JP5457554B2 - Oral health maintenance and improvement composition - Google Patents
Oral health maintenance and improvement composition Download PDFInfo
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- JP5457554B2 JP5457554B2 JP2012517361A JP2012517361A JP5457554B2 JP 5457554 B2 JP5457554 B2 JP 5457554B2 JP 2012517361 A JP2012517361 A JP 2012517361A JP 2012517361 A JP2012517361 A JP 2012517361A JP 5457554 B2 JP5457554 B2 JP 5457554B2
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- 230000002797 proteolythic effect Effects 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Birds (AREA)
- Cosmetics (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、口腔健康維持および改善用組成物に係り、さらに詳しくは、微生物バイオフィルム形成の抑制および除去効果に優れて口腔健康を効果的に維持および改善することが可能な組成物に関する。 The present invention relates to a composition for maintaining and improving oral health, and more particularly, to a composition capable of effectively maintaining and improving oral health with excellent microbial biofilm formation suppression and removal effects.
口腔の健康或いは正常な機能および状態は、全人類の日常生活に極めて大事なことであって、異常発生の際に生活の質を著しく阻害する。口腔の健康要素に関連して最も大きな影響を及ぼす要素は歯の腐食、虫歯、歯肉疾患、歯周疾患および不快な口臭の発生であるといえる。その根本原因は口腔に生息する病原菌によって形成されるバイオフィルムである(Allaker 2008)。これらの病原菌も、口腔面に付いていない状態では唾液などの溶液状態で存在し、歯磨きまたは飲料摂取と共に容易に除去できるため、口腔に疾患を起こす機会が非常に少ない。ところが、これらの病原菌は、効果的な生存のために口腔面にくっ付く性質があって、バイオフィルムを形成する。一旦バイオフィルムが形成されると、外部の物理・化学的刺激に対する防御力が著しく大きくなるから、一般な物理的方法で除去することは難しいうえ、一時的にでも除去するためには毒性の非常に強い抗菌剤を使用するか多量の抗菌剤を使用しなければならない。バイオフィルムは、放置される場合、さらに厚くかつ強くなって歯垢を形成して数多い細菌の生息地になり、各種口腔疾患をおこす。 Oral health or normal function and condition is extremely important to the daily life of all human beings, and significantly impairs the quality of life when an abnormality occurs. It can be said that the most influential factors related to oral health factors are tooth decay, dental caries, gingival disease, periodontal disease, and unpleasant bad breath. The root cause is a biofilm formed by pathogenic bacteria that inhabit the oral cavity (Allaker 2008). These pathogens are also present in a solution state such as saliva when they are not attached to the oral cavity surface, and can be easily removed with toothpaste or beverage intake, so there is very little chance of causing diseases in the oral cavity. However, these pathogens have the property of sticking to the oral surface for effective survival and form a biofilm. Once a biofilm is formed, the defense against external physical and chemical stimuli is remarkably increased, so it is difficult to remove by a general physical method, and it is extremely toxic to remove even temporarily. Must use a strong antibacterial agent or use a large amount of antibacterial agent. When left untreated, biofilms become thicker and stronger, forming plaque and becoming a habitat for many bacteria, causing various oral diseases.
このような過程を防ぐための代表的な方法として、歯科におけるスケーリングなどの物理的方法が勧められてきており、その効果も検証されてきている。ところが、歯科訪問の煩わしさ、恐ろしさおよび経済的な理由で、大部分の場合、十分な管理を受けていない。ところが、歯磨きによるバイオフィルムまたは歯垢の除去には限界があって、数多くの人口が口腔疾患に晒されている。これにより、クロルヘキシジン(chlorhexidine)、塩化セチルピリジニウム(cetylpridinium chloride)、トリクロサン(triclosan)などのように、虫歯菌および歯周炎菌を殺菌する成分の含有された製品が開発されて大衆化されている。ところが、このような合成抗生剤は、長期間使用したときは、口腔内粘膜を刺激し、正常菌も破壊して口腔内病原菌に対する耐性を増加させるうえ、歯を着色し、味覚を害するなど、人体に対する副作用が大きい。口腔洗浄剤に使用される代表的な抗菌物質としてのクロルヘキシジンの場合、ショック症状を示すことが確認されている。特に、口腔内に傷がある場合、さらに危険であると報告されている。これにより、最近、抗菌作用を有する植物由来成分の使用が増加しており、その例としてチモール(thymol)、オイカリプトール(eucalyptol)、メントール(menthol)、ＥＧＣＧ、キシリトール(xylitol)などが使用されているが、その効果は限定的である。 As a typical method for preventing such a process, a physical method such as scaling in dentistry has been recommended and its effect has been verified. However, due to the annoyance, horror and economic reasons of dental visits, in most cases they are not well managed. However, the removal of biofilm or plaque by brushing is limited, and many populations are exposed to oral diseases. This has led to the development and popularization of products containing ingredients that kill caries and periodontitis, such as chlorhexidine, cetylpridinium chloride, and triclosan. . However, such synthetic antibiotics, when used for a long period of time, stimulate the oral mucosa, destroy normal bacteria and increase resistance to oral pathogens, color the teeth, harm the taste, There are significant side effects on the human body. In the case of chlorhexidine as a typical antibacterial substance used for a mouthwash, it has been confirmed that it exhibits shock symptoms. It is reported to be even more dangerous, especially if there are wounds in the oral cavity. As a result, the use of plant-derived components having antibacterial activity has recently increased, and examples thereof include thymol, eucalyptol, menthol, EGCG, xylitol, and the like. However, the effect is limited.
本発明は、従来技術の問題点を解決するためのもので、その目的は、口腔健康の維持および改善において核心的要素といえる微生物によるバイオフィルムの生成抑制および除去効果が非常に著しいため、低い濃度でも口腔内不快感を効果的に防止し、優れた虫歯および歯肉疾患の改善機能を示すうえ、人体に安全な、口腔健康維持および改善用組成物を提供することにある。 The present invention is for solving the problems of the prior art, and its purpose is low because the effect of suppressing and removing biofilms by microorganisms, which can be said to be a core element in maintaining and improving oral health, is very significant. An object of the present invention is to provide a composition for maintaining and improving oral health that effectively prevents oral discomfort even at a concentration, exhibits an excellent ability to improve dental caries and gingival diseases, and is safe for the human body.
本発明は、ジベンゾ−ｐ−ジオキシン(dibenzo-p-dioxine)誘導体を有効成分として含む、口腔健康維持および改善用組成物を提供する。 The present invention provides a composition for maintaining and improving oral health, comprising a dibenzo-p-dioxine derivative as an active ingredient.
本発明に係るジベンゾ−ｐ−ジオキシン誘導体は、下記化学式１、化学式２、化学式３および化学式４よりなる群から選ばれる２種以上の化合物であってもよい。
式中、Ｒはそれぞれ独立に水素、Ｃ１〜Ｃ５のアルキル、Ｃ２〜Ｃ５のアルケニル、フェニル、Ｃ７〜Ｃ１２のフェニルアルキル、Ｃ２〜Ｃ２０のアルカノイル、Ｃ３〜Ｃ２０のアルケノイル、ヒドロキシフェニル、ジヒドロキシフェニルまたはトリヒドロキシフェニルである。 In which R is independently hydrogen, C1-C5 alkyl, C2-C5 alkenyl, phenyl, C7-C12 phenylalkyl, C2-C20 alkanoyl, C3-C20 alkenoyl, hydroxyphenyl, dihydroxyphenyl or tri Hydroxyphenyl.
本発明の口腔健康改善用組成物は、歯の腐食、歯垢形成、口臭の生成および歯肉疾患を生じさせる主要原因としてのバイオフィルムの生成抑制および除去効果に優れており、口腔および歯の状態を長時間爽快且つ清潔に維持することを可能にする。また、本発明は、虫歯および歯肉疾患を予防および改善することにより、口腔の健康を効果的に維持および改善させる効果を提供する。また、本発明の組成物は、天然成分から構成されて人体に安全であるから、年齢を問わず口腔健康の維持および改善に普遍的に使用できる。 The composition for improving oral health of the present invention is excellent in the suppression and removal effect of biofilm as a main cause of tooth corrosion, plaque formation, halitosis generation and gingival disease, and oral and dental conditions Can be kept refreshing and clean for a long time. In addition, the present invention provides an effect of effectively maintaining and improving oral health by preventing and improving dental caries and gingival diseases. Moreover, since the composition of the present invention is composed of natural ingredients and is safe for the human body, it can be universally used for maintaining and improving oral health regardless of age.
以下、本発明を詳細に説明する。
バイオフィルムの下部に存在するストレプトコッカス系の菌は、口腔内に存在する糖分を用いて乳酸を形成し、この乳酸が歯のエナメル質を腐食させて虫歯を発生させる。また、プラークが発達すると、歯肉に存在するＰ．ジンジバリス(P.gingivalis)菌が刺激性化合物を発生して歯肉組織に各種炎症を起こす。また、舌下に生息する嫌気性バクテリアからなるバイオフィルムは、口腔内に残るタンパク質を分解して硫黄またはアミン化合物を発生させ、口臭を引き起こして不快感を誘発する。
Hereinafter, the present invention will be described in detail.
