JP4630416B2 - Anti-caries, periodontal disease agent - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、オトギリソウ科マンゴスチン（Ｇａｒｃｉｎｉａ ｍａｎｇｏｓｔａｎａ Ｌ．）の果皮より得られる抽出物が、
一般式
【化１】
【０００２】
【従来の技術】
う蝕及び歯周病は、口腔内の二大疾患であり、いずれも特定の細菌による感染症であることが明らかにされている。グラム陽性、通性嫌気性細菌であるStreptococcus mutans（ストレプトコッカス・ミュータンス）及びStreptococcus sobrinus（ストレプトコッカス・ソブリヌス）は、人のう蝕病巣や歯垢の中に見出され、う蝕発生において重要な役割を果たしている。本菌種は、酵素グルコシルトランスフェラーゼを産生し、ショ糖を基質として粘着性の高い高分子多糖体グルカンを生成し、歯のエナメル質表層に強く定着して歯垢を形成する。形成された歯垢には、様々な口腔内細菌が棲息し、その糖代謝に伴い産生した酸により歯のエナメル質が脱灰されてう蝕が誘発されるのである。
【０００３】
一方、歯周病は、作用因子、環境及び宿主からなる多因性疾患であることが知られている。この中で最も病因的な役割を果たすのは細菌であり、近年の研究によりいくつかの病原菌及びその病原性因子等が報告されている。中でもグラム陰性、偏性嫌気性菌であるPorphyromonas gingivalis（ポルフィロモナス・ジンジバリス）は、歯周炎患者の歯周ポケットから高頻度に分離されることや、コラゲナーゼ、免疫グロブリン分解酵素、繊維芽細胞増殖阻害活性、揮発性硫化物の生成等の強い病原性因子を有することから、歯周病の中でも最も多い成人性歯周炎の病原菌として有力視されている。
【０００４】
従って、う蝕及び歯周病の予防や症状を改善するには、病巣においてこれらの細菌類の生育を抑制することや、これらの菌の病原性因子を抑制することが重要であることは明らかである。菌の抑制という観点からは、本菌類に対して有効な抗菌性物質を適用するのが効果的であり、これらの菌に対して有効なものとしてテトラサイクリン、ミノサイクリン等の抗生物質があげられる。しかし、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。
【０００５】
オトギリソウ科の植物であるマンゴスチン（Garcinia mangostana L.）は、その原産国のタイにおいてマンゴスチンの果皮を健胃生薬、抗炎症薬等として伝承的に用いられている。また、マンゴスチン果皮抽出物及びその主要成分であるα−、γ−マンゴスチンについては、抗アレルギー作用（特開平１０−７２３５７号公報）、美白・抗炎症作用（特開平４−２４４００４号公報）、テストステロン５α−レダクターゼ阻害作用（特開平５−１７３６５号公報）、抗酸化作用（特開平８−２２５７８３号公報）等の生理活性が知られている。さらに、ヘリコバクター・ピロリに対する抗菌活性（特開平８−２３１３９６号公報）、黄色ブドウ球菌やメシチリン耐性黄色ブドウ球菌（ＭＲＳＡ）に対する抗菌活性（特開平８−２５９４９３号公報、特開平９−９５４５３号公報、特開平９−１１０６８８号公報）等の様々な菌に対する抗菌活性が開示されている。しかし、現在までにマンゴスチンが、う蝕原因菌であるＳ・ミュータンス、Ｓ・ソブリヌス、また歯周病原因菌であるＰ・ジンジバリスに対する抗菌活性や、グルコシルトランスフェラーゼ阻害活性、さらにコラゲナーゼ阻害活性を有することについての報告はない。
【０００６】
【発明が解決しようとする課題】
本発明の課題は、安全性の高い天然物を原料として、その生産が容易であって、しかもう蝕、歯周病原因菌に対する抗菌活性が高く、う蝕原因菌であるＳ・ソブリヌスが産生するグルコシルトランスフェラーゼに対して阻害活性を有し、さらには歯周病原因菌であるＰ・ジンジバリスの産生するコラゲナーゼに対しても阻害活性を示し、う蝕、歯周病に対して予防効果や症状の軽減効果を有する抗う蝕、歯周病剤を提供することである。
【０００７】
【課題を解決するための手段】
本発明者等は、上記課題を解決するため、含そう剤、練り歯磨き等の衛生用品、チューインガム、キャンディ、錠菓等の食品のような口腔用組成物に配合して日常的に利用可能な、抗う蝕、歯周病剤を開発すべく種々の植物抽出物を用いて、う蝕原因菌であるＳ・ミュータンス、Ｓ・ソブリヌス、また歯周病原因菌であるＰ・ジンジバリスに対する抗菌活性、う蝕原因菌Ｓ・ソブリヌスの産生するグルコシルトランスフェラーゼに対する阻害活性、さらには歯周病原因菌Ｐ・ジンジバリスの産生するコラゲナーゼに対する阻害活性を指標としてスクリーニングを行った。その結果、オトギリソウ科の植物であるマンゴスチン（Garcinia mangostana L.）から得られる抽出物、特にその果皮から得られる抽出物、さらにはその成分であるα−マンゴスチン及びγ−マンゴスチンが、それらの強い効果を有することを見出し、本発明を完成させた。
【０００８】
