JP2018070463A - Anti-periodontal pathogen agent containing cyclic dipeptide - Google Patents
Anti-periodontal pathogen agent containing cyclic dipeptide Download PDFInfo
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Abstract
Description
本発明は、環状ジペプチドを含む抗歯周病原細菌剤に関する。 The present invention relates to an anti-periodontopathic bacterial agent comprising a cyclic dipeptide.
口腔内には700種をこえる細菌が生息している。これらの細菌は歯、歯肉溝、舌背など特有の生息場所でフローラを形成し、デンタルプラークを形成している。う蝕と並ぶ歯科の二大疾患の一つである歯周病は、プラーク中に存在する特定のグラム陰性嫌気性菌が原因となって発生する感染性疾患である。成人性歯周病の患者から高頻度で分離されるポルフィロモナス・ジンジバリス（Porphyromonas gingivalis; 以下、P. gingivalisと略記することがある）は口腔内細菌の中でも特に強い病原性を有しており、最も重要な歯周病原細菌であると考えられている（非特許文献１)。歯周病の予防・治療では、歯周病の原因細菌（歯周病原細菌）の減少や再発防止を目的として、抗菌薬や消毒薬を使用する。しかし、薬剤に対するアレルギーの可能性や薬剤耐性菌が出現するという問題がある。 There are over 700 species of bacteria in the mouth. These bacteria form flora and form dental plaques in specific habitats such as teeth, gingival sulcus and dorsum of tongue. Periodontal disease, one of the two major dental diseases along with dental caries, is an infectious disease caused by specific gram-negative anaerobic bacteria present in plaques. Porphyromonas gingivalis (hereinafter sometimes abbreviated as P. gingivalis) isolated from patients with adult periodontal disease has a particularly strong pathogenicity among oral bacteria. It is considered to be the most important periodontal pathogenic bacterium (Non-patent Document 1). In the prevention and treatment of periodontal disease, antibacterial drugs and disinfectants are used for the purpose of reducing periodontal disease-causing bacteria (periodontal pathogenic bacteria) and preventing recurrence. However, there is a problem that allergies to drugs and drug-resistant bacteria appear.
そこで近年、歯周病治療・予防においても、ビフィズス菌や乳酸菌に代表されるプロバイオティクスの有用性に注目が集まっている（非特許文献２）。プロバイオティクスとは「腸内細菌叢を改善し，宿主に有益な健康効果をもたらす微生物とそれらの増殖，促進をもたらす生きた微生物」であるとFullerにより定義されている（非特許文献３）。プロバイオティクスを直接口腔内に作用させてう蝕や歯周病などの口腔疾患を予防するいくつかの試みが行われている。例えば、Ishikawaらは、ラクトバチルス・サリバリウス（Lactobacillus salivarius）TI2711株の4週間の経口投与により、主要な歯周病原細菌であるP. gingivalis、プレボテラ・インターメディア（Prevotella intermedia）、及びプレボテラ・ニグレセンス（Prevotella nigrescens）が有意に減少したことを報告している（非特許文献４）。Vivekanandaらは、ラクトバチルス・ロイテリ（Lactobacillus reuteri）DSM17938株及びATCC PTA5289株の生菌が歯周病患者の口腔内P. gingivalisの菌数を減少させることを報告している（非特許文献５）。しかし、プロバイオティクスが口腔内細菌叢に与える影響に関する報告はまだ少ない。 Therefore, in recent years, attention has been focused on the usefulness of probiotics represented by bifidobacteria and lactic acid bacteria also in the treatment and prevention of periodontal disease (Non-patent Document 2). Probiotics are defined by Fuller as "microorganisms that improve the gut microbiota and provide beneficial health effects to the host, and live microorganisms that proliferate and promote them" (Non-patent Document 3) . Several attempts have been made to prevent oral diseases such as caries and periodontal disease by applying probiotics directly to the oral cavity. For example, Ishikawa et al., By oral administration of Lactobacillus salivarius strain TI2711 for 4 weeks, P. gingivalis, Prevotella intermedia, and Prevotella niglesens (Major periodontal pathogenic bacteria) (Prevotella nigrescens) has been significantly reduced (Non-patent Document 4). Vivekananda et al. Have reported that living bacteria of Lactobacillus reuteri DSM17938 strain and ATCC PTA5289 strain reduce the number of oral P. gingivalis bacteria in periodontal patients (Non-patent Document 5). . However, there are still few reports on the effects of probiotics on the oral flora.
乳酸菌が産生する抗菌物質として、これまでに乳酸や酢酸などの有機酸、過酸化水素、バクテリオシンが報告されている（非特許文献６〜８）。Takahashiらは、P. gingivalisは、酸感受性が高くpH6.5以下で増殖が抑制されることを報告している（非特許文献９）。MatsuokaらはP. gingivalisに対するL. salivarius TI2711株の抗菌活性は乳酸によるものと報告している（非特許文献１０）。しかし、酸によって口腔内の低pH状態を持続させることには、う蝕の発症や知覚過敏を誘発するリスクが考えられる。またバクテリオシンの多くは比較的高分子量のペプチドであり、製造技術に煩雑な工程が必要なため、労力、コスト、時間を要することから歯周病予防のためのバクテリオシンの使用は普及していない。そこで、歯周病の治療や予防のためにプロバイオティクス菌が産生する新たな抗菌物質が望まれている。
As antibacterial substances produced by lactic acid bacteria, organic acids such as lactic acid and acetic acid, hydrogen peroxide, and bacteriocin have been reported (
本発明は、歯周病原細菌に対する抗菌性低分子化合物を提供することを課題とする。 An object of the present invention is to provide an antibacterial low-molecular compound against periodontal pathogenic bacteria.
本発明者らは、上記課題を解決するため鋭意検討を重ねた結果、特定の乳酸菌株が、歯周病原細菌、特にポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）に対する強い抗菌作用を有するヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを産生することを見出し、その知見に基づいて本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a specific lactic acid strain has a strong antibacterial action against periodontopathic bacteria, particularly Porphyromonas gingivalis. It has been found that hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione is produced, and the present invention has been completed based on the findings.
すなわち、本発明は以下を包含する。
［１］下記式で表される化合物：
又はその塩を含む、抗歯周病原細菌剤。
［２］歯周病原細菌がポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）である、上記［１］に記載の抗歯周病原細菌剤。
［３］Ｒ１がヒドロキシル基であり、Ｒ２が水素原子である、上記［１］に記載の抗歯周病原細菌剤。
［４］ポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）の増殖を抑制するための、上記［１］〜［３］のいずれかに記載の抗歯周病原細菌剤。
［５］上記［１］〜［４］のいずれかに記載の抗歯周病原細菌剤を含む、抗歯周病剤。
［６］下記式で表される化合物：
又はその塩を含む、歯周病原細菌の増殖を抑制するための飲食品。
［７］下記式で表される化合物：
又はその塩を含む、歯周病の治療又は予防用の医薬。
That is, the present invention includes the following.
[1] A compound represented by the following formula:
Or an anti-periodontopathic bacterial agent comprising a salt thereof.
[2] The anti-periodontopathic bacterial agent according to the above [1], wherein the periodontopathic bacterium is Porphyromonas gingivalis.
[3] The anti-periodontal pathogenic bacterial agent according to the above [1], wherein R1 is a hydroxyl group and R2 is a hydrogen atom.
[4] The anti-periodontopathic bacterial agent according to any one of [1] to [3] above, which suppresses the growth of Porphyromonas gingivalis.
[5] An anti-periodontal disease agent comprising the anti-periodontopathic bacterial agent according to any one of [1] to [4].
[6] A compound represented by the following formula:
Or the food-drinks for suppressing the growth of periodontopathic bacteria containing the salt.
[7] A compound represented by the following formula:
Or the pharmaceutical for treatment or prevention of periodontal disease containing the salt.
本発明によれば、歯周病原細菌、特にポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）に対する高い抗菌活性をもたらすことができる。 ADVANTAGE OF THE INVENTION According to this invention, the high antimicrobial activity with respect to periodontopathic bacteria, especially Porphyromonas gingivalis (Porphyromonas gingivalis) can be brought about.
