JP4888750B2 - Extract of saba leaves effective for prevention and treatment of gastric ulcer, its use and process for obtaining berberine from the extract - Google Patents
Extract of saba leaves effective for prevention and treatment of gastric ulcer, its use and process for obtaining berberine from the extract Download PDFInfo
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- JP4888750B2 JP4888750B2 JP2001045047A JP2001045047A JP4888750B2 JP 4888750 B2 JP4888750 B2 JP 4888750B2 JP 2001045047 A JP2001045047 A JP 2001045047A JP 2001045047 A JP2001045047 A JP 2001045047A JP 4888750 B2 JP4888750 B2 JP 4888750B2
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- extract
- helicobacter pylori
- soba
- berberine
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- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- 239000003643 water by type Substances 0.000 description 1
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Description
【０００１】
【発明の属する技術分野】
本発明は胃潰瘍の予防及び治療に効果的な蘇葉抽出物とその用途及びそれからベルベリンを得る工程に関し、より具体的には、胃炎、胃潰瘍の原因菌として知られるヘリコバクターピロリに対して抗菌活性を有する胃潰瘍の予防及び治療に効果的な蘇葉抽出物とその用途及びそれからベルベリンを得る工程に関する。
【０００２】
【従来の技術】
ヘリコバクターピロリは１９８３年ウォレンとマーシャルにより分離された後（Ｌａｎｃｅｔ，１９８３，１，１２７３−１２７５）、胃炎及び十二指腸潰瘍の原因菌と明かされ（Ｊ．Ｉｎｆｅｃｔ．Ｄｉｓ．，１９９０，１６１，６２６−６３３；Ａｍ．Ｊ．Ｍｅｄ．，１９９１，９１，５６６−５７２）、現在は胃癌発病因子の一つと認められ全世界的な関心と研究の対象となっている。ヘリコバクターピロリは胃粘膜上皮細胞間の接合部に棲息するグラム陰性肝菌で、最適ｐＨは７．０〜７．４であり、３０〜３７℃の微好気的条件で生長するが、このような条件が充足されないか又は環境の変化が生ずると、コッコイド（球菌状）形態への変化が観察される。その病原性因子として最も代表的な特徴である強力なウレアーゼの生産能と胃粘膜層に対する付着及び移動を可能にするフラジェラ（鞭毛）などがあり、また、Ｖａｇ Ａ、Ｃａｇ Ａ、リポ多糖類などを含む細胞毒が研究されている。
【０００３】
これまでは、ヘリコバクターピロリによる消化器疾患の予防及び治療は３重化学療法に代表される多様な抗生物質に依存しているが、持続的な使用による耐性菌の出現又は再発性疾病の危険により依然としてその限界が指摘されている。これを克服するための努力としてワクチンの開発のための免疫学的方法又は乳酸菌を用いるアプローチ（Ｊ．Ａｐｐｌ．Ｂａｃｔｅｒｉａｌ．，１９９５，７９，４７５−４７９；Ｊ．Ｃｌｉｎ．Ｍｉｃｒｏｂｉａｌ．，１９８９，２７，２３２８−２３３０）などが試されている。
【０００４】
最近では、多様な天然物素材からヘリコバクターピロリを抑制し得る活性成分を探し出すための努力が持続されている。オオタ（Ｏｈｔａ）などはニンニク抽出物（ｇａｒｌｉｃ ｅｘｔｒａｃｔ）から多様な活性物質を分離報告し（Ａｎｔｉｍｉｃｒｏｂ． Ａｇｅｎｔｓ ａｎｄ Ｃｈｅｍｏｔｈｅｒ．，１９９９，４３，１８１１−１８１２）、マベ（Ｍａｂｅ）などは緑茶内のカテキン類化合物に対し、試験管内でだけでなく生体内水準でヘリコバクターピロリ抑制能を確認したことがある（Ａｎｔｉｍｉｃｒｏｂ．Ａｇｅｎｔｓ ａｎｄ Ｃｈｅｍｏｔｈｅｒ．，１９９９，４３，１７８８−１７９１）。そのほかにも、イブキジャコウソウ（Ｊ．Ａｐｐｌ．Ｂａｃｔｅｒｉａｌ．，１９９６，８０，６６７−６７２）又は多様なフラボノイド（Ａｒｚｎｅｉｍ．−Ｆｏｒｓｃｈ．／Ｄｒｕｇ Ｒｅｓ．，１９９５，４５，６９７−７００）からも強力な活性が報告されている。特に、最近には漢方素材から活性植物を探索するための努力が日本国と韓国を中心に活発に展開されている（Ｊ．Ｔｒａｄ．Ｍｅｄ．，１９９５，１２，１２９−１３６；新薬の臨床，１９９７，４６，４９−５３；Ｂｉｏｌ．Ｐｈａｒｍ．Ｂｕｌｌ．，１９９８，２１，９９０−９９２）。
【０００５】
本発明者らは胃潰瘍の予防と治療に食用と薬用に同時に使用される蘇葉を使用しようとした。蘇葉（Ｐｅｒｉｌｌａ ｆｒｕｔｅｓｃｅｎｓ ｖａｒ．ａｃｕｔａ ＫＵＤＯ）は紫蘇とも言われ、漢方では発汗、止血、鎮咳、風疾（中風）、鎮痛、鎮静、利尿などに使用してきた。蘇葉は紫蘇科（Ｌａｂｉａｔａｅ）に属する１年生草で、花は８〜９月に薄い紫色で咲く。日本国では“シソ”と言われ、香辛料及び食用色素として重要な位置を占めている。蘇葉には主としてアントシアニンが多様に含有されており、ペリロサイド（ｐｅｒｉｌｌｏｓｉｄｅ）などのグルコシド物質が多く報告されている（Ｐｈｙｔｏｃｈｅｍ．，１９９４，３７，５４３−５４６；Ｐｈｙｔｏｃｈｅｍ．，１９９２，３１，３２６５−３２６７）。
【０００６】
そこで、本発明者らは食用素材である蘇葉からヘリコバクターピロリに対する抗菌活性、ウレアーゼ活性などの直接的な除菌効果と胃内定着性、インターロイキン−８生成抑制などの効能を確認し、これに対する有効成分を同定しようとした。また、蘇葉抽出物と有効成分を用いる胃潰瘍予防と治療用食品及び薬品を提供しようとした。
【０００７】
【発明が解決しようとする課題】
本発明の主目的は、食用素材である蘇葉から胃炎及び胃、十二指腸潰瘍の原因菌であるヘリコバクターピロリに対して抗菌活性、ウレアーゼ活性、胃内定着性、インターロイキン−８生成抑制などの効能を確認し、これに対する有効成分を同定して提供することを目的とする。
【０００８】
また、本発明のほかの目的は、蘇葉抽出物と有効成分を用いる胃潰瘍予防及び治療用食品と薬品を提供することである。
【０００９】
【課題を解決するための手段】
本発明者らは昔から漢方で発汗、止血、鎮咳、風疾（中風）、鎮痛、鎮静、利尿などに使用してきながら食用にも使用した蘇葉を熱水抽出して、国内の胃潰瘍患者から分離したヘリコバクターピロリ（Ｈ．ｐｙｌｏｒｉ）ＫＳ ５１菌下部に対する抗菌活性を確認した。この蘇葉抽出物に対してヘリコバクターピロリウレアーゼ活性抑制と胃内定着抑制効果、そしてヘリコバクターピロリによる胃炎発生メカニズムに関与するインターロイキン−８の生成抑制効果を確認した。また、蘇葉からカラムクロマトグラフィ（ＸＡＤ−７，シリカ、セファデックス ＬＨ−２０）と薄層クロマトグラフィを実施して、その有効成分としてベルベリン（Ｂｅｒｂｅｒｉｎｅ）を分離、同定した。
【００１０】
本発明は、ヘリコバクターピロリに対する抗菌活性及びヘリコバクターピロリウレアーゼ活性抑制効果、胃上皮細胞定着性抑制効果、インターロイキン−８生成抑制効果を有するように、生薬素材である蘇葉を熱水抽出し濃縮して製造した熱水抽出物と、前記生薬素材である蘇葉を溶媒抽出し濃縮して製造した溶媒抽出物との中で１種以上を含むことを特徴とする胃潰瘍予防及び治療効果を有する蘇葉抽出物を提供する。
【００１１】
前記熱水抽出物は、前記蘇葉に一定量の水を添加し、高温で一定時間熱水抽出した後、その濾液を減圧濃縮することで製造される。好ましくは、蘇葉に添加される水の量は蘇葉の重量に対しておよそ５〜２０倍添加され、８０〜１２０℃で６〜１５時間熱水抽出され、４０〜７０ｃｍＨｇで減圧濃縮することで製造される。
【００１２】
前記溶媒抽出物は、前記蘇葉に一定量のエタノールを添加し、高温で一定時間抽出した後、その濾液を減圧濃縮することで製造される。好ましくは、エタノールは蘇葉の重量に対しておよそ５〜２０倍添加され、５０〜９０℃で４〜８時間抽出し、４０〜７０ｃｍＨｇで減圧濃縮することで製造される。
【００１３】
前記方法により製造された蘇葉抽出物（熱水抽出液及び／又は溶媒抽出液）を有効成分として含有して発酵乳又は飲料に製造できる。また、前記方法により製造された蘇葉抽出物を有効成分として含有し、これに薬剤学的に許容される及び補助剤を混合し、薬剤学的に許容される方法で剤形化して薬学製剤を製造することができる。
【００１４】
【発明の実施の形態】
蘇葉抽出物（特に、熱水抽出物）の製造
熱水抽出物は蘇葉に一定量の水を添加し、高温で一定時間熱水抽出した後、その濾液を減圧濃縮することで製造される。好ましくは、蘇葉に添加される水の量は蘇葉の重量に対しておよそ５〜２０倍添加され、８０〜１２０℃で６〜１５時間熱水抽出され、４０〜７０ｃｍＨｇで減圧濃縮することで製造される。
【００１５】
本発明の実施例１においては、蘇葉１５０ｇを加熱循環式抽出タンクに入れ、水１３５０ｍｌを加え９５℃で８時間抽出した後、１４９μｍの濾過布で濾過し、４０〜７０ｃｍＨｇで５０℃で減圧濃縮することにより、６５ｂｒｉｘ°の蘇葉濃縮液３７ｇを得た（実施例１参照）。
【００１６】
蘇葉溶媒抽出物の製造
溶媒抽出物は、蘇葉に一定量のエタノールを添加し、高温で一定時間抽出した後、その濾液を減圧濃縮することで製造される。好ましくは、エタノールは蘇葉の重量に対しておよそ５〜２０倍添加され、５０〜９０℃で４〜８時間抽出し、４０〜７０ｃｍＨｇで減圧濃縮することで製造される。
【００１７】
本発明の実施例２においては、蘇葉１ｋｇを加熱循環式抽出タンクに入れ、５０〜７０％のエタノール１０ｋｇを加え５５〜８５℃で４〜１５時間抽出した後、１４９μｍの濾過布で濾過し、４０〜７０ｃｍＨｇで減圧濃縮することにより、固形分が０．１〜２０％である抽出液を得た。これにデキストリンなどの副溶剤を混合して濃縮度１０〜７０ｂｒｉｘ°の蘇葉溶媒抽出物を得た（実施例２参照）。
【００１８】
蘇葉からのベルベリンの分離
蘇葉からベルベリンを分離するためには、蘇葉抽出物に蒸留水（水）と有機溶媒を加えて混合した後、有機溶媒層を除去し、水溶層をＸＡＤ−７カラムクロマトグラフィに入れ、アセトンとメタノールを加えて有機溶媒溶出液を得、前記有機溶媒溶出液からシリカカラムクロマトグラフィより分画液を得、前記分画液から予備ＴＬＣ（ｐｒｅｐａｒａｔｉｖｅ ＴＬＣ）によりベルベリンを分離することができる。
【００１９】
前記有機溶媒の好ましい例は、クロロホルム、ジクロロメタン、ジエチルエーテル及び酢酸エチルである。
【００２０】
多様な天然物が蘇葉の二次代謝産物として報告されたことがあるが、そのなかで抗ヘリコバクターピロリ活性を有する物質は知られていないため、抽出物に存在する種々の成分から抗ヘリコバクターピロリ活性物質をいろいろの分離技法で分離した。蘇葉抽出物に対する有機溶媒分画により非水溶性分画を分離した後、水溶性分画に対してＸＡＤ−７カラムクロマトグラフィを実施して水溶層内に存在する多量の糖成分を除去した。以後、シリカカラムクロマトグラフィと薄層クロマトグラフィ（ＴＬＣ）など分離技法により分離された活性分画から単一の有効成分を得ることができる。各分離段階で得られた分画に対して抑制環法で活性を追跡し、活性物質の分離程度はＴＬＣ及びＨＰＬＣ分析により確認した（実施例３参照）。
【００２１】
ベルベリンの同定
クロマトグラフィにより活性成分が単一物質として分離されることにより、ＥＳＩ−ＭＳ（ｅｌｅｃｔｒｏｓｐｒａｙ ｉｏｎｉｚａｔｉｏｎ ｍａｓｓ ｓｐｅｃｔｒｏｓｃｏｐｙ）とＮＭＲ（ｎｕｃｌｅａｒ ｍａｇｎｅｔｉｃ ｒｅｓｏｎａｎｃｅ）によりその構造を決定して活性物質の構造を確認し、この構造がイソキノリン系列のアルカロイド物質であるベルベリンと同定された。ＥＳＩ−ＭＳ測定で［Ｍ］＋が３３６と観察され、１Ｈ−ＮＭＲと１３Ｃ−ＮＭＲスペクトルで水素と炭素の数を確認して分子式がＣ21Ｈ21ＮＯ3であることが分かった。以下、１Ｈ−１Ｈ ＣＯＳＹ、ＤＥＰＴ、ＨＭＱＣ、ＨＭＢＣなどの２Ｄ−ＮＭＲ実験及びＦＴ−ＩＲスペクトルから物質の構造式を確認し、このような結果がベルベリン標準物質のスペクトルと正確に一致することを確認した（実施例４参照）。
【００２２】
抗菌活性
蘇葉抽出物とその有効成分であるベルベリンがヘリコバクターピロリに対して有する抗菌活性をヘリコバクターピロリの寒天培地と液体培地でそれぞれ確認した。寒天培地を用いる抑制環測定において、蘇葉抽出物は１０ｍｇ／ｍｌ以下の低濃度では抑制環を形成することができなかったが、２０ｍｇ／ｍｌの濃度では直径１８ｍｍの抑制環を形成することが観察された。ベルベリンの場合には、２．０ｍｇ／ｍｌの低濃度でも２５ｍｍの抑制環を形成することにより強力な抗菌活性を表した。前記実験で蘇葉抽出物及びベルベリンが強力な抗菌活性を確認することにより、ヘリコバクターピロリの液体培養を通じて抽出物とベルベリンのヘリコバクターピロリに対する最小阻止濃度（ＭＩＣ）を測定した。二倍希釈法により濃度の異なる素材を液体培地にそれぞれ溶かし、各培地に菌を接種した後、菌の生長抑制を、生菌数、ウレアーゼ活性、菌懸濁度の測定により観察した。三つの測定方法とも濃度依存的に素材が菌の生長を抑制することが観察され、蘇葉抽出物とベルベリンはそれぞれ２５０μｇ／ｍｌと１２．５μｇ／ｍｌが最小阻止濃度であることが測定された（実施例５、６参照）。
【００２３】
ヘリコバクターピロリ胃内定着抑制効果