すなわち、本発明は、オトギリソウ科マンゴスチン（Ｇａｒｃｉｎｉａ ｍａｎｇｏｓｔａｎａ Ｌ．）の果皮より得られる抽出物が、
一般式
【化１】
【０００９】
【発明の実施の形態】
本発明で使用するマンゴスチン（Garcinia mangostana L.）は、オトギリソウ科の植物であり、その果皮、葉、果肉、果実、材、樹皮、根、好ましくはその果皮または葉、特に好ましくはその果皮を乾燥させ、粉砕機等の適当な粉砕手段で粉砕する。この粉砕物に、エタノール、メタノール、ｎ−プロパノール、ｎ−ブタノール、アセトン、酢酸エチル及び水等の溶媒の１種類または2種以上の混合溶媒を加えて、従来一般に行われている抽出方法によって本発明のマンゴスチン抽出物及びマンゴスチン果皮抽出物を得ることができるが、本発明品は口腔に、または飲食品として用いるものであることを考慮すると、抽出溶媒としては安全性の高い水とエタノールとの組合せ、特にエタノール４０％以上の組合せが好ましい。得られた抽出物は、必要により減圧下、濃縮乾燥してもよい。
【００１０】
このようにして得られた抽出物を、さらに吸着クロマトグラフィー、分配クロマトグラフィー、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クロマトグラフィーのような適当な分離精製手段を用いて分離精製することにより、α−マンゴスチン及びγ−マンゴスチンを得ることができる。
【００１１】
このα−マンゴスチン及びγ−マンゴスチンは、一般式
【化２】
【００１２】
本発明の抗う蝕、歯周病剤として上記マンゴスチン抽出物、マンゴスチン果皮抽出物、α−マンゴスチン、またはγ−マンゴスチンを口腔内に投与する場合におけるその投与量としては、症状または個人差により一概に規定することは困難であるが、好ましくは口中または病巣局所での濃度が約０．００１重量％以上、好ましくは約０．０１重量％以上になるようにするとよい。
【００１３】
また、本発明の口腔用組成物は、上記したマンゴスチン抽出物、特にその果皮抽出物、あるいはα−マンゴスチン、γ−マンゴスチンを含有させることにより調製され、それらが、香り、呈味性に優れ、安全性が高いことから、例えば、含そう剤、練り歯磨等の口腔用組成物、チューインガム、キャンディ、錠菓、ジュース等の飲食品に配合し、日常的に利用することが可能である。添加量としては、口腔用組成物または飲食品に対して乾燥抽出物あるいはα−マンゴスチンもしくはγ−マンゴスチンを約０．００１重量％以上、好ましくは約０．０１重量％以上添加する。さらに飲食品においては、特に嗜好性の面を考慮すると約０．００１〜５重量％、好ましくは約０．０１〜１重量％の割合になるように添加するのが好適である。
【００１４】
マンゴスチン果皮の安全性については、その水溶性（または親水性）有機溶媒抽出物において種々の安全性試験が実施されている。例えば、マウスを用いて経口投与による急性毒性試験が行われ、前記抽出物１０ｇ／ｋｇを投与しても死亡例が認められず（特開平５−１７３６５号公報）、またマンゴスチン果皮抽出物の皮膚塗布による人に対する刺激性試験も行われており、皮膚に紅斑等の反応が全く認められていない（特開平４−２４４００４号公報）等、マンゴスチン果皮抽出物は哺乳動物及び人に対する投与においてその安全性は既に証明されている。従って、本発明品の坑う蝕、歯周病剤を人が口腔用組成物として口腔内で用いたり、飲食品として摂取する場合においても、安全性について問題はない。
【００１５】
以下に実施例、試験例、応用例を挙げて説明するが、これらの例は本発明の範囲を制限するものではない。
【００１６】
【実施例】
実施例１
マンゴスチンの果皮を乾燥後粉砕機で粉末化した。この乾燥粉末１ｋｇを１０リットルの水に浸漬し、４０℃で２４時間攪拌抽出した。抽出液を濾別し、濾液を減圧下、濃縮乾燥することにより、本発明品である赤褐色の抽出物１４０ｇを得た。
【００１７】
実施例２
マンゴスチンの果皮乾燥粉末１ｋｇを１０リットルの４０％エタノール水に浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である黄褐色の抽出物２０４ｇを得た。
【００１８】
実施例３
マンゴスチンの果皮乾燥粉末１ｋｇを１０リットルの７０％エタノール水に浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である黄褐色の抽出物２３６ｇを得た。
【００１９】
実施例４
マンゴスチンの果皮乾燥粉末１ｋｇを１０リットルのエタノールに浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である黄褐色の抽出物９１ｇを得た。
【００２０】
実施例５
マンゴスチンの果皮乾燥粉末１ｋｇを１０リットルのメタノールに浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である黄褐色の抽出物８８ｇを得た。
【００２１】
実施例６
マンゴスチンの果皮乾燥粉末１ｋｇを１０リットルの酢酸エチルに浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である黄褐色の抽出物４２ｇを得た。
