JP3749743B2 - Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same - Google Patents
Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same Download PDFInfo
- Publication number
- JP3749743B2 JP3749743B2 JP17457195A JP17457195A JP3749743B2 JP 3749743 B2 JP3749743 B2 JP 3749743B2 JP 17457195 A JP17457195 A JP 17457195A JP 17457195 A JP17457195 A JP 17457195A JP 3749743 B2 JP3749743 B2 JP 3749743B2
- Authority
- JP
- Japan
- Prior art keywords
- activity against
- antibacterial activity
- hemolytic
- present
- throat inflammation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Description
【０００１】
【産業上の利用分野】
本発明は、喉炎症起因菌（溶血性連鎖球菌）に対する抗菌活性および本菌種の産生する溶血毒に対する阻害活性剤並びにそれを含有する口腔用組成物及び飲食品に関する。
【０００２】
【従来の技術】
溶血性連鎖球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ：ストレプトコッカス・ピオゲネス）は、ヒトの口腔内や咽頭などに分布する通性嫌気性グラム陽性球菌である。本菌は、連鎖球菌の中で最も病原性が強く、粘膜では咽頭炎、扁桃炎、中耳炎等を起こし、皮膚では種々の化膿性炎症を引き起こすことが知られており、いずれも咽頭もしくは扁桃腺において増殖する。
【０００３】
本菌の病原性因子として、溶血毒（ストレプトリジンＯ）、ディック毒素、ストレプトキナーゼ、ＤＮＡ分解酵素、ヒアルロニダーゼ等が知られており、咽頭炎、扁桃炎、気管支炎等の呼吸器系疾患の病因的役割を果たしている。
【０００４】
これらの呼吸器系疾患を予防および治療するためには、病巣において本菌類の生育を阻害することや、菌の病原性因子を抑制することが重要であることはいうまでもない。
【０００５】
菌の抑制という観点では、本菌類に対して有効な抗菌性物質の応用が効果的である。従来では、ペニシリン系、セフェム系、セファロスポリン系の抗生物質が用いられていたが、これらは作用が強力である反面、耐性菌の出現、副作用等から日常的な利用に適しているとはいえない。
【０００６】
従って、日常的な利用に適する本菌類に有効な抗菌性物質としては、果実、野菜、その他の日常食用に供されている天然物由来のものが好適である。このようなものの例として従来、カリン果実が注目されている。
【０００７】
特開昭５９−１８７７６９号公報および特開平２−３１２５６５号公報は、それぞれ健康増進目的で、カリンを蜂蜜で抽出した果汁乃至エキスを利用することを開示しているが、対象物は蜂蜜で抽出された物に限られており、しかもその効果については甚だ抽象的である。
【０００８】
また、いずれも本出願人の出願による特開昭６２−６１５３８号公報および特開昭６２−２２８２３０号公報は、カリンの溶剤抽出エキスにＳｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎｓおよびＳｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓに対する抗菌作用があることを開示しているが、ストレプトコッカス・ピオゲネスに対する抗菌活性は明らかにされていない。
【０００９】
さらに、特公昭６３−２６０８３号公報および特開平１−２９０６１９号公報は、天然に広く存在するといわれるオレアノール酸等の五環性酸性トリテルペン化合物に抗う蝕作用、即ちＳ．ｍｕｔａｎｓに対する特異的抗菌作用があることを開示しているが、カリン果実にオレアノール酸他の特定の五環性酸性トリテルペン化合物が含まれていることおよびこれらのものにストレプトコッカス・ピオゲネスに対する抗菌活性があることは開示していない。
【００１０】
【発明が解決しようとする課題】
本発明者等は、天然物の溶媒抽出物を試料とし、ストレプトコッカス・ピオゲネスに対する抗菌活性および本菌の産生する溶血毒（ストレプトリジンＯ）阻害活性を指標としてスクリーニングを行った結果、カリン果実の溶媒抽出物にそれらの効果があることを見出し、本発明を完成させるに至った。
【００１１】
【課題を解決するための手段】
本発明は、カリン果実より得られるトリテルペン誘導体を含有してなり、喉炎症起因菌（溶血性連鎖球菌）に対する抗菌活性および本菌種の産生する溶血毒に対する阻害活性剤並びにそれを含有する口腔用組成物及び飲食品である。本発明は特に、カリン果実を５０〜１００％低級アルコール水溶液、アセトン、酢酸エチルまたはｎ−ヘキサン或いはこれらの２種以上の混合溶媒で抽出したエキスおよびこのエキスより得られるトリテルペン誘導体を含有してなり、喉炎症起因菌（溶血性連鎖球菌）に対する抗菌活性および本菌種の産生する溶血毒に対する阻害活性剤並びにそれを含有する口腔用組成物及び飲食品である。
【００１２】