前記抗菌活性とは別にヘリコバクターピロリの胃表皮細胞定着に対する蘇葉抽出物の抑制効果を観察するため、胃上皮腫瘍細胞であるＡＧＳ細胞培養液にヘリコバクターピロリ接種前と接種後にそれぞれ濃度別に素材を添加して予防と治療の２方法で抑制能を測定した。ヘリコバクターピロリ接種に先立って、素材を投与した予防効果測定群においては、２％の素材濃度で９８％の効果を示し、１％と０．５％濃度でも６０％以上の高い効果を示した。素材を接種後に投与した治療効果測定群においても、２％素材濃度で８０％、１％と０．５％の濃度では４０％以上の効果が観察されたが、予防効果よりは抑制能が低いことが判明した（実施例７参照）。
【００２４】
ヘリコバクターピロリ感染により誘導されるインターロイキン−８生成抑制効果
胃腸及び小腸の上皮細胞がバクテリアに露出されると、サイトカインの生成を誘導することになり、このようなサイトカインの増加はヘリコバクターの感染による炎症反応を起こす原因として知られている。ヘリコバクターピロリの場合、インターロイキン−８が胃上皮細胞に好中球又はマクロファージを活性化させる代表的なサイトカインとして知られている。したがって、感染された組織細胞のインターロイキン−８生成量の減少により素材による感染抑制の効果を確認することができる。ＡＧＳ細胞にヘリコバクターピロリを感染させた後、１％（ｗ／ｖ）濃度の蘇葉抽出物を投与し、インターロイキン−８の生成量を測定した結果、対照群に比べＡＧＳ細胞によるインターロイキン−８の生成量が８５％減少することが観察された（実施例８参照）。
【００２５】
動物実験によるヘリコバクターピロリ感染抑制効果の確認
ヘリコバクターピロリ感染に対する蘇葉抽出物の予防と治療効果を４週齢のＢＡＬＢ／ｃマウスで確認した。その結果、蘇葉熱水抽出物をまず投与してヘリコバクターピロリの感染を予防した場合、ヘリコバクターピロリのウレアーゼ活性測定とウレアーゼ活性測定の結果、それぞれ９２．５％と８７．５％の予防率を示した。
【００２６】
また、ヘリコバクターピロリをまず感染させた後、選択された乳酸菌を投与した治療の側面では、蘇葉熱水抽出物を投与した場合、生菌水とウレアーゼ活性測定の結果、それぞれ８２．５％と８０．０％の治療率を示した。
【００２７】
したがって、ヘリコバクターピロリの感染に対して蘇葉抽出物を使用すると、高い予防と治療効果が得られることを確認した（実施例９参照）。
【００２８】
以下、実施例に基づいて本発明をより詳細に説明する。しかし、つぎの実施例は本発明の範囲を限定するものではなく、本発明の技術的思想の範囲で当業者による通常の変化が可能である。
【００２９】
実施例１
蘇葉の熱水抽出
食用と薬用の両方に使用されている蘇葉から熱水抽出物を製造した。蘇葉１５０ｇを加熱循環式抽出タンクに入れ、水１３５０ｍｌを加えた後、およそ９５℃で８時間抽出して粗抽出液を得た。前記粗抽出液を１４９μｍの濾過布で濾過した後、５５ｃｍＨｇで減圧濃縮して６０ｂｒｉｘ°以上の蘇葉濃縮液３７ｇを得た。
【００３０】
実施例２
蘇葉の溶媒抽出
蘇葉１ｋｇを加熱循環式抽出タンクに入れ、６０％エタノール１０ｋｇを加え６０℃で１０時間抽出した後１４９μｍの濾過布で濾過し、５５ｃｍＨｇで減圧濃縮して固形分１０％の抽出液を得た。これにデキストリンなどの副溶剤を混合して濃縮度が４０ｂｒｉｘ°である生薬濃縮液を製造した。
【００３１】
実施例３
蘇葉からのベルベリン分離
蘇葉に存在する抗ヘリコバクターピロリ活性物質を同定するため、実施例１で製造された蘇葉抽出物に対し、図１に示すように、順次クロマトグラフィ技法を実施して単一の主活性物質を分離した。各分離段階で分けられた分画に対しては抑制環法で活性を確認し、分離の程度をＴＬＣ及びＨＰＬＣ分析で観察した。
【００３２】
１）抑制環方による抗菌活性の確認
活性を確認しようとする分画物を一定濃度でメタノールに溶かした後、０．４５μｍフィルタで濾過した。濾過液２０μｌを滅菌されたディスク（ＢａｃｔｏＣｏｎｃｅｎｔｒａｔｉｏｎ Ｄｉｓｋ， Ｄｉｆｃｏ， Ｕ．Ｓ．Ａ．）に吸収させ、ディスクが乾燥すると、２０μｌを更に滴下した後、溶媒が完全に除去されるまで無菌条件で放置した。培養２４〜４８時間を経たヘリコバクターピロリをブルセラ寒天培地に塗抹し、その上にディスクを置いた後、プレートを１０％ＣＯ2培養器で３７℃で７２時間培養した。分画物の活性はディスク周囲に形成された抑制環の大きさで確認した。
【００３３】
２）分画物の分析
分画物の分析はＴＬＣとＨＰＬＣにより行われた。ＴＬＣプレートはメルク社の製品（ｓｉｌｉｃａ ｇｅｌ ６０ Ｆ２５４， Ｇｅｒｍａｎｙ）を用いた。展開溶媒はＣＨＣｌ3／ＭｅＯＨ／ＡｃＯＨ＝７：１：０．５とトルエン／ＣＨＣｌ3＝１：１の２種を使用し、ヨード蒸気とＵＶ（２５４ｎｍ）で確認した。
【００３４】
ＨＰＬＣ分析に使用されたカラムはウォーターズ社の製品（ｍｉｃｒｏｂｏｎｄａｐａｋ，Ｃ１８，３００×４．６ｍｍ）であり、移動相は水／ＭｅＯＨを１．０ｍｌ／分の流速にして用いた。分析は２８５ｎｍで行い、２０μｌを注入して分析した。
【００３５】
３）有機溶媒による水溶性物質の分画
有機溶媒に対する溶解度と極性の差を用いて蘇葉抽出物内の物質を水溶層と有機溶媒層に分離した。適切な有機溶媒の選定のため、クロロホルム、ジクロロメタン、ジエチルエーテル、酢酸エチルの４種の有機溶媒を比較した結果、クロロホルムとジクロロメタンでは０．９％（ｗ／ｗ）の物質が移動し、残りの有機溶媒では、０．２％以下しか移動しなかった。また、ジクロロメタンがクロロホルムより人体にもっと有害であるので、クロロホルムを利用し、抽出物を２０ｍｇ／ｍｌの濃度で蒸留水に溶かした後、これに同じ容積のクロロホルムを分離ファネル（ｓｅｐａｒａｔｏｒｙ ｆｕｎｎｅｌ）で２回分画して水層から非極性物質を除去した。
【００３６】
４）ＸＡＤ−７カラムクロマトグラフィ
水溶層を濾過紙（Ｗｈａｔｍａｎ Ｎｏ．４）で濾過して沈殿物を除去した後、減圧濃縮し、再び蒸留水を加えて２５ｍｇ／ｍｌ濃度の抽出溶液を製造した。アンバーライトＸＡＤ−７（Ａｍｂｅｒｌｉｔｅ ＸＡＤ−７）（Ｎｏｎｉｏｎｉｃ ｐｏｌｙｍｅｒｉｃ ａｄｓｏｒｂｅｎｔ， Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．）をオープンカラム（ｏｐｅｎ ｃｏｌｕｍｎ）に充填した後、メタノールで十分に洗浄し、再び２倍容積の蒸留水を注いでメタノールを除去した。樹脂に対して１：１４（乾燥抽出物重量／樹脂重量）の抽出物溶液をカラムに滴下した後、標本容積の６０倍の蒸留水でカラムを溶出させて糖類を含む水溶性物質を除去した。標本容積の８０倍のメタノールを再び溶出させて非水溶性物質を得、最後にメタノール容積の半分のアセトンを加えて洗浄した。アセトン分画物とメタノール分画はＴＬＣで確認した結果、同じパターンを示し、抗菌活性も同じ水準であったので、２分画物を合わせた。
【００３７】
各溶離剤（ｅｌｕｅｎｔ ｆｒａｃｔｉｏｎ）を減圧濃縮した後、定量的にヘリコバクターピロリに対する抗菌活性を測定した結果を以下の表１に示した。水溶層では抗菌活性が全く表われなかったし、活性が有機溶媒層に全て移動されたことが確認できた。溶出液から得られた水溶性分画とメタノール分画に対して１．０ｍｌずつを取った後、フェノール−サルファリック法（ｐｈｅｎｏｌ−ｓｕｌｆｕｒｉｃ ｍｅｔｈｏｄ）を実施して２分画内の総当量を定量した。抽出液溶液をＸＡＤ−７により溶出させた場合、水層での糖含量は９７．０ｍｇ／Ｌと表われ、有機溶媒層では２．９ｍｇ／Ｌの糖含量が測定された。すなわち、ＸＡＤ−７カラムを用いて抽出物から９６％以上の糖を除去することができた。
【００３８】
【表１】
５）シリカカラムクロマトグラフィ
ＸＡＤ−７を通過した有機溶媒分画を少量のメタノールに溶かした後、十分な量のセライト（ｃｅｌｉｔｅ）（Ｙａｋｕｒｉ，Ｊａｐａｎ）を注ぎ、減圧濃縮器でメタノールを全て除去して抽出物をセライトに吸着させた。カラムにグラスウールを詰めた後、その上にセライトを表面が水平となるようにパッキングを行った。シリカゲル（Ｃａｔ．Ｎｏ．１．０９３８５，Ｍｅｒｃｋ，Ｇｅｒｍａｎｙ）を高さ８．０ｃｍとなるように注いだ後真空状態で２０分間圧力を加えて確実にパッキングした。減圧下で２００ｍｌのアセトンを注いで不純物を除去した後、アスピレータ（吸引器）を用いて１２時間乾燥させた。蘇葉抽出物が吸着されたセライトをシリカカラム上に充填した後、減圧下でメタノールの比率が０％から５０％まで段階的に増加したクロロホルム／メタノール溶液を順に溶出させた後、チューブに受けた。各分画をＴＬＣで確認した結果、６分画に分けることができ、このなかでＦｒ．５で高活性が観察された。
【００４０】
６）予備ＴＬＣ（Ｐｒｅｐａｒａｔｉｖｅ ＴＬＣ）
活性分画を減圧濃縮した後、ＴＬＣ板（ＴＬＣ ｐｌａｔｅ）（Ｃａｔ．Ｎｏ．，１．０５７４４，Ｓｉｌｉｃａ Ｇｅｌ ６０ Ｆ２５４，０．５ｍｍ，Ｍｅｒｃｋ，Ｇｅｒｍａｎｙ）の出発線上に吸着させ、ＭｅＯＨ／ＣＨＣｌ3／ＡｃＯＨ＝７：２：０．５に展開した。板を２８０ｎｍで観察したとき、Ｒｆの値がそれぞれ０．１１〜０．１５、０．１５〜０．２１、０．２２〜０．３７、０．３７〜０．６４、０．６９〜０．７１である五つのバンドを確認した後、スクレーパで掻いた。掻き出したシリカはアセトンとクロロホルム／ＭｅＯＨ（７：１，ｖ／ｖ）溶液で溶出して、吸着された物質を分離した。抗菌活性を確認した結果、Ｒｆ値が０．１５〜０．２１である分画（Ｆｒ．５−Ｍ）でのみ活性が表われた。
【００４１】
実施例４
ベルベリンの構造分析
クロマトグラフィの実施により活性成分が単一物質として分離されることによりＥＳＩ−ＭＳ（ｅｌｅｃｔｒｏｓｐｒａｙ ｉｏｎｉｚａｔｉｏｎ ｍａｓｓｓｐｅｃｔｒｏｓｃｏｐｙ）とＮＭＲ（ｎｕｃｌｅａｒ ｍａｇｎｅｔｉｃ ｒｅｓｏｎａｎｃｅ）によりその構造を決定して、活性物質が図２の構造を有することを確認し、この構造はイソキノリン系列のアルカロイド物質であるベルベリンであると同定された。ＥＳＩ−ＭＳ測定で［Ｍ］＋が３３６と観察され、１Ｈ−ＮＭＲ（図３、表２）と１３Ｃ−ＮＭＲスペクトル（図４、表３）で水素と炭素の数を確認して、分子式がＣ21Ｈ21ＮＯ3であることが分かった。以下、１Ｈ−１Ｈ ＣＯＳＹ（図５）、ＤＥＰＴ（図６）、ＨＭＱＣ（図７）、ＨＭＢＣ（図８）などの２Ｄ−ＮＭＲ実験及びＦＴ−ＩＲ（図９）スペクトルから物質の構造式が図２のようであることを確認し、このような結果はベルベリン標準物質のスペクトルと正確に一致した。
【００４２】
【表２】
【表３】
実施例５
培地分散（Ｗｅｌｌ ｄｉｆｆｕｓｉｏｎ）方法によるヘリコバクターピロリ抗菌活性比較
前記実施例１及び２から得られた蘇葉抽出物とベルベリンに対してヘリコバクターピロリに対する抗菌活性を培地分散（ｗｅｌｌ ｄｉｆｆｕｓｉｏｎ）法により確認した。まず滅菌されたシリンダをペトリ皿の適当な位置に立てた後、２５ｍｌのブルセラ寒天培地を注いで固めた。予め培養されたヘリコバクターピロリをブルセラブロスに懸濁して吸光度（４５０ｎｍ）を１．２となるようにした。菌懸濁液２００μｌを液体状態のソフト寒天（０．８％）培地に添加し、これをすぐシリンダ板に注いで均等に分散させた。この板を１０％の二酸化炭素インキュベータ（ＣＯ2 ｉｎｃｕｂａｔｏｒ）に入れて培地を固めた後、シリンダをピンセットで除去し、各試料をシリンダにより形成された培地に１７０μｌずつ加えた。この板を３７℃、１０％ＣＯ2の条件で７２時間培養した後、形成されたクリアゾーンの大きさを測定して抗菌効果を比較した。
【００４５】
培地分散法による蘇葉抽出物と蘇葉抽出物とベルベリンのヘリコバクターピロリに対する抗菌活性結果を表４に示した。蘇葉抽出物は２．０ｍｇ／ｍｌと２０．０ｍｇ／ｍｌの２濃度でブルセラブロスに溶かした後、フィルトレーションで除菌して使用し、ベルベリンは２．０ｍｇ／ｍｌの濃度でＤＭＳＯに溶かし除菌して使用した。対照群でＤＭＳＯの抗菌活性の有無を確認した。蘇葉抽出物は２．０ｍｇ／ｍｌの濃度ではクリアゾーンを形成することが全くできなかったが、２０．０ｍｇ／ｍｌの濃度では１８ｍｍ程度のクリアゾーンを形成して高い抗菌活性を表した。その有効成分であるベルベリンの場合は、２．０ｍｇ／ｍｌの濃度でクリアゾーンが２５ｍｍ程度と非常に高い活性を表わした。反面、対照群であるＤＭＳＯはクリアゾーンを全く形成できなかったので、ベルベリン活性に対する溶媒の影響がないことを確認した。
【００４６】
【表４】
実施例６
最小阻止濃度測定によるヘリコバクターピロリ（Ｈ．ｐｙｌｏｒｉ）抗菌活性の比較
前記実施例１及び２から得られた蘇葉抽出物とベルベリンに対してヘリコバクターピロリに対する抗菌活性を最小阻止濃度（ＭＩＣ：Ｍｉｎｉｍａｌ ｉｎｈｉｂｉｔｏｒｙ ｃｏｎｃｅｎｔｒａｔｉｏｎ）測定により確認した。
【００４８】
各試料を２４培地板（ｗｅｌｌ ｐｌａｔｅ）に２倍希釈法で希釈して９００μｌ／培地となるようにした。２４時間培養したヘリコバクターピロリブロス培養物から１．０ｍｌを取り、１０６ｃｆｕ／ｍｌの濃度に希釈し各培地に１００μｌずつ添加した（ｔｏｔａｌ ｖｏｌｕｍｅ，１．０ｍｌ）。板を１０％ＣＯ2条件でシェーキング培養しながら時間別に標本を取り、生菌数、ウレアーゼ活性、細胞懸濁度による最小阻止濃度（ＭＩＣ）を測定した。
【００４９】
１）生菌数の測定による最小阻止濃度（ＭＩＣ）
生菌数の測定は培養３６時間後に板から培地当たり１００μｌを取り１０３倍希釈した後、ブルセラ寒天板に塗抹し、１０％ＣＯ2インキュベータで３７℃、７２時間培養した後、現れた菌の集落を計数した。
【００５０】
２）ウレアーゼ活性測定による最小阻止濃度（ＭＩＣ）
ウレアーゼ活性の測定は培地当たり１００μｌを取り、１．０ｍｌのウレアＲブロスに添加し、これを１０％ＣＯ2インキュベータで６時間培養した後、５６０ｎｍで吸光度を測定した。
【００５１】
３）細胞懸濁度の測定による最小阻止濃度（ＭＩＣ）
細胞懸濁度の測定は、培養完了後、各培地からブロスを全てエッペンドルフチューブに移し、再度培地に１．０ｍｌの蒸留水で洗浄して加えた。チューブを１２，０００×ｇで５分間遠心分離した後、上澄み液を捨て、細胞ペレットを４００μｌの蒸留水に懸濁させた後、２００μｌを取り、９６培地板に移し６６０ｎｍで吸光度を測定した。
【００５２】
ヘリコバクターピロリに対する蘇葉抽出物とベルベリンの抗菌活性を生菌数の測定による最小阻止濃度（ＭＩＣ）結果を表５に示した。蘇葉抽出物の場合、１，０００μｇ／ｍｌ以上の濃度では２．０×１０８ｃｆｕ／ｍｌのヘリコバクターピロリが完全に阻止されることを確認し、ベルベリンの場合は２５μｇ／ｍｌ以上の濃度でヘリコバクターピロリが完全に阻止されることを確認し、１２．５μｇ／ｍｌの濃度でも５０％以上が阻止されることが分かった。