以下、本発明を詳細に説明する。
本発明は、下記構造式(I)で表される化合物（以下、化合物(I)と称する）又はその塩が抗歯周病原細菌活性を有することに基づき、その抗歯周病原細菌用途を提供する。化合物(I)は環状ジペプチド構造を有する低分子化合物である。
Hereinafter, the present invention will be described in detail.
The present invention provides the use of anti-periodontopathic bacteria based on the fact that a compound represented by the following structural formula (I) (hereinafter referred to as compound (I)) or a salt thereof has anti-periodontopathic bacterial activity. To do. Compound (I) is a low molecular compound having a cyclic dipeptide structure.
化合物(I)の上記式中、Ｒ１及びＲ２はそれぞれ独立に、水素原子又はヒドロキシル基である。Ｒ１及びＲ２が水素原子である場合、化合物(I)はプロリンとロイシンの環状ジペプチドである。Ｒ１が水素原子、Ｒ２がヒドロキシル基である場合、化合物（Ｉ）はヒドロキシプロリン（３−ヒドロキシプロリン）とロイシンの環状ジペプチドである。Ｒ１がヒドロキシル基、Ｒ２が水素原子である場合、化合物(I)はヒドロキシプロリン（４−ヒドロキシプロリン）とロイシンの環状ジペプチドである。化合物(I)は、好ましくは、ヒドロキシプロリン又はプロリンとロイシンとの環状ジペプチドであり、その場合、上記式中、Ｒ１及びＲ２の少なくとも一方は水素原子である。化合物(I)は、Ｒ１又はＲ２に関してシス体であってもトランス体であってもよい。化合物(I)の特に好ましい例は、ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンである。
In the above formula of compound (I),
本発明は、化合物(I)又はその塩を含む、抗歯周病原細菌剤を提供する。 The present invention provides an anti-periodontopathic bacterial agent comprising compound (I) or a salt thereof.
本発明の化合物(I)の塩は、毒性を有しない限り特に限定されないが、飲食品又は医薬に適した塩が好ましく、製薬上許容される塩がより好ましく、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、リチウム塩、カルシウム塩等が挙げられる。 The salt of the compound (I) of the present invention is not particularly limited as long as it has no toxicity, but is preferably a salt suitable for food or drink or a medicine, more preferably a pharmaceutically acceptable salt, for example, sodium salt, potassium salt, A magnesium salt, lithium salt, calcium salt, etc. are mentioned.
環状ジペプチドである化合物(I)又はその塩は、公知のペプチド製造法を用いて製造することができる。例えば、対応する直鎖ペプチド（直鎖ジペプチド）を合成し、それを環化し、必要に応じて精製することにより、当該化合物を製造することができる。 Compound (I) which is a cyclic dipeptide or a salt thereof can be produced using a known peptide production method. For example, the compound can be produced by synthesizing a corresponding linear peptide (linear dipeptide), cyclizing it, and purifying it as necessary.
具体的には、例えば、ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンは、特開２０１４−１２５４２７号公報に記載の方法を用いて合成できる。簡潔に説明すると、まず、ロイシンとヒドロキシプロリン（４−ヒドロキシプロリン）の直鎖ペプチドおよそ2mgを0.1Mリン酸緩衝液（pH8）100μlに溶解し、90℃で24時間インキュベートする。得られた環状ペプチドを逆相クロマトグラフィーにより精製する。逆相クロマトグラフィーは、例えばTSKgel ODS-80Tsカラム（4.6mm×150mm）を用い、溶媒A）H2O/TFA（100/0.1; v/v/v）、溶媒B）アセトニトリル/H2O/TFA（50/50/0.1; v/v/v）を用いて、カラム温度40℃、流速1ml/min、グラディエント条件:0〜15分 0％B、15〜30分 90%B、30〜33分 100%B、33〜35分 0%B、35〜50分 0%B、UV検出（214nm）で精製する。このようにして環状ペプチドであるヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを得ることができる。化合物(I)の範囲に含まれる他の化合物も出発物質となる直鎖ペプチドを変更することにより同様に合成することができる。なお、直鎖ペプチドはペプチド合成装置等を用いて合成することができ、コスモバイオ株式会社等に委託して合成したものを入手することもできる。
Specifically, for example, hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione is obtained by the method described in JP-A No. 2014-125427. Can be synthesized. Briefly, first, approximately 2 mg of a linear peptide of leucine and hydroxyproline (4-hydroxyproline) is dissolved in 100 μl of 0.1 M phosphate buffer (pH 8) and incubated at 90 ° C. for 24 hours. The resulting cyclic peptide is purified by reverse phase chromatography. Reverse phase chromatography uses, for example, a TSKgel ODS-80Ts column (4.6 mm × 150 mm), solvent A) H 2 O / TFA (100 / 0.1; v / v / v), solvent B) acetonitrile / H 2 O / Using TFA (50/50 / 0.1; v / v / v),
また、ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン等の化合物(I)は、それを産生する能力を有する微生物を培養し、培養物から回収することにより製造することもできる。ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン等の化合物(I)の産生能を有する微生物は、乳酸菌であることが好ましい。ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン等の化合物(I)の産生能を有する微生物としては、例えば、Lactobacillus fermentum ALAL020株が挙げられるが、これに限定されない。 In addition, compound (I) such as hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione is used to culture microorganisms having the ability to produce it. It can also be produced by recovering from the culture. The microorganism having the ability to produce compound (I) such as hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione is preferably a lactic acid bacterium. Examples of microorganisms capable of producing compound (I) such as hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione include Lactobacillus fermentum ALAL020 strain However, it is not limited to this.
一実施形態では、ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンの産生能を有する微生物を培養し、培養上清を採取し、酢酸エチル等の有機溶媒を用いた抽出を行い、水層を有機層から分離して採取し、クロマトグラフィー（例えば、ゲル濾過クロマトグラフィー）等の分画技術を用いて分画し、分子量226.131の環状ジペプチドであるヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを含む画分を取得し、そこからヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを取得することができる。ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを含む画分は、クロマトグラフィー（例えば、高速液体クロマトグラフィー（HPLC））等の分画技術を用いてさらに分画・精製を行ってもよい。分画技術としては、例えば、限外濾過や精密濾過等の膜ろ過、ゲル浸透クロマトグラフィーやゲル濾過クロマトグラフィー（例えば、高速液体クロマトグラフィー）等のサイズ排除クロマトグラフィー、分配クロマトグラフィー、吸着クロマトグラフィー、イオン交換クロマトグラフィー等が挙げられる。ヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンを含む画分を取得するに当たっては、各画分について抗菌性試験を行い、ポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）に対する抗菌活性を有することを確認したり抗菌活性を有する画分を選抜したりすることも好ましい。抽出に用いる有機溶媒としては、酢酸エチル、メタノール、エタノール、クロロホルム、アセトン、ヘキサン、ジクロロメタン、及びこれらの混合溶媒等が挙げられる。他の化合物(I)の範囲に含まれるヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン以外の化合物を、その産生能を有する微生物から同様の方法で取得してもよい。 In one embodiment, a microorganism capable of producing hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione is cultured, and the culture supernatant is collected. Extraction using an organic solvent such as ethyl acetate, the aqueous layer is separated from the organic layer and collected, fractionated using a fractionation technique such as chromatography (eg, gel filtration chromatography), and the molecular weight 226.131 A fraction containing hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione, which is a cyclic dipeptide of hexahydro-7-hydroxy- 3- (2-Methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione can be obtained. A fraction containing hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione can be used for chromatography (for example, high performance liquid chromatography (HPLC)), etc. Further fractionation / purification may be performed using fractionation technology. Examples of fractionation techniques include membrane filtration such as ultrafiltration and microfiltration, size exclusion chromatography such as gel permeation chromatography and gel filtration chromatography (for example, high performance liquid chromatography), distribution chromatography, and adsorption chromatography. And ion exchange chromatography. In obtaining fractions containing hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione, antibacterial tests were conducted on each fraction, It is also preferable to confirm that it has antibacterial activity against Porphyromonas gingivalis or to select a fraction having antibacterial activity. Examples of the organic solvent used for extraction include ethyl acetate, methanol, ethanol, chloroform, acetone, hexane, dichloromethane, and mixed solvents thereof. Has ability to produce compounds other than hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione included in the range of other compounds (I) You may acquire from a microorganism by the same method.