本発明の活性剤の原料となるカリン（Ｃｈａｅｎｏｍｅｌｅｓ ｓｉｎｅｎｓｉｓ，Ｃｙｄｏｎｉｓ ｓｉｎｅｎｓｉｓ，Ｐｓｅｕｄｏｃｙｄｏｎｉｓ ｓｉｎｅｎｓｉｓ）は、バラ科、中国原産の植物で世界各地で栽培されている落葉の高木であり、その果実は食品素材として用いられる他、漢方で木瓜（モッカ）と呼ばれ、焼酎で薬用酒としたものが鎮咳、去痰の目的で用いられている。しかしながらその有効成分は不明であり、またどのような作用機作により鎮咳、去痰の効果を発現するのかも不明である。
【００１３】
本発明の活性剤は、以下のようにしてカリン果実より抽出製造することができる。
【００１４】
先ず、カリン果実をそのまま或いは乾燥させた後、適当な手段で機械的に粉砕し、これに５０〜１００％低級アルコール水溶液、好ましくはエタノール水溶液、アセトン、酢酸エチルまたはｎ−ヘキサン或いはこれらの２種以上の混合溶媒を充分量適用して抽出操作を行う。これらの溶媒は市販されている通常のものを使用すればよく、また混合溶媒は、これらの２種以上を任意に選択し、任意の混合割合で混合して使用すればよい。抽出操作は、粉砕したカリン果実を、常温で前記溶媒に適当時間浸漬する方法によってもよいが、好ましくは５０〜１００℃の温度で１〜５時間、一層好ましくは約３時間かけて加熱還流する方法で行う。
【００１５】
次に、このようにして得られる抽出液を、減圧濃縮することにより本発明の活性剤が得られる。
【００１６】
このようにして得られた組成物を、さらにカラムクロマトグラフィーに付し、分離精製を行う。用いるカラムクロマトグラフィーは任意であり、吸着クロマトグラフィー、分配クロマトグラフィー、或いは高速液体クロマトグラフィー、薄層クロマトグラフィー等の技法を用いればよい。また、用いる展開液はメタノール等の低級アルコール、これの水溶液、アセトン、酢酸エチルまたはｎ−ヘキサン等でよい。
【００１７】
得られる分離精製物は、トリテルペン誘導体であり、次の構造を有している。
【００１８】
【化９】
【化１０】
【化１１】
【化１２】
本発明の活性剤では、これらの誘導体のいずれか１以上をも有効成分として使用することが可能である。
【００２３】
このようにして得られる溶媒抽出組成物およびトリテルペン誘導体を有効成分とする本発明の活性剤は、広範な用途に用いることができるが、特にその香り、呈味性が優れ、安全性が高いことを考慮すれば、チューインガム、キャンディ、錠菓、含そう剤などに配合して日常的な利用に供することもできる。このような用途に供する場合、本発明の活性剤を、これら口腔用組成物又は飲食品中に０．０１〜１０重量％の割合で含有するように添加すれば好適である。
【００２４】
以下、本発明の実施例、試験例および応用例を挙げて説明する。
【００２５】
【実施例】
実施例１
カリン乾燥果実粉砕物１ｋｇに５０％エタノール５リットルを加え、１００℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮し、溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤３２４ｇを得た。
【００２６】
実施例２
カリン乾燥果実粉砕物１ｋｇに１００％エタノール５リットルを加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮し、溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤７７ｇを得た。
【００２７】
実施例３
カリン乾燥果実粉砕物１ｋｇにアセトン５リットルを加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮し、溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤４５ｇを得た。
【００２８】
実施例４
カリン乾燥果実粉砕物１ｋｇに酢酸エチル５リットルを加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮し、溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤３５ｇを得た。
【００２９】
実施例５
カリン乾燥果実粉砕物１ｋｇにｎ−ヘキサン５リットルを加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより酸味および芳香を有する淡緑色の固形物である本発明製剤５ｇを得た。
【００３０】
実施例６
生食用カリン果実の粉砕物１ｋｇに５０％エタノール３リットルおよびアセトン２リットルの混合溶媒を加え、１００℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤２８０ｇを得た。
【００３１】
実施例７
生食用カリン果実の粉砕物１ｋｇに１００％エタノール３リットルおよび酢酸エチル２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤８２ｇを得た。
【００３２】
実施例８
生食用カリン果実の粉砕物１ｋｇに１００％エタノール３リットルおよびｎ−ヘキサン２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤７２ｇを得た。
【００３３】
実施例９
カリン乾燥果実粉砕物１ｋｇにアセトン３リットルおよび酢酸エチル２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤５８ｇを得た。
【００３４】
実施例１０
カリン乾燥果実粉砕物１ｋｇにアセトン３リットルおよびｎ−ヘキサン２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤４２ｇを得た。
【００３５】
実施例１１
カリン乾燥果実粉砕物１ｋｇに酢酸エチル３リットルおよびｎ−ヘキサン２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤３５ｇを得た。