【００２２】
実施例７
マンゴスチンの葉乾燥粉末１ｋｇを１０リットルのエタノールに浸漬し、４０℃で４時間攪拌抽出した。以下実施例１と同様の操作を行い本発明品である緑褐色の抽出物１９２ｇを得た。
【００２３】
実施例８
実施例４で得られた抽出物８０ｇを水３５０ｍｌに分散させた後、３５０ｍｌの酢酸エチルで分配抽出を２回行い、酢酸エチル画分を得た。酢酸エチル画分を減圧下で溶媒を留去させ、２０ｇの乾燥物を得た。この乾燥物をシリカゲルカラムクロマトグラフィー（８００ｇ）を用いて精製した。溶出はヘキサン−酢酸エチルの系により、徐々に酢酸エチルの構成比を増加させるグラジエント溶出法にて実施した。溶出物を薄層クロマトグラフィー（シリカゲル）分析により３つの画分に分けた。薄層クロマトグラフィーの展開溶媒はヘキサン−酢酸エチル（５０：５０もしくは３０：７０）を用いて実施し、検出にはアニスアルデヒドを用いた。最初の画分（５ｇ）をヘキサンで調製したシリカゲルカラムクロマトグラフィー（２００ｇ）にのせ、溶出はヘキサン−酢酸エチル（５０：５０→３０：７０→１０：９０）を用いて実施し、黄色針状結晶のα−マンゴスチン（Ｃ24Ｈ26Ｏ6、ｍｐ１８０〜１８１℃）を２ｇ得た。２つ目の画分（２ｇ）も同様に再度シリカゲルカラムクロマトグラフィー（１００ｇ）により精製した。溶出は、ヘキサン−酢酸エチル（５０：５０→３０：７０）、続いて酢酸エチルのみ、さらには酢酸エチル−メタノール（５０：５０）で実施した。これにより黄色非結晶状のγ−マンゴスチン（Ｃ23Ｈ24Ｏ6、ｍｐ２０６〜２１０℃）５００ｍｇを得た。これらの化合物の同定は、既に報告されている1Ｈ−ＮＭＲ、13Ｃ−ＮＭＲ、ＭＳ、ＵＶスペクトルデータとの比較により行った。
【００２４】
試験例１
実施例１〜８で調製した本発明品のう蝕及び歯周病原因菌に対する抗菌活性を、以下の方法により試験した。
【００２５】
Ｓ・ミュータンス、Ｓ・ソブリヌスの試験培地としてはブレイン・ハート・インフュージョン液体培地を、Ｐ・ジンジバリスの試験培地としてはトリプチケイス・ソイ液体培地にヘミン（５μｇ／ｍｌ）、メナジオン（０．５μｇ／ｍｌ）を添加したものを用いた。
【００２７】
２）抗菌活性試験法及び試験結果
う蝕原因菌２株及び歯周病原因菌1株をそれぞれの培地及び培養条件で２４〜４８時間培養し、菌を充分に生育させた。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倍希釈系列を調製した。試験系にて菌体量が１０５〜１０６個／ｍｌになるように、２倍濃度で調製した培地に各々の菌を接種し、その１００μlを各ウエルに添加した。２４〜４８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、試料の最小発育阻止濃度（ＭＩＣ）を求めた。試験結果を表1に示した。本発明品であるマンゴスチン果皮抽出物及びα−、γ−マンゴスチンは、いずれの菌に対しても抗菌活性を示し、その効果は葉抽出物（実施例７）より強いものであった。特に果皮の７０％以上のエタノール濃度での抽出や、メタノール、酢酸エチル等の抽出により、抗菌活性成分が効率よく抽出される事が分った。またα−、γ−マンゴスチンも非常に強い抗菌活性を有しており、マンゴスチン果皮抽出物中の抗菌活性の本体と考えられた。
【００２８】
【表１】
試験例２
実施例１〜８で調製した本発明剤のグルコシルトランスフェラーゼ阻害活性を、以下の方法により試験した。
【００３０】
１）グルコシルトランスフェラーゼ調製法
ストレプトコッカス・ソブリヌス６７１５株を１リットルのブレイン・ハート・インフュージョン液体培地中で３７℃、２４時間培養した。培養液を遠心分離し、上清を４℃下硫酸アンモニウム６０％飽和とし、遠心分離し、上清を除き、１０ｍＭリン酸カリウム緩衝液（ｐＨ６．５）３０ｍｌで沈殿を回収した。同緩衝液を用いて十分に透析を行い、透析内液を遠心分離し、その上清を試験用グルコシルトランスフェラーゼとした。
【００３１】
２）グルコシルトランスフェラーゼ阻害試験法及び試験結果
６２．５ｍＭリン酸カリウム緩衝液（ｐＨ６．５）に蔗糖１．２５％、アジ化ナトリウム０．０２５％を加えたものを基質溶液とした。試験試料規定量を５０％メタノールで溶解したものを試料溶液とした。基質溶液８００μｌ、試料溶液４０μｌ、グルコシルトランスフェラーゼ２０μｌ（試験系のグルコシルトランスフェラーゼ活性が試料無添加時の吸光度Ａ０＝１．５となるように調整したもの）、精製水１４０μｌをグラスチューブ中で混合し、水平面に対して３０度に傾け、３７℃、１６時間静置する。デカンテーションにより反応液を除去し、精製水３ｍｌを穏やかに加え、グルカンを洗浄し、洗浄液を除去する。精製水３ｍｌを加えて超音波処理し、グルカンを分散させた後、さらに精製水３ｍｌを加え、５５０ｎｍの吸光度を測定する。グルコシルトランスフェラーゼ阻害活性は以下の式により求めた。
【００３２】
グルコシルトランスフェラーゼ阻害活性 ＝（（Ａ０−Ａ）／Ａ０）×１００