得られた化合物がヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン又は化合物(I)の範囲に含まれる他の化合物であることの確認は、核磁気共鳴（NMR）分析を用いて行うことができる。またLC-MS等の質量分析やFT-IR（フーリエ変換赤外分光光度計）分析により当該化合物の構造についてさらに確認することもできる。このようにして得られたヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオン等の化合物(I)は、常法により塩の形態とすることもできる。 The resulting compound is hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione or other compound within the scope of compound (I) This confirmation can be performed using nuclear magnetic resonance (NMR) analysis. Further, the structure of the compound can be further confirmed by mass spectrometry such as LC-MS or FT-IR (Fourier transform infrared spectrophotometer) analysis. The compound (I) such as hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dione thus obtained can be converted into a salt form by a conventional method. It can also be.
このようにして得られる化合物(I)は、歯周病原細菌、特にポルフィロモナス・ジンジバリス（Porphyromonas gingivalis）に対する抗菌作用を有する。ここで歯周病原細菌とは、歯周病の原因となる細菌を指す。 The compound (I) thus obtained has an antibacterial action against periodontopathic bacteria, particularly Porphyromonas gingivalis. Here, periodontopathic bacteria refer to bacteria that cause periodontal disease.
本発明において化合物(I)及びそれを含む画分又は組成物等の抗菌活性は、公知の抗菌性試験によって確認することができるが、特に、後述の実施例に記載の抗菌性試験の方法に従って確認することができる。 In the present invention, the antibacterial activity of the compound (I) and the fraction or composition containing the compound (I) can be confirmed by a known antibacterial test. Can be confirmed.
具体的には、まず、ポルフィロモナス・ジンジバリスATCC 33277株を、ヘミンを5 μg/ml、メナジオンを1 μg/ml、酵母エキスを0.5 %、及び塩酸システインをBHI（Brain heart infusion）液体培地に0.05 %添加したBHI改変液体培地に接種して37℃で嫌気培養する（N2:80%, CO2:10%, H2:10%）。また、被験物質である化合物(I)又はそれを含む画分若しくは組成物を、BHI改変液体培地で段階希釈し、培地を調製する。調製した培地に、培養したポルフィロモナス・ジンジバリスATCC 33277株（1×107CFU/ml）を10μl接種し、37℃で48時間、嫌気培養する（N2:80%, CO2:10%, H2:10%）。培養後、菌体濃度とよく相関する620nmの吸光度を測定し、吸光度0.1以下を示した被験物質を、ポルフィロモナス・ジンジバリスの増殖を抑制する（抗菌活性有り）と判定することができる。さらに、抗菌活性が示された被験物質の最大希釈倍数が大きいほど、抗菌活性が高いことが示される。
Specifically, first, Porphyromonas gingivalis ATCC 33277, hemin 5 μg / ml,
抗菌性試験の指標菌株として用いることができるポルフィロモナス・ジンジバリスATCC 33277株（Porphyromonas gingivalis; P. gingivalis）は、ATCC（American Type Culture Collection）からカタログ番号ATCC 33277に基づいて入手することができる。 Porphyromonas gingivalis strain (P. gingivalis), which can be used as an indicator strain for antibacterial tests, can be obtained from ATCC (American Type Culture Collection) based on catalog number ATCC 33277.
以上のような抗菌性試験で確認できるとおり、化合物(I)又はその塩は、代表的な歯周病原細菌であるポルフィロモナス・ジンジバリスに対する高い抗菌活性を有することから、抗歯周病原細菌剤の有効成分として用いることができる。本発明において抗歯周病原細菌剤は、歯周病原細菌に対する抗菌活性をもたらす薬剤を意味する。好ましい一実施形態では、化合物(I)又はその塩を含む抗歯周病原細菌剤は、ポルフィロモナス・ジンジバリスに対する抗歯周病原細菌剤として用いることができる。一実施形態では、化合物(I)又はその塩を含む抗歯周病原細菌剤は、ポルフィロモナス・ジンジバリスの増殖を抑制するために好適に使用することができる。化合物(I)又はその塩を含む抗歯周病原細菌剤は、in vitro又はin vivoで使用することができる。本発明の抗歯周病原細菌剤は、抗歯周病効果をもたらす上で有効量の化合物(I)又はその塩を含む。 As can be confirmed by the antibacterial test as described above, the compound (I) or a salt thereof has a high antibacterial activity against Porphyromonas gingivalis, which is a typical periodontal pathogenic bacterium. It can be used as an active ingredient. In the present invention, the anti-periodontopathic bacterial agent means an agent that provides antibacterial activity against periodontal pathogenic bacteria. In a preferred embodiment, an anti-periodontopathic bacterial agent comprising compound (I) or a salt thereof can be used as an anti-periodontopathic bacterial agent against Porphyromonas gingivalis. In one embodiment, an anti-periodontopathic bacterial agent comprising compound (I) or a salt thereof can be suitably used for inhibiting the growth of Porphyromonas gingivalis. The anti-periodontopathic bacterial agent containing compound (I) or a salt thereof can be used in vitro or in vivo. The anti-periodontopathic bacterial agent of the present invention contains an effective amount of compound (I) or a salt thereof for providing an anti-periodontal disease effect.
本発明は、化合物(I)又はその塩を含む抗歯周病原細菌剤を含む抗歯周病剤を提供し、また、化合物(I)若しくはその塩、又はそれを含む抗歯周病原細菌剤の、抗歯周病剤としての使用を提供する。本発明において抗歯周病剤とは、歯周病原細菌（とりわけ、ポルフィロモナス・ジンジバリス）の増殖を抑制し、歯周病原細菌数（とりわけ、ポルフィロモナス・ジンジバリスの菌数）を減少させることにより、抗歯周病効果をもたらす薬剤を意味する。抗歯周病効果としては、例えば、歯周病の症状又は病的状態を改善若しくは回復させるか又はその進行（悪化）を抑制し、好ましくは歯周病の発症又は再発を防止することが挙げられる。なお一般に歯周病は歯肉炎と歯周炎を包含する。 The present invention provides an anti-periodontal disease agent comprising an anti-periodontopathic bacterial agent comprising compound (I) or a salt thereof, and also provides an anti-periodontopathic bacterial agent comprising compound (I) or a salt thereof, or the same. For use as an anti-periodontal disease agent. In the present invention, the anti-periodontal disease agent suppresses the growth of periodontopathic bacteria (particularly, Porphyromonas gingivalis) and decreases the periodontal pathogenic bacteria number (particularly, the number of Porphyromonas gingivalis). This means a drug that has an anti-periodontal disease effect. Examples of the anti-periodontal disease effect include improvement or recovery of periodontal disease symptoms or pathological conditions, or suppression of progression (deterioration) thereof, preferably prevention of onset or recurrence of periodontal disease. It is done. In general, periodontal disease includes gingivitis and periodontitis.
