用することができる。移動層として、含水有機溶媒、例えばメタノール：水：酢酸混液を用いて阻害活性の高い溶出区を得る。これを濃縮し前記化合物（Ｂ）をシラップ状にて得る。
【００２３】
請求項３記載の本発明に係るα−グルコシダーゼ阻害剤は、プロパン−１,２,３−トリオール誘導体である化合物（Ａ）および／または化合物（Ｂ）からなることを特徴とする。
α−グルコシダーゼ阻害剤において、有効成分である上記化合物の含有量は、使用目的などにより異なるが、天然物に由来する成分であるため、副作用の心配がなく、厳しく定める必要はない。
α−グルコシダーゼ阻害剤は、経口的に投与されるが、所望により医薬用担体とともに製剤化し、液剤、錠剤、カプセル剤、顆粒剤、散剤などの形態で用いることができる。その場合、賦形剤、滑沢剤、結合剤、崩壊剤、溶剤、懸濁化剤、安定化剤、着色料、甘味剤等の常用成分を適宜添加することができる。
【００２４】
その他、上記化合物の適量を食品や飲料に添加して用いることもできる。食品としては、例えばパン類、麺類、菓子・スナック類、乳製品などがあり、飲料としては、炭酸飲料、果実飲料、乳酸菌飲料などがある。
【００２５】
【実施例】
以下に実施例を示し，本発明を具体的に説明するが、本発明はこれらによって限定されるものではない。
【００２６】
実施例１ ヒソップ葉部からの化合物（Ａ）の抽出、分離
ヒソップ乾燥葉（１００ｇ）を含水メタノール溶液（メタノール：水＝７：３）を用いて抽出した。抽出液を濾紙を用い濾過して得た濾液を減圧下、約１．５リットルになるまで濃縮した。得られた濃緑色シラップを有機溶媒（酢酸エチル）可溶部と水可溶部に分配した。α−グルコシダーゼ阻害活性は両可溶部に観察された。
【００２７】
得られた有機溶媒（酢酸エチル）可溶部を濃縮し、濃黒色シラップを得た。次いで、このシラップをダイヤイオンHP-20（三菱化学(株)製）を担体に用いたカラムクロマトグラフィーに供し、水，含水メタノール溶液（メタノール：水＝１：１および＝７：３）およびメタノールを用いて溶出した。阻害活性の確認された含水メタノール溶液（メタノール：水＝１：１）溶出区を濃縮してシラップを得た。阻害活性の確認は、下記の文献記載の方法により実施した。すなわち、市販のラット腸アセトンパウダーよりα−グルコシダーゼ活性を有する粗酵素溶液を作り、これに基質としてシュクロースを加え、被検試料を反応溶液に加えて反応させ、反応後の反応溶液中のグルコース量を測定することにより行った。
【００２８】
Nishioka, T．;Kawabata, J.;Aoyama,Y. Baicalein, an β-glucosidase inhibitor from Scutellaria baicalensis. J. Nat. Prod.,(1989),61, p.1413-1415
Toda, M., Kawabata, J., and Kasai, T., Inhibitory effects of ellagi- and gallotannins on rat intestinalα-glucosidase complexes .Biosci.. Biotechnol. Biochem., 65, 542-547 (2001)
【００２９】
得られたシラップをシリカゲル（Wakogel C-200, 和光純薬製）を担体に用いたカラムクロマトグラフィーに供した。カラムはCHCl₃ 並びにMeOH:CHCl₃=５：９５, MeOH:CHCl₃＝５：９５,MeOH:CHCl₃＝１０：９０,MeOH:CHCl₃＝３０：７０,MeOH:CHCl₃＝４０：６０の５種類の溶媒で順次溶出した。阻害活性の確認されたMeOH: CHCl₃＝1：9， 3：7溶出区を減圧濃縮しシラップを得た。
得られたシラップをセファデッスクLH-20 (Amersham Pharmacia Biotech製)を用いたカラムクロマトグラフィーに供した。有機溶媒混液（MeOH：CHCl₃=７：３）を用いて溶出し、活性の確認された溶出区を濃縮し、シラップを得た。
【００３０】
得られたシラップをLpbar LiChroprep RP-18 (Merk社製)カラムを用いた中圧液体カラムクロマトグラフィーに供した。メタノール：水：酢酸＝５０：５０：０．１の溶液を用いてカラムを溶出し、活性の確認された溶出区を濃縮してシラップを得た。
次に、得られたシラップを高圧液体カラムクロマトグラフィー（HPLC）に供した。使用したカラムはYMC-Pack ODS-AMカラム(YMC)、UV吸収(210nm)、移動層にメタノール：水：酢酸＝８０：２０：０．１溶液を用いた。阻害活性の確認された溶出区を濃縮したところ、化合物（Ａ）がシラップ状で９０ｍｇ得られた。
【００３１】
以下に、得られた化合物（Ａ）の化学的諸性質を記載した。
化合物（Ａ）：(1-O-β-D-6'-O-cinnamoylglucopyranosyl-3-(3”,5”-dimethoxy-4”-hydroxyphenyl)propane-1, 2, 3-triol,９０ｍｇ):
[α]_D ²⁵ 31.5 ^o (c= 1.0, MeOH);
FDMS m/z (rel. int.): 536 [M]⁺ (100);
¹H-NMR (270 MHz, CD₃OD):δ 7.58 (1H, d, J= 16.1 Hz), 7.49 (2H, m), 7.33 (3H, complex), 6.62 (2H, s), 6.43 (1H, d, J= 16.1 Hz), 4.59 (1H, d, J= 6.5 Hz), 4.46 (1H, dd, J= 13.0, 2.1 Hz), 4.22 (2H, complex), 3.73 (6H, s), 3.73-3.59 (3H, complex), 3.56-3.24 (4H, complex);
¹³C-NMR (67.5 MHz, CD₃OD):δ 168.2, 148.8, 146.4, 135.7, 135.5, 133.5, 131.4, 129.9, 129.2, 118.4, 105.1, 104.9, 77.7, 75.9, 75.3, 75.2, 75.1, 72.0, 71.8, 64.9, 56.7.
