JP2007084534A - Composition for oral cavity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for oral cavity preventing Porphyromonas gingivalis as periodontal disease-related bacteria from deposition on oral cavity tissue. <P>SOLUTION: The composition for oral cavity contains a peptide where arginine and histidine are bound alternately to each other. Specifically, the above arginine is L-arginine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a composition for oral cavity, which has an effect of suppressing adhesion of oral bacteria to oral tissues and is used for prevention and treatment of periodontal disease.

Periodontal disease is caused by dental plaque, a bacterial clump that attaches to the teeth. Among these plaques, bacteria that are thought to be deeply involved in periodontal disease include Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia. Black pigment producing anaerobic bacilli (BPR), Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, etc. have been found. These bacteria produce various virulence factors (adhesins, capsular polysaccharides, tissue-degrading enzymes, organic acids, sulfides, endotoxins) and cause periodontal disease.
In particular, the Porphynomonas gingivalis produces arginine-specific proteolytic enzyme (Arg-gingipain) and lysine-specific proteolytic enzyme (Lys-gingipain) that destroy periodontal tissue. It is considered as a pathogen, and several research groups have pointed out its etiological relationship with adult periodontitis.

Preventing adhesion of Porphyromonas gingivalis, an important pathogen of periodontal disease, to oral tissues is considered to be effective in preventing periodontal disease.
Regarding the technique for preventing the adhesion of Porphyromonas gingivalis to oral tissues, for example, there is a report that lysine and arginine suppress the adhesion of Porphyromonas gingivalis to the buccal mucosa epithelial cells (Non-patent Document 1). . There is a report that a synthetic peptide of 8 to 24 residues, which is the primary structure of histatin, inhibits the hemagglutination activity of Porphyromonas gingivalis (Non-patent Document 2). There are reports that arginine and guanidylated albumin inhibit the hemagglutination activity of Exohemagglutinin of Porphyromonas gingivalis (Non-patent Document 3) and reports that lysine and arginine inhibit (Non-patent Document 4). is there.

  On the other hand, a report on an oral composition characterized in that a peptide in which two or more basic amino acids are continuously bonded in the molecule suppresses adhesion of Porphyromonas gingivalis to gingival epithelial cells or saliva-coated hydroxyapatite. (Patent Document 1).

  However, these have a problem in that they use a chemically synthesized long-chain peptide that has a weak aggregation-suppressing effect or is expensive.

  In addition, there is a report that a polyamino acid derived from microbial fermentation or a derivative thereof represented by the following formula (1) has antibacterial properties (Patent Document 2).

［式中、Ｘはアルギニン残基などを表し、Ｙはヒスチジン残基などを表す。］
特公平７-68111 WO2004/014944 A1 口腔衛生学会誌38：590-591、1988 Archs Oral Biol. Vol.35,No.9,p775-777(1990) Infection and Immunity,Vol.52 ,No.2,p421-427(1986) Infection and Immunity,Vol.54 ,No.3,p659-665(1986)

[Wherein, X represents an arginine residue and the like, and Y represents a histidine residue and the like. ]
7-68111 WO2004 / 014944 A1 Journal of Oral Hygiene 38: 590-591, 1988 Archs Oral Biol. Vol. 35, No. 9, p775-777 (1990) Infection and Immunity, Vol.52, No.2, p421-427 (1986) Infection and Immunity, Vol.54, No.3, p659-665 (1986)

  This invention makes it a subject to provide the composition for oral cavity which prevents that Porphyromonas gingivalis which is a periodontal disease bacterium adheres to an oral tissue.

（Ａｒｇはアルギニン、Ｈｉｓはヒスチジンを表す。また、Ｒ₁は水素、糖、アシル、ビオチニル、チオール、フェノール、又はインドールを表し、Ｒ₂は水酸基、糖、アシル、ビオチニル、チオール、フェノール、又はインドールを表し、ｎは２以上の整数を示す。）