本発明は、化合物(I)又はその塩を含む、歯周病の治療又は予防用の医薬も提供する。本発明の歯周病の治療又は予防用の医薬は、抗歯周病効果をもたらす上で有効量の化合物(I)又はその塩を含む。本発明の医薬は、医薬品又は医薬組成物であってよい。本発明に係る医薬は、経口用又は口腔用であることが好ましい。本発明に係る医薬は、錠剤、カプセル剤、トローチ剤、ゲル剤、フィルム剤、顆粒剤、細粒剤、散剤、徐放性製剤、懸濁液、エマルジョン剤、シロップ剤、エリキシル剤、液剤、軟膏剤、貼付剤、含嗽剤、スプレー剤等の剤形でありうるが、これらに限定されない。本発明に係る医薬は、保存剤、賦形剤、緩衝剤、界面活性剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤、溶解補助剤、懸濁化剤、コーティング剤などの製薬上許容される担体又は添加剤をさらに含んでよい。本発明に係る医薬はまた、他の薬効成分等を含有してもよい。 The present invention also provides a medicament for treating or preventing periodontal disease, which comprises compound (I) or a salt thereof. The medicament for treatment or prevention of periodontal disease of the present invention comprises an effective amount of compound (I) or a salt thereof for providing an anti-periodontal disease effect. The medicament of the present invention may be a pharmaceutical product or a pharmaceutical composition. The medicament according to the present invention is preferably for oral or oral use. The medicament according to the present invention includes tablets, capsules, troches, gels, films, granules, fine granules, powders, sustained-release preparations, suspensions, emulsions, syrups, elixirs, liquids, The dosage form may be, but is not limited to, an ointment, a patch, a gargle, and a spray. The medicament according to the present invention includes preservatives, excipients, buffers, surfactants, binders, disintegrants, lubricants, colorants, flavoring agents, solubilizers, suspending agents, coating agents, etc. Further pharmaceutically acceptable carriers or additives may be included. The medicament according to the present invention may also contain other medicinal ingredients.
本発明は、化合物(I)又はその塩を含む、飲食品も提供する。本発明の飲食品は、抗歯周病効果をもたらす上で有効量の化合物(I)又はその塩を含むことが好ましい。本発明の飲食品は飲食品組成物であってもよい。本発明の飲食品は、化合物(I)又はその塩を飲食品に添加したものであってもよい。本発明に係る飲食品は、食品及び飲料を包含する。本発明に係る飲食品は、任意のカテゴリー又は種類であってよく、菓子（ガム、キャンディー、グミ、ゼリー等）、飲料、乳製品、惣菜、パン、米・麺類、調味料等であってもよい。本発明に係る飲食品は、機能性食品であってもよい。「機能性食品」とは、生体に対して通常の栄養摂取ではない一定の効果を付与することを目的とした食品を意味し、例えば、特定保健用食品（条件付きトクホ[特定保健用食品]を含む）や栄養機能食品を含む保健機能食品、機能性表示食品、特別用途食品（介護用食品、乳幼児用食品、妊産婦用食品、病者用食品、乳児用調製乳、高齢者用食品等）、健康食品、サプリメント（例えば、錠剤、被覆錠、糖衣錠、カプセル及び液剤などの各種剤形のもの）及び美容食品（例えばダイエット食品）などを包含する。本発明に係る機能性食品はまた、コーデックス（FAO/WHO合同食品規格委員会）の食品規格に基づく健康強調表示（Health claim）が適用される健康食品を包含する。本発明に係る飲食品の形状は特に限定されず、固形、液状、流動食状、ゼリー状、フィルム状、タブレット状、顆粒状、カプセル状など、任意の飲食品形状をとることができる。本発明に係る飲食品は、化合物(I)又はその塩に加えて、様々な食品素材を含んでよく、例えば、糖質、タンパク質、脂質、食物繊維、ビタミン類、ミネラル類、及び食品添加物（例えば、香料、保存剤、pH調整剤等）などを含んでよい。 The present invention also provides a food or drink containing compound (I) or a salt thereof. The food and drink of the present invention preferably contains an effective amount of compound (I) or a salt thereof for providing an anti-periodontal disease effect. The food or drink of the present invention may be a food or drink composition. The food or drink of the present invention may be one obtained by adding compound (I) or a salt thereof to the food or drink. The food and drink according to the present invention includes food and beverages. The food or drink according to the present invention may be of any category or type, and may be a confectionery (gum, candy, gummi, jelly, etc.), beverage, dairy product, prepared food, bread, rice / noodles, seasoning, etc. Good. The food or drink according to the present invention may be a functional food. “Functional food” means food intended to give a certain effect to the living body that is not normal nutrition intake, such as food for specified health use (conditional tokuho [food for specified health use] Health foods including functional foods, functional labeling foods, special-purpose foods (food for nursing care, food for infants, food for pregnant women, food for the sick, infant formula, food for the elderly, etc.) , Health foods, supplements (for example, in various dosage forms such as tablets, coated tablets, dragees, capsules and liquids) and beauty foods (for example, diet foods). The functional food according to the present invention also includes health foods to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied. The shape of the food / beverage products according to the present invention is not particularly limited, and can be any food / beverage product shape such as solid, liquid, liquid food, jelly, film, tablet, granule or capsule. The food and drink according to the present invention may contain various food materials in addition to compound (I) or a salt thereof, such as carbohydrates, proteins, lipids, dietary fibers, vitamins, minerals, and food additives. (For example, a fragrance | flavor, a preservative, a pH adjuster etc.) etc. may be included.
本発明に係る医薬及び飲食品は、歯周病原細菌（とりわけ、ポルフィロモナス・ジンジバリス）の増殖を抑制するために用いることができ、及び／又は歯周病原細菌に起因する疾患、好ましくは歯周病の治療又は予防用に用いることができる。本発明に係る医薬及び飲食品は、以下に限定するものではないが、例えば１用量当たり１mg〜1000mgの化合物(I)又はその塩を含んでいてもよい。ここで１用量とは、歯周病原細菌、特にポルフィロモナス・ジンジバリスに対する増殖抑制効果をもたらすために摂取すべき１回当たりの投与量又は摂取量を指す。 The medicament and food and drink according to the present invention can be used for suppressing the growth of periodontopathic bacteria (in particular, Porphyromonas gingivalis) and / or diseases caused by periodontopathic bacteria, preferably teeth. It can be used for treatment or prevention of perinatal disease. Although the medicine and food-drinks which concern on this invention are not limited to the following, they may contain 1 mg-1000 mg of compound (I) or its salt per dose, for example. Here, one dose refers to a single dose or intake to be taken in order to bring about a growth-inhibiting effect against periodontal pathogenic bacteria, particularly Porphyromonas gingivalis.
本発明に係る医薬、飲食品、及び抗歯周病原細菌剤は、本発明の化合物(I)又はその塩を産生する能力を有する微生物（例えば、Lactobacillus fermentum ALAL020株などのラクトバチルス・ファーメンタム菌）を含んでもよいが、含まなくてもよい。 The medicament, food and drink, and anti-periodontal pathogenic bacterial agent according to the present invention are microorganisms having the ability to produce the compound (I) of the present invention or a salt thereof (for example, Lactobacillus fermentum ALAL020 strain and other Lactobacillus fermentum strains). ) May or may not be included.
本発明に係る医薬及び飲食品は、化合物(I)又はその塩を有効成分として用いて、常法により製造することができる。 The medicament and food and drink according to the present invention can be produced by a conventional method using Compound (I) or a salt thereof as an active ingredient.
本発明において「歯周病の治療」とは、歯周病原細菌（とりわけ、ポルフィロモナス・ジンジバリス）の増殖等を抑制し、口腔内の歯周病原細菌数（とりわけ、ポルフィロモナス・ジンジバリスの菌数）を減少させることにより、歯周病の症状又は病的状態を改善若しくは回復させるか又はその進行（悪化）を抑制することを意味する。本発明において「歯周病の予防」とは、歯周病原細菌（とりわけ、ポルフィロモナス・ジンジバリス）の増殖等を抑制し、口腔内の歯周病原細菌数（とりわけ、ポルフィロモナス・ジンジバリスの菌数）を減少させることにより、歯周病の発症又は再発を防止することを意味する。 In the present invention, “treatment of periodontal disease” refers to the suppression of the growth of periodontopathic bacteria (particularly porphyromonas gingivalis) and the like, and the number of periodontal pathogenic bacteria in the oral cavity (particularly porphyromonas gingivalis). By reducing the number of bacteria, it means that the symptoms or pathological state of periodontal disease is improved or recovered, or the progression (deterioration) is suppressed. In the present invention, “prevention of periodontal disease” means that the growth of periodontopathic bacteria (especially Porphyromonas gingivalis) is suppressed, and the number of periodontopathic bacteria in the oral cavity (especially Porphyromonas gingivalis) By reducing the number of bacteria), it means preventing the onset or recurrence of periodontal disease.