【００３２】
実施例２ ヒソップ葉部からの化合物（Ｂ）の抽出、分離
実施例１で得られた粗抽出液の水可溶部についてさらに精製を進めた。
得られた水可溶部を約５００ｍｌまで濃縮し、この溶液に４倍量のエタノールを加え、８０％エタノール溶液になるように調製した。
得られた溶液を−２５℃の条件で２日間静置した。この工程により沈殿が生じたので、濾紙を用いて該沈殿を除去して濾液を得、これを濃縮した。
濃縮後、得られたシラップをダイヤイオンHP-20（三菱化学(株)製）を担体に用いたカラムクロマトグラフィーに供した。水，含水メタノール溶液（メタノール：水＝７：３）を用いて溶出した。活性の確認された含水メタノール溶液（メタノール：水＝７：３）溶出区を濃縮し、シラップを得た。
【００３３】
次いで、得られたシラップをセファデッスクLH-20 (Amersham Pharmacia Biotech製)を用いたカラムクロマトグラフィーに供した。含水メタノール溶液（メタノール：水＝７：３）を用いて溶出し、阻害活性の確認された溶出区を濃縮してシラップを得た。
得られたシラップをLpbar LiChroprep RP-18 (Merk社製)を用いた中圧液体カラムクロマトグラフィーに供した。メタノール：水：酢酸＝５０：５０：０．１の溶液を用いカラムを溶出した。活性の確認された溶出区を濃縮しシラップを得た。
得られたシラップを高圧液体カラムクロマトグラフィー（HPLC）に供した。使用したカラムはInertsil ODS カラム（GL Science社製）、UV吸収(210nm)、移動層にメタノール：水：酢酸＝８０：２０：０．１溶液を用いた。活性の確認された溶出区を濃縮したところ、化合物（Ｂ）がシラップ状で５６５ｍｇ得られた。
【００３４】
以下に、得られた化合物（Ｂ）の化学的諸性質を記載した。
化合物（Ｂ）：[α]_D ²⁵ 17.7 ^o (c= 0.5, MeOH);
FABMS (positive, matrix: glycerol) m/z (rel. int.): 429 [M+Na]⁺ (100);
FABMS (negative, matrix: TEA) m/z (rel. int.): 405 [M-H]^- (10.8);
¹H-NMR (270 MHz, CD₃OD):δ 6.62 (2H, s), 4.54 (1H, d, J= 6.2 Hz), 4.15 (1H, d, J= 7.6 Hz), 3.77 (6H, s), 3.73 (2H, m), 3.63 (1H, m), 3.55 (1H, dd, J= 11.6, 5.4 Hz), 3.47 (1H, dd, J= 10.0, 3.5 Hz), 3.3-3.1 (4H, complex);
¹³C-NMR (67.5 MHz, CD₃OD):δ 148.9, 135.9, 133.6, 105.3, 104.6, 78.0, 77.9, 76.0, 75.5, 75.2, 72.0, 71.7, 62.8, 56.9.