Ａ：試料添加時の吸光度 Ａ０：試料無添加時の吸光度
【００３３】
試験結果を表２に示した。本発明品であるマンゴスチン果皮、葉抽出物及びα−、γ−マンゴスチンは、いずれも強いグルコシルトランスフェラーゼ阻害活性を有することが分った。
【００３４】
【表２】
試験例３
実施例１〜８で調製した本発明品のコラゲナーゼ阻害活性を、以下の方法により試験した。
【００３６】
１）コラゲナーゼ調製法
ポルフィロモナス・ジンジバリスＦＤＣ３８１株をヘミン（５μｇ／ｍｌ）、メナジオン（０．５μｇ／ｍｌ）を添加したトリプチケイス・ソイ液体培地１．５リットルに接種し３７℃で３日間嫌気培養した。培養液を遠心分離し、上清を４℃で硫酸アンモニウム８０％飽和とし、遠心分離し、上清を除き、５ｍＭ塩化カルシウムを含む０．０５Ｍトリス−塩酸緩衝液（ｐＨ７．５）１００ｍｌにて沈殿を回収した。同緩衝液を用いて十分に透析を行い、透析内液を遠心分離し、その上清を試験用コラゲナーゼとした。
【００３７】
２）コラゲナーゼ阻害試験法及び試験結果
コラゲナーゼ阻害活性の測定はコラゲノキットＣＬＮ−１００（コラーゲン技術研修会）を用いて行った。試料溶液１００μｌ、ＦＩＴＣ標識コラーゲン溶液２００μｌ、コラゲナーゼ２００μｌ（コラゲナーゼ活性が１ｕｎｉｔ/ｍｌとなるよう希釈したもの、ここで１ｕｎｉｔは１分間にコラーゲン１μｇを分解するコラゲナーゼの活性）をグラスチューブ中で混合し、３５℃で２時間反応を行った。コラゲナーゼにより分解したＦＩＴＣ標識コラーゲンを抽出し、その蛍光強度によりコラゲナーゼ活性の測定を行った。コラゲナーゼ阻害活性は以下の式により求めた。
【００３８】
コラゲナーゼ阻害活性 ＝（（Ｆ０−Ｆ）／Ｆ０）×１００
Ｆ：試料添加時の蛍光強度 Ｆ０：試料無添加時の蛍光強度
【００３９】
【表３】
試験結果を表３に示した。本発明品であるマンゴスチン果皮、葉抽出物及びα−、γ−マンゴスチンは、いずれもコラゲナーゼ阻害活性を有することが分った。特に果皮の水抽出物及び含水エタノール抽出物が強い阻害活性を有することが分った。
【００４１】
以上の試験結果より、本発明品であるマンゴスチン抽出物、特にその果皮抽出物及びα−マンゴスチン及びγ−マンゴスチンは、う蝕、歯周病原因菌に対する抗菌活性、う蝕原因菌のグルコシルトランスフェラーゼ及び歯周病原因菌のコラゲナーゼ阻害活性を併せ持つことが今回初めて見出された。
【００４２】
【発明の効果】
本発明品は安全性の高いマンゴスチン（Garcinia mangostana L.）、特にその果皮から得られる抽出物、その精製物であるα−及び／またはγ−マンゴスチンを有効成分とする抗う蝕、歯周病剤であり、う蝕、歯周病原因菌に対して強い抗菌活性を有し、また、う蝕原因菌ストレプトコッカス・ソブリヌスが産生するグルコシルトランスフェラーゼに対する阻害活性及び歯周病原因菌ポルフィロモナス・ジンジバリスの産生するコラゲナーゼに対する阻害活性を併せ持つものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an extract obtained from the skin of a mangosteen of the family Hypericaceae (Garcinia mangostana L.) .
General formula
[Chemical 1]
[0002]
[Prior art]
Caries and periodontal disease are two major diseases in the oral cavity, both of which have been shown to be infections caused by specific bacteria. Gram-positive, facultative anaerobic bacteria, Streptococcus mutans and Streptococcus sobrinus, are found in human caries lesions and plaque and play an important role in caries development Plays. This bacterial species produces the enzyme glucosyltransferase, produces high-viscosity high-molecular-weight polysaccharide glucan using sucrose as a substrate, and firmly settles on the tooth enamel surface layer to form plaque. Various oral bacteria live in the formed plaque, and the enamel of the teeth is decalcified by the acid produced by the sugar metabolism to induce caries.