（３） 前記ペプチドが下記式（Ｉ）で表される構造であることを特徴とする、（１）または（２）に記載の口腔用組成物。

（４） 前記アルギニンのＤ体アルギニンとＬ体アルギニンの比率が、Ｄ体アルギニン：Ｌ体アルギニン＝１０：９０〜０：１００であることを特徴とする、（１）〜（３）のいずれか一に記載の口腔用組成物。
（５） 前記アルギニンがＬ体アルギニンであることを特徴とする、（１）〜（４）のいずれか一に記載の口腔用組成物。
（６） 前記ヒスチジンのＤ体ヒスチジンとＬ体ヒスチジンの比率が、Ｄ体ヒスチジン：Ｌ体ヒスチジン＝１００：０〜７０：３０であることを特徴とする、（１）〜（５）のいずれか一に記載の口腔用組成物。
（７） 前記ヒスチジンがＤ体ヒスチジンであることを特徴とする、（１）〜（６）のいずれか一に記載の口腔用組成物。
（８） 前記ペプチドが（アルギニン−ヒスチジン）の１〜２０量体であることを特徴とする、（１）〜（７）のいずれか一項に記載の口腔用組成物。
（９） 前記ペプチドが（アルギニン−ヒスチジン）の５量体であることを特徴とする、（１）〜（８）のいずれか一に記載の口腔用組成物。
（１０） 前記ペプチドが、微生物の発酵生産により製造されたペプチドであることを特徴とする、（１）〜（９）のいずれか一に記載の口腔用組成物。
（１１） 前記ペプチドの含有量が０．００１〜１０重量％であることを特徴とする、（１）〜（１０）のいずれか一に記載の口腔用組成物。
（１２） 前記ペプチドの含有量が０．０１〜１重量％であることを特徴とする、（１）〜（１１）のいずれか一に記載の口腔用組成物。
（１３） ポルフィロモナス・ジンジバリスの赤血球凝集活性、ポルフィロモナス・ジンジバリスと唾液被覆ハイドロキシアパタイトとの付着、及びポルフィロモナス・ジンジバリスとストレプトコッカス・オラリス（Streptococcus oralis）との共凝集を抑制することを特徴とする、（１）〜（１２）のいずれか一に記載の口腔用組成物。
（１４） 歯周病予防用であることを特徴とする、（１）〜（１３）のいずれか一に記載の口腔用組成物。
（１５） 歯周病治療用であることを特徴とする、（１）〜（１３）のいずれか一に記載の口腔用組成物。 The present inventors have sought for a composition in the oral cavity that prevents periodontal disease bacteria from adhering to oral tissues, and as a result of earnest research, a specific peptide produced by fermentation of certain microorganisms is porphyromonas.・ Although there is no antibacterial activity (growth inhibitory activity) against gingivalis, hemagglutination activity of porphyromonas gingivalis, adhesion of porphyromonas gingivalis and saliva-coated hydroxyapatite, and Porphyromonas gingivalis and Streptococcus oralis and found to suppress coaggregation with oralis). Therefore, the use of peptides produced by certain microorganisms as oral compositions was examined. That is, the present invention is as follows.
(1) An oral composition containing a peptide in which arginine and histidine are alternately bound.
(2) The composition for oral cavity according to (1), wherein the peptide has a structure represented by the following formula (I) or (II).

(Arg represents arginine, His represents histidine. R ₁ represents hydrogen, sugar, acyl, biotinyl, thiol, phenol, or indole, and R ₂ represents a hydroxyl group, sugar, acyl, biotinyl, thiol, phenol, or indole. And n represents an integer of 2 or more.)

(3) The composition for oral cavity according to (1) or (2), wherein the peptide has a structure represented by the following formula (I).

(4) The ratio of the D-form arginine and the L-form arginine of the arginine is D-form arginine: L-form arginine = 10: 90 to 0: 100, any one of (1) to (3) The composition for oral cavity according to 1.
(5) The composition for oral cavity according to any one of (1) to (4), wherein the arginine is L-form arginine.
(6) The ratio of D-histidine to L-histidine in the histidine is D-histidine: L-histidine = 100: 0 to 70:30, any one of (1) to (5) The composition for oral cavity according to 1.
(7) The composition for oral cavity according to any one of (1) to (6), wherein the histidine is D-form histidine.
(8) The composition for oral cavity according to any one of (1) to (7), wherein the peptide is a 1-20 mer of (arginine-histidine).
(9) The composition for oral cavity according to any one of (1) to (8), wherein the peptide is a pentamer of (arginine-histidine).
(10) The composition for oral cavity according to any one of (1) to (9), wherein the peptide is a peptide produced by fermentation production of a microorganism.
(11) The composition for oral cavity according to any one of (1) to (10), wherein the content of the peptide is 0.001 to 10% by weight.
(12) The composition for oral cavity according to any one of (1) to (11), wherein the content of the peptide is 0.01 to 1% by weight.
(13) It suppresses the hemagglutination activity of Porphyromonas gingivalis, adhesion of Porphyromonas gingivalis and saliva-coated hydroxyapatite, and co-aggregation of Porphyromonas gingivalis and Streptococcus oralis The composition for oral cavity according to any one of (1) to (12), which is characterized.
(14) The composition for oral cavity according to any one of (1) to (13), which is used for preventing periodontal disease.
(15) The composition for oral cavity according to any one of (1) to (13), which is used for treatment of periodontal disease.