Streptococcus bacteria present in the lower part of the biofilm form lactic acid using sugars present in the oral cavity, and this lactic acid corrodes the enamel of the teeth to generate caries. In addition, when plaque develops, P. cerevisiae present in the gingiva. P. gingivalis bacteria produce irritating compounds and cause various inflammations in the gingival tissue. In addition, a biofilm composed of anaerobic bacteria that live under the tongue decomposes proteins remaining in the oral cavity to generate sulfur or amine compounds, causing bad breath and causing discomfort.
一般人が歯磨き粉または口腔清浄剤の形で容易、安全かつ効果的に口腔の健康を維持および改善するのに適した理想的な活性成分の要件は、（１）低濃度における抗菌活性、（２）抗炎活性の持続性、（３）無刺激性、（４）持続的使用時の安全性、（５）不快感の不在などであるといえる。これにより、本発明者は、合成抗生剤の欠点、すなわち耐性増加の問題、歯牙着色、毒性、味覚損失などの副作用のおそれがない化学的構造を持つ天然由来成分であって、バイオフィルムの生成抑制および除去活性において公知の植物成分より低濃度で効果的であるうえ、持続的な効果を示す成分を見出そうと研究した。 The requirements for an ideal active ingredient suitable for the average person to maintain and improve oral health easily, safely and effectively in the form of toothpaste or oral cleanser are: (1) antibacterial activity at low concentrations, (2) It can be said that the anti-inflammatory activity is persistent, (3) non-irritating, (4) safety during continuous use, and (5) absence of discomfort. As a result, the inventor is a naturally-occurring component having a chemical structure that has no risk of side effects such as the disadvantages of synthetic antibiotics, that is, the problem of increased resistance, tooth coloring, toxicity, taste loss, and the like. Researchers have sought to find ingredients that are effective in inhibiting and eliminating activity at lower concentrations than known plant ingredients, and that also show sustained effects.
文献(Rosan et al, Microbes and Infection 2000, 2:1599-1607)によれば、バイオフィルムの形成は口腔組織の表面にバクテリアが付着する初期付着段階(initial attachment)と、数週にわたってバクテリアとバクテリア間の結合が膨大に形成される相互付着(co-adhesion)段階とからなる。第１段階は、約１週間にわたって起こり、ほぼ全体的にストレプトコッカス属の菌が付着する。第２段階は、数週間にわたって起こり、７００余種のバクテリアが関与しうるが、主にＰ．ジンジバリス菌が関与するものと知られている。また、初期付着段階および相互付着段階の両方ともにおいて付着を媒介する特定のタンパク質が存在することにより、バイオフィルムの形成を可能にするということが知られている。よって、既存の抗菌成分の効果が制限的な理由は、付着段階を効果的に防ぐことができないながら、抗菌成分が、３次元溶液上に存在するバクテリアの除去に主に使用されることにより、一時的な効果のみを示すためである。よって、高濃度或いは毒性の強い抗菌成分が必要となり、これにより人体に対する毒性増加、味覚損失、バクテリアの耐性増加の欠点が発生する。よって、既存の成分を用いた製品としては、一般人が通常の方法（１日１〜２回の歯磨きおよび／或いは口腔洗浄液の使用など）ではバイオフィルムの生成を十分に予防および除去することができないうえ、副作用により、予防次元で経済的、便利、根本的かつ安全に口腔健康の維持および改善を追求することが難しい。 According to the literature (Rosan et al, Microbes and Infection 2000, 2: 1599-1607), the formation of biofilms is an initial attachment stage where bacteria attach to the surface of oral tissues and bacteria and bacteria over several weeks. It consists of a co-adhesion stage in which a lot of bonds are formed. The first stage occurs over a period of about one week, and almost entirely adheres to the genus Streptococcus. The second stage takes place over several weeks and can involve over 700 species of bacteria, but mainly P. aeruginosa. It is known that gingivalis bacteria are involved. It is also known that the presence of specific proteins that mediate attachment in both the initial and inter-adhesion stages allows for the formation of biofilms. Therefore, the reason why the effect of the existing antibacterial component is limited is that the antibacterial component is mainly used for removing bacteria present on the three-dimensional solution, while the adhesion stage cannot be effectively prevented, This is to show only a temporary effect. Therefore, a highly concentrated or highly toxic antibacterial component is required, which causes the disadvantages of increased toxicity to the human body, loss of taste, and increased bacterial resistance. Therefore, as a product using existing components, a general person cannot sufficiently prevent and remove the formation of a biofilm by a usual method (such as use of a toothpaste and / or a mouth washing solution once or twice a day). Moreover, due to side effects, it is difficult to pursue maintenance and improvement of oral health in a preventive dimension economically, conveniently, fundamentally and safely.
バイオフィルム形成の核心要因として付着段階に対する重要性を認知した状態で、本発明者は、既存の３次元溶液上の抗菌作用ではなく、口腔面における２次元的な抗菌作用が可能な成分或いは組成を見出すことにより、そのような組成がより効果的に口腔健康の維持および改善に使用できるようにするために努力した。より具体的に、溶液上で優れた抗菌活性を有する天然化合物であって、口腔組織の表面にバクテリアが付着するときに使用される媒介タンパク質と効果的に結合できる成分を見出し、口腔内のバクテリアの付着部位を不活性化させると同時に、口腔組織の表面に抗菌成分を２次元的に配列させることにより、低い濃度でも口腔内のバイオフィルムの生成を根本的に遮断し、効果の持続性を極大化させることができるようにする。より具体的に、（１）口腔面に付着するバクテリアとしてのストレプトコッカス属の病原菌に対する抗菌効果に優れた天然化合物であって、口腔面に存在するバクテリア付着部位と効果的に結合することが可能な成分を探す。（２）バイオフィルム形成の第２段階に主に関与して炎症を引き起こすＰ．ジンジバリスなどのバクテリアなどに対する抗菌作用に優れた成分であって、バクテリア間の相互結合を媒介するタンパク質と結合することが可能な成分を探す。このような成分はそれ以上のバイオフィルムの蓄積を抑制するだけでなく、２次元的に配列されることにより抗菌成分の実際濃度を極大化させ、その結果としてバイオフィルムの効果的な消滅を可能にする。 Recognizing the importance of the adhesion stage as a core factor for biofilm formation, the present inventor is not able to antibacterial action on the existing three-dimensional solution, but a component or composition capable of two-dimensional antibacterial action on the oral surface. Efforts were made to enable such compositions to be used more effectively to maintain and improve oral health. More specifically, a natural compound having an excellent antibacterial activity on a solution and found a component capable of effectively binding to a mediator protein used when bacteria adhere to the surface of oral tissues. At the same time as inactivating the attachment site, the antibacterial component is two-dimensionally arranged on the surface of the oral tissue, thereby fundamentally blocking the production of the biofilm in the oral cavity even at low concentrations, and maintaining the durability of the effect. To be able to maximize. More specifically, (1) a natural compound having an excellent antibacterial effect against pathogenic bacteria belonging to the genus Streptococcus as a bacterium adhering to the oral cavity surface, which can effectively bind to a bacterial adhesion site present on the oral cavity surface. Search for ingredients. (2) P. cerevisiae that is primarily involved in the second stage of biofilm formation and causes inflammation. A component that has an excellent antibacterial action against bacteria such as Gingivalis and that can bind to a protein that mediates the mutual bond between bacteria is searched for. Such components not only prevent further biofilm accumulation, but also two-dimensionally arraying them maximizes the actual concentration of the antimicrobial component and, as a result, effectively eliminates biofilm. To.
したがって、本発明が既存の技術と差別化される点は、既存の抗菌剤はストレプトコッカスおよびＰ．ジンジバリスの病原菌と抗菌剤の相互作用が３次元溶液上で起こるようにするが、これに対し、本発明の抗菌成分は口腔面上でバクテリアが付着できる部位に特異的に結合することにより、各種口腔疾患発生の中心を効果的に遮断するうえ、２次元上で抗菌作用が起こるようにすることである。本発明の利点は、バイオフィルムの発生点を特異的に遮断し、これと同時にその部位に抗菌力を提供することにより、従来不可能であった水準のバイオフィルム抑制効果を示すことができることである。本発明の他の利点は、２次元上で作用するので、少量の抗菌成分でバイオフィルムまたはプラークの形成を抑制し消滅させることができることである。その上、付着能力に優れた成分と抗菌活性に優れた成分との組み合わせによって多様な組成を提供することができる。 Therefore, the present invention is differentiated from existing technologies in that existing antibacterial agents are Streptococcus and P. The interaction between the gingivalis pathogen and the antibacterial agent occurs on the three-dimensional solution. On the other hand, the antibacterial component of the present invention binds variously to the site where bacteria can adhere on the oral surface. In addition to effectively blocking the center of oral disease development, antibacterial action occurs in two dimensions. The advantage of the present invention is that the biofilm suppression effect can be shown at a level that has been impossible in the past by specifically blocking the generation point of the biofilm and simultaneously providing antibacterial power to the site. is there. Another advantage of the present invention is that it acts in two dimensions, so that the formation of biofilms or plaques can be suppressed and eliminated with a small amount of antimicrobial components. In addition, various compositions can be provided by a combination of a component having excellent adhesion ability and a component having excellent antibacterial activity.