【００５３】
【表５】
△対照群（Ｃｏｎｔｒｏｌ）：２．０×１０８ｃｆｕ／ｍｌ
＋＋：菌数が多くて計数不可
蘇葉抽出物とベルベリンによるヘリコバクターピロリウレアーゼ活性抑制程度による最小阻止濃度（ＭＩＣ）結果を図１０に示した。同図に示すように、各素材のヘリコバクターピロリに対する最小阻止濃度（ＭＩＣ）を決定するため、ヘリコバクターピロリ培養６０時間後、ウレアーゼの活性をＲブロスにより測定した。
【００５５】
図１０に示すように、蘇葉抽出物の場合、ウレアーゼ分析試験により測定された最小阻止濃度（ＭＩＣ）は２５０ｍｇ／ｍｌの濃度と決定され、これは以前の実験結果である１００ｍｇ／ｍｌと違いを表す。このことは、測定時、培養時間（４８ｈｒ ｖｓ．６０ｈｒ）の違いと測定方法（Ｖｉａｂｌｅ ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｖｓ．ｕｒｅａｓｅ ａｓｓａｙ）によって違いを表すことで説明される。ベルベリンの最小阻止濃度（ＭＩＣ）は１２．５μｇ／ｍｌと決定された。ベルベリンに対する溶媒として使用されたＤＭＳＯも高濃度では細胞成長に影響を及ぼすことが分かるが、分画物の活性が依然として存在する低濃度では全く阻害効果が表われないので、分画物の最小阻止濃度（ＭＩＣ）の決定には影響を及ぼさないことが分かる。
【００５６】
そして、細胞懸濁度による最小阻止濃度（ＭＩＣ）の決定は蘇葉抽出物とベルベリンにより培養されて６０時間後に現われた増殖抑制程度を細胞懸濁度により測定したもので、その結果は図１１に示されたとおりである。蘇葉抽出物は０．５ｍｇ／ｍｌの濃度までは完全な抗菌活性を表し、２５０、１２５ｍｇ／ｍｌの濃度ではそれぞれ５０、３０％の阻止率を表わした。この結果は、以前に生菌数の測定により確認された最小阻止濃度（ＭＩＣ）（１００μｇ／ｍｌ）と一致する結果と考えられる。また、ベルベリンの最小阻止濃度（ＭＩＣ）は１２．５μｇ／ｍｌで、ウレアーゼ分析試験での結果と同じに決定された。
【００５７】
実施例７
ヘリコバクターピロリ胃内定着抑制効果
ヘリコバクターピロリの胃内定着抑制効果をつぎのように予防治療の観点で確認した。
【００５８】
Ａ．予防効果の測定
ＡＧＳ細胞（ＡＧＳ ｃｅｌｌ，１０６ｃｆｕ／ｗｅｌｌ）を６−培地板に付着させた後、各素材とＲＰＭＩ １６４０（１０％ ＦＢＳ）培地を入れ３時間インキュベーションを行った。ヘリコバクターピロリを１０７細胞／ウエル濃度で摂取し再び１時間インキュベーションを行った。板シェーカーで３回ウォッシングした後、滅菌蒸留水１．０ｍｌを入れ１時間反応してＡＧＳ細胞をリシス（溶離）させた。細胞を収集して遠心分離した細胞ペレットにウレアＲブロス１．０ｍｌを入れ、３７℃で１時間反応させてＯ．Ｄ（５６０ｎｍ）を測定した。
【００５９】
Ｂ．処理（Ｔｒｅａｔｍｅｎｔ）
ＡＧＳ細胞（１０６細胞／ウエル）を６培地板に付着させた後、ヘリコバクターピロリを１０７ｃｆｕ／ウエル（０．１ｍｌ）接種して３時間インキュベーションを行った。０．２ｍｌの素材と２．７ｍｌのＲＰＭＩ １６４０（１０％ ＦＢＳ）培地を入れ、１時間反応させた。板シェーカーで３回ウォッシングした後、滅菌蒸留水１．０ｍｌを入れ１時間ＡＧＳ細胞をリシス（溶離）させた。細胞を収集して遠心分離したセルペレットにウレアＲブロス１．０ｍｌを入れ３７℃で１時間反応させてＯ．Ｄ（５６０ｎｍ）を測定した。
【００６０】
蘇葉抽出物をそれぞれヘリコバクターピロリ接種前と接種後にＡＧＳ細胞培地に添加し、その付着阻害率を測定した結果を図１２に示した。同図に示すように、０．５％、１％、２％の添加時、濃度の異なる実験群でヘリコバクターピロリ感染以前に蘇葉を投与した群が感染以後に蘇葉を投与した群よりは高い活性を示すことが分かった。特に、予防と治療群とも２％では９８％と８２％の高い阻害効果を示し、その以下の濃度では活性に大きい違いがないことが示された。
【００６１】
実施例８
ヘリコバクターピロリ感染により誘導されるインターロイキン−８生成抑制効果
ヘリコバクターピロリの感染により誘導される胃上皮細胞でのインターロイキン−８の生成量の変化により蘇葉抽出物の感染抑制効果を測定した。
【００６２】
まず寒天培地で培養されたヘリコバクターピロリを１０ｍｌのＰＢＳ緩衝液で洗浄した後、４℃、９，０００ｒｐｍで１０分間遠心分離した。沈殿された細胞を１ｍｌのハンクス液 Ｆ１２培地に懸濁し、懸濁液の少量を取り１：５０に希釈した後、６６０ｎｍで懸濁度を測定して下記のように濃度を実験条件に合わせた。
【００６３】
細胞濃度（細胞集団）＝１６．９５×Ａ６６０×（希釈因子）×１０８／ｍｌヘリコバクターピロリ＝１．０８４×１６．９５×５０×１０８＝９．１８×１０８
ＡＧＳ培養液はＡ、Ｂ、Ｃ、Ｄの４群に分け２回繰り返して処理した。Ａ群はＡＧＳのみを培養した群であり、Ｂ群とＣ群はそれぞれＡＧＳに蘇葉抽出物とヘリコバクターピロリのみをそれぞれ処理した。Ｄ群は培地に溶かした蘇葉抽出物を１％（ｗ／ｖ）となるように添加し、１時間後にヘリコバクターピロリ懸濁液１００μｌを７．５×１０８ｃｆｕ／ｍｌで再び添加した。全群から、培養後０、２、４、６、９時間にそれぞれ培地を取り、４℃、１２，０００ｒｐｍで３分間遠心分離し、その上澄み液を取った後、エリザ法（ＱｕａｎｔｉｋｉｎｅＴＭＨｕｍａｎ ＩＬ−８ Ｉｍｍｕｎｏａｓｓａｙ ｋｉｔ，Ｒ＆Ｄ Ｓｙｓｔｅｍ Ｅｕｒｏｐｅ Ｌｔｄ．）でインターロイキン−８の量を測定した。
【００６４】
Ａ：対照群
Ｂ：蘇葉抽出物処理群
Ｃ：ヘリコバクターピロリ処理群
Ｄ：蘇葉抽出物＋ヘリコバクターピロリ処理群
１）サンプリング
板培地（Ｐｌａｔｅ ｗｅｌｌ）内の培地を収集した後、４℃、１２，０００ｒｐｍで３分間遠心分離した。上澄み液をエペンドルフチューブに移した後、−７０℃で貯蔵した。
【００６５】
２）標準溶液
ＲＤ５Ｐ（５×）２．０ｍｌをＤ．Ｗ．で５倍に希釈した後、５．０ｍｌを取り、ＩＬ−８標準溶液バイアルに添加して２，０００ｐｇ／ｍｌのストック（ｓｔｏｃｋ）を製造した。２倍希釈により８濃度の１００μｌ標準溶液を準備した。
【００６６】
３）分析試験（Ａｓｓａｙ）
分析希釈液（Ａｓｓａｙ ｄｉｌｕｅｎｔ）（ＲＤ１−８）１００μｌを板培地に入れ、５０μｌの標本と標準溶液を添加した。共役（Ｃｏｎｊｕｇａｔｅ）溶液１００μｌを加えた後、常温で２時間半の間シェーキングを行った。板を４００μｌのウォッシング緩衝液で６回洗浄し、これに試薬Ａと試薬Ｂをそれぞれ１：１で混ぜ合わせた基質溶液２００μｌを添加した。板を３０分間定置した後、色変化を観察しつつ４９２ｎｍで吸光度を観察した。
【００６７】
ヘリコバクターピロリが胃粘膜の上皮細胞と相互作用するとき、多様なサイトカインの生成を促進することになり、このなかでもＩＬ−８の分泌が最も著しく現れる。そして、ＩＬ−８は胃炎の直接的な原因となる好中球又はマクロファージまで誘導することになる。したがって、ヘリコバクターピロリ感染により誘導されるＩＬ−８の生成を抑制することが胃炎発生を抑制するための１方法となり得る。
【００６８】
図１３に培養時間による４群でのインターロイキン−８の生成量の変化を示した。ヘリコバクターピロリに感染されていない群（Ａ、Ｂ群）では、９時間経過後にもインターロイキン−８の生成が殆どなかったが、ヘリコバクターピロリ感染群（Ｃ群）では、６時間経過時まで急に増加し、その以後におよそ２，５００ｐｇ／ｍｌ水準でインターロイキン−８の量が維持された。反面、ＡＧＳを蘇葉抽出物とともに１時間培養した後、ヘリコバクターピロリを感染させたとき（Ｄ群）、インターロイキン−８の生成量は著しく減少して、培養９時間後にも３００ｐｇ／ｍｌ程度とＣ群の１５％水準に止まることが現れた。したがって、蘇葉抽出物がヘリコバクターピロリ感染により誘導、生成されるインターロイキン−８の生成量をおよそ８５％程度減少させることにより胃炎の発生率も減少させることを確認することができた。
【００６９】
実施例９
動物実験によるヘリコバクターピロリ感染の予防と治療効果の確認
ヘリコバクターピロリの感染に対する蘇葉抽出物の予防と治療効果をマウスにより確認した。実験に使用されたＢＡＬＢ／ｃマウスはマウスの胃腸管内に存在してウレアーゼ活性を表すヘリコバクターフェリス（Ｈｅｌｉｃｏｂａｃｔｅｒｆｅｌｉｓ）が存在しないものだけを選別して使用した。使用したＢＡＬＢ／ｃマウスは４週齢の雌性で、予防と治療各群当たり４０匹を使用し、陽性、陰性対照群には群当たり２０匹を使用した。マウスは２５℃、５０％湿度、ＳＰＦ条件で飼育しながら実験した。この際に、陰性対照群としては、ヘリコバクターピロリ懸濁液の代わりに７．５％重炭酸ナトリウム緩衝液のみを０．５ｍｌずつ経口投与した１５匹のマウスを使用し、陽性対照群としては、ヘリコバクターピロリを７．５％重炭酸ナトリウム緩衝液に２〜４×１０９ｃｆｕ／ｍｌの濃度に懸濁して経口投与した１５匹のマウスを使用した。
【００７０】
１）予防効果
マウスに蘇葉熱水抽出物を飲用水に１ｍｇ／ｍｌの濃度で混合して一日に１匹当たり１０ｍｌずつ１４日間投与した後、７．５％重炭酸ナトリウム緩衝液に懸濁した２〜４×１０９ｃｆｕ／ｍｌのヘリコバクターピロリ菌液を２日間マウス当たり０．５ｍｌずつ６回にわたり感染させた。感染６週後、各実験群の胃を摘出し、胃組織内のヘリコバクターピロリ生菌数とウレアーゼ活性有無を測定することで、ヘリコバクターピロリ感染に対する予防効果を確認した。
【００７１】
２）治療効果
ヘリコバクターピロリを７．５％重炭酸ナトリウム緩衝液に２〜４×１０９ｃｆｕ／ｍｌの濃度に懸濁し、この菌液を２日間マウスに０．５ｍｌずつ６回にわたり感染させた。ヘリコバクターピロリがマウスの胃内に完全に定着した感染６週後、蘇葉熱水抽出物を飲用水に１ｍｇ／ｍｌの濃度で混合して１匹当たり１日に１０ｍｌずつ３週間投与した。投与後、各実験群の胃を摘出し、胃組織内のヘリコバクターピロリ生菌数とウレアーゼ活性を測定することで、ヘリコバクターピロリ感染に対する治療効果を確認した。
【００７２】
３）胃組織内のヘリコバクターピロリ生菌数とウレアーゼ活性の測定
摘出した胃組織は、胃内固形分を除去した後、迅速に２等分して１／２は１０％馬血清の添加されたブルセラブロスに浸漬させた後、均質化してブルセラ寒天板に分散させておいた。板は湿度の高い１０％ＣＯ2培養器で３７℃で５〜７日間培養した後、現れたコロニーを確認してヘリコバクターピロリの存在有無を確認する。残りの１／２はウレアブロスに浸漬させ、湿度の高い１０％ＣＯ2培養器で３７℃で５日間培養しつつ色の変化を観察してヘリコバクターピロリの存在有無を判断した。
【００７３】
ヘリコバクターピロリの感染に対する蘇葉熱水抽出物の予防と治療効果を下記の表６に示した。陰性対照群の場合、感染６週後にもヘリコバクターピロリは全く感染しておらず、陽性対照群の場合は、感染６週後に１００％ヘリコバクターピロリ感染率を示した。しかし、蘇葉熱水抽出物をまず投与してヘリコバクターピロリの感染を予防した場合、ヘリコバクターピロリのウレアーゼ活性の測定結果とウレアーゼ活性測定の結果、それぞれ９２．５％と８７．５％の予防率を示した。
【００７４】
また、ヘリコバクターピロリをまず感染させた後、選択された乳酸菌を投与した治療の側面において、蘇葉熱水抽出物を投与した場合、生菌数とウレアーゼ活性の測定結果、それぞれ８２．５％と８０．０％の治療率を示した。
【００７５】
したがって、ヘリコバクターピロリの感染に対して蘇葉熱水抽出物を使用すると、高い予防、治療効果が得られることを確認した。
【００７６】
【表６】
実施例１０
熱、ｐＨ及び消化酵素に対する安定性
蘇葉抽出物が実際胃内でヘリコバクターピロリを阻害するためには、胃内の消化酵素及び胃酸による酸性条件で安定性を維持しなければならない。また、発酵乳のような低酸性条件の食品への利用性を高めるためには、製品自体が低酸性条件で素材の活性が長時間維持されなければならない。また、食品又は薬品の製造工程上加わる熱処理条件でもその活性が維持されなければならない。したがって、蘇葉抽出物に対して熱、ｐＨ、消化酵素に対する安定性を確認した。
【００７８】
１）熱安定性
蘇葉抽出物を５ｍｇ／ｍｌの濃度で蒸留水に溶解し五つのキャップチューブ（ｃａｐ ｔｕｂｅ）に移した。熱処理を行わない対照群を除くそれぞれのキャップチューブを６０、８０及び１００℃で１時間ずつ、１２１℃で１５分間処理し、各サンプルを０．４５μｍ濾過膜で除菌した。除菌されたサンプルをディスク分散テスト（ｄｉｓｃ ｄｉｆｆｕｓｉｏｎ ｔｅｓｔ）を用いて抗ヘリコバクターピロリ活性を測定した。
【００７９】
２）ｐＨ安定性
蘇葉抽出物７０ｍｇを、ｐＨをそれぞれ２．０、３．０、４．０、５．０に調製した酢酸ナトリウム緩衝液とｐＨ７．０のＰＢＳ緩衝液６．０ｍｌに溶かした後、酢酸と水酸化ナトリウムにてそれぞれのｐＨを合わせ、最終容積が７．０ｍｌとなるようにした。緩衝溶液を３７℃で２時間反応させた後、各サンプルを酢酸と水酸化ナトリウムでｐＨ７に中和させて容積が１０．０ｍｌとなるようにした。濾過膜にて除菌した後、ディスク分散テストにより活性の変化を測定した。
【００８０】
３）胃液安定性
胃液は４人の成人からそれぞれ食事後３０、６０、９０、１２０分後、そして空腹状態で採取し冷凍させたものを解凍して使用した。胃液４００μｌの入ったテストチューブに１００ｍｇ／ｍｌの濃度で蒸留水に溶かした素材５０μｌを添加した。対照群は胃液の代わりにＰＢＳ緩衝液を使用した。各テストチューブを３７℃の水浴で３０分間反応させた後、５％炭酸ナトリウムでｐＨ７に合わせた（最終容積、５００μｌ）。各サンプルに対する活性変化をディスク分散テストで測定した。
【００８１】
蘇葉抽出物をそれぞれの熱処理条件に置いた場合、図１４に示すように、ヘリコバクターピロリに対する抗菌活性は対照群と比較して、変化がないものとして現れた。特に、１２１℃、１５分の殺菌条件でも活性は全然減少しなかったので、蘇葉の活性は製品の生産工程上の加熱処理条件により影響を受けないことを確認した。
【００８２】
また、ｐＨ安定性の場合、ｐＨ２〜５の条件で蘇葉抽出物の活性をｐＨ７の条件で対照群と比較した結果、図１５に示すように、蘇葉抽出物のヘリコバクターピロリに対する抗菌活性は全てのｐＨ条件で安定した。
【００８３】