【００３６】
実施例１２
カリン乾燥果実粉砕物１ｋｇに１００％エタノール１リットル、アセトン１リットル、酢酸エチル１リットルおよびｎ−ヘキサン２リットルの混合溶媒を加え、８０℃の温度で３時間加熱還流抽出を行った。得られた抽出液を濾過し、濾液を減圧下、濃縮して溶媒を除去することにより、酸味および芳香を有する赤褐色の固形物である本発明製剤６３ｇを得た。
【００３７】
実施例１３
実施例２で得られた本発明製剤の６０ｇに水を加えて分散させた後、酢酸エチルで分配し、酢酸エチル可溶画分（１７．１ｇ）を得た。
【００３８】
この酢酸エチル可溶画分をシリカゲル（ワコーゲルＣ−２００、和光純薬）１ｋｇを充填したカラムに吸着させた。このカラムをｎ−ヘキサン酢酸エチル（１０：１〜１：１）にて溶出させ、分画を行った。ＴＣＬプレート（６０Ｆ２５４、メルク）を用いてｎ−ヘキサン−酢酸エチル（２：１）で展開した時、Ｒｆ値０．２〜０．５付近に１０％硫酸により青または紫色のスポットを呈する画分を集めた。
【００３９】
この画分を高速液体クロマトグラフィー（ＨＰＬＣ）用のオクタデシルシラノール型（ＯＤＳ）分取カラム（センシュー科学）を用いて、メタノール−水（９０：１０〜１００：０）で溶出させ、２０５ナノメータのＵＶ吸収をモニターしながら各ピークを分取する。
【００４０】
それぞれのピークはさらにＨＰＬＣ用ＯＤＳカラムを用いてアセトニトリル−水（９５：５〜１００：０）、メタノール−水（９０：１０〜１００：０）で繰り返し精製することにより、本発明製剤である４種類の無色結晶性化合物１（２８５０ｍｇ）、化合物２（２５０ｍｇ）、化合物３（２５ｍｇ）、化合物４（２５ｍｇ）を得た。
【００４１】
これらの化合物について、融点、マススペクトル、赤外線吸収スペクトル、核磁気共鳴スペクトルにより分析した結果、前記化合物１〜４はオレアノール酸、３−アセチルオレアノール酸、エリスロジオールおよびウバオール等のトリテルペン誘導体であることを確認した。
【００４２】
試験例１
実施例１〜５および１３で示した方法により調製した本発明製剤の溶血性連鎖球菌、黄色ブドウ球菌およびう蝕原因菌に対する抗菌活性を以下の方法により試験した。
【００４３】
試験方法
１）供試菌株、使用培地および培養条件
【００４４】
【表１】
２）抗菌活性試験法
各菌株をそれぞれの培地および培養条件で２４時間培養し、菌を充分に生育させる。９６穴平底マイクロプレートを用い、各ウエル中で試料溶液（１００μｌ）の２倍希釈系列を調製する。２倍濃度で調製した培地に各々の菌を接種し、その１００μｌを各ウエルに添加する。２４時間培養した後、マイクロプレートリーダーを用いて各ウエルの濁度（５５０ｎｍ）を測定し、菌の生育を判定し、その最小発育阻止濃度（ＭＩＣ）を求める。
【００４６】
試験結果
試験結果を表２に示す。本発明製品（実施例１〜５および化合物１〜４）は、黄色ブドウ球菌やう蝕原因菌に対しても抗菌活性を示すが、溶血性連鎖球菌に対する抗菌活性は、さらに強いものである。カリンの水や３０％エタノール抽出物では本発明製品のような抗菌活性はみられなかったことから、カリンを抽出する場合のエタノール濃度は、５０％以上でなくてはならないことが明らかである。
【００４７】
【表２】
試験例２
実施例１〜５および１３で示した方法により調製した本発明製剤の溶血毒（ストレプトリジンＯ）阻害活性を以下の方法により試験した。
【００４９】
試験方法
ウサギ脱繊維血液２５ｍｌを緩衝食塩水（ｐＨ６．５）で３回洗浄（３０００ｒｐｍ×１０分間）し、赤血球を集める。緩衝食塩水で２００ｍｌに希釈し、ウサギ赤血球浮遊液とする。試料規定量をエタノールに溶解してから、さらに緩衝食塩水に溶解し（エタノール２％）、試料溶液とする。試料溶液６００μｌとストレプトリジンＯ（栄研）水溶液（１結合力価）３００μｌを１３ｍｍ径の試験管中で混合し、３７℃で３０分反応させる。ウサギ赤血球浮遊液３００μｌを添加し、３７℃で４５分間反応させる。反応終了後、１５００ｒｐｍで１分間遠心分離し、上清の溶血度合を肉眼で判定する。判定基準は次のようにした。
【００５０】
判定基準
−：溶血のみられないもの、＋：不完全溶血、＋＋：完全溶血
【００５１】
試験結果
試験結果を表３に示す。本発明製剤（実施例１〜５および化合物１〜４）は、溶血毒に対する阻害活性を示し、１０００μｇ／ｍｌ濃度では、いずれの試料でも溶血を完全に阻害した。１００μｇ／ｍｌ濃度においても阻害活性を示し、完全溶血を起こすものはみられなかった。抗菌活性と同様に、カリンの水や３０％エタノール抽出物では本発明製剤のような溶血毒に対する阻害活性はみられなかったことから、カリンを抽出する場合のエタノール濃度は、５０％以上でなくてはならないことが明らかである。
【００５２】
【表３】
応用例
実施例１、２および１３で示した方法により調製した本発明製剤を用いて、次の処方によりチューインガム、キャンディ、錠菓、含そう剤を製造した。
【００５４】
【表４】
【表５】
【表６】
【表７】
【発明の効果】
本発明の喉炎症起因菌に対する抗菌活性及び溶血毒に対する阻害活性剤は、喉炎症起因菌である溶血性連鎖球菌（ストレプトコッカス・ピオゲネス）に対して特異的に強い生育阻害効果を有するばかりでなく、本菌種の産生する溶血毒（ストレプトリジンＯ）に対する阻害効果を併せ有しており、喉の炎症および化膿性疾患を始め種々の感染症に対して有効に作用する。
【００５９】
本発明の喉炎症起因菌に対する抗菌活性及び溶血毒に対する阻害活性剤の原料であるカリンは食用素材や薬用酒として用いられており、その安全性については問題がないので本発明の活性剤の安全性にも問題がなく、従って抗生物質を応用した場合にみられる副作用等の心配はないと認識される。
【００６０】