【００３５】
実施例３ 化合物（Ａ）および化合物（Ｂ）のα−グルコシダーゼ阻害活性の測定
ラット腸アセトンパウダー(Sigma Aldrich Japan 社製)を用いて阻害活性試験に必要な粗酵素溶液を調製した。α−グルコシダーゼ阻害活性の測定方法は、西岡らの文献(Nishioka, T.; Kawabata, J.; Aoyama, Y, Baicalein, an β-glucosidase inhibitor from Scutellaria baicalensis. J. Nat. Prod.，(1998), 61,p.1413-1415)に準じて実施した。すなわち、化合物（Ａ）および化合物（Ｂ）のそれぞれを３×１０^-3 Ｍ、１×１０^-3 Ｍおよび３×１０^-4Ｍの濃度に調整し、Positive controlとして1-deoxynojirimycin（和光純薬製）を用い、各化合物の阻害活性を評価した。その結果を図１に示した。なお、1-deoxynojirimycinの１×１０^-8 Ｍおよび３×１０^-7 Ｍでの阻害活性は、それぞれ２１％と５８％である。
したがって、化合物（Ａ）および化合物（Ｂ）は、３×１０^-3 Ｍの濃度でコントロールの３×１０^-7 Ｍと同等な活性を有することが分かる。
【００３６】
実施例４ ヒソップ葉部抽出画分におけるグルコース吸収抑制作用
本発明に係るヒソップ葉部抽出画分のグルコース吸収に対する作用を反転腸管法を用いて試験した。
動物はWistar/ST系雄性ラット（SPF，約３００ｇ）を使用した。エーテル麻酔下において小腸を摘出し、冷却したKRB（Krebs-ringer bicarbonate solusion）中にて反転腸管を作製した。すなわち、トライツ靭帯から下部の小腸約２５ｃｍをKRB中にて約２ｃｍ断片とし、一方の断端を縫合糸で結紮した後、腸管を反転（粘膜側と奨膜側を反転）し、腸管内に１０ｍＭグルコースを含むKRBを満たして袋状の反転腸管を作製した。
【００３７】
次に、ヒソップ葉部抽出画分（終濃度１．０，０．５および０．１ｍｇ／ｍｌ）を含むKRB液中に浸漬し、３７℃で１０分間の前処置の後、最終濃度５ｍＭマルトースあるいは１０ｍＭグルコースとなるように添加し、３７℃で１時間の反応を行った。
反応終了後、反転腸管外液（粘膜側）および反転腸管内液（奨膜側）のグルコース濃度を測定（グルコースB-testワコー，和光製）した。なお、実験中は混合ガス（９５％酸素および５％二酸化炭素含有）を通気しながら行った。
【００３８】
結果を図２および３に示した。腸管外液にマルトース（５ｍＭ）を添加し、１時間の反応を行うと、対照において腸管内グルコース濃度は約２５０ｍｇ／ｄｌを示し、有意なグルコース濃度の上昇が認められた。これに対し、ヒソップ葉部抽出画分は０．５および１ｍｇ／ｍｌの濃度で腸管内グルコース濃度の上昇を完全に抑制した（図２）。
一方、腸管外液にグルコース（１０ｍＭ）を添加した場合、グルコース濃度上昇に対する抑制効果は認められなかった（図３）。
すなわち、化合物（Ａ）および（Ｂ）を含むヒソップ葉部抽出画分は、マルトースから単糖であるグルコースへの分解を抑制（グルコシダーゼ阻害作用）し、グルコース濃度の上昇を抑制することが明らかとなった。
【００３９】
実施例５ 糖負荷マウスにおけるヒソップ葉部抽出画分の血糖値上昇抑制作用
本発明に係るヒソップ抽出画分の食後血糖値上昇抑制効果をショ糖負荷試験により検討を行った。
動物はICR系雄性マウス（SPF，5週齢）を使用し、２４時間の絶食後、尾静脈採血による空腹時血糖値を測定した。その後、ヒソップ葉部抽出画分（１００ｍｇ／ｋｇまたは３００ｍｇ／ｋｇ）を強制経口投与し、２０分後にショ糖（１ｇ／ｋｇ）を同様に経口投与した。
【００４０】
以下、投与３０，６０，９０および１２０分後に尾静脈採血を行って血糖値を測定した。なお、血糖値はメディセーフ血糖測定システム（テルモ）を用いて測定した。
図４にショ糖負荷マウスの血糖値上昇の経時的変化を示した。対照において、ショ糖負荷３０分後に血糖値は約２１０ｍｇ／ｄｌを示し、その後血糖値は徐々に減少して２時間後には空腹時血糖値とほぼ同じ値に回復した。これに対し、ヒソップ抽出画分の投与群は、３０分後の値を有意に抑制した。
すなわち、化合物（Ａ）および（Ｂ）を含むヒソップ葉部抽出画分は、ショ糖負荷による血糖値の上昇を抑制し、糖尿病や食後血糖値の上昇が気になるヒトにとって有用であることが示唆される。なお、図中の*はP＜0.05を、**はP＜0.01を表している。
【００４１】
【発明の効果】
本発明により、プロパン−１,２,３−トリオール誘導体、化合物ＡおよびＢが提供される。
この化合物は、シソ科植物であるヒソップから抽出することによって得ることができる。当該プロパン−１,２,３−トリオール誘導体は、α-グルコシターゼ阻害活性効果を有しており、食品や飲料などに配合することにより、肥満を抑制したり、糖尿病、虚血性心疾患、高血圧症、高脂血症等の疾患の予防を図ることが期待される。しかも、この化合物は天然物由来であるので、副作用の心配がなく、安全性に優れている。
【図面の簡単な説明】
【図１】 本発明に係る化合物ＡおよびＢのα-グルコシターゼ阻害活性の測定結果を示す図である。
【図２】 腸管内におけるヒソップ抽出画分のマルトースに対するα-グルコシターゼ阻害作用を示す図である。
【図３】 腸管内におけるヒソップ抽出画分のグルコースに対するα-グルコシターゼ阻害作用を示す図である。
【図４】 ショ糖負荷マウスにおけるヒソップ抽出画分の血糖値上昇抑制効果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a propane-1,2,3-triol derivative, a method for producing the same, and an α-glucosidase inhibitor containing the same.