本発明は、化合物(I)若しくはその塩、それを含む抗歯周病原細菌剤、抗歯周病剤、又は飲食品若しくは医薬を対象に投与することにより、被験体に抗歯周病効果をもたらす方法も提供する。したがって、この方法は歯周病を治療又は予防するために用いることができ、本発明は歯周病の治療又は予防方法も提供する。本発明における投与量は、被験体の年齢、体重、歯周病の状態等にも依存し、以下に限定するものではないが、例えば１用量当たり１mg〜1000mgの化合物(I)又はその塩を被験体に投与してもよい。ここで１用量とは、歯周病原細菌、特にポルフィロモナス・ジンジバリスに対する増殖抑制効果をもたらすために投与すべき１回当たりの投与量又は摂取量を指す。本発明に係る化合物(I)若しくはその塩、それを含む抗歯周病原細菌剤、抗歯周病剤、又は飲食品若しくは医薬は、剤形、投与経路等により異なるが、１日１回又は複数回投与してもよいし、さらに低い頻度、例えば、１週間〜１年に１回〜数回投与してもよい。 The present invention provides an anti-periodontal disease effect to a subject by administering to a subject compound (I) or a salt thereof, an anti-periodontopathic bacterial agent containing the same, an anti-periodontal disease agent, a food or drink, or a medicine. It also provides a way to bring about. Therefore, this method can be used for treating or preventing periodontal disease, and the present invention also provides a method for treating or preventing periodontal disease. The dose in the present invention depends on the age, body weight, periodontal disease state, etc. of the subject, and is not limited to the following. For example, 1 mg to 1000 mg of Compound (I) or a salt thereof is administered per dose. It may be administered to a subject. Here, one dose refers to a single dose or intake to be administered in order to bring about a growth inhibitory effect against periodontopathic bacteria, particularly Porphyromonas gingivalis. Compound (I) or a salt thereof according to the present invention, an anti-periodontopathic bacterial agent containing the compound, an anti-periodontal disease agent, or a food or drink, or a drug varies depending on the dosage form, administration route, etc., but once a day or It may be administered multiple times, or may be administered at a lower frequency, for example, once a week to once a year to several times.
本発明において化合物(I)若しくはその塩、それを含む抗歯周病原細菌剤、抗歯周病剤、又は飲食品若しくは医薬を投与される又は摂取する対象は、歯を有する動物であり、好ましくは、ヒト、家畜（ウマ、ウシ、ヒツジ、ヤギ、ラバ、ラクダ等）、愛玩動物（イヌ、ネコ、フェレット、ウサギなど）、実験動物（ラット、マウス、サル）、動物園等で飼育されている動物（ライオン、チーター、ゾウ、カンガルーなど）等を含む哺乳動物である。特に、歯周病の兆候又は素因（遺伝的又は環境的要因）を有する対象、う蝕や歯周病に罹患している対象、う蝕や歯周病の治療が完了した対象、歯周病の予防が特に望まれる対象（例えば、高齢者、乳幼児、妊産婦、宇宙飛行士）等が、本発明の投与対象としてより好ましい。 In the present invention, the subject to be administered or ingested with the compound (I) or a salt thereof, an anti-periodontopathic bacterial agent containing the same, an anti-periodontal disease agent, or a food or drink or a medicament is an animal having teeth, preferably Is bred in humans, livestock (horses, cows, sheep, goats, mules, camels, etc.), pets (dogs, cats, ferrets, rabbits, etc.), laboratory animals (rats, mice, monkeys), zoos, etc. Mammals including animals (lions, cheetahs, elephants, kangaroos, etc.). In particular, subjects who have signs or predisposition (genetic or environmental factors) of periodontal disease, subjects who have caries or periodontal disease, subjects who have completed treatment of caries or periodontal disease, periodontal disease Subjects (for example, elderly people, infants, pregnant women, astronauts) and the like for which prevention is particularly desired are more preferable as administration subjects of the present invention.
以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
［実施例１］抗菌活性を有する乳酸菌のスクリーニング
本実施例では、鶴見大学歯学部口腔微生物講座で保有する参照株6株、食品由来株12株、口腔由来株21株と、エイ・エル・エイ中央研究所で保有する豆乳発酵食品由来株11株の計50株の乳酸菌（表１参照）を抗菌活性についてスクリーニングした。
[Example 1] Screening for Lactic Acid Bacteria with Antibacterial Activity In this example, 6 reference strains, 12 food-derived strains, 21 oral-derived strains held at Tsurumi University Dental School Oral Microbiology Course, A total of 50 lactic acid bacteria (see Table 1) of 11 strains derived from fermented soymilk held by the laboratory were screened for antibacterial activity.
表１に示す各乳酸菌はMRS（de Man-Rogosa-Sharpe）液体培地（Difco, Becton Dickinson and Company, Sparks, MD, USA）にて37℃で24時間嫌気培養した（N2:80%, CO2:10%, H2:10%）。
Each lactic acid bacterium shown in Table 1 was anaerobically cultured at 37 ° C. for 24 hours in an MRS (de Man-Rogosa-Sharpe) liquid medium (Difco, Becton Dickinson and Company, Sparks, MD, USA) (N 2 : 80%,
抗菌性を試験するための指標菌株としてはポルフィロモナス・ジンジバリス（Porphyromonas gingivalis; 以下、P. gingivalisとも称する）ATCC(R)33277TM株を使用した。P. gingivalis ATCC 33277株は、BHI（Brain heart infusion）液体培地（Difco, Becton Dickinson and Company, Sparks, MD, USA）にヘミン（Sigma, St. Louis, MO, USA）を5 μg/ml、メナジオン（Wako Pure Chemical Industries, Ltd., Osaka, Japan）を1 μg/ml、酵母エキス（Difco, Becton Dickinson and Company, Sparks, MD, USA）を0.5%、及び塩酸システイン（Wako Pure Chemical Industries, Ltd., Osaka, Japan）を0.05%添加したBHI改変液体培地を用い、37℃で48時間嫌気培養した（N2:80%, CO2:10%, H2:10%）。 As an indicator strain for testing antibacterial properties, Porphyromonas gingivalis (hereinafter also referred to as P. gingivalis) ATCC (R) 33277 TM strain was used. P. gingivalis ATCC 33277 strain is BHI (Brain heart infusion) liquid medium (Difco, Becton Dickinson and Company, Sparks, MD, USA), hemin (Sigma, St. Louis, MO, USA) 5 μg / ml, menadione (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 1 μg / ml, yeast extract (Difco, Becton Dickinson and Company, Sparks, MD, USA) 0.5%, and cysteine hydrochloride (Wako Pure Chemical Industries, Ltd. , Osaka, Japan) was anaerobically cultured at 37 ° C. for 48 hours using BHI modified liquid medium supplemented with 0.05% (N 2 : 80%, CO 2 : 10%, H 2 : 10%).
得られた乳酸菌培養液を、それぞれ、7000×gで4℃、20分間遠心分離して培養上清を得た。この培養上清を10N NaOHにてpH 7.0に調整後、0.22 μmのメンブレンフィルターでろ過したものを被験サンプルとした。 The obtained lactic acid bacteria cultures were each centrifuged at 7000 × g at 4 ° C. for 20 minutes to obtain culture supernatants. The culture supernatant was adjusted to pH 7.0 with 10N NaOH and then filtered through a 0.22 μm membrane filter as a test sample.