本発明の喉炎症起因菌に対する抗菌活性及び溶血毒に対する阻害活性剤は、副作用等の心配はなく、香り、呈味性が優れているので口腔用組成物又は飲食品への添加使用等、広範な利用に供することが可能な汎用性の高いものである。[0001]
[Industrial application fields]
The present invention relates to an antibacterial activity against a throat inflammation-causing bacterium (hemolytic streptococcus) and an inhibitory active agent against a hemolytic toxin produced by the bacterium species, and an oral composition and food or drink containing the same .
[0002]
[Prior art]
Streptococcus pyogenes (Streptococcus pyogenes) is a facultative anaerobic Gram-positive cocci that are distributed in the human oral cavity and pharynx. This bacterium is the most pathogenic among streptococci and is known to cause pharyngitis, tonsillitis, otitis media, etc. in the mucosa, and various suppurative inflammations in the skin. Proliferate in
[0003]
Known virulence factors of this bacterium include hemolysin (streptolysin O), dick toxin, streptokinase, DNA-degrading enzyme, hyaluronidase, etc., etiology of respiratory diseases such as pharyngitis, tonsillitis, bronchitis Plays an important role.
[0004]
Needless to say, in order to prevent and treat these respiratory diseases, it is important to inhibit the growth of this fungus in the lesion and to suppress the pathogenic factor of the fungus.
[0005]
From the viewpoint of controlling the fungus, it is effective to apply an antibacterial substance effective against the fungus. Conventionally, penicillin, cephem, and cephalosporin antibiotics were used, but they are powerful, but they are suitable for daily use due to the appearance of resistant bacteria and side effects. I can't say that.
[0006]
Therefore, as an antibacterial substance effective for the fungus suitable for daily use, fruits, vegetables, and other natural products derived from daily food are suitable. Conventionally, karin fruit has attracted attention as an example of such a thing.
[0007]
Japanese Patent Application Laid-Open Nos. 59-187769 and 2-312565 disclose use of fruit juice or extract obtained by extracting karin with honey for the purpose of promoting health, but the object is extracted with honey. It is limited to what has been done, and its effect is very abstract.
[0008]
In addition, Japanese Patent Application Laid-Open Nos. 62-61538 and 62-228230, both of which are filed by the present applicant, disclose that the solvent extract of Karin has antibacterial activity against Streptococcus mutans and Staphylococcus aureus. However, antibacterial activity against Streptococcus pyogenes has not been clarified.