胃液に対する蘇葉抽出物の安定性を各胃液サンプルと素材を３０分間反応させた後、ヘリコバクターピロリに対する抗菌活性を確認した。表７に示すように、蘇葉抽出物の抗菌活性は胃液により全然影響を受けないことが分かり、このことは、実際の胃腸環境でも蘇葉抽出物がヘリコバクターピロリの増殖を抑制することができることを意味する。
【００８４】
【表７】
実施例１１
乳酸菌に対する影響
蘇葉抽出物を発酵乳に添加する場合、蘇葉抽出物が有する抗菌活性が乳酸菌の生育には影響を及ぼさず、一方ヘリコバクターピロリにのみ選択的に作用できなければならない。
【００８６】
発酵乳の種菌として使用されるラクトバシルス（乳酸菌）、ストレプトコッカス（連鎖球菌）、ビフィドバクテリウムに対する蘇葉抽出物の影響をつぎのように確認した。まず、ラクトバシルスアシドフィラス、ラクトバシルスカセイ、ストレプトコッカスサーモフィルス、ビフィドバクテリウムロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｌｏｎｇｕｍ）を１２％スキムミルクに接種し３７℃で９時間混合培養した。培養の完了後、それぞれの選択培地を用いて初期生菌数を測定した。蘇葉抽出物は減圧濃縮して６０ｂｒｉｘ°の濃縮液を製造した後、１．０％（ｖ／ｖ）の濃度で乳酸菌培養液に添加した。蘇葉抽出物が添加された乳酸菌培養液を１０日間４℃で冷蔵保管しながら２日間隔で各乳酸菌に対する選択培地を用いて生菌数を測定した。各乳酸菌に対する生菌数の測定条件は表８のようである。
【００８７】
【表８】
乳酸菌培養液の初期生菌数はラクトバシルス５×１０６ｃｆｕ／ｍｌ、ビフィドバクテリウム３×１０６ｃｆｕ／ｍｌ、エス．サームフィリウス３．０×１０９ｃｆｕ／ｍｌであり、６０ｂｒｉｘ°蘇葉抽出物１．０％（ｖ／ｖ）を乳酸菌培養液に添加し１０日間冷蔵保管した後にも、それぞれの乳酸菌数が初期生菌数をそのまま維持していることを確認した。したがって、蘇葉抽出物のヘリコバクターピロリに対する抗菌活性は乳酸菌の影響がなく、ただヘリコバクターピロリに対してのみ選択性を持っていることを確認した。
【００８９】
実施例１２
蘇葉抽出物を用いる発酵乳の製造
脱脂粉乳で無脂乳固形分含有量を８〜２０重量％に調整した原料乳を７２〜７５℃で１５秒間殺菌した。殺菌された原料乳を一定温度に冷却した後、ラクトバシルスカセイＨＹ ２７８２を１０６ｃｆｕ／ｍｌの濃度で接種し、培養液がｐＨ４〜５となるまで３７℃で培養し冷却させた。シロップは果汁濃縮液０．１〜５０重量％、食餌繊維０．１〜２０重量％、ブドウ糖０．５〜３０重量％、オリゴ糖０．１〜１５重量、カルシウム０．００１〜１０重量％、ビタミン０．０００１〜５重量％、蘇葉抽出物０．１〜５重量％などを溶かして製造し殺菌してから冷却した。こうして製造されたシロップを前記培養液と一定割合で混合、撹拌して均質化した後、容器に包装して発酵乳を製造した。製造された発酵乳は官能検査の結果、風味、物性、全体的な味において良好な結果を示した。
【００９０】
実施例１３
蘇葉抽出物を用いる胃潰瘍予防、治療用飲料の製造
蘇葉抽出物１４重量％、ナツメのエキス１．５重量％、当帰（馬芹）濃縮液７重量％に液状果糖７．４重量％を混合し、これに精製水を添加、混合してドリンク用瓶に充填し９０〜９５℃で殺菌した後、室温で冷却して飲料を製造した。
【００９１】
実施例１４
蘇葉抽出物を用いる胃潰瘍予防、治療用生薬製剤の製造
蘇葉抽出物０．０１〜３０重量％に栄養補助成分（ビタミンＢ1、Ｂ2、Ｂ5、Ｂ6、Ｅ及び酢酸エステル、ニコチン酸アミド）、オリゴ糖、５０％エタノールを助成分の５〜１０％となるように添加して高速回転混合器で混合した。前記混合物に滅菌精製水１０重量％を添加、混合し、直径１〜２ｍｍの顆粒状に成形した。前記成形された顆粒は４０〜５０℃の真空乾燥機で乾燥させた後、１２〜１４メッシュを通過させて均一に顆粒を製造した。このように製造された顆粒は適量ずつ押出成形されて錠剤又は粉末となるか、硬質カプセルに充填されて硬質カプセル製品に製造できる。
【００９２】
実施例１５
ベルベリンを用いる胃潰瘍予防、治療用生薬製剤の製造
蘇葉抽出物から得られたベルベリン０．００１〜１０重量％に栄養補助成分（ビタミンＢ1、Ｂ2、Ｂ5、Ｂ6、Ｅ及び酢酸エステル、ニコチン酸アミド）、オリゴ糖、５０％エタノールを助成分の５〜１０％となるように添加して高速回転混合器で混合した。前記混合物に滅菌精製水１０重量％を添加、混合し、直径１〜２ｍｍの顆粒状に成形した。前記成形された顆粒は４０〜５０℃の真空乾燥機で乾燥させた後、１２〜１４メッシュを通過させて均一に顆粒を製造した。この製造された顆粒は適量ずつ押出成形されて錠剤又は粉末となるか、硬質カプセルに充填されて硬質カプセル製品に製造できる。
【００９３】
【発明の効果】
以上説明したように、本発明は胃潰瘍の予防及び治療に効果がある蘇葉抽出物とその用途及びそれからベルベリンを得る工程に関するもので、生薬素材である蘇葉に対しておよそ５〜２０倍重量の水を添加した後、８０〜１２０℃で６〜１５時間熱水抽出した後、その濾液を４０〜７０ｃｍＨｇで減圧濃縮して製造した熱水抽出物と、前記生薬素材添加量に対しておよそ５〜１０倍重量の５０〜７０％のエタノールを添加した後、５０〜９０℃で４〜８時間抽出し、その濾液を４０〜７０ｃｍＨｇで減圧濃縮して製造した溶媒抽出物の１種以上を含むことを特徴として、ヘリコバクターピロリに対する抗菌活性及びヘリコバクターピロリウレアーゼ活性抑制効果、胃上皮細胞定着性抑制効果、インターロイキン−８精製抑制効果を有する。
【００９４】
また、本発明のベルベリンは前記蘇葉抽出物からクロロホルム、ジクロロメタン、ジエチルエーテル、酢酸エチルの有機溶媒を用いて得た水溶層をＸＡＤ−７、シリカカラムクロマトグラフィにより分画し、予備ＴＬＣで分離してなったもので、前記蘇葉抽出物の主要有効成分としてベルベリンを容易に得ることができる効果を有する。
【図面の簡単な説明】
【図１】 蘇葉抽出物からクロマトグラフィによるベルベリンの分離過程を示す図
【図２】 ベルベリンの化学構造を示す図
【図３】 分離された活性物質の１Ｈ−ＮＭＲスペクトルを示す図
【図４】 分離された活性物質の１３Ｃ−ＮＭＲスペクトルを示す図
【図５】 分離された活性物質の１Ｈ−１Ｈ ＣＯＳＹ（２Ｄ ＮＭＲ）スペクトルを示す図
【図６】 分離された活性物質のＤＥＰＴ（２Ｄ ＮＭＲ）スペクトルを示す図
【図７】 分離された活性物質のＨＭＱＣ（２Ｄ ＮＭＲ）スペクトルを示す図
【図８】 分離された活性物質のＨＭＢＣ（２Ｄ ＮＭＲ）スペクトルを示す図
【図９】 分離された活性物質のＦＴ−ＩＲスペクトルを示す図
【図１０】 蘇葉抽出物とベルベリンのヘリコバクターピロリウレアーゼ活性抑制効果を示す図
【図１１】 細胞懸濁度測定による蘇葉抽出物とベルベリンのヘリコバクターピロリ増殖抑制効果の比較を示す図
【図１２】 ヘリコバクターピロリ胃上皮細胞付着に対する蘇葉抽出物の抑制効果を示す図
【図１３】 ヘリコバクターピロリ感染により誘導生成されるＩＬ−８に対する蘇葉抽出物の生成抑制効果を示す図
【図１４】 蘇葉抽出物の熱安定性を示す図
【図１５】 蘇葉抽出物のｐＨ安定性を示す図
【図１６】 ラクトバシルスの生育に対する蘇葉抽出物の影響を示す図
【図１７】 ビフィドバクテリウムの生育に対する蘇葉抽出物の影響を示す図
【図１８】 ストレプトコッカスサーモフィルスの生育に対する蘇葉抽出物の影響を示す図[0001]
BACKGROUND OF THE INVENTION
TECHNICAL FIELD The present invention relates to a soba extract effective for the prevention and treatment of gastric ulcer, its use and a process for obtaining berberine therefrom. More specifically, it has antibacterial activity against Helicobacter pylori known as a causative agent of gastritis and gastric ulcer. The present invention relates to a soy leaf extract effective for the prevention and treatment of gastric ulcer, its use, and a process for obtaining berberine therefrom.
[0002]
[Prior art]
Helicobacter pylori was isolated by Warren and Marshall in 1983 (Lancet, 1983, 1, 1273-1275) and then revealed to be the causative agent of gastritis and duodenal ulcer (J. Infect. Dis., 1990, 161, 626-633) Am. J. Med., 1991, 91, 566-572), currently recognized as one of the factors causing gastric cancer and is the subject of worldwide interest and research. Helicobacter pylori is a Gram-negative hepatobacterium that inhabits the junction between gastric mucosal epithelial cells, and has an optimum pH of 7.0 to 7.4 and grows under microaerobic conditions of 30 to 37 ° C. If the new conditions are not met or if environmental changes occur, a change to a coccoidal morphology is observed. There are flagella (flagella) etc. that enable strong urease production ability and the adhesion and movement to the gastric mucosa layer, which are the most typical features as the pathogenic factor, etc. Also, Vag A, Cag A, lipopolysaccharide, etc. Cytotoxins containing are being studied.
[0003]
Until now, prophylaxis and treatment of gastrointestinal diseases caused by Helicobacter pylori has depended on various antibiotics represented by triple chemotherapy, but it still remains due to the emergence of resistant bacteria or the risk of recurrent disease due to continuous use. The limit is pointed out. In an effort to overcome this, immunological methods for vaccine development or approaches using lactic acid bacteria (J. Appl. Bacterial., 1995, 79, 475-479; J. Clin. Microbial., 1989, 27, 2328-2330) have been tried.
[0004]
Recently, efforts to find an active ingredient capable of suppressing Helicobacter pylori from various natural product materials have been continued. Ota and others have reported and reported various active substances from garlic extract (Antimicrob. Agents and Chemother., 1999, 43, 1811-1812), Mabe and others are catechins in green tea. The compound has been confirmed to inhibit Helicobacter pylori in vivo as well as in vitro (Antimicrob. Agents and Chemother., 1999, 43, 1788-1791). In addition, strong activity from Ibusuki Sou (J. Appl. Bacterial., 1996, 80, 667-672) or various flavonoids (Arzneim.-Forsch./Drug Res., 1995, 45, 697-700). Has been reported. In particular, recently, efforts to search for active plants from Kampo materials have been actively developed mainly in Japan and Korea (J. Trad. Med., 1995, 12, 129-136; 1997, 46, 49-53; Biol. Pharm. Bull., 1998, 21, 990-992).
[0005]