[0002]
[Prior art]
The onset of diabetes is closely related to lifestyle habits, and one of the causes includes obesity and hyperglycemia due to overeating. Obesity is estimated to be the cause of many diseases other than diabetes, such as ischemic heart disease, hypertension, and hyperlipidemia. One method for preventing diabetes and preventing and eliminating obesity is to suppress a rapid increase in blood glucose level and to suppress the amount of absorbed energy by ingesting a substance that inhibits the activity of α-glucosidase, which is a digestive enzyme.
Nojirimycin, acarbose and voglibose are known as typical α-glucosidase inhibitory activators. However, since these have a high inhibitory activity in a very small amount, the dosage is required to be strict and is desirably used under the observation of a doctor. In addition, it often causes side effects such as abdominal distension, increased paralysis, loose stool, and diarrhea, which is a safety problem.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a substance having an α-glucosidase inhibitory action that is easy to handle and has no safety concerns, and contributes to the prevention and elimination of the above diseases. Therefore, the present inventors have conducted extensive studies to search for a natural product-derived α-glucosidase inhibitory active substance, and found that a component in a specific plant is useful. That is, in addition to investigating that the component extracted from the above-ground part of Hyssopus (Hyssopus officinalis) used as a spice in plants belonging to the family Lamiaceae has an α-glucosidase inhibitory action, the active ingredient is separated. Succeeded in doing. In addition, it has been found that when foods and beverages containing this substance are ingested, the increase in blood glucose level becomes slow, and the amount of absorbed energy is reduced to improve obesity and to prevent diabetes. The present invention has been completed based on these findings.
[0004]
[Means for Solving the Problems]
The present invention according to claim 1, a propane-1,2,3-triol derivative represented by the following chemical formula (1).
[0005]
[Chemical 6]
(2S, 3S) 1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (1 )

[0006]
According to the present invention, the organic solvent-soluble part is extracted with ethyl acetate and water after extraction with a hydrous methanol solution, a hydrous ethanol solution, or a hydrous butanol solution from the above-ground part of a Hymenoptera plant Hyssopus officinalis. It is a method for producing a propane-1,2,3-triol derivative represented by the following chemical formula ( 1 ) or ( 2 ) , characterized by being distributed to a water-soluble part .
[0007]
[Chemical 7]
(2S, 3S) 1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (1 )

[0008]
[Chemical 8]
(2S, 3S) 1- O- β-D-glucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (2)

[0009]
The present invention according to claim 3 is an α-glucosidase inhibitor comprising one or both of propane-1,2,3-triol derivatives represented by the following chemical formula ( 1 ) or ( 2 ) .
[0010]
[Chemical 9]
(2S, 3S) 1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (1 )

[0011]
Embedded image
(2S, 3S) 1- O -β -D-glucopyranosyl-3- (3 '', 5 '' - dimethoxy-4 '' - hydroxyphenyl) propane-1,2,3-triol (2)

[0012]
The present invention according to claim 4 is a food or beverage characterized by adding the propane-1 , 2 , 3-triol derivative according to claim 1 .
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The novel compound propane-1,2,3-triol derivative according to the present invention is represented by the above chemical formulas (1) and (2). Hereinafter, the former is referred to as compound (A), and the latter is referred to as compound (B).
[0014]
Compound (A) and compound (B) according to the present invention can be obtained from Hyssop above-ground part. Examples of varieties of hyssop (Japanese name: willow mint) that can be used in the present invention include hyssop, white hyssop, pink hyssop, but are not limited to these varieties. Further, examples of the above-ground part include leaves, stems, buds and the like, but the use of the leaf part is suitable for processing the active ingredient such as powdering.
[0015]
As a method for extracting a target substance from the above-mentioned plant, for example, a method exemplified below is possible, but is not limited thereto. Further, depending on the purpose of use and the concentration used, the crudely extracted fraction at each fractionation stage can be used.
[0016]
As a rough extraction process, raw materials such as dried hyssop are extracted using a water-containing organic solvent such as acetone, ethanol, butanol, acetonitrile, and tetrahydrofuran. The extraction temperature may be room temperature, and the extraction time is about 1 week to 10 days in the case of dried hyssop leaves.