本発明者は、このような新しい成分或いは組成を実現するために文献を研究する中、米国特許第５０１３５４２号(Method to inhibit adhesion of disease-causing microorganisms to teeth)および文献(Rosan et al., Microbes and Infection 2000, 2:1599-1607)より、口腔病原菌が歯の表面および他の病原菌に付着する過程において口腔面および病原菌の表面に存在すること、および、プロリンに富む(proline-rich)ペプチドが大きく関与するという事実を確認した。その事実に着目し、抗菌力、およびプロリンに富むペプチドとの結合力が同時に優れた成分を検索したところ、ジベンゾ−ｐ−ジオキシン骨格を有する天然化合物およびこれらを含有した組成が抗菌力、およびプロリンに富むペプチドとの結合力の面で最も優れることを見出した。また、これらの成分およびこれらからなる組成物の微生物フィルム生成抑制および除去効果が低濃度でも卓越することを見出し、これを適用した試験製品に対する効能実験によって、その優れた実用的価値を確認することにより、本発明を完成した。 While the inventor has studied the literature to realize such new components or compositions, US Pat. No. 5,013,542 (Method to inhibit adhesion of disease-causing microorganisms to teeth) and literature (Rosan et al., Microbes and Infection 2000, 2: 1599-1607) that oral pathogens are present on the oral and pathogenic surfaces in the process of adhering to the tooth surface and other pathogens, and proline-rich peptides Confirmed the fact that it was heavily involved. Focusing on this fact, we searched for components having excellent antibacterial activity and binding ability to proline-rich peptides, and found that natural compounds having a dibenzo-p-dioxin skeleton and compositions containing these have antibacterial activity and proline It has been found that it is most excellent in terms of the binding force with a peptide rich in. In addition, it has been found that the effects of inhibiting and removing the microbial film of these components and compositions comprising them are excellent even at low concentrations, and confirming their excellent practical value through efficacy experiments on test products to which they are applied. Thus, the present invention was completed.
本発明は、ジベンゾ−ｐ−ジオキシン誘導体を有効成分として含む、口腔健康維持および改善用組成物に関する。 The present invention relates to a composition for maintaining and improving oral health comprising a dibenzo-p-dioxin derivative as an active ingredient.
好ましくは、本発明に係るジベンゾ−ｐ−ジオキシン誘導体が下記化学式１、下記化学式２、下記化学式３および下記化学式４よりなる群から選ばれる２種以上の化合物であってもよい。 Preferably, the dibenzo-p-dioxin derivative according to the present invention may be two or more compounds selected from the group consisting of the following chemical formula 1, the following chemical formula 2, the following chemical formula 3, and the following chemical formula 4.
式中、Ｒはそれぞれ独立に水素、Ｃ１〜Ｃ５のアルキル、Ｃ２〜Ｃ５のアルケニル、フェニル、Ｃ７〜Ｃ１２のフェニルアルキル、Ｃ２〜Ｃ２０のアルカノイル、Ｃ３〜Ｃ２０のアルケノイル、ヒドロキシフェニル、ジヒドロキシフェニルまたはトリヒドロキシフェニルである。 Wherein hydrogen each R is independently alkyl of C 1 -C 5, alkenyl C 2 -C 5, phenyl, C 7 -C 12 phenylalkyl, alkanoyl of C 2 ~C 20, C 3 ~C 20 Alkenoyl, hydroxyphenyl, dihydroxyphenyl or trihydroxyphenyl.
好ましくは、Ｒはそれぞれ独立に水素；メチル；エテニル；ベンジル；アセチルまたはオレオイル(oleoyl)；４−ヒドロキシフェニル；２，４−ヒドロキシフェニル；または２，４，６−トリヒドロキシフェニルである。さらに好ましくは水素である。 Preferably, each R is independently hydrogen; methyl; ethenyl; benzyl; acetyl or oleoyl; 4-hydroxyphenyl; 2,4-hydroxyphenyl; or 2,4,6-trihydroxyphenyl. More preferred is hydrogen.
前記口腔健康維持および改善用組成物は、組成物の全体重量に対し、化学式１の化合物３〜７０重量％、化学式２の化合物１〜７５重量％、化学式３の化合物２〜６０重量％、および化学式４の化合物２〜６０重量％を含有することが好ましい。さらに好ましくは、組成物の全体重量に対し、化学式１の化合物１５〜６０重量％、化学式２の化合物６〜５０重量％、化学式３の化合物１５〜５０重量％、および化学式４の化合物１２〜５０重量％を含有する。 The oral health maintenance and improvement composition comprises 3 to 70% by weight of the compound of Formula 1, 1 to 75% by weight of the compound of Formula 2, 2 to 60% by weight of the compound of Formula 3 with respect to the total weight of the composition, and It is preferable to contain 2 to 60% by weight of the compound of Chemical Formula 4. More preferably, the compound of formula 1 is 15 to 60% by weight, the compound of formula 2 is 6 to 50% by weight, the compound of formula 3 is 15 to 50% by weight, and the compound of formula 4 is 12 to 50% by weight based on the total weight of the composition. Contains% by weight.
前記化学式１〜４の化合物は、口腔健康維持および改善に役立つ様々な優れた効能を有するが、特に次の観点で優れた効能を示す。 The compounds of Chemical Formulas 1 to 4 have various excellent effects that are useful for maintaining and improving oral health, and particularly exhibit excellent effects from the following viewpoints.
前記化学式１の化合物は、特にバイオフィルムの初期形成過程に関与する虫歯原因多種菌に対する抑制効能に非常に優れた特性を持つ。よって、前述したような含量の範囲内で含まれる場合、特に、組成物のバイオフィルム形成抑制効能、および虫歯原因菌などのバクテリアに対する抗菌活性効能を強化させる。 The compound of Chemical Formula 1 has a very excellent characteristic of suppressing the causative fungi that are involved in the initial biofilm formation process. Therefore, when contained within the range of the content as described above, the biofilm formation inhibitory effect of the composition and the antibacterial activity effect against bacteria such as caries-causing bacteria are enhanced.
前記化学式２の化合物は、特に歯周細胞の炎症抑制活性（ＴＮＦ−α抑制活性およびＩＬ−１β抑制活性）に非常に優れた特性を持つ。したがって、前述したような含量の範囲内で組成物に含まれる場合、特に歯周炎抑制および改善効果を強化させる。 The compound of the chemical formula 2 has particularly excellent characteristics in periodontal cell inflammation inhibitory activity (TNF-α inhibitory activity and IL-1β inhibitory activity). Therefore, when it is contained in the composition within the range of the content as described above, the periodontitis suppression and improvement effect are particularly enhanced.
前記化学式３の化合物は、特に、バイオフィルムの初期形成過程に関与する虫歯原因多種菌に対する抑制効能、およびバイオフィルムの第２段階の発達に関与する歯周炎原因菌に対する優れた抗菌活性効能を有する。したがって、前述したような含量の範囲内で組成物に含まれる場合、特に、バイオフィルム形成抑制効能、および虫歯原因菌や歯周炎原因菌などのバクテリアに対する抗菌活性効能を強化させる。 The compound of Formula 3 has an excellent antibacterial activity efficacy against periodontitis causative bacteria involved in the development of the second stage of biofilm, and particularly an inhibitory effect against various caries causative bacteria involved in the initial biofilm formation process. Have. Therefore, when it is contained in the composition within the content range as described above, in particular, the biofilm formation inhibitory effect and the antibacterial activity effect against bacteria such as caries-causing bacteria and periodontitis-causing bacteria are enhanced.
前記化学式４の化合物は、特に、バクテリアが付着する初期付着段階(initial attachment)で口腔組織の表面に対するバクテリアの付着を防止する効能に非常に優れた特性を持つ。したがって、前述したような含量の範囲内で組成物に含まれる場合、特にバイオフィルム形成抑制効能を強化させる。 The compound of Formula 4 has a particularly excellent property in preventing the attachment of bacteria to the surface of the oral tissue at the initial attachment stage where bacteria attach. Therefore, when it is contained in the composition within the content range as described above, the biofilm formation inhibitory effect is particularly enhanced.