The present inventors tried to use the resuscitation used simultaneously for food and medicine for the prevention and treatment of gastric ulcer. The soba (Perilla frucescens var. Acuta KUDO) is also referred to as shiso and has been used in Chinese medicine for sweating, hemostasis, cough suppression, wind (medium wind), analgesia, sedation, diuresis and the like. Soba is an annual grass belonging to Labiatae, and the flowers bloom in pale purple from August to September. In Japan, it is called “shiso” and occupies an important position as a spice and food coloring. Soba mainly contains a variety of anthocyanins, and many glucoside substances such as periloside have been reported (Phytochem., 1994, 37, 543-546; Phytochem., 1992, 31, 3265-3267). ).
[0006]
Therefore, the present inventors have confirmed the direct antibacterial effect against the Helicobacter pylori, the activity of urease, and the like, the gastric colonization, and the effects such as the suppression of the production of interleukin-8 from the edible material, Soba. An attempt was made to identify the active ingredients for. In addition, the present inventors sought to provide foods and medicines for preventing and treating gastric ulcer using a soya extract and active ingredients.
[0007]
[Problems to be solved by the invention]
The main object of the present invention is to provide antibacterial activity, urease activity, gastric colonization, and inhibition of interleukin-8 production against gastricitis, stomach, and Helicobacter pylori, which is a causative agent of duodenal ulcers It is an object to identify and provide an active ingredient for this.
[0008]
Another object of the present invention is to provide a food and a medicine for preventing and treating gastric ulcer using a soya extract and an active ingredient.
[0009]
[Means for Solving the Problems]
The inventors of the present invention extract hot leaves from traditional Japanese herbal medicines that have been used for food, while sweating, hemostasis, antitussives, wind (medium wind), analgesia, sedation, diuresis, etc. Antibacterial activity against the lower part of the isolated H. pylori KS 51 bacteria was confirmed. It confirmed the inhibitory effect of Helicobacter pyloriurease activity and gastric colonization, and the inhibitory effect of Helicobacter pylori on the production of interleukin-8 involved in the gastritis generation mechanism. Further, column chromatography (XAD-7, silica, Sephadex LH-20) and thin layer chromatography were carried out from Soba, and berberine was separated and identified as its active ingredient.
[0010]
The present invention extracts a herbal medicinal raw material from hot water so as to have antibacterial activity against Helicobacter pylori and Helicobacter pylori urease activity inhibitory effect, gastric epithelial cell anchorage inhibitory effect, and interleukin-8 production inhibitory effect. A hot water extract produced by the above-mentioned method and a solvent extract produced by extracting and concentrating the above-mentioned herbal raw material, the solvent extract, comprising at least one kind, and having an effect of preventing and treating gastric ulcer Provide leaf extract.
[0011]
The hot water extract is produced by adding a certain amount of water to the soba leaf, extracting the hot water at a high temperature for a certain time, and then concentrating the filtrate under reduced pressure. Preferably, the amount of water added to the leaf is about 5 to 20 times the weight of the leaf, extracted with hot water at 80 to 120 ° C. for 6 to 15 hours, and concentrated under reduced pressure at 40 to 70 cmHg. Manufactured by.
[0012]
The solvent extract is produced by adding a certain amount of ethanol to the soba leaf, extracting at a high temperature for a certain time, and then concentrating the filtrate under reduced pressure. Preferably, ethanol is added by approximately 5 to 20 times with respect to the weight of the leaf, extracted at 50 to 90 ° C. for 4 to 8 hours, and concentrated under reduced pressure at 40 to 70 cmHg.
[0013]
It can be produced into fermented milk or beverage by containing as an active ingredient the soba extract (hot water extract and / or solvent extract) produced by the above method. In addition, it contains as an active ingredient the soba extract produced by the above method, mixed with pharmaceutically acceptable and adjuvants, and formulated into a pharmaceutical formulation by a pharmaceutically acceptable method. Can be manufactured.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Manufacture of soba extract (especially hot water extract)
The hot water extract is produced by adding a certain amount of water to soba leaves, extracting the hot water at a high temperature for a certain time, and then concentrating the filtrate under reduced pressure. Preferably, the amount of water added to the leaf is about 5 to 20 times the weight of the leaf, extracted with hot water at 80 to 120 ° C. for 6 to 15 hours, and concentrated under reduced pressure at 40 to 70 cmHg. Manufactured by.
[0015]
In Example 1 of the present invention, 150 g of reed leaves are placed in a heated circulation extraction tank, 1350 ml of water is added and extracted at 95 ° C. for 8 hours, then filtered through a filter cloth of 149 μm, and the pressure is reduced at 50 ° C. at 40 to 70 cmHg. By concentrating, 37 g of a 65-brix ° reef concentrate was obtained (see Example 1).
[0016]
Manufacture of soba solvent extract
The solvent extract is produced by adding a certain amount of ethanol to the soba leaf, extracting at a high temperature for a certain time, and then concentrating the filtrate under reduced pressure. Preferably, ethanol is added by approximately 5 to 20 times with respect to the weight of the leaf, extracted at 50 to 90 ° C. for 4 to 8 hours, and concentrated under reduced pressure at 40 to 70 cmHg.
[0017]
In Example 2 of the present invention, 1 kg of soba leaves are placed in a heated circulation type extraction tank, 10 kg of 50 to 70% ethanol is added and extracted at 55 to 85 ° C. for 4 to 15 hours, and then filtered through a 149 μm filter cloth. The extract with a solid content of 0.1 to 20% was obtained by concentration under reduced pressure at 40 to 70 cmHg. This was mixed with a co-solvent such as dextrin to obtain a soy solvent extract having a concentration of 10 to 70 brix ° (see Example 2).