[0003]
On the other hand, it is known that periodontal disease is a multifactorial disease composed of an agent, an environment and a host. Among them, bacteria play the most pathogenic role, and recent studies have reported several pathogenic bacteria and their pathogenic factors. Among them, Porphyromonas gingivalis, a Gram-negative, obligate anaerobic bacterium, is frequently isolated from the periodontal pockets of periodontitis patients, collagenase, immunoglobulin-degrading enzymes, fibroblasts Because it has strong pathogenic factors such as growth inhibitory activity and generation of volatile sulfides, it is regarded as a promising pathogen of adult periodontitis, the most common periodontal disease.
[0004]
Therefore, it is clear that to prevent caries and periodontal disease and improve symptoms, it is important to suppress the growth of these bacteria in the lesion and to control the pathogenic factors of these bacteria. It is. From the viewpoint of suppression of bacteria, it is effective to apply an antibacterial substance effective against this fungus, and antibiotics such as tetracycline and minocycline are effective against these bacteria. However, these have a strong action, but cannot be said to be suitable for daily use due to the appearance of resistant bacteria and side effects.
[0005]
Mangosteen (Garcinia mangostana L.), a plant of the Hypericaceae family, is traditionally used in Thailand, the country of origin, as mangosteen pericarp, anti-inflammatory drugs and the like. As for mangosteen peel extract and α-, γ-mangosteen which are the main components thereof, antiallergic action (JP-A-10-72357), whitening / anti-inflammatory action (JP-A-4-244004), testosterone Physiological activities such as 5α-reductase inhibitory action (JP-A-5-17365) and antioxidant action (JP-A-8-225783) are known. Furthermore, antibacterial activity against Helicobacter pylori (JP-A-8-231396), antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) (JP-A-8-259493, JP-A-9-95453), JP, 9-110688, A) and the like have disclosed antibacterial activity against various bacteria. However, to date, mangosteen has antibacterial activity, glucosyltransferase inhibitory activity, and collagenase inhibitory activity against caries-causing S. mutans, S. sobrinus, and periodontal disease-causing P. gingivalis. There is no report about this.
[0006]
[Problems to be solved by the invention]
The object of the present invention is to produce a highly safe natural product as a raw material, which is easy to produce, has high antibacterial activity against caries and periodontal disease causing bacteria, and is produced by S. sobrinus, the caries causing bacteria. Has an inhibitory activity against glucosyltransferase, and also shows an inhibitory activity against collagenase produced by P. gingivalis, a periodontal disease-causing bacterium, and has preventive effects and symptoms against caries and periodontal disease It is to provide an anti-caries and periodontal disease agent having an alleviating effect.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors can use it daily by blending it into a composition for oral use such as a mouthwash, a sanitary product such as toothpaste, foods such as chewing gum, candy, and tablet confectionery. Antibacterial activity against various caries-causing bacteria, S. mutans, S. sobrinus, and periodontal disease-causing P. gingivalis, using various plant extracts to develop anti-carious and periodontal disease agents Screening was carried out using as an index the inhibitory activity against the glucosyltransferase produced by the caries-causing bacteria S. sobrinus, and the collagenase produced by the periodontal disease-causing bacteria P. gingivalis. As a result, an extract obtained from the mangosteen (Garcinia mangostana L.) which is a member of the family Hypericaceae, particularly an extract obtained from the pericarp, as well as α-mangostin and γ-mangostin, which are components thereof, exert their strong effects. The present invention was completed.
[0008]
That is, the present invention provides an extract obtained from the pericarp of Mangosteen (Garcinia mangostana L.) ,
General formula [Chemical formula 1]
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Mangosteen (Garcinia mangostana L.) used in the present invention is a Hypericaceae plant, and its pericarp, leaves, flesh, fruit, wood, bark, root, preferably pericarp or leaves, particularly preferably pericarp is dried. And pulverizing with an appropriate pulverizing means such as a pulverizer. One or a mixture of two or more solvents such as ethanol, methanol, n-propanol, n-butanol, acetone, ethyl acetate and water is added to the pulverized product, and the mixture is extracted by a conventional extraction method. The mangosteen extract and mangosteen peel extract of the invention can be obtained, but considering that the product of the present invention is used in the oral cavity or as a food or drink, the extraction solvent is made of highly safe water and ethanol. A combination, particularly a combination of 40% or more ethanol is preferable. The obtained extract may be concentrated and dried under reduced pressure if necessary.
[0010]
The extract thus obtained is further separated and purified using an appropriate separation and purification means such as chromatography such as adsorption chromatography, partition chromatography, high performance liquid chromatography, and thin layer chromatography. α-Mangosteen and γ-mangosteen can be obtained.