本発明の口腔健康維持および改善用組成物において、前記化学式１、化学式２、化学式３および化学式４よりなる群から選ばれる２種以上の化合物が化学式１の化合物、化学式３の化合物および化学式４の化合物であれば、バイオフィルム生成抑制活性に特に優れる。 In the composition for maintaining and improving oral health of the present invention, two or more compounds selected from the group consisting of Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 and Chemical Formula 4 are compounds of Chemical Formula 1, Compounds of Chemical Formula 3, and Chemical Formula 4 If it is a compound, it will be especially excellent in biofilm formation inhibitory activity.
本発明の口腔健康維持および改善用組成物は、下記化学式５、化学式６、化学式７、化学式８、化学式９および化学式１０よりなる群から選ばれる少なくとも１種のジベンゾ−ｐ−ジオキシン(dibenzo-p-dioxine)誘導体をさらに含むことができる： The composition for maintaining and improving oral health of the present invention comprises at least one dibenzo-p-dioxin (dibenzo-p) selected from the group consisting of the following chemical formula 5, chemical formula 6, chemical formula 7, chemical formula 8, chemical formula 9 and chemical formula 10. -dioxine) derivatives may further include:
式中、Ｒはそれぞれ独立に水素、Ｃ１〜Ｃ５のアルキル、Ｃ２〜Ｃ５のアルケニル、フェニル、Ｃ７〜Ｃ１２のフェニルアルキル、Ｃ２〜Ｃ２０のアルカノイル、Ｃ３〜Ｃ２０のアルケノイル、ヒドロキシフェニル、ジヒドロキシフェニルまたはトリヒドロキシフェニルである。 Wherein hydrogen each R is independently alkyl of C 1 -C 5, alkenyl C 2 -C 5, phenyl, C 7 -C 12 phenylalkyl, alkanoyl of C 2 ~C 20, C 3 ~C 20 Alkenoyl, hydroxyphenyl, dihydroxyphenyl or trihydroxyphenyl.
好ましくは、Ｒはそれぞれ独立に水素；メチル；エテニル；ベンジル；アセチルまたはオレオイル(oleoyl)；４−ヒドロキシフェニル；２，４−ヒドロキシフェニル；または２，４，６−トリヒドロキシフェニルである。さらに好ましくは水素である。 Preferably, each R is independently hydrogen; methyl; ethenyl; benzyl; acetyl or oleoyl; 4-hydroxyphenyl; 2,4-hydroxyphenyl; or 2,4,6-trihydroxyphenyl. More preferred is hydrogen.
化学式５、化学式６、化学式７、化学式８、化学式９および化学式１０よりなる群から選ばれる少なくとも１種の本発明に係るジベンゾ−ｐ−ジオキシン(dibenzo-p-dioxine)誘導体は、本発明の組成物に組成物の全体重量に対し０．１〜５０重量％で含有できる。 At least one dibenzo-p-dioxine derivative according to the present invention selected from the group consisting of Chemical Formula 5, Chemical Formula 7, Chemical Formula 7, Chemical Formula 8, Chemical Formula 9, and Chemical Formula 10 is a composition of the present invention. The composition can contain 0.1 to 50% by weight based on the total weight of the composition.
本発明の口腔健康維持および改善用組成物にさらに含まれる化学式５、化学式６、化学式７、化学式８、化学式９および化学式１０よりなる群から選ばれる少なくとも１種のジベンゾ−ｐ−ジオキシン誘導体が化学式７の化合物を含む場合に、さらに好ましい活性を示す。 At least one dibenzo-p-dioxin derivative selected from the group consisting of Chemical Formula 5, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, Chemical Formula 9, and Chemical Formula 10 further included in the composition for maintaining and improving oral health of the present invention has the chemical formula When the compound of 7 is included, more preferable activity is shown.
前記化学式７の化合物は、特に、バイオフィルムの初期形成過程に関与する虫歯原因多種菌に対する抑制効能、およびバイオフィルムの第２段階の発達に関与する歯周炎原因菌に対する優れた抗菌活性効能を有する。したがって、本発明の組成物に含まれる場合、特に、バイオフィルム形成抑制効能、および虫歯原因菌や歯周炎原因菌などのバクテリアに対する抗菌活性効能を強化させる役目をする。 In particular, the compound of Formula 7 has an inhibitory effect on various caries-causing bacteria involved in the initial biofilm formation process and an excellent antibacterial activity effect on periodontitis-causing bacteria involved in the second stage development of the biofilm. Have. Therefore, when it is contained in the composition of the present invention, it serves to reinforce the biofilm formation inhibiting effect and the antibacterial activity efficacy against bacteria such as caries-causing bacteria and periodontitis-causing bacteria.
本発明の口腔健康維持および改善用組成物が化学式１、化学式３および化学式７の化合物を含む場合に、バイオフィルム除去活性に特に優れた特性を持つ。 When the composition for maintaining and improving oral health of the present invention contains the compounds of Chemical Formula 1, Chemical Formula 3 and Chemical Formula 7, the composition has particularly excellent biofilm removal activity.
本発明の口腔健康維持および改善用組成物がジベンゾ−ｐ−ジオキシン誘導体として前記化学式１〜化学式１０の化合物を全て含む場合に、化学式１の化合物３〜５０重量％、化学式２の化合物１〜１５重量％、化学式３の化合物２〜４０重量％、化学式４の化合物２〜３０重量％、化学式５の化合物０．１〜１０重量％、化学式６の化合物０．１〜１０重量％、化学式７の化合物２〜４０重量％、化学式８の化合物０．１〜１０重量％、化学式９の化合物０．１〜６重量％、および化学式１０の化合物０．１〜２０重量％で含むことが好ましい。 When the composition for maintaining and improving oral health according to the present invention contains all the compounds of Chemical Formula 1 to Chemical Formula 10 as dibenzo-p-dioxin derivatives, 3 to 50% by weight of the compound of Chemical Formula 1 and 1 to 15 of Chemical Formula 2 2% to 40% by weight of the compound of Formula 3, 2 to 30% by weight of the compound of Formula 4, 0.1 to 10% by weight of the compound of Formula 5, 0.1 to 10% by weight of the compound of Formula 6, It is preferable to contain 2 to 40% by weight of the compound, 0.1 to 10% by weight of the compound of Formula 8, 0.1 to 6% by weight of the compound of Formula 9, and 0.1 to 20% by weight of the compound of Formula 10.
本発明の口腔健康維持および改善用組成物は、薬学的製剤や健康補助食品などとして製造できる。薬学的製剤として製造される場合は、当該分野で通常使用される、薬学的に許容できる担体をさらに含むことができる。また、前記薬学的組成物は補助活性物質をさらに含むことができる。 The composition for maintaining and improving oral health according to the present invention can be produced as a pharmaceutical preparation or a health supplement. When manufactured as a pharmaceutical preparation, it can further contain a pharmaceutically acceptable carrier that is usually used in the art. The pharmaceutical composition may further include an auxiliary active substance.
本発明の組成物による薬学的組成物は、錠剤、トローチ、カプセル、エリキシル剤、サスペンション、シロップ、ウエハースなどの形で製造できる。また、本発明の組成物による健康補助食品は、錠剤、粉末、カプセル、サスペンション、シロップ、飲料、食品(例えば、バー(bar)またはパン)、歯磨き粉、チューインガム、キャンデーなどの形で製造できる。 The pharmaceutical composition according to the composition of the present invention can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, health supplements according to the composition of the present invention can be produced in the form of tablets, powders, capsules, suspensions, syrups, beverages, foods (eg, bars or breads), toothpastes, chewing gums, candy and the like.
本発明の口腔健康維持および改善用組成物の一日使用量は０．０１〜１００ｍｇであることが好ましい。本発明の口腔健康維持および改善用組成物は、口腔内におけるバイオフィルムの発生抑制および除去、歯の腐食予防、歯垢形成予防、歯肉疾患の予防および改善、並びに口臭改善の用途で好ましく使用できる。 The daily use amount of the composition for maintaining and improving oral health of the present invention is preferably 0.01 to 100 mg. The composition for maintaining and improving oral health according to the present invention can be preferably used for the purpose of suppressing and removing biofilms in the oral cavity, preventing dental corrosion, preventing plaque formation, preventing and improving gingival diseases, and improving bad breath. .
以下、実施例によって本発明をより詳細に説明する。ところが、下記の実施例は、本発明さらに具体的に説明するためのもので、本発明の範囲を限定するものではない。下記の実施例は本発明の範囲内で当業者によって適切に修正および変更できる。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for explaining the present invention more specifically, and do not limit the scope of the present invention. The following examples can be appropriately modified and changed by those skilled in the art within the scope of the present invention.
本発明に係るジベンゾ−ｐ−ジオキシン誘導体は、通常の任意の方法によって得ることができ、市販の試薬を用いて合成することができ、天然物、特に海藻類から抽出および分離して得ることができる。 The dibenzo-p-dioxin derivative according to the present invention can be obtained by any ordinary method, can be synthesized using a commercially available reagent, and can be obtained by extraction and separation from natural products, particularly seaweeds. it can.