[0009]
Further, Japanese Patent Publication No. 63-26083 and Japanese Patent Application Laid-Open No. 1-290619 have an anti-cariogenic effect on pentacyclic acidic triterpene compounds such as oleanolic acid, which are said to exist widely in nature, Discloses specific antibacterial activity against mutans, but karin fruit contains oleanolic acid and other specific pentacyclic acidic triterpene compounds and these have antibacterial activity against Streptococcus pyogenes That is not disclosed.
[0010]
[Problems to be solved by the invention]
As a result of screening using natural solvent extract as a sample, the present inventors conducted screening using antibacterial activity against Streptococcus pyogenes and hemolytic toxin (streptolysin O) inhibitory activity produced by this bacterium as an index. The inventors have found that the extract has these effects, and have completed the present invention.
[0011]
[Means for Solving the Problems]
The present invention contains a triterpene derivative obtained from a karin fruit , and has an antibacterial activity against a throat inflammation-causing bacterium (hemolytic streptococcus) and an inhibitory active agent against a hemolytic toxin produced by the bacterium species, and an oral cavity containing the same It is a composition and food and drink. In particular, the present invention comprises an extract obtained by extracting karin fruit with 50-100% lower alcohol aqueous solution, acetone, ethyl acetate or n-hexane or a mixed solvent of two or more thereof, and a triterpene derivative obtained from the extract. Antibacterial activity against throat inflammation-causing bacteria (hemolytic streptococci) and an inhibitory active agent against hemolytic toxins produced by this bacterial species, and oral compositions and foods and drinks containing the same .
[0012]
Karin (Chaenomeles sinensis, Cydonis sinensis, Pseudocydonis sinensis), which is a raw material of the active agent of the present invention, is a deciduous high tree grown in various parts of the world, a plant native to the Rosaceae family, and its fruit is used as a food material In addition, it is called Mokka in Chinese medicine, and medicinal liquor made from shochu is used for coughing and expectoration. However, the active ingredient is unknown, and it is also unclear what action mechanism exerts antitussive and expectorant effects.
[0013]
The active agent of the present invention can be extracted and produced from the karin fruit as follows.
[0014]
First, the carin fruit is left as it is or dried, and then mechanically pulverized by an appropriate means, and then 50 to 100% lower alcohol aqueous solution, preferably ethanol aqueous solution, acetone, ethyl acetate or n-hexane or two of them. The extraction operation is performed by applying a sufficient amount of the above mixed solvent. These solvents may be ordinary ones that are commercially available, and two or more of these solvent mixtures may be arbitrarily selected and mixed at an arbitrary mixing ratio. The extraction operation may be performed by immersing the pulverized quince fruit in the above-mentioned solvent at room temperature for an appropriate period of time, but is preferably heated to reflux at a temperature of 50 to 100 ° C. for 1 to 5 hours, more preferably about 3 hours. By the way.
[0015]
Next, the extract obtained in this way is concentrated under reduced pressure to obtain the activator of the present invention.
[0016]
The composition thus obtained is further subjected to column chromatography for separation and purification. The column chromatography to be used is arbitrary, and a technique such as adsorption chromatography, partition chromatography, high performance liquid chromatography, thin layer chromatography or the like may be used. The developing solution used may be a lower alcohol such as methanol, an aqueous solution thereof, acetone, ethyl acetate or n-hexane.
[0017]
Separating purified product obtained is triterpene derivatives der is, has the following structure.
[0018]
[Chemical 9]
[Chemical Formula 10]
Embedded image
Embedded image
In the active agent of the present invention, any one or more of these derivatives can be used as an active ingredient.
[0023]
The active agent of the present invention comprising the solvent extraction composition and the triterpene derivative thus obtained as active ingredients can be used in a wide range of applications, but particularly has excellent fragrance and taste and high safety. In consideration of the above, it can be blended into chewing gum, candy, tablet confectionery, gargle and the like for daily use. When using for such a use, it is suitable if the active agent of this invention is added so that it may contain in the ratio for 0.01 to 10 weight% in these compositions for oral cavity or food-drinks .
[0024]
Hereinafter, examples, test examples, and application examples of the present invention will be described.
[0025]
【Example】
Example 1
5 kg of 50% ethanol was added to 1 kg of the pulverized dried fruits of karin, and the mixture was subjected to reflux extraction at 100 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 324 g of the preparation of the present invention which is a reddish brown solid having a sour taste and aroma.
[0026]
Example 2
5 kg of 100% ethanol was added to 1 kg of pulverized dried fruits of karin, and the mixture was subjected to reflux extraction at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 77 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0027]
Example 3
5 kg of acetone was added to 1 kg of the pulverized dried karin fruit, and the mixture was refluxed and extracted at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 45 g of the preparation of the present invention which is a reddish brown solid having a sour taste and aroma.