[0011]
The α-mangosteen and γ-mangosteen are represented by the general formula:
[0012]
When the above-mentioned mangosteen extract, mangosteen peel extract, α-mangosteen, or γ-mangostin is administered into the oral cavity as an anti-cariogenic and periodontal disease agent of the present invention, the dose is generally determined depending on symptoms or individual differences. Although it is difficult to define, it is preferable that the concentration in the mouth or the lesion is about 0.001% by weight or more, preferably about 0.01% by weight or more.
[0013]
Moreover, the composition for oral cavity of the present invention is prepared by containing the above-mentioned mangosteen extract, in particular, the pericarp extract, or α-mangosteen and γ-mangostin, and they are excellent in aroma and taste. Because of its high safety, it can be used on a daily basis, for example, by blending it into an oral composition such as a mouthwash, a toothpaste, etc., or a food or drink such as chewing gum, candy, tablet confectionery or juice. As an addition amount, about 0.001% by weight or more, preferably about 0.01% by weight or more of a dry extract or α-mangostin or γ-mangostin is added to the composition for oral cavity or food and drink. Furthermore, in the case of food / beverage products, it is preferable to add so that the ratio is about 0.001 to 5% by weight, preferably about 0.01 to 1% by weight, particularly in view of palatability.
[0014]
Regarding the safety of mangosteen peel, various safety tests have been conducted on water-soluble (or hydrophilic) organic solvent extracts. For example, an acute toxicity test was conducted by oral administration using mice, and no death was observed even when the above extract was administered at 10 g / kg (JP-A-5-17365), and the skin of mangosteen peel extract Mangosteen skin extract is safe for administration to mammals and humans, such as irritant tests on humans by application, and no reaction such as erythema has been observed on the skin (Japanese Patent Laid-Open No. Hei 4-244004). Sex has already been proven. Therefore, even when a person uses the anti-caries and periodontal disease agent of the present invention as an oral composition in the oral cavity or ingests it as a food or drink, there is no problem with safety.
[0015]
Examples, test examples, and application examples will be described below, but these examples do not limit the scope of the present invention.
[0016]
【Example】
Example 1
Mangosteen peel was dried and pulverized with a pulverizer. 1 kg of this dry powder was immersed in 10 liters of water and extracted by stirring at 40 ° C. for 24 hours. The extract was separated by filtration, and the filtrate was concentrated and dried under reduced pressure to obtain 140 g of a reddish brown extract as the product of the present invention.
[0017]
Example 2
1 kg of dried mangosteen peel powder was immersed in 10 liters of 40% ethanol water and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 204 g of a yellowish brown extract as the product of the present invention.
[0018]
Example 3
1 kg of dried mangosteen peel powder was immersed in 10 liters of 70% ethanol water and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 236 g of a yellowish brown extract as the product of the present invention.
[0019]
Example 4
1 kg of dried mangosteen peel skin powder was immersed in 10 liters of ethanol and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 91 g of a yellowish brown extract as the product of the present invention.
[0020]
Example 5
1 kg of dried mangosteen peel powder was immersed in 10 liters of methanol and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 88 g of a yellowish brown extract as the product of the present invention.
[0021]
Example 6
1 kg of dried mangosteen peel powder was immersed in 10 liters of ethyl acetate, and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 42 g of a yellowish brown extract as the product of the present invention.
[0022]
Example 7
1 kg of dried mangosteen leaf powder was immersed in 10 liters of ethanol and extracted by stirring at 40 ° C. for 4 hours. Thereafter, the same operation as in Example 1 was performed to obtain 192 g of a greenish brown extract as the product of the present invention.
[0023]
Example 8
80 g of the extract obtained in Example 4 was dispersed in 350 ml of water and then partitioned and extracted twice with 350 ml of ethyl acetate to obtain an ethyl acetate fraction. The solvent was distilled off from the ethyl acetate fraction under reduced pressure to obtain 20 g of a dried product. This dried product was purified using silica gel column chromatography (800 g). Elution was performed by a gradient elution method in which the composition ratio of ethyl acetate was gradually increased by a hexane-ethyl acetate system. The eluate was divided into three fractions by thin layer chromatography (silica gel) analysis. The developing solvent for thin layer chromatography was hexane-ethyl acetate (50:50 or 30:70), and anisaldehyde was used for detection. The first fraction (5 g) was applied to silica gel column chromatography (200 g) prepared with hexane, and elution was carried out using hexane-ethyl acetate (50: 50 → 30: 70 → 10: 90). 2 g of crystalline α-mangostin (C 24 H 26 O 6 , mp 180-181 ° C.) was obtained. The second fraction (2 g) was again purified by silica gel column chromatography (100 g). Elution was performed with hexane-ethyl acetate (50: 50 → 30: 70), followed by ethyl acetate alone, and further ethyl acetate-methanol (50:50). As a result, 500 mg of yellow amorphous γ-mangosteen (C 23 H 24 O 6 , mp 206-210 ° C.) was obtained. These compounds were identified by comparison with previously reported 1 H-NMR, 13 C-NMR, MS, and UV spectrum data.