被験サンプルをBHI改変液体培地で2倍毎に256倍まで段階希釈し、希釈した被験サンプル2 mlに対して、1×107CFU/mlに調製した指標菌株P. gingivalis ATCC 33277の培養液を10 μl接種し、37℃で48時間、上記と同じ条件で嫌気培養した。培養後620nmの吸光度を測定し、吸光度0.1以下を示した被験サンプルを、P. gingivalisの増殖を抑制した（抗菌活性有り）と判定した。結果を表１に示す。 Test samples are serially diluted up to 256 times every 2 times with BHI modified liquid medium, and 2 ml of diluted test sample is diluted with 1 × 10 7 CFU / ml of indicator strain P. gingivalis ATCC 33277 10 μl was inoculated and anaerobically cultured at 37 ° C. for 48 hours under the same conditions as described above. After culturing, the absorbance at 620 nm was measured, and a test sample exhibiting an absorbance of 0.1 or less was determined to suppress the growth of P. gingivalis (with antibacterial activity). The results are shown in Table 1.
表１に示すように、豆乳発酵物由来のラクトバチルス・ファーメンタム（Lactobacillus fermentum; 以下、L. fermentumとも称する） ALAL020株はP. gingivalisに対して特に強い抗菌活性を示し、最大希釈倍数は16であった。一方で、ALAL020株と同種のヒト口腔由来のL. fermentum 103株の最大希釈倍数は2であり、菌株により抗菌活性に大幅な差が認められた。 As shown in Table 1, Lactobacillus fermentum (hereinafter also referred to as L. fermentum) ALAL020 strain derived from a fermented soymilk exhibits particularly strong antibacterial activity against P. gingivalis, with a maximum dilution factor of 16 Met. On the other hand, the maximum dilution factor of L. fermentum 103 strain derived from human oral cavity of the same type as ALAL020 strain was 2, and a large difference in antibacterial activity was recognized depending on the strain.
ラクトバチルス・ロイテリ（L. reuteri） DSM 17938株及びL. reuteri ATCC PTA 5289株についてはその生菌が歯周病患者の口腔内P. gingivalisの菌数を減少させることをVivekanandaらが報告している（Vivekananda MR., et al., J. Oral Microbiol., (2010) 2:5344）。また、ラクトバチルス・サリバリウス（L. salivarius） LS1についてもin vitro試験によりP. gingivalisの菌数を減少させる作用が報告されている（Ishikawa H., et al., J. Jpn. Soc. Periodontol., (2003) 45:105-112）。しかしLactobacillus fermentum ALAL020株はこれらの既報乳酸菌株よりもかなり強い増殖抑制作用を示した。したがってLactobacillus fermentum ALAL020株はP. gingivalisの抑制に非常に有用であると考えられた。 Lactobacillus reuteri (L. reuteri) DSM 17938 and L. reuteri ATCC PTA 5289 have reported that viable bacteria reduce the number of P. gingivalis in the oral cavity of patients with periodontal disease. (Vivekananda MR., Et al., J. Oral Microbiol., (2010) 2: 5344). In addition, L. salivarius LS1 has been reported to reduce the number of P. gingivalis bacteria by in vitro tests (Ishikawa H., et al., J. Jpn. Soc. Periodontol. , (2003) 45: 105-112). However, Lactobacillus fermentum ALAL020 strain showed a significantly stronger growth inhibitory action than these previously reported lactic acid strains. Therefore, Lactobacillus fermentum ALAL020 strain was considered to be very useful for suppression of P. gingivalis.
乳酸菌が産生する抗菌物質として、これまでに乳酸や酢酸などの有機酸、過酸化水素、バクテリオシンが報告されている。さらに、Takahashiらは、P. gingivalisは、酸感受性が高くpH6.5以下で増殖が抑制されることを報告している（Takahashi N. et al., Oral Microbiol. Immunol., (1997) 12(6):323-328）。しかし口腔内で低いpH状態を持続することには、う蝕の発症や知覚過敏を誘発するリスクが考えられる。そこで本実施例では、pHの影響を除外するため水酸化ナトリウムでpHを7に調整してP. gingivalisに対する抗菌試験を行った。したがって表１は、Lactobacillus fermentum ALAL020株が、pHが中性であるにもかかわらず、P. gingivalisに対して強い増殖抑制効果を発揮したことを示している。この結果から、この菌株が、pH低下をもたらす有機酸以外の、抗菌物質を産生している可能性が示された。 As antibacterial substances produced by lactic acid bacteria, organic acids such as lactic acid and acetic acid, hydrogen peroxide, and bacteriocin have been reported so far. Furthermore, Takahashi et al. Reported that P. gingivalis is highly acid sensitive and inhibits growth at pH 6.5 or lower (Takahashi N. et al., Oral Microbiol. Immunol., (1997) 12 ( 6): 323-328). However, sustaining a low pH state in the oral cavity may be associated with the risk of developing caries and hypersensitivity. Therefore, in this example, an antibacterial test against P. gingivalis was conducted by adjusting the pH to 7 with sodium hydroxide in order to exclude the influence of pH. Therefore, Table 1 shows that the Lactobacillus fermentum ALAL020 strain exerted a strong growth inhibitory effect against P. gingivalis even though the pH was neutral. From this result, it was shown that this strain is producing an antibacterial substance other than the organic acid which brings about the pH fall.
［実施例２］Lactobacillus fermentum ALAL020株の培養上清からの抗菌性フラクションの分離
１）培養上清の分画
実施例１のようにして得たLactobacillus fermentum ALAL020株の培養上清に、同量の酢酸エチルを加えて振とう後、水層と有機層に分け、有機層を除去した水層に酢酸エチルを加え、この作業を合計3回繰り返した。水層を分取し、エバポレーターにて減圧濃縮後、分子篩カラムSephadex G-25（100 mmφ×350 mm, GE Healthcare, Uppsala, Sweden）にアプライし、イオン交換水で溶出（流速5 ml/min）した（ゲル濾過クロマトグラフィー）。併せて糖の発色もモニタリングした。溶出液を5つのフラクション（分画物）に分け（図１Ａ）、210 nmでの吸収（吸光度）から溶出ピークを検出した。フラクションにアセトンを加え、振とう後1晩静置し、アセトン上清及び沈殿についての抗菌活性の確認を行った。活性画分の精製は高速液体クロマトグラフィー（JASCO Corporation、Tokyo, Japan）を用い、分取用逆相ODS(C18)カラム（Wakoshil II 5C18 AR Prep20.0 mmφ×250 mm、Wako Pure Chemical Industries, Ltd., Osaka, Japan）にて0.05%(v/v) トリフルオロ酢酸含有10%(v/v) アセトニトリル溶液と50%(v/v) アセトニトリル溶液を用いて、流速5 ml/minにて10 %〜30 ％までのグラディエント溶出を20分間行い、その後10分間50%にて溶出を行った。210 nmの吸収から溶出ピークを検出し、溶出液を分取し、それぞれのフラクションを凍結乾燥して抗菌性試験に供した。
[Example 2] Separation of antibacterial fraction from culture supernatant of Lactobacillus fermentum ALAL020 strain 1) Fractionation of culture supernatant The same amount was added to the culture supernatant of Lactobacillus fermentum ALAL020 strain obtained as in Example 1. After adding ethyl acetate and shaking, it was divided into an aqueous layer and an organic layer, ethyl acetate was added to the aqueous layer from which the organic layer was removed, and this operation was repeated a total of 3 times. The aqueous layer was collected, concentrated under reduced pressure using an evaporator, applied to a molecular sieve column Sephadex G-25 (100 mmφ × 350 mm, GE Healthcare, Uppsala, Sweden), and eluted with ion-exchanged water (flow
２）フラクションの乳酸の定量
上記で得たフラクションについて、高速液体クロマトグラフィーNANOSPACE SI-1 system（Shiseido, Tokyo, Japan）を用いて乳酸（乳酸ナトリウム）を定量した。CAPCELL PAC C18 TYPE MGカラム（1.5×250mm； Shiseido, Tokyo, Japan）を用い、50mM NH4H2PO4-H3PO4溶液（pH 4.2）にて溶出（流速100 μl/min）し、210nmにて検出した。
2) Quantification of lactic acid in fractions Lactic acid (sodium lactate) was quantified in the fractions obtained above using high performance liquid chromatography NANOSPACE SI-1 system (Shiseido, Tokyo, Japan). Using a CAPCELL PAC C18 TYPE MG column (1.5 x 250 mm; Shiseido, Tokyo, Japan), elute with 50 mM NH 4 H 2 PO 4 -H 3 PO 4 solution (pH 4.2) (
３）フラクションの抗菌性試験
96ウェルプレートに、ヘミンを5 μg/ml、メナジオンを1 μg/ml添加したGAM（日水製薬株式会社，東京）液体培地を160μlずつ分注し、凍結乾燥したフラクションの水溶液をpH7.0に調整後に20μl加え、指標菌株P. gingivalis ATCC 33277株の培養液（1×106〜107 CFU/ml）を20μl接種し、37℃で2日間嫌気培養した。対照として凍結乾燥したフラクションの水溶液を添加しないこと以外は同様にして指標菌株P. gingivalis ATCC 33277株を嫌気培養した。培養後プレートリーダーで650nmの濁度を測定し、対照の濁度との差を求めた。
3) Antibacterial test of fraction
Dispense 160 μl of GAM (Nissui Pharmaceutical Co., Ltd., Tokyo) liquid medium supplemented with 5 μg / ml hemin and 1 μg / ml menadion to a 96-well plate, and adjust the lyophilized fraction aqueous solution to pH 7.0. After adjustment, 20 μl was added, 20 μl of a culture solution (1 × 10 6 to 10 7 CFU / ml) of the indicator strain P. gingivalis ATCC 33277 was inoculated, and anaerobically cultured at 37 ° C. for 2 days. As a control, the indicator strain P. gingivalis ATCC 33277 was anaerobically cultured in the same manner except that the aqueous solution of the lyophilized fraction was not added. After incubation, the turbidity at 650 nm was measured with a plate reader, and the difference from the turbidity of the control was determined.