[0018]
Isolation of berberine from soba
In order to separate berberine from Soba, distilled water (water) and an organic solvent are added to and mixed with the Soba extract, the organic solvent layer is removed, the aqueous layer is put into XAD-7 column chromatography, and acetone is added. And methanol are added to obtain an organic solvent eluate, and a fraction liquid is obtained from the organic solvent eluate by silica column chromatography, and berberine can be separated from the fraction liquid by preparative TLC (preparative TLC).
[0019]
Preferred examples of the organic solvent are chloroform, dichloromethane, diethyl ether and ethyl acetate.
[0020]
A variety of natural products have been reported as secondary metabolites of soba, but there are no known substances with anti-Helicobacter pylori activity. The active substance was separated by various separation techniques. After the water-insoluble fraction was separated by organic solvent fractionation for the Soba extract, XAD-7 column chromatography was performed on the water-soluble fraction to remove a large amount of sugar components present in the aqueous layer. Thereafter, a single active ingredient can be obtained from the active fraction separated by a separation technique such as silica column chromatography and thin layer chromatography (TLC). The activity of the fractions obtained in each separation step was monitored by the inhibition ring method, and the degree of separation of the active substance was confirmed by TLC and HPLC analysis (see Example 3).
[0021]
Identification of berberine
By separating the active component as a single substance by chromatography, the structure of the active substance is confirmed by determining its structure by ESI-MS (electrospray ionization mass spectroscopy) and NMR (nuclear magnetic resonance), and this structure is isoquinoline. It was identified as berberine, a family of alkaloid substances. [M] + was observed to be 336 by ESI-MS measurement, and the number of hydrogen and carbon was confirmed by 1H-NMR and 13C-NMR spectra. twenty one H twenty one NO Three It turns out that. In the following, the structural formula of the substance is confirmed from 2D-NMR experiments such as 1H-1H COSY, DEPT, HMQC, and HMBC and the FT-IR spectrum, and it is confirmed that such a result exactly matches the spectrum of the berberine standard substance. (See Example 4).
[0022]
Antibacterial activity
The antibacterial activity of Soba extract and its active ingredient berberine against Helicobacter pylori was confirmed in Helicobacter pylori agar and liquid medium, respectively. In the suppression ring measurement using an agar medium, the Soyo Extract could not form a suppression ring at a low concentration of 10 mg / ml or less, but it could form a suppression ring with a diameter of 18 mm at a concentration of 20 mg / ml. Observed. In the case of berberine, strong antibacterial activity was exhibited by forming a 25 mm inhibitory ring even at a low concentration of 2.0 mg / ml. By confirming the strong antibacterial activity of the Soba extract and berberine in the experiment, the minimum inhibitory concentration (MIC) of the extract and berberine against Helicobacter pylori was measured through liquid culture of Helicobacter pylori. Materials with different concentrations were dissolved in a liquid medium by the 2-fold dilution method, and each medium was inoculated with bacteria, and then the growth inhibition of the bacteria was observed by measuring the number of viable bacteria, urease activity, and the degree of suspension of the bacteria. In all three measurement methods, it was observed that the material suppressed the growth of the fungus in a concentration-dependent manner, and it was determined that the leaf inhibitory extract and berberine had minimum inhibitory concentrations of 250 μg / ml and 12.5 μg / ml, respectively. (See Examples 5 and 6).
[0023]
Helicobacter pylori suppression effect in gastric colonization
In addition to the antibacterial activity, in order to observe the inhibitory effect of the soy extract on gastric epidermal cell colonization of Helicobacter pylori, materials were added to the AGS cell culture solution, which is gastric epithelial tumor cells, before and after inoculation with Helicobacter pylori. Thus, the suppression ability was measured by two methods of prevention and treatment. Prior to the Helicobacter pylori inoculation, the preventive effect measurement group to which the material was administered showed an effect of 98% at the material concentration of 2%, and a high effect of 60% or more at the 1% and 0.5% concentrations. In the treatment effect measurement group administered after inoculation of the material, the effect of 2% material concentration was 80%, 1% and 0.5% were observed to be 40% or more, but the inhibitory ability was lower than the preventive effect (See Example 7).
[0024]
Inhibition of interleukin-8 production induced by Helicobacter pylori infection
When the gastrointestinal and small intestinal epithelial cells are exposed to bacteria, they induce the production of cytokines, and the increase in such cytokines is known to cause an inflammatory response due to Helicobacter infection. In the case of Helicobacter pylori, interleukin-8 is known as a representative cytokine that activates neutrophils or macrophages in gastric epithelial cells. Therefore, the effect of the infection suppression by a raw material can be confirmed by the reduction | decrease of the production amount of interleukin-8 of the infected tissue cell. After infecting ALS cells with Helicobacter pylori, a 1% (w / v) concentration of soy extract was administered, and the amount of interleukin-8 produced was measured. It was observed that the production of 8 was reduced by 85% (see Example 8).
[0025]
Confirmation of Helicobacter pylori infection suppression effect by animal experiment
The prophylactic and therapeutic effects of the soba extract against Helicobacter pylori infection were confirmed in 4-week-old BALB / c mice. As a result, when the infection with Helicobacter pylori was prevented by first administering the Soba hot water extract, as a result of the measurement of urease activity and urease activity of Helicobacter pylori, the prevention rates were 92.5% and 87.5%, respectively. Indicated.
[0026]
In addition, in the aspect of treatment in which Helicobacter pylori was first infected and then administered with the selected lactic acid bacteria, when the soba hot water extract was administered, the results of the measurement of viable water and urease activity were 82.5% respectively. It showed a treatment rate of 80.0%.
[0027]
Therefore, it was confirmed that high preventive and therapeutic effects can be obtained by using the soba extract against Helicobacter pylori infection (see Example 9).
[0028]
Hereinafter, the present invention will be described in more detail based on examples. However, the following examples do not limit the scope of the present invention, and ordinary changes can be made by those skilled in the art within the scope of the technical idea of the present invention.
[0029]
Example 1
Hot water extraction of Soba
A hot water extract was produced from the soba leaves used both edible and medicinal. 150 g of soba was placed in a heated circulation extraction tank, and 1350 ml of water was added, followed by extraction at approximately 95 ° C. for 8 hours to obtain a crude extract. The crude extract was filtered through a filter cloth of 149 μm, and then concentrated under reduced pressure at 55 cmHg to obtain 37 g of a reed concentrate at 60 brix ° or higher.
[0030]
Example 2
Soyo solvent extraction
1 kg of soba leaves are placed in a heated circulation extraction tank, 10 kg of 60% ethanol is added and extracted at 60 ° C. for 10 hours, filtered through a filter cloth of 149 μm, and concentrated under reduced pressure at 55 cmHg to obtain an extract with a solid content of 10%. . This was mixed with a co-solvent such as dextrin to produce a crude drug concentrate having a concentration of 40 brix °.
[0031]
Example 3
Berberine separation from soba
In order to identify the anti-Helicobacter pylori active substance present in Soba, the single main active substance is obtained by sequentially performing a chromatographic technique on the Soyo extract produced in Example 1 as shown in FIG. separated. For the fractions separated at each separation step, the activity was confirmed by the inhibition ring method, and the degree of separation was observed by TLC and HPLC analysis.
[0032]
1) Confirmation of antibacterial activity by inhibition ring
The fraction to be confirmed for activity was dissolved in methanol at a constant concentration, and then filtered through a 0.45 μm filter. 20 μl of the filtrate was absorbed on a sterilized disc (BactoConcentration Disk, Difco, USA) and when the disc dried, 20 μl was further added dropwise and left under aseptic conditions until the solvent was completely removed. . After 24 to 48 hours of culture, Helicobacter pylori was smeared on Brucella agar medium, and a disk was placed on it. 2 The cells were cultured in an incubator at 37 ° C. for 72 hours. The activity of the fraction was confirmed by the size of the inhibition ring formed around the disk.
[0033]
2) Analysis of fractions
Analysis of the fractions was performed by TLC and HPLC. The TLC plate used was a Merck product (
[0034]
The column used for the HPLC analysis was a Waters product (microbondapak, C18, 300 × 4.6 mm), and the mobile phase was water / MeOH at a flow rate of 1.0 ml / min. Analysis was performed at 285 nm and 20 μl was injected for analysis.
[0035]
3) Fractionation of water-soluble substances with organic solvents
Using the difference in solubility and polarity with respect to organic solvent, the substance in the sorghum extract was separated into an aqueous layer and an organic solvent layer. As a result of comparing four types of organic solvents, chloroform, dichloromethane, diethyl ether, and ethyl acetate, in order to select an appropriate organic solvent, 0.9% (w / w) of the substance moved in chloroform and dichloromethane, and the rest In the organic solvent, only 0.2% or less migrated. In addition, since dichloromethane is more harmful to human body than chloroform, chloroform is used and the extract is dissolved in distilled water at a concentration of 20 mg / ml, and then the same volume of chloroform is separated by 2 in a separation funnel. Fractionation was performed to remove non-polar substances from the aqueous layer.
[0036]
4) XAD-7 column chromatography
The aqueous layer was filtered through a filter paper (Whatman No. 4) to remove precipitates, concentrated under reduced pressure, and distilled water was added again to produce an extraction solution having a concentration of 25 mg / ml. After filling Amberlite XAD-7 (Anonlite polymer adsorbent, Sigma Chemical Co.) into an open column, thoroughly wash with methanol and pour again 2 volumes of distilled water. Methanol was removed. After dropping an extract solution of 1:14 (dry extract weight / resin weight) to the resin, the column was eluted with distilled
[0037]
Table 1 below shows the results of quantitative measurement of antibacterial activity against Helicobacter pylori after concentration of each eluent under reduced pressure. It was confirmed that the antibacterial activity did not appear at all in the aqueous layer, and that all the activity was transferred to the organic solvent layer. After taking 1.0 ml each of the water-soluble fraction and methanol fraction obtained from the eluate, the phenol-sulfuric method (phenol-sulfuric method) is performed to determine the total equivalent amount in the two fractions. did. When the extract solution was eluted with XAD-7, the sugar content in the aqueous layer was 97.0 mg / L, and the sugar content of 2.9 mg / L was measured in the organic solvent layer. That is, 96% or more of sugars could be removed from the extract using an XAD-7 column.
[0038]
[Table 1]
5) Silica column chromatography
After the organic solvent fraction that has passed through XAD-7 is dissolved in a small amount of methanol, a sufficient amount of celite (Yakuri, Japan) is poured, and all the methanol is removed with a vacuum concentrator to extract the extract into celite. Adsorbed. After packing glass wool into the column, Celite was packed thereon so that the surface was horizontal. Silica gel (Cat. No. 1.09385, Merck, Germany) was poured to a height of 8.0 cm and then packed in a vacuum by applying pressure for 20 minutes. After removing impurities by pouring 200 ml of acetone under reduced pressure, it was dried for 12 hours using an aspirator (aspirator). After the celite adsorbed with the soba extract is packed on a silica column, a chloroform / methanol solution in which the methanol ratio is gradually increased from 0% to 50% is eluted in order under reduced pressure, and then received in a tube. It was. As a result of confirming each fraction by TLC, it can be divided into 6 fractions. High activity was observed at 5.
[0040]
6) Preparative TLC
The active fraction was concentrated under reduced pressure and then adsorbed on the starting line of a TLC plate (Cat. No., 1.05744,
[0041]
Example 4
Structural analysis of berberine
When the active ingredient is separated as a single substance by performing chromatography, its structure is determined by ESI-MS (electrospray ionization mass spectroscopy) and NMR (nuclear magnetic resonance), and the active substance has the structure of FIG. This structure was identified and identified as berberine, an isoquinoline series alkaloid substance. [M] + was observed as 336 by ESI-MS measurement, and the number of hydrogen and carbon was confirmed by 1H-NMR (FIG. 3, Table 2) and 13C-NMR spectrum (FIG. 4, Table 3). C twenty one H twenty one NO Three It turns out that. Hereinafter, the structural formula of the substance is shown from 2D-NMR experiments and FT-IR (FIG. 9) spectra such as 1H-1H COSY (FIG. 5), DEPT (FIG. 6), HMQC (FIG. 7), HMBC (FIG. 8), etc. 2 and confirmed that these results were in good agreement with the spectrum of the berberine standard.
[0042]
[Table 2]
[Table 3]
Example 5
Comparison of antibacterial activity of Helicobacter pylori by the medium diffusion method
The antibacterial activity against Helicobacter pylori was confirmed by the well diffusion method against the soya extract obtained from Examples 1 and 2 and berberine. First, the sterilized cylinder was placed at an appropriate position on the Petri dish, and then 25 ml of Brucella agar medium was poured and hardened. Helicobacter pylori cultured in advance was suspended in Brucella broth so that the absorbance (450 nm) was 1.2. 200 μl of the bacterial suspension was added to a liquid soft agar (0.8%) medium, which was immediately poured into a cylinder plate and dispersed evenly. This plate is placed in a 10% carbon dioxide incubator (CO 2 After the medium was solidified in an incubator), the cylinder was removed with tweezers, and each sample was added to the medium formed by the cylinder by 170 μl. This plate is 37 ° C, 10% CO 2 After culturing for 72 hours under the above conditions, the size of the formed clear zone was measured to compare the antibacterial effect.
[0045]
Table 4 shows the antibacterial activity results of the leaf extract, the leaf extract and berberine against Helicobacter pylori by the medium dispersion method. Soybean extract was dissolved in Brucella broth at 2 concentrations of 2.0 mg / ml and 20.0 mg / ml, and then sterilized by filtration. Berberine was dissolved in DMSO at a concentration of 2.0 mg / ml. Dissolved and sterilized before use. The presence or absence of antimicrobial activity of DMSO was confirmed in the control group. The soy extract could not form a clear zone at a concentration of 2.0 mg / ml, but formed a clear zone of about 18 mm at a concentration of 20.0 mg / ml and exhibited high antibacterial activity. In the case of berberine, which is the active ingredient, the clear zone exhibited a very high activity of about 25 mm at a concentration of 2.0 mg / ml. On the other hand, DMSO as a control group could not form a clear zone at all, so that it was confirmed that there was no influence of the solvent on the berberine activity.