実施例１．ジベンゾ−ｐ−ジオキシン誘導体の分離精製
実施例１−１．サガラメからポリフェノール混合物の精製
サガラメ(Eisenia arborea)１ｋｇから新鮮な状態で搾汁器を用いて繊維質を除去した後、９５％のエチルアルコール（４Ｌ）を加えて常温で３０分間攪拌し、溶液のみを濾過した後、乾燥させて５８ｇの褐色粉末を得た。得られた乾燥粉末を２０倍量の蒸留水（５０℃）に溶解させた後、ＰＶＰＰ(Polyvinylpyrrolidone)樹脂（乾燥粉末の１０倍）と混合して攪拌した後（５０℃、１ｈｒ）、溶液を濾過して除去し、十分な量の蒸留水（ＰＶＰＰ樹脂の５倍以上）で洗浄した。洗浄されたＰＶＰＰ樹脂に９５％エタノール（ＰＶＰＰ樹脂の３倍量）を加えて常温で３０分間攪拌した後、溶液を濾取して乾燥させて８ｇの黒褐色粉末を得た。フォリン試薬を用いて総ポリフェノール含量を測定した結果、９５．６％であった。
Example 1. Separation and purification of dibenzo-p-dioxin derivative Example 1-1. Purification of polyphenol mixture from garlic After removing fiber from 1 kg of garlic (Eisenia arborea) using a squeezer, add 95% ethyl alcohol (4 L) and stir at room temperature for 30 minutes. Was filtered and dried to obtain 58 g of brown powder. The obtained dry powder was dissolved in 20 times the amount of distilled water (50 ° C.), then mixed with PVPP (Polyvinylpyrrolidone) resin (10 times the dry powder) and stirred (50 ° C., 1 hr). The solution was removed by filtration and washed with a sufficient amount of distilled water (more than 5 times that of PVPP resin). After adding 95% ethanol (3 times the amount of PVPP resin) to the washed PVPP resin and stirring at room temperature for 30 minutes, the solution was collected by filtration and dried to obtain 8 g of a blackish brown powder. As a result of measuring the total polyphenol content using a forin reagent, it was 95.6%.
実施例１−２．精製されたポリフェノール混合物からジベンゾ−ｐ−ジオキシン誘導体の分離
前記実施例１で得たポリフェノール混合物を０．２μｍの膜濾過紙で濾過して高速液体クロマトグラフィーにロードした。高速液体クロマトグラフィーにおいて、カラムとしてはＨＰ ＯＤＳ Ｈｙｐｅｒｓｉｌカラムを使用し、溶媒としては蒸留水とメタノールを使用した。１．０ｍＬ／分の流速で溶媒を供給し、メタノール１５％〜７０％を用いて３０分間にわたって線形勾配(linear gradient)をかけて１０個の活性物質を分離した。各化合物は化学式１〜１０のジベンゾ−ｐ−ジオキシン誘導体であることを確認した。
Example 1-2. Separation of Dibenzo-p-dioxin Derivative from Purified Polyphenol Mixture The polyphenol mixture obtained in Example 1 was filtered through 0.2 μm membrane filter paper and loaded on high performance liquid chromatography. In high performance liquid chromatography, a HP ODS Hypersil column was used as the column, and distilled water and methanol were used as the solvent. Solvent was fed at a flow rate of 1.0 mL / min and 10 active substances were separated using a 15% to 70% methanol over a 30 minute linear gradient. Each compound was confirmed to be a dibenzo-p-dioxin derivative represented by Chemical Formulas 1-10.
式中、Ｒは水素である。 In the formula, R is hydrogen.
実施例２．プロリンに富むペプチドとの結合(binding)／不活性化(inactivation)実験
本発明のジベンゾ−ｐ−ジオキシン化合物、および抗菌活性がよく知られている成分がバイオフィルムの形成において決定的な役目をする付着段階を抑制する能力を評価した。付着活性は、同じ濃度で、固定化されたプロリンに富むタンパク質１に試料が結合する度合いからＥＬＩＳＡ法で決定した。より詳しくは次のとおり行われた。
Example 2 Binding / inactivation experiments with proline-rich peptides The dibenzo-p-dioxin compounds of the present invention and components with well-known antibacterial activity play a crucial role in biofilm formation The ability to suppress the adhesion stage was evaluated. The adhesion activity was determined by the ELISA method from the degree of binding of the sample to the immobilized proline-rich protein 1 at the same concentration. The details were as follows.
ＰＲＰ１(Proline-rich protein 1)を、３名の男性から提供された唾液からＲａｍａｓｕｂｂｕ ｅｔ ａｌ(Ramasubbu, N., M. S. Reddy, E. J. Bergey, G. G. Haraszthy, S.-D. Soni, and M. J. Levine. 1991. Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem. J. 280:341-352.)の方法で分離精製した。精製されたＰＲＰ１（２ｍｍｏＬ／ｍＬ、リン酸緩衝溶液ｐＨ７．４）を、２％グルタルアルデヒド（リン酸緩衝溶液、ｐＨ７．４）で活性化された９６−ウェルプレートに処理した後、４℃で一晩培養(overnight incubation)させて固定させた。０．１％ Ｔｗｅｅｎ２０の含まれたリン酸緩衝溶液（ＰＢＳＴ溶液）で３回洗浄し、カゼイン溶液を処理して常温で１時間放置した後、さらにＰＢＳＴ溶液で３回洗浄した。化学式１〜１０および比較試料（１００μｇ／ｍＬ）を各ウェルに添加し、常温で３時間放置した後、ＰＢＳＴ溶液で３回洗浄し、しかる後に、カゼイン溶液を処理した。Ｒａｂｂｉｔ Ａｎｔｉ−Ｈｕｍａｎ ＰＲＡＰ１ Ｐｏｌｙｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ（Ａｂｃａｍ、英国）を１：１０，０００で希釈して各ウェルに添加した後、４℃で一晩放置し、しかる後に、ＰＢＳＴ溶液で３回洗浄することにより、結合してしない抗体を除去した。結合した抗体は、ＡＰ−ｃｏｎｊｕｇａｔｅｄ Ｇｏａｔ ａｎｔｉ−ｒａｂｂｉｔ ＩｇＧ（Ｈ＋Ｌ）（Ａｎａｓｐｅｃ、ＵＳＡ）を２次抗体として用い、ｐ−ニトロフェニルホスフェート(p-Nitrophenyl phosphate)を基質として用いて４０５ｎｍで検出し、次の式を用いて計算した。陰性対照試料としてアルブミン(bovine serum albumin)を使用した。 PRP1 (Proline-rich protein 1) was obtained from the saliva provided by three men by Ramasubbu et al (Ramasubbu, N., MS Reddy, EJ Bergey, GG Haraszthy, S.-D. Soni, and MJ Levine. 1991 Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem. J. 280: 341-352.) Purified PRP1 (2 mmol / mL, phosphate buffer solution pH 7.4) was treated in 96-well plates activated with 2% glutaraldehyde (phosphate buffer solution, pH 7.4) and then at 4 ° C. It was fixed by overnight incubation. After washing three times with a phosphate buffer solution (PBST solution) containing 0.1% Tween 20, the casein solution was treated and allowed to stand at room temperature for 1 hour, and further washed three times with a PBST solution. Chemical formulas 1 to 10 and a comparative sample (100 μg / mL) were added to each well, allowed to stand at room temperature for 3 hours, washed 3 times with PBST solution, and then treated with the casein solution. Rabbit Anti-Human PRAP1 Polyclonal Antibody (Abcam, UK) was diluted 1: 10,000 and added to each well, then left at 4 ° C. overnight, and then washed three times with PBST solution. Unbound antibody was removed. The bound antibody was detected at 405 nm using AP-conjugated Goat anti-rabbit IgG (H + L) (Anaspec, USA) as a secondary antibody and p-nitrophenyl phosphate as a substrate. This was calculated using the following formula. Albumin (bovine serum albumin) was used as a negative control sample.
ＰＲＰ１に対する付着活性（％）＝１００×（陰性対照試料処理時の吸光度−試料処理時の吸光度）／陰性対照試料処理時の吸光度
実験結果：化学式４の化合物が最も優れた効果を示した。
Adhesive activity (%) for PRP1 = 100 × (absorbance at the time of negative control sample treatment−absorbance at the time of sample treatment) / absorbance at the time of negative control sample treatment Experimental results: The compound of Formula 4 showed the most excellent effect.
実施例３．溶液上における抗菌活性の評価
実施例３−１．バイオフィルムの初期形成過程に関与する虫歯原因菌に関する溶液上における抗菌活性の測定
口腔面のバクテリア付着部位に結合して付着部位を封鎖すると同時に、付着するバクテリアに対する抗菌活性まで兼備すれば、バイオフィルム生成抑制効果がさらに大きくなる。よって、付着段階に対する抑制と独立して溶液上における抗菌活性を測定し、活性に優れた成分を探索した。
Example 3 Evaluation of antibacterial activity on solution Example 3-1. Measurement of antibacterial activity on solution related to causative agents of dental caries involved in the initial formation process of biofilm. Biofilms can be combined with antibacterial activity against adhering bacteria at the same time as binding to and blocking the bacterial adhesion site on the oral surface. The generation suppression effect is further increased. Therefore, the antibacterial activity on the solution was measured independently of the suppression to the adhesion stage, and a component having excellent activity was searched.