[0028]
Example 4
5 kg of ethyl acetate was added to 1 kg of the pulverized dried karin fruit, and the mixture was subjected to reflux extraction at 80 ° C. for 3 hours. The obtained extract was filtered, the filtrate was concentrated under reduced pressure, and the solvent was removed to obtain 35 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0029]
Example 5
5 liters of n-hexane was added to 1 kg of the pulverized dried fruit of karin, and the mixture was heated and refluxed at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 5 g of the preparation of the present invention which was a light green solid having a sour taste and aroma.
[0030]
Example 6
A mixed solvent of 3 liters of 50% ethanol and 2 liters of acetone was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 100 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 280 g of the preparation of the present invention which is a reddish brown solid having a sour taste and aroma.
[0031]
Example 7
A mixed solvent of 3 liters of 100% ethanol and 2 liters of ethyl acetate was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 82 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0032]
Example 8
A mixed solvent of 3 liters of 100% ethanol and 2 liters of n-hexane was added to 1 kg of ground quince fruits, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 72 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0033]
Example 9
A mixed solvent of 3 liters of acetone and 2 liters of ethyl acetate was added to 1 kg of pulverized dried fruit of karin, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 58 g of the preparation of the present invention which was a reddish brown solid having a sour taste and aroma.
[0034]
Example 10
A mixed solvent of 3 liters of acetone and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and the mixture was heated and refluxed at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 42 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0035]
Example 11
A mixed solvent of 3 liters of ethyl acetate and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and extraction under heating was performed at a temperature of 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 35 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0036]
Example 12
A mixed solvent of 1 liter of 100% ethanol, 1 liter of acetone, 1 liter of ethyl acetate and 2 liters of n-hexane was added to 1 kg of pulverized dried fruits of karin, and the mixture was refluxed and heated at 80 ° C. for 3 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, thereby obtaining 63 g of the preparation of the present invention as a reddish brown solid having a sour taste and aroma.
[0037]
Example 13
Water was added to and dispersed in 60 g of the preparation of the present invention obtained in Example 2, and then partitioned with ethyl acetate to obtain an ethyl acetate soluble fraction (17.1 g).
[0038]
This ethyl acetate soluble fraction was adsorbed on a column packed with 1 kg of silica gel (Wakogel C-200, Wako Pure Chemical Industries). This column was eluted with n-hexane ethyl acetate (10: 1 to 1: 1) and fractionated. Fraction that shows blue or purple spots with 10% sulfuric acid around Rf value 0.2-0.5 when developed with n-hexane-ethyl acetate (2: 1) using TCL plate (60F254, Merck) Collected.
[0039]
This fraction was eluted with methanol-water (90:10 to 100: 0) using an octadecylsilanol-type (ODS) preparative column (Senshu Scientific) for high-performance liquid chromatography (HPLC), and 205 nm UV Separate each peak while monitoring absorption.
[0040]
Each of the peaks is the preparation of the present invention by repeatedly purifying with acetonitrile-water (95: 5 to 100: 0) and methanol-water (90:10 to 100: 0) using an ODS column for HPLC. Various kinds of colorless crystalline compound 1 (2850 mg), compound 2 (250 mg), compound 3 (25 mg), and compound 4 (25 mg) were obtained.
[0041]
These compounds were analyzed by melting point, mass spectrum, infrared absorption spectrum, and nuclear magnetic resonance spectrum. As a result, the compounds 1 to 4 were triterpene derivatives such as oleanolic acid, 3-acetyloleanolic acid, erythrodiol and ubaol. confirmed.
[0042]
Test example 1
The antibacterial activity of the preparations of the present invention prepared by the methods shown in Examples 1 to 5 and 13 against hemolytic streptococci, Staphylococcus aureus and caries-causing bacteria was tested by the following method.
[0043]
Test method 1) Test strain, culture medium and culture conditions
[Table 1]
2) Antibacterial activity test method Each strain is cultured for 24 hours in the respective medium and culture conditions to sufficiently grow the bacteria. Using a 96-well flat bottom microplate, prepare a 2-fold dilution series of sample solution (100 μl) in each well. Each culture is inoculated into a medium prepared at a double concentration, and 100 μl thereof is added to each well. After culturing for 24 hours, the turbidity (550 nm) of each well is measured using a microplate reader, the growth of the bacteria is determined, and the minimum growth inhibitory concentration (MIC) is obtained.
[0046]
Test results The test results are shown in Table 2. The products of the present invention (Examples 1 to 5 and compounds 1 to 4) show antibacterial activity against Staphylococcus aureus and caries-causing bacteria, but the antibacterial activity against hemolytic streptococci is even stronger. Since antibacterial activity as in the product of the present invention was not observed in water of Karin or 30% ethanol extract, it is clear that the ethanol concentration when extracting Karin must be 50% or more.