[0046]
[Table 4]
Example 6
Comparison of H. pylori antibacterial activity by measuring minimum inhibitory concentration
Antibacterial activity against Helicobacter pylori was confirmed by measuring the minimum inhibitory concentration (MIC) against the soy extract obtained from Examples 1 and 2 and berberine.
[0048]
Each sample was diluted to a volume of 900 μl / medium on a 24 medium plate (well plate) by a 2-fold dilution method. 1.0 ml was taken from the Helicobacter pylori broth culture cultured for 24 hours, diluted to a concentration of 106 cfu / ml, and 100 μl was added to each medium (total volume, 1.0 ml). 10% CO 2 Samples were taken according to time while shaking culture under conditions, and the minimum inhibitory concentration (MIC) based on the viable cell count, urease activity, and cell suspension was measured.
[0049]
1) Minimum inhibitory concentration (MIC) by measuring the number of viable bacteria
The number of viable cells was measured after 36 hours of culturing by taking 100 μl per medium from the plate, diluting it 103 times, and smearing it on a Brucella agar plate. 2 After culturing at 37 ° C. for 72 hours in an incubator, colonies of the bacteria that appeared were counted.
[0050]
2) Minimum inhibitory concentration (MIC) by measuring urease activity
Urease activity is measured by taking 100 μl per medium and adding to 1.0 ml of urea R broth, which is added to 10
[0051]
3) Minimum inhibitory concentration (MIC) by measuring cell suspension
The cell suspension was measured by transferring all broth from each medium to an Eppendorf tube after completion of the culture, and again washing the medium with 1.0 ml of distilled water. After centrifuging the tube at 12,000 × g for 5 minutes, the supernatant was discarded, the cell pellet was suspended in 400 μl of distilled water, 200 μl was taken, transferred to a 96 medium plate, and the absorbance was measured at 660 nm.
[0052]
Table 5 shows the minimum inhibitory concentration (MIC) results obtained by measuring the number of viable bacteria for the antibacterial activity of Soba extract and berberine against Helicobacter pylori. In the case of the soba extract, it was confirmed that 2.0 × 10 8 cfu / ml of Helicobacter pylori was completely blocked at a concentration of 1,000 μg / ml or more. In the case of berberine, Helicobacter pylori at a concentration of 25 μg / ml or more. It was confirmed that 50% or more was blocked even at a concentration of 12.5 μg / ml.
[0053]
[Table 5]
ΔControl: 2.0 × 10 8 cfu / ml
++: The number of bacteria is large and cannot be counted
FIG. 10 shows the minimum inhibitory concentration (MIC) result according to the degree of inhibition of Helicobacter pyloriurease activity by the soba extract and berberine. As shown in the figure, in order to determine the minimum inhibitory concentration (MIC) of each material against Helicobacter pylori, the activity of urease was measured with R broth after 60 hours of Helicobacter pylori culture.
[0055]
As shown in FIG. 10, in the case of the soya extract, the minimum inhibitory concentration (MIC) measured by the urease analysis test was determined to be 250 mg / ml, which is different from the previous experimental result of 100 mg / ml. Represents. This is explained by expressing the difference according to the difference in culture time (48 hr vs. 60 hr) and the measurement method (Viable cell counting vs. urea assay) during measurement. The minimum inhibitory concentration (MIC) of berberine was determined to be 12.5 μg / ml. DMSO, used as a solvent for berberine, can also be seen to affect cell growth at high concentrations, but there is no inhibitory effect at low concentrations where the fraction activity is still present, so minimal inhibition of the fraction It can be seen that the determination of concentration (MIC) has no effect.
[0056]
The determination of the minimum inhibitory concentration (MIC) based on the degree of cell suspension was determined by measuring the degree of growth inhibition after 60 hours after cultivating with the leaf extract and berberine based on the degree of cell suspension. It is as shown in. The soba extract exhibited complete antibacterial activity up to a concentration of 0.5 mg / ml and 50 and 30% inhibition rates at concentrations of 250 and 125 mg / ml, respectively. This result is considered to be consistent with the minimum inhibitory concentration (MIC) (100 μg / ml) previously confirmed by viable counts. The minimum inhibitory concentration (MIC) of berberine was 12.5 μg / ml, which was the same as the result in the urease analysis test.
[0057]
Example 7
Helicobacter pylori suppression effect in gastric colonization
The inhibitory effect of Helicobacter pylori on gastric colonization was confirmed from the viewpoint of preventive treatment as follows.
[0058]
A. Measurement of preventive effect
After attaching AGS cells (AGS cell, 106 cfu / well) to the 6-medium plate, each material and RPMI 1640 (10% FBS) medium were added and incubated for 3 hours. Helicobacter pylori was ingested at a concentration of 107 cells / well and incubated again for 1 hour. After washing three times with a plate shaker, 1.0 ml of sterile distilled water was added and reacted for 1 hour to lyse (elute) AGS cells. Cells were collected and centrifuged, and 1.0 ml of urea R broth was added to the cells and allowed to react at 37 ° C. for 1 hour. D (560 nm) was measured.
[0059]
B. Treatment (Treatment)
After AGS cells (106 cells / well) were attached to 6 medium plates, Helicobacter pylori was inoculated with 107 cfu / well (0.1 ml) and incubated for 3 hours. 0.2 ml of raw material and 2.7 ml of RPMI 1640 (10% FBS) medium were added and allowed to react for 1 hour. After washing three times with a plate shaker, 1.0 ml of sterile distilled water was added and AGS cells were lysed (eluted) for 1 hour. Cells were collected and centrifuged, and 1.0 ml of urea R broth was added to the cell pellet and allowed to react at 37 ° C. for 1 hour. D (560 nm) was measured.
[0060]
FIG. 12 shows the results of adding the Soba extract to the AGS cell medium before and after inoculation with Helicobacter pylori and measuring the adhesion inhibition rate. As shown in the figure, when 0.5%, 1%, and 2% were added, in the experimental groups with different concentrations, the group that administered resuscitation before Helicobacter pylori infection was more effective than the group that received resuscitation after infection. It was found to show high activity. In particular, the prevention and treatment groups showed high inhibitory effects of 98% and 82% at 2%, and it was shown that there was no significant difference in activity at concentrations below that.
[0061]
Example 8
Inhibition of interleukin-8 production induced by Helicobacter pylori infection
The infection inhibitory effect of the soba extract was measured by changing the amount of interleukin-8 produced in gastric epithelial cells induced by Helicobacter pylori infection.
[0062]
First, Helicobacter pylori cultured on an agar medium was washed with 10 ml of PBS buffer, and then centrifuged at 4 ° C. and 9,000 rpm for 10 minutes. The precipitated cells were suspended in 1 ml of Hank's solution F12 medium, a small amount of the suspension was taken and diluted 1:50, and then the degree of suspension was measured at 660 nm and the concentration was adjusted to the experimental conditions as follows. .
[0063]
Cell concentration (cell population) = 16.95 x A660 x (dilution factor) x 108 / ml Helicobacter pylori = 1.084 x 16.95 x 50 x 108 = 9.18 x 108
The AGS culture solution was divided into 4 groups of A, B, C, and D and treated twice. Group A was a group in which only AGS was cultured, and Group B and Group C were each treated with AGS extract only with a soy extract and Helicobacter pylori. In group D, the leaf extract extracted in the medium was added to 1% (w / v), and 1 hour later, 100 μl of Helicobacter pylori suspension was added again at 7.5 × 10 8 cfu / ml. From all groups, the culture medium was taken at 0, 2, 4, 6, and 9 hours after culturing, centrifuged at 4 ° C. and 12,000 rpm for 3 minutes, and the supernatant was collected, followed by the Eliza method (Quantikine ™ Human IL-8). (Immunoassay kit, R & D System Europe Ltd.). The amount of interleukin-8 was measured.
[0064]
A: Control group
B: Soyo extract treatment group
C: Helicobacter pylori treatment group
D: Soyo extract + Helicobacter pylori treatment group
1) Sampling
After collecting the medium in the plate medium, it was centrifuged at 12,000 rpm for 3 minutes at 4 ° C. The supernatant was transferred to an Eppendorf tube and stored at -70 ° C.
[0065]
2) Standard solution
2.0 ml of RD5P (5 ×) W. Then, 5.0 ml was taken and added to an IL-8 standard solution vial to produce a 2,000 pg / ml stock. A 100 μl standard solution of 8 concentrations was prepared by 2-fold dilution.
[0066]
3) Analytical test (Assay)
100 μl of assay diluent (RD1-8) was placed in the plate medium, and 50 μl of sample and standard solution were added. After adding 100 μl of the conjugate solution, the mixture was shaken at room temperature for 2.5 hours. The plate was washed 6 times with 400 μl of washing buffer, and 200 μl of a substrate solution in which reagent A and reagent B were mixed 1: 1 respectively was added thereto. After the plate was placed for 30 minutes, the absorbance was observed at 492 nm while observing the color change.
[0067]
When Helicobacter pylori interacts with epithelial cells of the gastric mucosa, it will promote the production of various cytokines, among which the secretion of IL-8 is most prominent. And IL-8 induces to neutrophils or macrophages which are the direct cause of gastritis. Therefore, suppressing the production of IL-8 induced by Helicobacter pylori infection can be one method for suppressing the occurrence of gastritis.
[0068]
FIG. 13 shows changes in the production amount of interleukin-8 in the four groups depending on the culture time. In the group not infected with Helicobacter pylori (groups A and B), there was almost no production of interleukin-8 even after 9 hours, but in the group infected with Helicobacter pylori (group C), it suddenly reached 6 hours. The amount of interleukin-8 was maintained at approximately 2,500 pg / ml level thereafter. On the other hand, when AGS was cultured for 1 hour with the soy extract and then infected with Helicobacter pylori (group D), the amount of interleukin-8 produced was markedly reduced to about 300 pg / ml even after 9 hours of culture. It appeared to remain at the 15% level of Group C. Therefore, it was confirmed that the incidence of gastritis was reduced by reducing the production amount of interleukin-8 induced and produced by Helicobacter pylori infection by about 85%.
[0069]
Example 9
Confirmation of prevention and treatment effect of Helicobacter pylori infection by animal experiments
The prevention and treatment effect of the soy extract against Helicobacter pylori infection was confirmed by mice. The BALB / c mice used in the experiment were selected and used only in mice that do not have Helicobacterferis, which is present in the gastrointestinal tract of mice and exhibits urease activity. The BALB / c mice used were 4 week old females, 40 per prevention and treatment group were used, and 20 per group for positive and negative control groups. The mice were tested while being reared at 25 ° C., 50% humidity and SPF conditions. At this time, as a negative control group, 15 mice that were orally administered with 0.5% sodium bicarbonate buffer alone in place of Helicobacter pylori suspension were used, and as a positive control group, Fifteen mice orally administered with Helicobacter pylori suspended in 7.5% sodium bicarbonate buffer at a concentration of 2-4 × 10 9 cfu / ml were used.
[0070]
1) Preventive effect
The mouse was mixed with the hot spring extract of soba at a concentration of 1 mg / ml in drinking water and administered 10 ml per mouse for 14 days, then suspended in 7.5% sodium bicarbonate buffer. 4 × 10 9 cfu / ml Helicobacter pylori solution was infected 6 times with 0.5 ml per mouse for 2 days. Six weeks after infection, the stomach of each experimental group was removed, and the preventive effect against Helicobacter pylori infection was confirmed by measuring the number of living Helicobacter pylori and the presence or absence of urease activity in the stomach tissue.
[0071]
2) Therapeutic effect
Helicobacter pylori was suspended in 7.5% sodium bicarbonate buffer at a concentration of 2-4 × 10 9 cfu / ml, and the bacterial solution was infected 6 times with 0.5 ml of mice for 2 days. Six weeks after infection, when Helicobacter pylori was completely established in the stomach of the mouse, the Soba hot water extract was mixed with drinking water at a concentration of 1 mg / ml and administered at 10 ml per mouse for 3 weeks. After administration, the stomach of each experimental group was removed, and the therapeutic effect on Helicobacter pylori infection was confirmed by measuring the number of viable Helicobacter pylori and urease activity in the stomach tissue.
[0072]
3) Measurement of viable Helicobacter pylori and urease activity in stomach tissue
The removed stomach tissue is quickly divided into two parts after removing the solid content in the stomach, and 1/2 is immersed in Brucella broth supplemented with 10% horse serum, then homogenized and dispersed on Brucella agar plates. I was allowed to. The board is 10% CO with high humidity 2 After culturing at 37 ° C. for 5 to 7 days in an incubator, the appearing colonies are confirmed to confirm the presence or absence of Helicobacter pylori. The other half is immersed in urea broth, and the humidity is 10% CO2. 2 While culturing at 37 ° C. for 5 days in an incubator, the color change was observed to determine the presence or absence of Helicobacter pylori.
[0073]
Table 6 below shows the preventive and therapeutic effects of the soba hot water extract against Helicobacter pylori infection. In the case of the negative control group, Helicobacter pylori was not infected at all even 6 weeks after the infection, and in the case of the positive control group, a 100% Helicobacter pylori infection rate was exhibited 6 weeks after the infection. However, when infection with Helicobacter pylori was prevented by first administering the Soba hot water extract, the results of measurement of urease activity and measurement of urease activity of Helicobacter pylori were 92.5% and 87.5%, respectively. showed that.
[0074]
In addition, in the aspect of treatment in which Helicobacter pylori was first infected and then administered with a selected lactic acid bacterium, when the soba hot water extract was administered, the measurement results of viable cell count and urease activity were 82.5%, respectively. It showed a treatment rate of 80.0%.