前記実施例２で製造された試験物および比較サンプルに対して、虫歯の原因菌としてのストレプトコッカスミュータンス(Streptococcus mutans、ＡＴＣＣ２５２７５)、ストレプトコッカスサングイス(S.sanguis ＡＴＣＣ１０５５６)（Ａｍｅｒｉｃａｎ ｔｙｐｅ ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ、Ｒｏｃｋｗｉｌｌｅ、ＭＤ、ＵＳＡ）、アクチノミセスビスコサス(Actinomyces viscosus ＫＣＣＭ １２０７４、Ｋｏｒｅａ Ｃｕｌｔｕｒｅ ｅｎｔｅｒ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ、ソウル)からなる多種菌に対する抗菌活性を測定した。全ての菌は嫌気性条件の下に３７℃で２４時間ＢＨＩ（brain heart infusion broth, ＤＩＦＣＯ ｌａｂｏｒａｔｏｒｉｅｓ、Ｄｉｔｒｏｉｔ、ＭＩ、ＵＳＡ）培養液内で培養された。各試料の抗菌活性は、培養液微細希釈法を用いた最小阻害濃度（Minimum inhibitory concentration、ＭＩＣ）測定法を用いて測定した。より詳しくは、前記組成物の標準溶液を、１，０００μｇ／ｍＬの濃度で１０％ ＤＭＳＯ溶液を溶媒として製造した後、これを、ＢＨＩ培養液を用いて段階的に２倍ずつ連続希釈し、０．２５〜１２５μｇ／ｍＬ溶液を製造した。９６−ポリスチレンプレートの各ウェル当たり２０μＬの多腫菌を接種させ、細胞濃度が１×１０６ＣＦＵ／ｍＬとなるように培養液を用いて濃度を調節し後、連続希釈して製造された様々な濃度の前記試料を添加し、３７℃で２４時間培養した。各ウェル内のバクテリアの成長程度を、５９６ｎｍで吸光度を測定して求めた。最小阻害濃度（ＭＩＣ）は、対照群（試料を処理していないセル）と比較してバクテリアの成長が９０％以上阻害される試料の最小濃度で決定した。全ての実験を３回繰り返し行い、その各平均値を使用した。各試料の最小阻害濃度（ＭＩＣ）を下記表２に示す。 For the test sample and comparative sample prepared in Example 2, Streptococcus mutans (ATCC 25275) and Streptococcus sanguis (S. sanguis ATCC 10556) (American type culture Collection, Rockwill, MD, USA), antibacterial activity against various bacteria consisting of Actinomyces viscosus KCCM 12074, Korea Culture of the Microorganisms, Seoul). All the bacteria were cultured in BHI (brain heart infusion broth, DIFCO laboratories, Ditroit, MI, USA) culture medium at 37 ° C. for 24 hours under anaerobic conditions. The antibacterial activity of each sample was measured using a minimum inhibitory concentration (MIC) measurement method using a culture broth fine dilution method. More specifically, after preparing a standard solution of the above composition at a concentration of 1,000 μg / mL using a 10% DMSO solution as a solvent, this was serially diluted stepwise by 2 times using a BHI culture solution, A 0.25-125 μg / mL solution was prepared. 96-polystyrene plates were inoculated with 20 μL of multifilament bacteria, adjusted to a concentration of 1 × 10 6 CFU / mL using a culture solution, and then serially diluted. The sample was added at various concentrations and incubated at 37 ° C. for 24 hours. The degree of bacterial growth in each well was determined by measuring the absorbance at 596 nm. The minimum inhibitory concentration (MIC) was determined at the minimum concentration of the sample at which bacterial growth was inhibited by 90% or more compared to the control group (cell not treated with sample). All experiments were repeated 3 times and their average values were used. The minimum inhibitory concentration (MIC) for each sample is shown in Table 2 below.
実施例３−２．バイオフィルムの第２段階の発達に関与する歯周炎原因菌に対する抗菌活性の測定
前記実施例２で製造された試験物を用いて、歯周炎の原因菌としてのポルフィロモナスジンジバリス(Porphyromonas gingivalis ＡＴＣＣ ５３９７８)、ポルフィロモナスエンドドンターリス(Porphyromonas endodotalis ＡＴＣＣ ３５４０６)、プレボテラインターメディア(Provotella intermedia ＡＴＣＣ ２５６１１)に対する抗菌活性を測定した。全てのバクテリアは、嫌気性条件の下（９０％Ｎ２、５％ＣＯ２、５％Ｈ２）に３７℃で２４時間培養液（３０ｇ／Ｌ トリプティックソイブロス(trypticase soy broth、５ｇ／Ｌ酵母エキス、０．５ｇ／Ｌ Ｌ−システイン、５μｇ／ｍＬヘミンおよび０．２μｇ／ｍＬビタミンＫ１)で培養された。前記組成物の歯周炎菌に対する抗菌活性は、培養液微細希釈法を用いた最小阻害濃度（ＭＩＣ）測定法を用いて測定した。より詳しくは、前記組成物の標準溶液を、１，０００μｇ／ｍＬの濃度で１０％ ＤＭＳＯ溶液を溶媒として製造した後、これをＢＨＩ培養液を用いて段階的に２倍ずつ連続希釈して０．２５〜１２５μｇ／ｍＬの溶液を製造した。９６−ポリスチレンプレートの各ウェルあたり２０μＬのバクテリアを接種させ、前記菌株の細胞数が１×１０６ＣＦＵ／ｍＬとなるように培養液を用いて濃度を調節した後、連続希釈して製造された様々な濃度の前記組成物を添加し、３７℃で２４時間培養した。各ウェル内のバクテリアの成長程度を、５９６ｎｍで吸光度を測定して求めた。最小阻害濃度（ＭＩＣ）は、対照群（試料を処理してないセル）と比較して、バクテリアの成長が９０％以上阻害される試料の最小濃度で決定した。全ての実験を３回繰り返し行い、その各平均値を使用した。各組成物の最小阻害濃度（ＭＩＣ）を下記表２に示す。
Example 3-2. Measurement of antibacterial activity against periodontitis causative bacteria involved in the development of the second stage of biofilm Using the test product produced in Example 2, Porphyromonas gingivalis as a causative agent of periodontitis Antibacterial activity against ATCC 53978), Porphyromonas endodotalis ATCC 35406, and Prevotella intermedia ATCC 25611 was measured. All bacteria were cultured for 24 hours at 37 ° C. under anaerobic conditions (90% N 2 , 5% CO 2 , 5% H 2 ) (30 g / L trypticase soy broth, 5 g / L yeast). The extract was cultured with 0.5 g / L L-cysteine, 5 μg / mL hemin and 0.2 μg / mL vitamin K1). More specifically, a standard solution of the composition was prepared using a 10% DMSO solution as a solvent at a concentration of 1,000 μg / mL, and this was then used as a BHI culture solution. A 0.25-125 μg / mL solution was prepared by serially doubling in a stepwise manner using a 20 to 20 microliter of bacteria per well of a 96-polystyrene plate. After the number has to adjust the concentration using the culture solution so that 1 × 10 6 CFU / mL, was added the composition of various concentrations prepared by serial dilution, and incubated for 24 hours at 37 ° C.. The extent of bacterial growth in each well was determined by measuring the absorbance at 596 nm, and the minimum inhibitory concentration (MIC) was 90% bacterial growth compared to the control group (cells without sample treatment). The minimum concentration of the sample to be inhibited was determined as described above, and all the experiments were repeated three times, and the average value was used, and the minimum inhibitory concentration (MIC) of each composition is shown in Table 2 below.
グルコン酸クロルヘキシジンが溶液上で最も強力な抗菌作用を示した。天然成分の中では、化学式３および７が最も優れた抗菌作用を示した。 Chlorhexidine gluconate showed the strongest antibacterial action on the solution. Of the natural ingredients, Chemical Formulas 3 and 7 showed the best antibacterial action.
実施例４．Ｐ．ジンジバリス菌から分泌されるＬＰＳによって誘導される歯周細胞の炎症抑制活性の評価
歯周炎とは、歯肉炎症とこれによる歯周細胞の破壊により起こる一連の感染疾患をいう。歯周炎と関連のある様々なバクテリアのうち、ポルフィロモナスジンジバリス(Porphyromonas gingivalis)は、これらの中でも最もよく知られているバクテリアである。このようなバクテリアにより生成されるＬＰＳ、タンパク質、或いはタンパク質分解酵素などは、代表的な細胞毒性物質であって、歯周疾患と密接した関係がある。
Example 4 P. Evaluation of Periodontal Inflammation Inhibitory Activity Induced by LPS Secreted from Gingivalis Periodontitis refers to a series of infectious diseases caused by gingival inflammation and periodontal cell destruction. Of the various bacteria associated with periodontitis, Porphyromonas gingivalis is the best known of these. LPS, protein, or proteolytic enzyme produced by such bacteria is a typical cytotoxic substance and has a close relationship with periodontal diseases.