[0017]
After completion of the extraction operation, the extract is separated into a filtrate and a residue by solid-liquid separation means using a filter paper, a glass filter or the like. The filtrate is then concentrated under reduced pressure. The obtained concentrated liquid is distributed into an organic solvent soluble part and a water soluble part. That is, the organic solvent soluble part and the water soluble part are distributed using an organic solvent such as ethyl acetate and water. The α-glucosidase inhibitory activity is observed in both soluble parts, and the compound (A) is obtained from the organic solvent-soluble part and the compound (B) is obtained from the water-soluble part. In addition, the process distributed to this organic-solvent soluble part and water soluble part can be skipped, and can be used for this invention with the form of the mixture containing both compounds.
[0018]
The method for separating the compound (A) will be described. As the first stage of extraction, the organic solvent soluble part was concentrated, and this was used as a carrier using activated carbon or Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation). Subject to adsorption column chromatography. Next, elution is performed using water, a water-containing methanol solution, and methanol, and the water-containing methanol solution elution zone having inhibitory activity is concentrated.
Then, this silica gel (wakogelc-200, manufactured by Wako Pure Chemical) was subjected to carrier column chromatography organic solvent mixtures, for example CHCl ₃ and MeOH: eluting with a mixture of CHCl _3. The eluate where the target inhibitory activity is confirmed is reduced in pressure and concentrated.
[0019]
As a second stage of extraction of the above compound (A), this fraction is subjected to gel filtration chromatography, for example, Sephadex LH-20 (manufactured by Amersham Pharmacia Biotech), and eluted with an organic solvent, with high inhibitory activity. Concentrate the ward.
Subsequently, this fraction is subjected to reverse phase medium pressure liquid column chromatography as the third stage of extraction of compound (A). As the column, for example, Lpbar LiChroprep RP-18 (manufactured by Merck) or the like can be used. Elution is performed using a water-containing organic solvent such as methanol: water: acetic acid as the moving layer, and the elution zone having high inhibitory activity is concentrated.
Further, this fraction is subjected to reversed-phase high-pressure liquid column chromatography as the fourth stage of extraction of compound (A). As the column, for example, a YMC-Pack ODS-AM column (manufactured by YMC) or the like can be used. As the moving layer, a water-containing organic solvent such as methanol: water: acetic acid mixture is used to obtain an elution zone with high inhibitory activity. This is concentrated to obtain the compound (A) in the form of syrup.
[0020]
Next, a method for separating the compound (B) will be described. As the first stage of extraction of the compound (B), the water-soluble part is concentrated and then prepared to an appropriate organic solvent concentration using an organic solvent such as ethanol.
The obtained concentrated liquid is allowed to stand at a low temperature, preferably about −25 ° C. for 1 to 4 days, preferably 2 days. When precipitation occurs, the precipitate is removed by solid-liquid separation means using a filter paper or a glass filter.
[0021]
As a second stage of extraction of the compound (B), the concentrated solution is subjected to adsorption column chromatography using activated carbon or Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) as a carrier. The adsorbed substance is eluted with a water-containing organic solvent, for example, a water-containing methanol solution, and the elution zone having the target inhibitory activity is concentrated.
Next, as a third stage of extraction of compound (B), this fraction is subjected to gel filtration chromatography, for example, Sephadex LH-20 (manufactured by Amersham Pharmacia Biotech), and using a water-containing organic solvent such as ethyl acetate. Elute to obtain a fraction with high inhibitory activity.
[0022]
In addition, as a fourth stage of extraction of compound (B), this fraction is subjected to reverse phase medium pressure liquid column chromatography. As the column, for example, Lpbar LiChroprep RP-18 (manufactured by Merck) or the like can be used. Elution with a water-containing organic solvent such as methanol: water: acetic acid mixture to obtain a fraction with high antioxidant activity.
Further, as a fifth stage of extraction of compound (B), this fraction is concentrated and subjected to reverse phase high pressure liquid column chromatography. As a column used at this time, for example, an Inertsil ODS column (manufactured by GL Science) or the like can be used. As the moving layer, a water-containing organic solvent such as methanol: water: acetic acid mixture is used to obtain an elution zone having high inhibitory activity. This is concentrated to obtain the compound (B) in the form of syrup.
[0023]
The α-glucosidase inhibitor according to the present invention described in claim 3 is characterized by comprising a compound (A) and / or a compound (B) which is a propane-1,2,3-triol derivative.
In the α-glucosidase inhibitor, the content of the above-mentioned compound, which is an active ingredient, varies depending on the purpose of use, but since it is a component derived from a natural product, there is no concern about side effects and it is not necessary to strictly determine it.
The α-glucosidase inhibitor is administered orally, but if desired, it can be formulated with a pharmaceutical carrier and used in the form of a solution, tablet, capsule, granule, powder or the like. In that case, conventional components such as excipients, lubricants, binders, disintegrants, solvents, suspending agents, stabilizers, colorants, sweeteners and the like can be added as appropriate.
[0024]
In addition, an appropriate amount of the above compound can be added to foods and beverages. Examples of food include breads, noodles, confectionery and snacks, and dairy products. Examples of beverages include carbonated drinks, fruit drinks, and lactic acid bacteria drinks.