[0047]
[Table 2]
Test example 2
The hemolysin (streptolysin O) inhibitory activity of the preparations of the present invention prepared by the methods shown in Examples 1 to 5 and 13 was tested by the following method.
[0049]
Test method Rabbit defibrillated blood (25 ml) is washed 3 times with buffered saline (pH 6.5) (3000 rpm × 10 minutes), and erythrocytes are collected. Dilute to 200 ml with buffered saline to make a rabbit erythrocyte suspension. Dissolve the specified amount of sample in ethanol, and then dissolve in buffered saline (ethanol 2%) to obtain a sample solution. 600 μl of the sample solution and 300 μl of streptolysin O (Eiken) aqueous solution (1 binding titer) are mixed in a 13 mm diameter test tube and reacted at 37 ° C. for 30 minutes. Add 300 μl of rabbit erythrocyte suspension and react at 37 ° C. for 45 minutes. After completion of the reaction, the mixture is centrifuged at 1500 rpm for 1 minute, and the degree of hemolysis of the supernatant is determined with the naked eye. Judgment criteria were as follows.
[0050]
Judgment criteria- : hemolysis is not observed, +: incomplete hemolysis, ++: complete hemolysis
Test results The test results are shown in Table 3. The preparations of the present invention (Examples 1 to 5 and Compounds 1 to 4) showed inhibitory activity against hemolysin, and at a concentration of 1000 μg / ml, hemolysis was completely inhibited in all samples. No inhibitory activity was observed even at a concentration of 100 μg / ml, and no complete hemolysis was observed. Similar to the antibacterial activity, no inhibitory activity against hemolysin such as the preparation of the present invention was found with water of Karin or 30% ethanol extract, so the ethanol concentration when extracting Karin is not more than 50% It is clear that it should not be.
[0052]
[Table 3]
Application Examples Using the preparations of the present invention prepared by the methods shown in Examples 1, 2, and 13, chewing gums, candy, tablet confectionery, and gargles were produced according to the following formulation.
[0054]
[Table 4]
[Table 5]
[Table 6]
[Table 7]
【The invention's effect】
The antibacterial activity against throat inflammation-causing bacteria of the present invention and the inhibitory activity against hemolysin are not only have a strong growth-inhibiting effect specifically against hemolytic streptococci (Streptococcus pyogenes), which are throat inflammation-causing bacteria, It also has an inhibitory effect on hemolysin (streptolysin O) produced by this species and effectively acts on various infections including throat inflammation and purulent diseases.
[0059]
Karin, which is a raw material for an antibacterial activity against throat inflammation-causing bacteria and a hemolytic toxin of the present invention, is used as an edible material and medicinal liquor, and there is no problem with its safety, so the active agent of the present invention It is recognized that there is no problem with side effects, etc. that are observed when antibiotics are applied.
[0060]
Inhibitory active agent to antimicrobial activity and hemolysin against throat inflammation caused bacteria of the present invention is not fear of such side effects, fragrance, oral composition so taste is excellent or additives used, etc. to food and drink, extensive It is highly versatile and can be used for various purposes.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17457195A JP3749743B2 (en) | 1995-07-11 | 1995-07-11 | Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17457195A JP3749743B2 (en) | 1995-07-11 | 1995-07-11 | Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2005106944A Division JP4457035B2 (en) | 2005-04-01 | 2005-04-01 | Oral composition for antibacterial against throat inflammation-causing bacteria (hemolytic streptococci) and for inhibiting hemolytic toxins produced by this species |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0920674A JPH0920674A (en) | 1997-01-21 |
| JP3749743B2 true JP3749743B2 (en) | 2006-03-01 |
Family
ID=15980894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17457195A Expired - Lifetime JP3749743B2 (en) | 1995-07-11 | 1995-07-11 | Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3749743B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103054972A (en) * | 2012-12-20 | 2013-04-24 | 蚌埠丰原涂山制药有限公司 | Method of extracting oleanolic acid and ursolic acid from pawpaw |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE60114708T2 (en) | 2000-06-05 | 2006-07-20 | Loders Croklaan B.V. | Ursolic acid and oleanolic acid containing mixtures |
| EP1161879B1 (en) * | 2000-06-05 | 2005-11-09 | Loders Croklaan B.V. | Blends of ursolic acid/oleanolic acid |
| WO2002078685A1 (en) * | 2001-03-30 | 2002-10-10 | The Nisshin_Oillio, Ltd. | Drugs for vascular lesion |
| KR100615389B1 (en) * | 2002-08-23 | 2006-08-25 | (주)헬릭서 | Health food comprising the extract of Actinidia arguta and related species for the prevention and improvement of allergic disease and non-allergic inflammatory disease |
| JP5374911B2 (en) * | 2008-04-18 | 2013-12-25 | 日成興産株式会社 | Oral protein flocculant |