Ｐ．ジンジバリスによって生成されたＬＰＳは、インターロイキン−１（ＩＬ−１β）或いは腫瘍壊死因子（ＴＮＦ−α）などの炎症因子の生成を促進して、これらの炎症因子は各種歯周疾患を起こす原因として知られている。 P. LPS produced by Gingivalis promotes the production of inflammatory factors such as interleukin-1 (IL-1β) or tumor necrosis factor (TNF-α), and these inflammatory factors cause various periodontal diseases. Are known.
したがって、前記組成物のＰ．ジンジバリスによって生成されたＬＰＳにより発現されるインターロイキン−１（ＩＬ−１β）と腫瘍壊死因子（ＴＮＦ−α）の生成抑制効果から歯周炎予防効果を測定した。 Therefore, the P.O. Periodontitis preventive effect was measured from the production inhibitory effect of interleukin-1 (IL-1β) and tumor necrosis factor (TNF-α) expressed by LPS produced by gingivalis.
Ｐ．ジンジバリス（Ａ７Ａ１−２８）は、ＢＨＩ培養液（５ｍｇのヘミンおよび０．５ｍｇのビタミンＫ／ｍＬ含有）を用いて３７℃で嫌気性条件の下で（９０％Ｎ２、５％Ｈ２および５％ＣＯ２）２４時間培養した。Ｐ．ジンジバリスによって生成されたＬＰＳをフェノール蒸留水方法を用いて抽出した。より詳しくは、培養されたバクテリアセルを蒸留水にサスペンションさせ、同量の９０％フェノールを６０℃で添加した後、２０分間攪拌した。水溶液層を遠心分離（４℃で７０００ｒｐｍにて１５分間）して分離した後、非イオン化水を用いて４℃で３日間透析し、しかる後に、これをさらに遠心分離（４℃で４０，０００ｒｐｍにて１．５時間）して沈殿物を得た。この沈殿物を透析によって精製した後、これを真空乾燥させて４℃に保管した。マウス由来のマクロファージ（ＲＡＷ ２６４．７）を、ブドウ糖４．５ｇ／ｌ、１０％血清および１％抗生剤が含有されたＤＭＥＭ培地に接種して３７℃で２４時間培養した。これらの細胞を分注して４８時間培養した後、化学式１〜１０および比較試料を５０μｇ／ｍＬの濃度でＤＭＥＭ培地に希釈させ、これを培養されたマクロファージに処理し、３７℃で３日間培養した。培養の後、培地を全て除去し、溶菌バッファ（０．１Ｍリン酸カリウムバッファ、ｐＨ７．８／１％Ｔｒｉｔｏｎ Ｘ−１００／１ｍＭ ＤＴＴ／２ｍＭ ＥＤＴＡ）を用いて培養液を処理した後、培養液内の生成されたインターロイキン−１βと腫瘍壊死因子（ＴＮＦ−α）の量を酵素免疫アッセイ法（Enzyme-linked immunosorbent assay、ＥＬＩＳＡ）によって測定した。その結果を下記表３に示す。 P. Gindivaris (A7A1-28) was used under anaerobic conditions (90% N 2 , 5% H 2 and 5) at 37 ° C. using BHI culture medium (containing 5 mg hemin and 0.5 mg vitamin K / mL). % CO 2 ) for 24 hours. P. LPS produced by Gingivalis was extracted using the phenol distilled water method. More specifically, the cultured bacterial cell was suspended in distilled water, and the same amount of 90% phenol was added at 60 ° C., followed by stirring for 20 minutes. The aqueous layer was separated by centrifugation (15 minutes at 7000 rpm at 4 ° C.), then dialyzed for 3 days at 4 ° C. using non-ionized water, and then further centrifuged (40,000 rpm at 4 ° C.). For 1.5 hours) to obtain a precipitate. After the precipitate was purified by dialysis, it was vacuum dried and stored at 4 ° C. Mouse-derived macrophages (RAW 264.7) were inoculated into DMEM medium containing 4.5 g / l of glucose, 10% serum and 1% antibiotic and cultured at 37 ° C. for 24 hours. After dispensing these cells and culturing for 48 hours, the chemical formulas 1 to 10 and the comparative sample were diluted in DMEM medium at a concentration of 50 μg / mL, treated with cultured macrophages, and cultured at 37 ° C. for 3 days. did. After culturing, all the medium was removed and the culture solution was treated with a lysis buffer (0.1 M potassium phosphate buffer, pH 7.8 / 1% Triton X-100 / 1 mM DTT / 2 mM EDTA). The amount of interleukin-1β and tumor necrosis factor (TNF-α) produced was measured by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3 below.
表３に示すように、グルコン酸クロルヘキシジンは、培養された細胞を消滅させたので測定が不可であり、化学式２が最も優れた活性を示した。 As shown in Table 3, chlorhexidine gluconate was unable to be measured because the cultured cells were extinguished, and chemical formula 2 showed the most excellent activity.
実施例５．組成物１〜１２の製造
前記実施例３および４から分かるように、ジベンゾ−ｐ−ジオキシン成分がバイオフィルムの生成を抑制するのに必要なそれぞれの活性において最も優れた。各活性の優れた成分を選定して組成物１〜１０を製造した。各組成物の化学的組成を下記表４に示す。
Example 5 FIG. Production of Compositions 1-12 As can be seen from Examples 3 and 4 above, the dibenzo-p-dioxin component was most excellent in each activity required to inhibit biofilm formation. Compositions 1 to 10 were produced by selecting ingredients having excellent activity. The chemical composition of each composition is shown in Table 4 below.
実施例６．バイオフィルム生成予防効果の評価
前記実施例５で製造された組成物と比較試料に対してバイオフィルム生成抑制活性を測定した。前記組成物は、１００％ジメチルスルホキシドに溶かして０．５−４０μＬ／ｍＬの濃度でムチンを用いて希釈した。人工唾液は、１％タイプＩＩＩムチン（mucin from porcine stomach、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ）を付着緩衝溶液に希釈させた後、１２１℃で１５分間滅菌させたものを使用した。
Example 6 Evaluation of biofilm production preventing effect Biofilm production inhibitory activity was measured for the composition produced in Example 5 and a comparative sample. The composition was dissolved in 100% dimethyl sulfoxide and diluted with mucin at a concentration of 0.5-40 μL / mL. Artificial saliva was prepared by diluting 1% type III mucin (mucin from porcine stomach, Sigma-Aldrich) in an adhesion buffer solution and then sterilizing at 121 ° C. for 15 minutes.
菌株としては、虫歯原因菌としてのストレプトコッカスミュータンス(Streptococcus mutans、ＡＴＣＣ２５２７５)、ストレプトコッカスサングイス(S.sanguis ＡＴＣＣ１０５５６)（Ａｍｅｒｉｃａｎ ｔｙｐｅ ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ、ｒｏｃｋｗｉｌｌｅ、ＭＤ、ＵＳＡ）、アクチノミセスビスコサス（Actinomyces viscosus ＫＣＣＭ １２０７４、Ｋｏｒｅａ Ｃｕｌｔｕｒｅ ｅｎｔｅｒ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ、ソウル）を使用し、これらを接種して多種(multi-species)接種溶液を製造した。より詳しくは、培養された各バクテリアを遠心分離機によって分離した後（４５００、５ｍｉｎ）、ＰＢＳ（phosphate-buffered saline、ｐＨ７．２）で洗浄した。これを付着緩衝溶液（１０ｍＭ ＫＰＯ４、５０ｍＭ ＫＣｌ、１ｍＭ ＣａＣｌ２、０．１ｍＭ ＭｇＣｌ２、ｐＨ７．０）にさらに１×１０６ＣＦＵ／ｍＬ濃度でサスペンションさせた。接種細胞溶液は各バクテリアを同じ濃度で含んだ。前記組成物のバイオフィルム生成抑制活性を測定するために、ＲｕｋａｙａｄｉとＨｗａｎｇの方法を使用した。より詳しくは、９６ウェルポリスチレンマイクロプレートの各ウェルに０．５〜５０μｇ／ｍＬの試料を入れて常温でシェーカーを用いて３時間培養した後、空気中で乾燥させた。試料がコートされた９６−ウェルプレートに２０μＬのバクテリア接種液を添加し、全体体積が２００μｌとなるように培養液を満たした後（最終濃度１×１０５ＣＦＵ／ｍＬ）、３７℃で２４時間培養した。残っている培養液を除去した後、２００μＬ ５０ｍＭ ＰＢＳを用いて、残ったバイオフィルムを形成していないセルを洗浄した。バイオフィルムを形成したセルを０．４％クリスタルバイオレット溶液を３０分間着色した後、蒸留水を用いて、残っている溶液を綺麗に洗浄した。着色されたセルを９５％エタノール２００μＬに溶かした後、その量を吸光度（５９６ｎｍ）を用いて測定した。本試料のバイオフィルム抑制効果は次の式を用いて測定し、その結果を下記表５に示す。 Examples of strains include Streptococcus mutans (ATCC 25275) and S. sanguis ATCC 10556 (American type culture Collection, rockwill, MD, USA), Actinoscus Cactus v. These were inoculated to produce a multi-species inoculation solution using the Korea Culture enter of Microorganisms, Seoul). More specifically, each cultured bacterium was separated by a centrifuge (4500, 5 min) and then washed with PBS (phosphate-buffered saline, pH 7.2). This was further suspended in an attachment buffer solution (10 mM KPO4, 50 mM KCl, 1 mM CaCl 2 , 0.1 mM MgCl 2 , pH 7.0) at a concentration of 1 × 10 6 CFU / mL. The inoculated cell solution contained the same concentration of each bacterium. In order to measure the biofilm formation inhibitory activity of the composition, the method of Rukayadi and Hwang was used. More specifically, a sample of 0.5 to 50 μg / mL was placed in each well of a 96-well polystyrene microplate, cultured at room temperature using a shaker for 3 hours, and then dried in air. After adding 20 μL of bacterial inoculum to the sample-coated 96-well plate and filling the culture solution to a total volume of 200 μl (final concentration 1 × 10 5 CFU / mL), at 37 ° C. for 24 hours Cultured. After removing the remaining culture, 200 μL 50 mM PBS was used to wash the remaining cells that had not formed a biofilm. The cell in which the biofilm was formed was colored with a 0.4% crystal violet solution for 30 minutes, and then the remaining solution was washed cleanly using distilled water. The colored cell was dissolved in 200 μL of 95% ethanol, and the amount thereof was measured using absorbance (596 nm). The biofilm suppression effect of this sample was measured using the following formula, and the results are shown in Table 5 below.