[0025]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.
[0026]
Example 1 Extraction and Separation of Compound (A) from Hyssop Leaf Part Hyssop dry leaf (100 g) was extracted using a water-containing methanol solution (methanol: water = 7: 3). The filtrate obtained by filtering the extract using filter paper was concentrated under reduced pressure to about 1.5 liters. The obtained dark green syrup was distributed into an organic solvent (ethyl acetate) soluble part and a water soluble part. α-Glucosidase inhibitory activity was observed in both soluble regions.
[0027]
The obtained organic solvent (ethyl acetate) soluble part was concentrated to obtain dark black syrup. Next, the syrup was subjected to column chromatography using Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) as a carrier, and water, a water-containing methanol solution (methanol: water = 1: 1 and = 7: 3), and methanol Was used to elute. The hydrated methanol solution (methanol: water = 1: 1) elution zone in which the inhibitory activity was confirmed was concentrated to obtain syrup. The inhibitory activity was confirmed by the method described in the following literature. That is, a crude enzyme solution having α-glucosidase activity is prepared from commercially available rat intestinal acetone powder, sucrose is added as a substrate to this, and a test sample is added to the reaction solution for reaction, and glucose in the reaction solution after the reaction is reacted. This was done by measuring the amount.
[0028]
Nishioka, T. ; Kawabata, J .; Aoyama, Y. Baicalein, an β-glucosidase inhibitor from Scutellaria baicalensis. J. Nat. Prod., (1989), 61 , p.1413-1415
Toda, M., Kawabata, J., and Kasai, T., Inhibitory effects of ellagi- and gallotannins on rat intestinalα-glucosidase complexes .Biosci .. Biotechnol. Biochem., 65 , 542-547 (2001)
[0029]
The obtained syrup was subjected to column chromatography using silica gel (Wakogel C-200, manufactured by Wako Pure Chemical Industries) as a carrier. The columns are CHCl _{3 and} MeOH: CHCl ₃ = 5: 95, MeOH: CHCl ₃ = 5: 95, MeOH: CHCl ₃ = 10: 90, MeOH: CHCl ₃ = 30: 70, MeOH: CHCl ₃ = 40: 60 Elution was performed sequentially with 5 types of solvents. The MeOH: CHCl ₃ = 1: 9, 3: 7 elution zone in which the inhibitory activity was confirmed was concentrated under reduced pressure to obtain syrup.
The obtained syrup was subjected to column chromatography using Sephadex LH-20 (manufactured by Amersham Pharmacia Biotech). Organic solvent mixture _{(MeOH: CHCl 3 = 7:} 3) and eluted with, and concentrated confirmed elution fraction of activity, to obtain a syrup.
[0030]
The obtained syrup was subjected to medium pressure liquid column chromatography using an Lpbar LiChroprep RP-18 (Merk) column. The column was eluted with a solution of methanol: water: acetic acid = 50: 50: 0.1, and the eluate with confirmed activity was concentrated to obtain syrup.
Next, the obtained syrup was subjected to high pressure liquid column chromatography (HPLC). The column used was a YMC-Pack ODS-AM column (YMC), UV absorption (210 nm), and methanol: water: acetic acid = 80: 20: 0.1 solution was used for the moving bed. When the elution zone in which the inhibitory activity was confirmed was concentrated, 90 mg of compound (A) was obtained in the form of syrup.
[0031]
The chemical properties of the obtained compound (A) are described below.
Compound (A): (1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 ", 5" -dimethoxy-4 "-hydroxyphenyl) propane-1, 2, 3-triol, 90mg):
[α] _D ²⁵ 31.5 ^o ( c = 1.0, MeOH);
FDMS m / z (rel. Int.): 536 [M] ⁺ (100);
¹ H-NMR (270 MHz, CD ₃ OD): δ 7.58 (1H, d, J = 16.1 Hz), 7.49 (2H, m), 7.33 (3H, complex), 6.62 (2H, s), 6.43 (1H , d, J = 16.1 Hz), 4.59 (1H, d, J = 6.5 Hz), 4.46 (1H, dd, J = 13.0, 2.1 Hz), 4.22 (2H, complex), 3.73 (6H, s), 3.73 -3.59 (3H, complex), 3.56-3.24 (4H, complex);
¹³ C-NMR (67.5 MHz, CD ₃ OD): δ 168.2, 148.8, 146.4, 135.7, 135.5, 133.5, 131.4, 129.9, 129.2, 118.4, 105.1, 104.9, 77.7, 75.9, 75.3, 75.2, 75.1, 72.0, 71.8, 64.9, 56.7.
[0032]
Example 2 Extraction and Separation of Compound (B) from Hyssop Leaf Part The water-soluble part of the crude extract obtained in Example 1 was further purified.
The obtained water-soluble part was concentrated to about 500 ml, and 4-fold amount of ethanol was added to this solution to prepare an 80% ethanol solution.