| JP5835877B2 (en) * | 2010-08-19 | 2015-12-24 | 株式会社ロッテ | Salivary secretion promoter |
| CN104069332A (en) * | 2014-06-26 | 2014-10-01 | 济南伟传信息技术有限公司 | Traditional Chinese medicine for treating amygdalitis |
| CN104147418A (en) * | 2014-08-22 | 2014-11-19 | 孙元盛 | Traditional Chinese medicine granule for clearing heat and relieving sore throat and preparation method thereof |
| CN104645059A (en) * | 2015-03-19 | 2015-05-27 | 许兰兰 | Medicine for treating infantile acute acute suppurative tonsillitis at infantile medicine center |
| CN104784453A (en) * | 2015-04-26 | 2015-07-22 | 于巧媛 | Traditional Chinese medicine composition for treating phlegm-heat accumulation type acute pharyngitis |
-
1995
- 1995-07-11 JP JP17457195A patent/JP3749743B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103054972A (en) * | 2012-12-20 | 2013-04-24 | 蚌埠丰原涂山制药有限公司 | Method of extracting oleanolic acid and ursolic acid from pawpaw |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0920674A (en) | 1997-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100343486B1 (en) | Plant extract having antibacterial adhesion inhibition activity, its preparation method and composition containing the same | |
| Santos et al. | Brazilian propolis: physicochemical properties, plant origin and antibacterial activity on periodontopathogens | |
| Policegoudra et al. | Antimicrobial, antioxidant, cytotoxicity and platelet aggregation inhibitory activity of a novel molecule isolated and characterized from mango ginger (Curcuma amada Roxb.) rhizome | |
| US7214392B2 (en) | Process and composition for inhibiting growth of microorganisms | |
| JP3749743B2 (en) | Antibacterial activity against throat inflammation-causing bacteria and hemolytic toxin inhibitor, and oral composition and food and drink containing the same | |
| US20110059193A1 (en) | Method for obtaining an extract of cranberry marc that can be used in particular in the prevention and treatment of conditions such as caries, gingivitis or sore throats | |
| KR101937345B1 (en) | A composition for anti-bacterial effect and anti-inflammation comprising unripe apple extracts and baicalin | |
| JP6514684B2 (en) | Oral bacterial growth inhibitor | |
| JP4201898B2 (en) | Antibacterial preparation | |
| JP4630416B2 (en) | Anti-caries, periodontal disease agent | |
| JPH0232255B2 (en) | ||
| JP2804232B2 (en) | Anti-caries, periodontal agent and oral composition containing it | |
| JP3547835B2 (en) | Prophylactic / therapeutic agent for throat inflammation and hemolytic toxin and oral composition containing the same | |
| JP4457035B2 (en) | Oral composition for antibacterial against throat inflammation-causing bacteria (hemolytic streptococci) and for inhibiting hemolytic toxins produced by this species | |
| JP3837172B2 (en) | Inhibitor of adhesion to periodontal tissue of Porphyromonas gingivalis containing high molecular weight polyphenol as an active ingredient | |
| KR100454226B1 (en) | Processing method of licorice extract and its' utilization on developing anti-cariogenic products | |
| US20080175934A1 (en) | Novel herbal composition of extracts of quercus infectoria, process for preparing the same and use thereof | |
| JPWO2004052898A1 (en) | Method for producing hop koji polyphenol | |
| Zhou et al. | Characterization and quantification of two acylated flavonol glycosides from Camellia sinensis and their antibacterial effect on oral pathogens | |
| KR101211669B1 (en) | Antimicrobial compounds from Scilla scilloides and composition containing thereof | |
| KR100388357B1 (en) | Antimicrobial Active Compound Isolated from the Leaves and Stems of Hovenia dulcis Thunb and Isolation Method Thereof | |
| KR20050035954A (en) | Antibacterial agent and oral composition for preventing and treating caries and periodontal disease containing lignan derivatives | |
| JPH01283227A (en) | Antimutagen agent | |
| CN114736110B (en) | Anthraquinone compound, preparation method and application thereof | |
| KR20020087225A (en) | Antibacterial agent for oral pathogens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040702 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20040817 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040907 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20041108 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20050302 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20050401 |
|
| A911 | Transfer of reconsideration by examiner before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20050519 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20051109 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20051205 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20081209 Year of fee payment: 3 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20081209 Year of fee payment: 3 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20081209 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111209 Year of fee payment: 6 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20141209 Year of fee payment: 9 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| EXPY | Cancellation because of completion of term |