[0075]
Therefore, it was confirmed that a high preventive and therapeutic effect can be obtained by using the Soba hot water extract against Helicobacter pylori infection.
[0076]
[Table 6]
Example 10
Stability to heat, pH and digestive enzymes
In order for the Soba extract to actually inhibit Helicobacter pylori in the stomach, it must remain stable under acidic conditions due to digestive enzymes and gastric acid in the stomach. Moreover, in order to improve the utilization to the food of low acid conditions like fermented milk, the product itself must maintain the activity of a raw material for a long time in low acid conditions. Moreover, the activity must be maintained even under heat treatment conditions applied in the production process of food or medicine. Therefore, heat, pH, and stability against digestive enzymes were confirmed for the soya extract.
[0078]
1) Thermal stability
The soya extract was dissolved in distilled water at a concentration of 5 mg / ml and transferred to five cap tubes. Each cap tube except the control group not subjected to heat treatment was treated at 60, 80 and 100 ° C. for 1 hour at 121 ° C. for 15 minutes, and each sample was sterilized with a 0.45 μm filter membrane. The sterilized sample was measured for anti-Helicobacter pylori activity using a disc dispersion test.
[0079]
2) pH stability
70 mg of the soy extract was dissolved in sodium acetate buffer adjusted to pH 2.0, 3.0, 4.0, 5.0 and 6.0 ml PBS buffer pH 7.0, and then acetic acid and The pH was adjusted with sodium hydroxide so that the final volume was 7.0 ml. After the buffer solution was reacted at 37 ° C. for 2 hours, each sample was neutralized to
[0080]
3) Gastric fluid stability
The gastric juices were collected from four adults at 30, 60, 90, 120 minutes after meals, and frozen and collected after hunger. 50 μl of a material dissolved in distilled water at a concentration of 100 mg / ml was added to a test tube containing 400 μl of gastric juice. In the control group, PBS buffer was used instead of gastric juice. Each test tube was reacted in a 37 ° C. water bath for 30 minutes and then adjusted to
[0081]
As shown in FIG. 14, when the soya extract was placed under each heat treatment condition, the antibacterial activity against Helicobacter pylori appeared to be unchanged compared to the control group. In particular, since the activity did not decrease at all even under sterilization conditions at 121 ° C. for 15 minutes, it was confirmed that the activity of the leaf was not affected by the heat treatment conditions in the product production process.
[0082]
In addition, in the case of pH stability, as a result of comparing the activity of the soba extract with the control group under the condition of
[0083]
The stability of the soy extract against gastric juice was reacted with each gastric juice sample for 30 minutes, and then the antibacterial activity against Helicobacter pylori was confirmed. As shown in Table 7, it can be seen that the antibacterial activity of the soy extract is not affected at all by the gastric juice, which means that the soy extract can suppress the growth of Helicobacter pylori even in the actual gastrointestinal environment. Means.
[0084]
[Table 7]
Example 11
Effects on lactic acid bacteria
When adding a soba extract to fermented milk, the antibacterial activity of the soba extract must not affect the growth of lactic acid bacteria, but must be able to selectively act only on Helicobacter pylori.
[0086]
The effect of the soba extract on Lactobacillus (lactic acid bacteria), Streptococcus (Streptococcus), and Bifidobacterium used as seeds for fermented milk was confirmed as follows. First, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium longum were inoculated into 12% skim milk and mixed and cultured at 37 ° C. for 9 hours. After completion of the culture, the initial viable cell count was measured using each selective medium. The soy extract was concentrated under reduced pressure to produce a 60-brix ° concentrate, and then added to the lactic acid bacteria culture solution at a concentration of 1.0% (v / v). The number of viable bacteria was measured using a selective medium for each lactic acid bacterium at intervals of 2 days while refrigerated and storing the lactic acid bacterium culture solution to which the soy extract was added at 4 ° C. for 10 days. Table 8 shows the measurement conditions for the number of viable bacteria for each lactic acid bacterium.
[0087]
[Table 8]
The initial viable cell count of the lactic acid bacteria culture solution was
[0089]
Example 12
Manufacture of fermented milk using soba extract
The raw material milk which adjusted skim milk solid content to 8-20 weight% with skim milk powder was sterilized at 72-75 degreeC for 15 second (s). The sterilized raw material milk was cooled to a certain temperature, and then inoculated with Lactobacillus seisei HY2782 at a concentration of 106 cfu / ml, and cultured at 37 ° C. until the culture solution became pH 4-5 and cooled. Syrup is 0.1-50% by weight of juice concentrate, 0.1-20% by weight of dietary fiber, 0.5-30% by weight of glucose, 0.1-15% by weight of oligosaccharides, 0.001-10% by weight of calcium, Vitamin 0.0001 to 5% by weight, soy leaf extract 0.1 to 5% by weight, etc. were dissolved, manufactured, sterilized and then cooled. The syrup produced in this manner was mixed with the culture solution at a certain ratio, stirred and homogenized, and then packaged in a container to produce fermented milk. The produced fermented milk showed good results in sensory tests, flavor, physical properties and overall taste.
[0090]
Example 13
Manufacture of beverages for the prevention and treatment of gastric ulcers using the soba extract
Mix 14% by weight of soba extract, 1.5% by weight of jujube extract, 7% by weight of Toki (Majiro) concentrate and 7.4% by weight of liquid fructose, and add purified water to this. After filling the bottle for drinks and sterilizing at 90-95 degreeC, it cooled at room temperature and manufactured the drink.
[0091]
Example 14
Production of herbal medicines for the prevention and treatment of gastric ulcers using the soba extract
Soy leaf extract 0.01 to 30% by weight, supplemented with nutritional supplements (vitamin B 1 , B 2 , B Five , B 6 , E and acetate ester, nicotinic acid amide), oligosaccharide, and 50% ethanol were added so as to be 5 to 10% of the auxiliary components and mixed with a high-speed rotary mixer. To the mixture, 10% by weight of sterilized purified water was added and mixed to form granules having a diameter of 1 to 2 mm. The formed granule was dried with a vacuum dryer at 40 to 50 ° C. and then passed through 12 to 14 mesh to uniformly produce the granule. The granules thus produced can be extruded into appropriate amounts to form tablets or powders, or filled into hard capsules to produce hard capsule products.
[0092]
Example 15
Production of herbal medicines for the prevention and treatment of gastric ulcer using berberine
Nutritional supplements (vitamin B) from 0.001 to 10% by weight of berberine obtained from the soba extract 1 , B 2 , B Five , B 6 , E and acetate ester, nicotinic acid amide), oligosaccharide, and 50% ethanol were added so as to be 5 to 10% of the auxiliary components and mixed with a high-speed rotary mixer. To the mixture, 10% by weight of sterilized purified water was added and mixed to form granules having a diameter of 1 to 2 mm. The formed granule was dried with a vacuum dryer at 40 to 50 ° C. and then passed through 12 to 14 mesh to uniformly produce the granule. The produced granules can be extruded by appropriate amounts to form tablets or powders, or filled into hard capsules to produce hard capsule products.
[0093]
【Effect of the invention】
As described above, the present invention relates to a soy leaf extract effective for the prevention and treatment of gastric ulcer, its use, and a process for obtaining berberine therefrom. After adding hot water at 80 to 120 ° C. for 6 to 15 hours, the filtrate was concentrated under reduced pressure at 40 to 70 cmHg, and the amount of the crude drug material added was about the same. After adding 5 to 10 times the weight of 50 to 70% ethanol, extract at 50 to 90 ° C. for 4 to 8 hours, and concentrate the filtrate under reduced pressure at 40 to 70 cmHg to obtain one or more solvent extracts. Including the antibacterial activity against Helicobacter pylori and Helicobacter pyloriurease activity inhibitory effect, gastric epithelial cell fixing inhibitory effect, and interleukin-8 purification inhibitory effect.
[0094]
In addition, the berberine of the present invention was obtained by fractionating the aqueous layer obtained from the Soba extract using an organic solvent such as chloroform, dichloromethane, diethyl ether and ethyl acetate by XAD-7 and silica column chromatography, and separating by preparative TLC. Thus, berberine can be easily obtained as a main active ingredient of the soya extract.
[Brief description of the drawings]
FIG. 1 is a diagram showing the separation process of berberine from a soba extract by chromatography.
FIG. 2 shows the chemical structure of berberine
FIG. 3 is a diagram showing a 1H-NMR spectrum of a separated active substance.
FIG. 4 is a diagram showing a 13C-NMR spectrum of a separated active substance.
FIG. 5 shows a 1H-1H COSY (2D NMR) spectrum of the separated active substance.
FIG. 6 shows a DEPT (2D NMR) spectrum of the separated active substance.
FIG. 7 is a diagram showing an HMQC (2D NMR) spectrum of the separated active substance.
FIG. 8 is a diagram showing an HMBC (2D NMR) spectrum of the separated active substance.
FIG. 9 is a diagram showing an FT-IR spectrum of the separated active substance
FIG. 10 is a graph showing the inhibitory effect of Soba extract and berberine on Helicobacter pyloriurease activity.
FIG. 11 is a graph showing a comparison of the inhibitory effect of Soba extract and berberine on Helicobacter pylori growth by measuring cell suspension
FIG. 12 is a graph showing the inhibitory effect of a soy extract on adhesion of Helicobacter pylori gastric epithelial cells.
FIG. 13 is a graph showing the inhibitory effect of the soy extract on IL-8 induced by Helicobacter pylori infection.
FIG. 14 is a diagram showing the thermal stability of a soba extract
FIG. 15 is a graph showing the pH stability of a soba extract.
FIG. 16 is a graph showing the effect of a soy extract on the growth of Lactobacillus
FIG. 17 is a graph showing the effect of a soy extract on the growth of Bifidobacterium.
FIG. 18 is a graph showing the effect of a soy extract on the growth of Streptococcus thermophilus
Claims (2)
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| Application Number | Priority Date | Filing Date | Title |
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| KR2000-13074 | 2000-03-15 | ||
| KR1020000013074A KR100353262B1 (en) | 2000-03-15 | 2000-03-15 | Perilla frutescence extract Method which effects to prevention and treatment of a stomach ulcer and its use and process of obtaining for berberine therefrom |
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| JP2001270835A JP2001270835A (en) | 2001-10-02 |
| JP4888750B2 true JP4888750B2 (en) | 2012-02-29 |
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| KR100512403B1 (en) * | 2002-05-23 | 2005-09-07 | 송도원 | Manufacturing method of beefsteak plant extracted composition |
| KR100506955B1 (en) * | 2002-05-23 | 2005-08-11 | 송도원 | Manufacturing method of sesame extracted composition |
| KR100496146B1 (en) * | 2002-06-12 | 2005-06-20 | 주식회사 뉴로넥스 | Composition comprising the extract of Glycyrrhiza uralensis FISCH., Curcuma longa L. and Perilla frutescens BRITT. for the prevention and treatment of gastritis and gastric ulcer |
| KR100892180B1 (en) * | 2007-04-23 | 2009-04-10 | 강릉원주대학교산학협력단 | Composition comprising powder of tangerine peel or the extract thereof for treating and preventing gastrointestinal disease |
| CN102215852B (en) * | 2008-10-30 | 2014-06-11 | 国立大学法人琉球大学 | Agent having anti-helicobacter pylori activity |
| KR101127675B1 (en) | 2009-09-28 | 2012-03-22 | 영남대학교 산학협력단 | Beefsteak plant extract and beverage using the same |
| KR20140033998A (en) * | 2012-09-11 | 2014-03-19 | 씨제이제일제당 (주) | Composition for preventing or treating a gastrointestinal disorder, comprising saikosaponin a, berberine and licoisoflavone b |
| CN103254189B (en) * | 2013-04-23 | 2015-11-04 | 广西民族大学 | Application berberine hydrochloride molecular imprinting column is separated the method for berberine hydrochloride |
| CA2916972A1 (en) * | 2013-07-18 | 2015-01-22 | Amino Up Chemical Co., Ltd. | A composition having a prebiotic effect |
| KR101650522B1 (en) | 2014-11-03 | 2016-08-25 | 경상북도(농업기술원) | A method for extracting myristicin from the Perilla frutescens |
| CN109187841B (en) * | 2018-08-31 | 2020-11-13 | 成都大学 | Thin-layer identification method of coptidis flos |
| CN112684093B (en) * | 2020-12-08 | 2022-11-25 | 广西中医药大学 | Quality detection method of eleven-prescription medicinal liquor |
| KR102416803B1 (en) * | 2021-12-21 | 2022-07-05 | 주식회사 유담 | Development of fermented vegetable lactic acid bacteria beverage packed with spout pouch for room temperature distribution using batch type sterilization and after-sterilization process |
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| JP3059795B2 (en) * | 1991-08-27 | 2000-07-04 | 鐘紡株式会社 | Mental stability food |
| US5340833A (en) * | 1992-05-01 | 1994-08-23 | Eisai Co., Ltd. | Urokinase inhibitors |
| JP3547782B2 (en) * | 1993-12-28 | 2004-07-28 | ロート製薬株式会社 | Anti-Helicobacter pylori activator |
| JPH08295632A (en) * | 1995-03-02 | 1996-11-12 | Takeda Chem Ind Ltd | Antibacterial agent |
| JPH0987189A (en) * | 1995-09-19 | 1997-03-31 | Ichimaru Pharcos Co Ltd | Antiallergic agent containing isodon japonicus hara, paeonia suffruticosa andrews, perilla frutescens britton var. acuta kudo, and/or arunica montana linne |
| JP3326589B2 (en) * | 1996-10-26 | 2002-09-24 | 祥之 亀山 | Health food made from mulberry leaves, plum meat, umenin, shiso leaves, etc. |
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