The obtained solution was allowed to stand at −25 ° C. for 2 days. Since precipitation occurred in this step, the precipitate was removed using filter paper to obtain a filtrate, which was concentrated.
After concentration, the obtained syrup was subjected to column chromatography using Diaion HP-20 (Mitsubishi Chemical Corporation) as a carrier. Elution was performed using water and a water-containing methanol solution (methanol: water = 7: 3). A water-containing methanol solution with confirmed activity (methanol: water = 7: 3) was concentrated to obtain syrup.
[0033]
Subsequently, the obtained syrup was subjected to column chromatography using Sephadex LH-20 (manufactured by Amersham Pharmacia Biotech). Elution was carried out using a hydrous methanol solution (methanol: water = 7: 3), and the eluate where the inhibitory activity was confirmed was concentrated to obtain syrup.
The obtained syrup was subjected to medium pressure liquid column chromatography using Lpbar LiChroprep RP-18 (manufactured by Merk). The column was eluted using a solution of methanol: water: acetic acid = 50: 50: 0.1. The eluate with confirmed activity was concentrated to obtain syrup.
The obtained syrup was subjected to high pressure liquid column chromatography (HPLC). The column used was an Inertsil ODS column (GL Science), UV absorption (210 nm), and methanol: water: acetic acid = 80: 20: 0.1 solution was used for the moving bed. When the elution section where the activity was confirmed was concentrated, 565 mg of compound (B) was obtained in the form of syrup.
[0034]
The chemical properties of the obtained compound (B) are described below.
Compound (B): [α] _D ²⁵ 17.7 ^o ( c = 0.5, MeOH);
FABMS (positive, matrix: glycerol) m / z (rel. Int.): 429 [M + Na] ⁺ (100);
(.. Rel int): FABMS (negative, matrix TEA) m / z: 405 [MH] - (10.8);
¹ H-NMR (270 MHz, CD ₃ OD): δ 6.62 (2H, s), 4.54 (1H, d, J = 6.2 Hz), 4.15 (1H, d, J = 7.6 Hz), 3.77 (6H, s ), 3.73 (2H, m), 3.63 (1H, m), 3.55 (1H, dd, J = 11.6, 5.4 Hz), 3.47 (1H, dd, J = 10.0, 3.5 Hz), 3.3-3.1 (4H, complex);
¹³ C-NMR (67.5 MHz, CD ₃ OD): δ 148.9, 135.9, 133.6, 105.3, 104.6, 78.0, 77.9, 76.0, 75.5, 75.2, 72.0, 71.7, 62.8, 56.9.
[0035]
Example 3 Measurement of α-Glucosidase Inhibitory Activity of Compound (A) and Compound (B) A crude enzyme solution required for the inhibitory activity test was prepared using rat intestine acetone powder (manufactured by Sigma Aldrich Japan). The method for measuring α-glucosidase inhibitory activity is described in Nishioka et al. (Nishioka, T .; Kawabata, J .; Aoyama, Y, Baicalein, an β- glucosidase inhibitor from Scutellaria baicalensis . J. Nat. Prod., (1998). , 61 , p.1413-1415). That is, each of the compound (A) and the compound (B) is adjusted to a concentration of 3 × 10 ⁻³ M, 1 × 10 ⁻³ M and 3 × 10 ⁻⁴ M, and 1-deoxynojirimycin (Wako Pure Chemical) is used as a positive control. The inhibitory activity of each compound was evaluated. The results are shown in FIG. The inhibitory activity of 1-deoxynojirimycin at 1 × 10 ⁻⁸ M and 3 × 10 ⁻⁷ M is 21% and 58%, respectively.
Therefore, it can be seen that the compound (A) and the compound (B) have an activity equivalent to that of the control 3 × 10 ⁻⁷ M at a concentration of 3 × 10 ⁻³ M.
[0036]
Example 4 Glucose Absorption Inhibitory Action in Hyssop Leaf Extraction Fractionation of Hyssop leaf extract fraction according to the present invention on glucose absorption was tested using the inverted intestinal tract method.
The animal used was a Wistar / ST male rat (SPF, about 300 g). The small intestine was removed under ether anesthesia, and an inverted intestinal tract was prepared in cooled KRB (Krebs-ringer bicarbonate solusion). That is, about 25 cm of the small intestine below the Tritz ligament is cut into about 2 cm pieces in the KRB, and one of the stumps is ligated with a suture, and then the intestinal tract is inverted (the mucosa side and the scholar membrane side are inverted) A bag-shaped intestinal tract was filled with KRB containing 10 mM glucose.
[0037]
Next, it is immersed in a KRB solution containing Hyssop leaf extract fractions (final concentrations of 1.0, 0.5 and 0.1 mg / ml), and after pretreatment at 37 ° C. for 10 minutes, a final concentration of 5 mM maltose is obtained. Or it added so that it might become 10 mM glucose, and reaction was performed at 37 degreeC for 1 hour.