バイオフィルム生成予防効果（％）＝（１−試料がコートされたセルの吸光度／対照群セルの吸光度）×１００
試料がコートされていないセルを対照群として使用し、５０％予防効果を示す濃度（ＩＣ５０）を求めた。
Biofilm formation prevention effect (%) = (1−absorbance of cell coated with sample / absorbance of control group cell) × 100
A cell that was not coated with a sample was used as a control group, and a concentration (IC 50 ) showing a 50% preventive effect was determined.
実施例７．バイオフィルム除去効果の評価
口腔内に既に形成されたバイオフィルムを効果的に除去することは、口腔の健康を改善させるに際して、生成を抑制することと同様に重要である。したがって、前記実施例５で製造された組成物と比較サンプルに対してバイオフィルムの除去効果を測定した。製造された組成物と比較試料に対するバクテリアバイオフィルム除去効果を測定するために、９６−ウェルプレートに様々なバクテリアバイオフィルムをＳｔｅｐａｎｏｖｉｃの方法によって次のようにコートした。各プレートに２００μＬの人工唾液を入れ、常温で３時間軽く振とうしながら培養した後、付着していない残りの人工唾液を除去した後、プレートを空気中で乾燥させる。プレートにバクテリア接種液２０μＬを入れ、３％スクロース含有ＢＨＩ培養液を用いて３７℃で２４時間培養してバイオフィルムを形成させた。残っている培養液を除去した後、２００μＬ ５００ｍＭ ＰＢＳを用いて、残ったバイオフィルムを形成していないセルを洗浄した。各セルに試験物または比較試料を０．５〜５０μｇ／ｍＬの濃度で処理し、１時間培養した。バイオフィルムを形成したセルを、０．４％クリスタルバイオレット溶液を用いて３０分間着色した後、蒸留水を用いて、残っている溶液を綺麗に洗浄した。着色されたセルを９５％エタノール２００μＬに溶かした後、吸光度を測定した（５９６ｎｍ）。各試料のバイオフィルム除去活性は処理前後の吸光度を比較して決定した。
Example 7 Evaluation of Biofilm Removal Effect Effective removal of a biofilm already formed in the oral cavity is as important as suppressing production when improving oral health. Therefore, the biofilm removal effect was measured on the composition prepared in Example 5 and the comparative sample. In order to measure the bacterial biofilm removal effect on the prepared composition and comparative samples, 96-well plates were coated with various bacterial biofilms by Stepanovic method as follows. 200 μL of artificial saliva is added to each plate, cultured while gently shaking at room temperature for 3 hours, the remaining artificial saliva not adhered is removed, and then the plate is dried in air. 20 μL of the bacterial inoculum was placed on the plate and cultured at 37 ° C. for 24 hours using a 3% sucrose-containing BHI culture to form a biofilm. After removing the remaining culture, 200 μL 500 mM PBS was used to wash the remaining cells that had not formed a biofilm. Each cell was treated with a test sample or a comparative sample at a concentration of 0.5 to 50 μg / mL and cultured for 1 hour. The cell on which the biofilm was formed was colored with a 0.4% crystal violet solution for 30 minutes, and then the remaining solution was washed cleanly with distilled water. After the colored cell was dissolved in 200 μL of 95% ethanol, the absorbance was measured (596 nm). The biofilm removal activity of each sample was determined by comparing the absorbance before and after treatment.
バイオフィルム除去効果は次の式によって計算した。５０％除去効果を示す濃度（ＩＣ５０）を求め、下記表６に示す。 The biofilm removal effect was calculated by the following formula. The concentration (IC 50 ) showing the 50% removal effect was determined and shown in Table 6 below.
バイオフィルム除去効果（％）＝（１−試料の吸光度／試料を処理していないセルの吸光度）×１００ Biofilm removal effect (%) = (1−absorbance of sample / absorbance of cell not treated with sample) × 100
実施例８．歯周疾患に対する予防および治療効果の評価
前記組成物の歯周疾患予防および治療効果を、下記表７に記載の方法に従う臨床試験によって評価した。
Example 8 FIG. Evaluation of preventive and therapeutic effects on periodontal diseases The preventive and therapeutic effects of the composition on periodontal diseases were evaluated by clinical trials according to the methods described in Table 7 below.
チューインガムの製造成分を下記表８に示す。 The production components of chewing gum are shown in Table 8 below.
前記評価結果、図１に示すように、本発明の組成物７を含むチューインガムを使用した試験群の歯周状態の改善効果および歯周疾患の予防効果が、対照群と比較して非常に優れることを確認した。 As a result of the evaluation, as shown in FIG. 1, the periodontal condition improving effect and the periodontal disease preventing effect of the test group using the chewing gum containing the composition 7 of the present invention are very excellent compared to the control group. It was confirmed.
実施例９．前記組成物の虫歯予防効果
前記組成物６の虫歯予防および虫歯内に存在するバクテリアに対する抗菌効果を、下記表９に記載の方法による臨床試験によって評価した。
Example 9 Caries preventive effect of the composition The antibacterial effect of the composition 6 and the antibacterial effect against bacteria present in the caries were evaluated by a clinical test according to the method described in Table 9 below.
前記評価結果、下記表１０に示すように、本発明の組成物６の虫歯改善効果および予防効果が優れることを確認した。 As a result of the evaluation, as shown in Table 10 below, it was confirmed that the caries improving effect and the preventive effect of the composition 6 of the present invention were excellent.
実施例１０．歯磨き効果の持続性の比較実験
一般人が日常生活で最も大きい不便を感じる口臭の発生など、口腔内不快度に及ぼす影響を、歯磨き粉を用いて下記表１１に記載の方法で評価した。対照歯磨き粉としては、口臭除去効果に優れるものと知られているカジメ抽出物およびクロルヘキシジンの含まれた歯磨き粉を使用した。
Example 10 Comparative experiment of persistence of toothpaste effect The effects on oral discomfort, such as the occurrence of bad breath, which ordinary people feel the most inconvenient in daily life, were evaluated by the method described in Table 11 below using toothpaste. As a control toothpaste, a toothpaste containing a scab extract and chlorhexidine, which is known to have an excellent bad breath removal effect, was used.
前記実験結果によれば、図２に示すように、本発明の組成物５を含む歯磨き粉を使用した実験群は歯磨き効果が長時間持続されたが、活性成分としてカジメ抽出物を含む歯磨き粉またはクロルヘキシジンを含む歯磨き粉を使用した実験群は歯磨き効果が長く持続されなかった 。
According to the experimental results, as shown in FIG. 2, the experimental group using the toothpaste containing the composition 5 of the present invention maintained the toothpaste effect for a long time, but the toothpaste or chlorhexidine containing the Kajime extract as an active ingredient. In the experimental group using toothpaste containing, the toothpaste effect was not sustained for a long time.
Claims (9)
2. Maintenance of oral health according to claim 1, characterized by having the functions of inhibiting and removing occurrence of oral biofilm, preventing dental corrosion, preventing plaque formation, preventing and improving gingival diseases, or improving bad breath. And improving compositions.
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