[0024]
Test example 1
The antibacterial activity against the caries and periodontal disease causative bacteria of the products of the present invention prepared in Examples 1 to 8 was tested by the following method.
[0025]
Brain heart infusion liquid medium is used as the test medium for S. mutans and S. sobrinus, and hemin (5 μg / ml) and menadione (0.5 μg / ml) are used as the test medium for P. gingivalis in trypticase soy liquid medium. ml) was used.
[0027]
2) Antibacterial activity test method and test results Two caries-causing fungi and one periodontal disease-causing fungus were cultured for 24 to 48 hours in their respective media and culture conditions, and the fungi were fully grown. Using a 96-well flat-bottom microplate, a 2-fold dilution series of the sample solution (100 μl) was prepared in each well. Each bacterium was inoculated into a medium prepared at a double concentration so that the amount of cells in the test system was 10 5 to 10 6 cells / ml, and 100 μl thereof was added to each well. After culturing for 24-48 hours, the turbidity (550 nm) of each well was measured using a microplate reader, the growth of the bacteria was determined, and the minimum inhibitory concentration (MIC) of the sample was determined. The test results are shown in Table 1. The mangosteen peel extract and α-, γ-mangosteen, which are the products of the present invention, exhibited antibacterial activity against any fungus, and the effect was stronger than that of the leaf extract (Example 7). In particular, it was found that the antibacterial active ingredient was efficiently extracted by extracting the skin with an ethanol concentration of 70% or more, or extracting with methanol, ethyl acetate or the like. Α- and γ-mangosteen also have very strong antibacterial activity, and were considered to be the main body of antibacterial activity in mangosteen peel extract.
[0028]
[Table 1]
Test example 2
The glucosyltransferase inhibitory activity of the agents of the present invention prepared in Examples 1 to 8 was tested by the following method.
[0030]
1) Glucosyltransferase preparation method Streptococcus sobrinus 6715 strain was cultured at 37 ° C. for 24 hours in 1 liter of brain heart infusion liquid medium. The culture solution was centrifuged, the supernatant was made saturated with ammonium sulfate 60% at 4 ° C., centrifuged, the supernatant was removed, and the precipitate was collected with 30 ml of 10 mM potassium phosphate buffer (pH 6.5). Dialysis was sufficiently performed using the same buffer, the dialyzed internal solution was centrifuged, and the supernatant was used as a test glucosyltransferase.
[0031]
2) Glucosyltransferase inhibition test method and test results A substrate solution was prepared by adding 1.25% sucrose and 0.025% sodium azide to a 62.5 mM potassium phosphate buffer (pH 6.5). A sample solution was prepared by dissolving a prescribed amount of a test sample with 50% methanol. Mix 800 μl of the substrate solution, 40 μl of the sample solution, 20 μl of glucosyltransferase (adjusted so that the glucosyltransferase activity of the test system is the absorbance A 0 = 1.5 when no sample is added) and 140 μl of purified water in a glass tube. Inclined at 30 degrees with respect to the horizontal plane and allowed to stand at 37 ° C. for 16 hours. The reaction solution is removed by decantation, 3 ml of purified water is gently added, the glucan is washed, and the washing solution is removed. After adding 3 ml of purified water and sonicating to disperse the glucan, 3 ml of purified water is further added and the absorbance at 550 nm is measured. The glucosyltransferase inhibitory activity was determined by the following formula.
[0032]
Glucosyltransferase inhibitory activity = ((A 0 -A) / A 0 ) × 100
A: Absorbance when sample is added A 0 : Absorbance when sample is not added
The test results are shown in Table 2. It was found that the mangosteen peel, leaf extract and α-, γ-mangosteen, which are the products of the present invention, all have strong glucosyltransferase inhibitory activity.
[0034]
[Table 2]
Test example 3
The collagenase inhibitory activity of the products of the present invention prepared in Examples 1 to 8 was tested by the following method.
[0036]
1) Method for preparing collagenase Porphyromonas gingivalis strain FDC381 was inoculated into 1.5 liter of Trypticase soy liquid medium supplemented with hemin (5 μg / ml) and menadione (0.5 μg / ml) and anaerobically cultured at 37 ° C. for 3 days. did. The culture solution is centrifuged, the supernatant is made 80% saturated with ammonium sulfate at 4 ° C., centrifuged, and the supernatant is removed and precipitated with 100 ml of 0.05 M Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride. Was recovered. Dialysis was sufficiently performed using the same buffer solution, the dialyzed internal solution was centrifuged, and the supernatant was used as a test collagenase.
[0037]
2) Collagenase inhibition test method and test results Collagenase inhibitory activity was measured using collageno kit CLN-100 (collagen technical workshop). 100 μl of sample solution, 200 μl of FITC-labeled collagen solution, 200 μl of collagenase (diluted so that the collagenase activity becomes 1 unit / ml, where 1 unit is the activity of collagenase that degrades 1 μg of collagen per minute) Reaction was performed at 35 degreeC for 2 hours. FITC-labeled collagen decomposed by collagenase was extracted, and collagenase activity was measured by its fluorescence intensity. Collagenase inhibitory activity was determined by the following formula.