After completion of the reaction, the glucose concentrations of the inverted intestinal fluid (mucosal side) and the inverted intestinal fluid (scholar membrane side) were measured (glucose B-test Wako, manufactured by Wako). During the experiment, a mixed gas (containing 95% oxygen and 5% carbon dioxide) was aerated.
[0038]
The results are shown in FIGS. When maltose (5 mM) was added to the extraintestinal fluid and reacted for 1 hour, the intestinal glucose concentration in the control was about 250 mg / dl, and a significant increase in glucose concentration was observed. In contrast, the Hyssop leaf extract fraction completely suppressed the increase in intestinal glucose concentration at concentrations of 0.5 and 1 mg / ml (FIG. 2).
On the other hand, when glucose (10 mM) was added to the extra-intestinal fluid, no inhibitory effect on the increase in glucose concentration was observed (FIG. 3).
That is, it is clear that the hyssop leaf extract fraction containing compounds (A) and (B) suppresses the degradation of maltose to monosaccharide glucose (glucosidase inhibitory action) and suppresses the increase in glucose concentration. became.
[0039]
Example 5 Suppressing effect on blood glucose level increase of Hyssop leaf extract fraction in glucose-loaded mice The postprandial blood glucose level increase suppressing effect of Hyssop extract fraction according to the present invention was examined by a sucrose tolerance test.
The animals used were ICR male mice (SPF, 5 weeks old). After fasting for 24 hours, fasting blood glucose levels were measured by blood collection from the tail vein. Thereafter, the hyssop leaf fraction (100 mg / kg or 300 mg / kg) was forcibly administered orally, and 20 minutes later, sucrose (1 g / kg) was orally administered in the same manner.
[0040]
Thereafter, blood was collected from tail veins at 30, 60, 90 and 120 minutes after administration to measure blood glucose levels. The blood glucose level was measured using a Medisafe blood glucose measurement system (Terumo).
FIG. 4 shows the change over time in the increase in blood glucose level of sucrose-loaded mice. In the control, the blood glucose level was about 210 mg / dl after 30 minutes of sucrose loading, and then the blood glucose level gradually decreased and recovered to almost the same value as the fasting blood glucose level after 2 hours. On the other hand, the administration group of the hyssop extract fraction significantly suppressed the value after 30 minutes.
That is, the hyssop leaf extract fraction containing compounds (A) and (B) suppresses an increase in blood glucose level due to sucrose load, and is useful for humans who are concerned about diabetes or an increase in postprandial blood glucose level. It is suggested. In the figure, * represents P <0.05, and ** represents P <0.01.
[0041]
【The invention's effect】
According to the present invention, propane-1,2,3-triol derivatives, compounds A and B are provided.
This compound can be obtained by extracting from Hyssop, a Labiatae plant. The propane-1,2,3-triol derivative has an α-glucosidase inhibitory activity effect, and can be added to foods and beverages to suppress obesity, diabetes, ischemic heart disease, hypertension. It is expected to prevent diseases such as hyperlipidemia. Moreover, since this compound is derived from a natural product, there is no worry about side effects and it is excellent in safety.
[Brief description of the drawings]
FIG. 1 is a graph showing the measurement results of α-glucosidase inhibitory activity of compounds A and B according to the present invention.
FIG. 2 is a view showing an α-glucosidase inhibitory action on maltose of a hyssop extract fraction in the intestinal tract.
FIG. 3 is a diagram showing an α-glucosidase inhibitory action on glucose in a hyssop extract fraction in the intestinal tract.
FIG. 4 is a graph showing the blood glucose level increase inhibitory effect of Hyssop extract fraction in sucrose-loaded mice.

Claims (4)

Propane-1,2,3-triol derivative represented by the following chemical formula (1).
[Chemical 1]
(2S, 3S) 1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (1 )

Extraction from the above-ground part of Hyssopus officinalis with a water-containing methanol solution, a water-containing ethanol solution or a water-containing butanol solution , followed by partitioning into an organic solvent soluble part and a water soluble part using ethyl acetate and water A method for producing a propane-1,2,3-triol derivative represented by the following chemical formula ( 1 ) or ( 2 ) :
[Chemical formula 2]
(2S, 3S) 1- O- β-D-6'- O- cinnamoylglucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (1 )

[Chemical 3]
(2S, 3S) 1- O- β-D-glucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (2)

An α-glucosidase inhibitor comprising one or both of propane-1,2,3-triol derivatives represented by the following chemical formula ( 1 ) or ( 2 ) .
[Formula 4]
(2S, 3S) 1- O -β -D-6'- O -cinnamoylglucopyranosyl-3- (3 '', 5 '' - dimethoxy-4 '' - hydroxyphenyl) propane-1,2,3-triol (1 )

[Chemical formula 5]
(2S, 3S) 1- O- β-D-glucopyranosyl-3- (3 '', 5 ''-dimethoxy-4 ''-hydroxyphenyl) propane-1,2,3-triol (2)

A food or beverage comprising the propane-1 , 2 , 3-triol derivative according to claim 1 added thereto.

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