各フラクションの抗菌活性は、乳酸当量（sodium lactic acid equivalence; SLU）で表した。SLUは以下の式で算出した。 The antibacterial activity of each fraction was expressed in terms of lactic acid equivalent (sodium lactic acid equivalence; SLU). SLU was calculated by the following formula.
ＳＬＵ＝（１／Ｃ）／（１／Ｌ）＝Ｌ／Ｃ SLU = (1 / C) / (1 / L) = L / C
Ｃは、P. gingivalisが検出限界を下回るまで増殖を抑制したフラクション濃度である。Ｌは、P. gingivalisに対する乳酸（乳酸ナトリウム）のMIC（最小発育阻止濃度; minimum inhibitory concentration）であり、ここでは20 mg/mlを用いた。 C is the fraction concentration at which growth was inhibited until P. gingivalis fell below the detection limit. L is the MIC (minimum inhibitory concentration) of lactic acid (sodium lactate) against P. gingivalis, and 20 mg / ml was used here.
その結果、最も抗菌活性の高いフラクション3は1.82 SLUを示した（図１Ｂ）。フラクション3、フラクション4及びフラクション5に含まれる乳酸量はそれぞれ1.3%（w/w）、5.3%（w/w）、2.4%（w/w）であった（図１Ｂ）。フラクション3、フラクション4及びフラクション5中の乳酸の終濃度はそれぞれ0.1％、0.5％、0.2％であり、いずれのフラクションも、ポジティブコントロールとして用いた乳酸が抗菌活性を示した濃度2.0％よりも低い乳酸濃度を示した。
As a result,
さらに最も強い抗菌活性を示したフラクション3の凍結乾燥物の水溶液に４倍量のアセトンを加え、得られたアセトン上清と沈殿についての抗菌活性を調べたところ、アセトン上清の抗菌活性が最も強く、8.0 SLUであった（図１Ｃ）。このため、培養上清のアセトン沈殿を行い、アセトン上清をSephadex G-25カラムにて分離溶出し、ピーク面積のフラクションのみを取得した（図２Ａ）。その溶出したピーク面積のフラクションをODS(C18)逆相HPLCにてH1、H2、H3、H4、H5、H6、H7、H8-1、及びH-2のフラクションに分けた（図２Ｂ）。各フラクションのP. gingivalisに対する抗菌活性については、H8-1が最も強く、5.0 SLUであり、次いでH8-2が3.08 SLUであった（図２Ｃ）。
Furthermore, when 4 times the amount of acetone was added to the aqueous solution of the freeze-dried product of
［実施例３］抗菌物質の同定
フラクションH8-1及びH-2についてそれぞれ液体クロマトグラフィー質量分析法（LC-MS）により分析を行った。HPLCについて、フラクションH8-1及びH-2を、UltiMate 3000 UHPLC System（Thermo Fisher Scientific, Inc. Massachusetts, USA）を用い、Shim-pack VP-ODS（150mm x φ4.6mm, Shimadzu, Tokyo, Japan）カラムに20μLアプライし、溶離液（10〜50%アセトニトリル、0.05%ギ酸）を用いてグラディエント溶出した。質量分析（MS）は、Q ExactionTM（四重極/Orbitrap ハイブリッド質量分析計、Thermo Fisher Scientific, Inc.、Massachusetts、USA）を用いて行った。イオン化はESI方式で行い、ポジティブモードで検出した。検出範囲は分子量50〜750 Daであった。得られた分析結果を図３に示す。この精密質量分析の結果、H8-1、H8-2にそれぞれ対応する化合物は共に分子量226.131、分子式C11H18O3N2と推定された。なおH8-1とH8-2にそれぞれ対応する化合物はHPLCにおいて異なる保持時間を示したことから、構造異性体（シス体及びトランス体）と考えられた。
[Example 3] Identification of antibacterial substances Fractions H8-1 and H-2 were analyzed by liquid chromatography mass spectrometry (LC-MS), respectively. For HPLC, fractions H8-1 and H-2 were used with an UltiMate 3000 UHPLC System (Thermo Fisher Scientific, Inc. Massachusetts, USA) and Shim-pack VP-ODS (150mm x φ4.6mm, Shimadzu, Tokyo, Japan) 20 μL was applied to the column, and gradient elution was performed using an eluent (10 to 50% acetonitrile, 0.05% formic acid). Mass spectrometry (MS) was performed using a Q Exaction ™ (quadrupole / Orbitrap hybrid mass spectrometer, Thermo Fisher Scientific, Inc., Massachusetts, USA). Ionization was performed by ESI method and detected in positive mode. The detection range was a molecular weight of 50 to 750 Da. The analysis results obtained are shown in FIG. As a result of this accurate mass spectrometry, the compounds corresponding to H8-1 and H8-2 were both estimated to have a molecular weight of 226.131 and a molecular formula of C 11 H 18 O 3 N 2 . Since the compounds corresponding to H8-1 and H8-2 showed different retention times in HPLC, they were considered to be structural isomers (cis isomer and trans isomer).
さらに、H8-1及びH8-2に含まれる抗菌物質の構造解析を、核磁気共鳴（Nuclear Magnetic Resonance; NMR）を用いた測定により行った。NMR装置AVANCE700（Bruker Biospin）を用いて、共鳴周波数700.33329828MHz、測定温度10℃でNMR分析を行った。全てのスペクトルは、一次元1H NMRと13C NMR、二次元1H-1H COSY（correlation spectroscopy; 相関分光）法、1H-13C HSQC（hetero nuclear single quantum coherence; 異種核一量子相関）法、1H-13C HMBC（heteronuclear multiple bond correlation; 異種核多結合相関）法の結果を照らし合わせて解析した。 Furthermore, structural analysis of antibacterial substances contained in H8-1 and H8-2 was performed by measurement using nuclear magnetic resonance (NMR). NMR analysis was performed using a NMR apparatus AVANCE700 (Bruker Biospin) at a resonance frequency of 700.33329828 MHz and a measurement temperature of 10 ° C. All spectra are one-dimensional 1 H NMR and 13 C NMR, two-dimensional 1 H- 1 H COZY (correlation spectroscopy), 1 H- 13 C HSQC (hetero nuclear single quantum coherence) ) Method, 1 H- 13 C HMBC (heteronuclear multiple bond correlation) method was compared and analyzed.