[0038]
Collagenase inhibitory activity = ((F 0 −F) / F 0 ) × 100
F: Fluorescence intensity when sample is added F 0 : Fluorescence intensity when sample is not added
[Table 3]
The test results are shown in Table 3. It was found that the mangosteen peel, leaf extract and α-, γ-mangosteen, which are the products of the present invention, all have collagenase inhibitory activity. In particular, it was found that the water extract of fruit skin and the water-containing ethanol extract have strong inhibitory activity.
[0041]
From the above test results, the mangosteen extract, particularly the pericarp extract, and α-mangosteen and γ-mangosteen, of the present invention are caries, antibacterial activity against periodontal disease-causing bacteria, caries-causing glucosyltransferase and It was discovered for the first time that it has a collagenase inhibitory activity of periodontal disease-causing bacteria.
[0042]
【The invention's effect】
The product of the present invention is a highly safe mangosteen (Garcinia mangostana L.), especially an extract obtained from its peel, an anti-cariogenic agent and a periodontal disease agent containing α- and / or γ-mangostin, which is a purified product, as an active ingredient. It has strong antibacterial activity against caries and periodontal disease-causing bacteria, and also has inhibitory activity against glucosyltransferase produced by the caries-causing fungus Streptococcus sobrinus and periodontal disease-causing bacteria Porphyromonas gingivalis. It also has inhibitory activity against the collagenase produced.
Claims (1)
一般式
General formula
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| CN1889967A (en) * | 2003-11-13 | 2007-01-03 | Dbc有限责任公司 | Nutraceutical mangosteen tea |
| JP2007511526A (en) * | 2003-11-13 | 2007-05-10 | ディービーシー エルエルシー | Dissolvable thin film xanthone supplement |
| CN100413868C (en) * | 2004-05-21 | 2008-08-27 | 台湾森本生物科技开发股份有限公司 | Compounds separated from gamboges with activities of inhibiting tumour/cancer cell growth and pharmaceutical compositions containing same |
| JP2009203193A (en) * | 2008-02-28 | 2009-09-10 | Michiya Suzuki | External wash, oral cavity wash product and food or beverage product having effect of oral cavity wash |
| BR112012013473A2 (en) * | 2009-12-04 | 2016-05-17 | Colgate Palmolive Co | oral hygiene composition and method for treating an oral cavity soft tissue disease or condition |
| WO2011068814A1 (en) * | 2009-12-04 | 2011-06-09 | Colgate-Palmolive Company | Oral compositions containing extracts of garcinia mangostana l. and related methods |
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| SG10201900541QA (en) * | 2011-09-08 | 2019-02-27 | Agency Science Tech & Res | Derivatives of xanthone compounds |
| CN102618071A (en) * | 2012-03-08 | 2012-08-01 | 桂林理工大学 | Method for extracting garcinia mangostana pericarp pigment by ultrasonic wave method |
| CN102702215B (en) * | 2012-05-25 | 2014-08-06 | 南方医科大学 | Compound mangostenone F, preparation method and application in preparation of antitumor drugs thereof |
| KR101775613B1 (en) * | 2014-09-16 | 2017-09-07 | 주식회사 메디바이오랩 | Composition for preventing, alleviating or treating periodontal diseases comprising extract of Garcinia Mangostana or Alpha, Gamma-mangostins |
| CN114796298A (en) * | 2022-04-28 | 2022-07-29 | 广西卫生职业技术学院 | Preparation process and test method of water decoction of mangosteen fruit shell for inhibiting gastrointestinal movement |
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| JPH11124322A (en) * | 1997-10-22 | 1999-05-11 | Lion Corp | Composition for oral cavity |
| JPH11279039A (en) * | 1998-03-31 | 1999-10-12 | Saiseido Yakuhin Kk | Composition for oral cavity |
| JPH11302142A (en) * | 1998-04-24 | 1999-11-02 | Sunstar Inc | Food composition for prevention and treatment of periodontal disease, oral composition and pharmaceutical composition |
| JPH11343247A (en) * | 1998-05-29 | 1999-12-14 | Sumitomo Forestry Co Ltd | Glucosyltransferase inhibitor |
-
2000
- 2000-03-02 JP JP2000057839A patent/JP4630416B2/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1072357A (en) * | 1996-08-30 | 1998-03-17 | Lotte Co Ltd | Antiallergic agent |
| JPH11124322A (en) * | 1997-10-22 | 1999-05-11 | Lion Corp | Composition for oral cavity |
| JPH11279039A (en) * | 1998-03-31 | 1999-10-12 | Saiseido Yakuhin Kk | Composition for oral cavity |
| JPH11302142A (en) * | 1998-04-24 | 1999-11-02 | Sunstar Inc | Food composition for prevention and treatment of periodontal disease, oral composition and pharmaceutical composition |
| JPH11343247A (en) * | 1998-05-29 | 1999-12-14 | Sumitomo Forestry Co Ltd | Glucosyltransferase inhibitor |
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| JP2001247469A (en) | 2001-09-11 |
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