一次元NMRのチャートから、17個の水素原子（プロトン）と11個の炭素原子の存在が確認でき、また1個のH原子は交換性プロトンの可能性が高いことが示された（図４）。 The one-dimensional NMR chart confirmed the presence of 17 hydrogen atoms (protons) and 11 carbon atoms, and one H atom was highly likely to be an exchangeable proton (FIG. 4). ).
二次元NMRの結果を図５〜７に示す。COSY測定（図５）では隣接するプロトン−プロトン間の相関が明らかになり、２つのユニットが導かれた。HSQC測定（図６）ではプロトンとそれに直接結合している炭素原子との相関、相関の正負、及びプロトンの積分値により炭素の級数を決定した。 The results of two-dimensional NMR are shown in FIGS. The COZY measurement (FIG. 5) revealed an adjacent proton-proton correlation, leading to two units. In the HSQC measurement (FIG. 6), the series of carbons was determined based on the correlation between protons and carbon atoms directly bonded to them, the sign of the correlation, and the integral value of the protons.
さらにHMBC測定（図７）ではプロトンとそのプロトンに直接結合している炭素原子の隣や２つ隣に位置する炭素原子との相関が観測された。 Furthermore, in the HMBC measurement (FIG. 7), a correlation was observed between the proton and the carbon atom located next to or adjacent to the carbon atom directly bonded to the proton.
以上の解析の結果、フラクションH8-1及びH8-2に相当する抗菌物質はいずれも、下記構造式で表されるヘキサヒドロ-7-ヒドロキシ-3-(2-メチルプロピル)ピロロ[1,2-a]ピラジン-1,4-ジオンであること、またこの抗菌物質がヒドロキシプロリンとロイシンの環状ジペプチド構造を有することが明らかとなった（図８）。 As a result of the above analysis, all the antibacterial substances corresponding to the fractions H8-1 and H8-2 were hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2- a] pyrazine-1,4-dione, and this antibacterial substance has a cyclic dipeptide structure of hydroxyproline and leucine (FIG. 8).
H8-1の抗菌物質について得られたNMRスペクトルは、以下のとおりである。
1H NMR (700.33MHz, CD3COCD3)δ 0.92 (d, J=6.58Hz 3H) 0.96 (d, J=6.65 3H) 1.53-1.57 (m, 1H) 1.65-1.69 (m, 1H) 1.75-1.82 (m, 1H) 2.22-2.27 (m, 1H) 2.38-2.42 (m, 1H) 3.37 (dd, J=11.91, 5.39Hz 1H). 3.65 (ddd, J=11.94, 3.68, 0.77Hz 1H) 3.81-3.84 (m, 1H) 4.28 (dd, J=7.07, 8.61Hz 1H) 4.42-4.45 (m, 1H) 7.49 (s, 1H)
13C NMR (176.12MHz, CD3COCD3)δ 21.83 (C-12), 23.17 (C-13), 25.01 (C-11), 37.61 (C-8), 43.09 (C-10), 53.90 (C-6), 56.46 (C-3), 56.91 (C-9), 68.46 (C-7), 167.28 (C-4), 169.29 (C-1)。
The NMR spectrum obtained for the antimicrobial substance of H8-1 is as follows.
1 H NMR (700.33MHz, CD 3 COCD 3 ) δ 0.92 (d, J = 6.58Hz 3H) 0.96 (d, J = 6.65 3H) 1.53-1.57 (m, 1H) 1.65-1.69 (m, 1H) 1.75- 1.82 (m, 1H) 2.22-2.27 (m, 1H) 2.38-2.42 (m, 1H) 3.37 (dd, J = 11.91, 5.39
13 C NMR (176.12MHz, CD 3 COCD 3 ) δ 21.83 (C-12), 23.17 (C-13), 25.01 (C-11), 37.61 (C-8), 43.09 (C-10), 53.90 ( C-6), 56.46 (C-3), 56.91 (C-9), 68.46 (C-7), 167.28 (C-4), 169.29 (C-1).
H8-2の抗菌物質について得られたNMRスペクトルは、以下のとおりである。
1H NMR (700.33MHz, H2O+D2O) δ 0.75 (d, J=6.37Hz 6H) 1.43-1.47 (m, 1H) 1.60-1.65 (m, 1H) 1.64-1.69 (m, 1H) 1.98-2.02 (m, 1H) 2.15 (dd, J=13.45, 6.37 Hz, 1H) 3.31 (d, J=13.17Hz, 1H) 3.57 (dd, J=13.17, 4.41Hz, 1H) 4.15-4.16 (m, 1H) 4.41-4.42 (m, 1H) 4.43 (t, J=4.45Hz 1H)
13C NMR (176.12MHz, H2O+D2O) 20.61 (C-12), 21.99 (C-13), 23.72 (C-11), 35.92 (C-8), 37.22 (C-10), 53.23 (C-3), 53.61 (C-6), 57.04 (C-9), 67.68 (C-7), 168.58 (C-4), 172.23 (C-1)。
The NMR spectrum obtained for the antibacterial substance of H8-2 is as follows.
1 H NMR (700.33MHz, H 2 O + D 2 O) δ 0.75 (d, J = 6.37Hz 6H) 1.43-1.47 (m, 1H) 1.60-1.65 (m, 1H) 1.64-1.69 (m, 1H) 1.98-2.02 (m, 1H) 2.15 (dd, J = 13.45, 6.37 Hz, 1H) 3.31 (d, J = 13.17Hz, 1H) 3.57 (dd, J = 13.17, 4.41Hz, 1H) 4.15-4.16 (m , 1H) 4.41-4.42 (m, 1H) 4.43 (t, J = 4.45Hz 1H)
13 C NMR (176.12MHz, H 2 O + D 2 O) 20.61 (C-12), 21.99 (C-13), 23.72 (C-11), 35.92 (C-8), 37.22 (C-10), 53.23 (C-3), 53.61 (C-6), 57.04 (C-9), 67.68 (C-7), 168.58 (C-4), 172.23 (C-1).
本発明は、歯周病原細菌に対する高い抗菌作用を有する薬剤を提供するために用いることができる。 The present invention can be used to provide a drug having a high antibacterial action against periodontopathic bacteria.
Claims (7)
又はその塩を含む、抗歯周病原細菌剤。 A compound represented by the following formula:
Or an anti-periodontopathic bacterial agent comprising a salt thereof.
又はその塩を含む、歯周病原細菌の増殖を抑制するための飲食品。 A compound represented by the following formula:
Or the food-drinks for suppressing the growth of periodontopathic bacteria containing the salt.
又はその塩を含む、歯周病の治療又は予防用の医薬。 A compound represented by the following formula:
Or the pharmaceutical for treatment or prevention of periodontal disease containing the salt.
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Citations (3)
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|---|---|---|---|---|
| JP2014000039A (en) * | 2012-06-19 | 2014-01-09 | Lion Corp | Lactobacillus and substance from cultivation thereof, and composition containing these |
| KR20140045075A (en) * | 2012-10-08 | 2014-04-16 | 서울대학교산학협력단 | Antibacterial and antiviral composition comprising cis??cyclo (l??leu??l??pro) or cis??cyclo (l??phe??pro) and methods of preparation thereof |
| KR20150057636A (en) * | 2013-11-20 | 2015-05-28 | 서울대학교산학협력단 | Composition comprising cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014000039A (en) * | 2012-06-19 | 2014-01-09 | Lion Corp | Lactobacillus and substance from cultivation thereof, and composition containing these |
| KR20140045075A (en) * | 2012-10-08 | 2014-04-16 | 서울대학교산학협력단 | Antibacterial and antiviral composition comprising cis??cyclo (l??leu??l??pro) or cis??cyclo (l??phe??pro) and methods of preparation thereof |
| KR20150057636A (en) * | 2013-11-20 | 2015-05-28 | 서울대학교산학협력단 | Composition comprising cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents |
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| Title |
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