  Since the composition for oral cavity of the present invention can prevent periodontal disease bacteria from adhering to the oral tissue, it is used, for example, as an agent for preventing or treating periodontal disease.

  The oral composition of the present invention contains a peptide as described above. The peptide is a peptide in which arginine and histidine are alternately bound, and is represented by the following general formula (2) or (3). A preferred structure is the following general formula (2), that is, a structure in which arginine is N-terminal and arginine and histidine are alternately linked. The composition for oral cavity of the present invention may contain one or a combination of two or more of the peptides.

In the above general formulas (2) and (3), Arg represents arginine and His represents histidine. R ₁ represents hydrogen, sugar, acyl, biotinyl, thiol, phenol, or indole, R ₂ represents a hydroxyl group, sugar, acyl, biotinyl, thiol, phenol, or indole, and n represents an integer of 2 or more. .
The peptides represented by the general formulas (2) and (3) are generally referred to as polyarginyl histidine and are also referred to in the present specification. Polyarginyl histidine can also be abbreviated as “pRH”. “P” is an abbreviation for poly, “R” is a single letter of arginine, and “H” is a single letter of histidine.

  The arginine may be D-form arginine or L-form arginine. A preferable ratio of arginine in the general formula (2) or (3) is D-form: L-form = 10: 90 to 0: 100. More preferably, all are L-form arginine. This ratio is a value based on analysis of D-form and L-form of arginine and histidine described later.

  The histidine may be D-form histidine or L-form histidine. In the general formula (2) or (3), the preferred ratio of histidine is D-form: L-form = 100: 0 to 70:30. More preferred are all D-form histidine. This ratio is a value based on analysis of D-form and L-form of arginine and histidine described later.

Analysis of the D-form and L-form of arginine and histidine constituting the polyarginyl histidine, that is, the optical purity test of arginine and histidine is shown below.
Polyarginyl histidine is hydrolyzed by heating in 6N hydrochloric acid solution at 100 ° C. for 20 hours to prepare a mixture of arginine and histidine. The prepared mixture of arginine and histidine was mixed with an optical resolution column (Daicel Kogyo CROWNPAK CR (+), mobile phase was pH 1.5 perchloric acid aqueous solution, column temperature was 4 ° C). (HPLC). Detection measures the absorption at 200 nm. As control experiments, four amino acid standards of D-histidine, L-histidine, D-arginine and L-arginine, two chemically synthesized polyamino acids (N-terminal L-Arg-D-His-L-Arg) -D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His C-terminal and N-terminal L-Arg-L-His-L-Arg-L-His-L -Arg-L-His-L-Arg-L-His-L-Arg-L-His The standard product hydrolyzed by heating in 6N hydrochloric acid solution at 100 ° C for 20 hours under the same conditions. Analyze by HPLC. Thereby, D-form and L-form of arginine and histidine are analyzed, and a ratio is determined.

In the general formula (2) and (3), as mentioned above, R ₁ of the N-terminus, hydrogen, sugars, acyl, biotinyl, thiol, phenol, or indole, R ₂ of the C-terminal hydroxy group, a sugar, Acyl, biotinyl, thiol, phenol, or indole. Preferably, the N-terminal R ₁ is hydrogen and the C-terminal R ₂ is a hydroxyl group.

  In the present invention, the polymerization degree n of polyarginyl histidine is not particularly limited, but a preferable polymerization degree n is 1 to 20, and a more preferable polymerization degree is 5. Polyarginyl histidine having a polymerization degree of 5 is particularly preferably used because it can be produced at low cost by microbial fermentation.

The degree of polymerization is measured as follows.
The molecular weight of polyarginyl histidine is measured by a MALDI-TOF Mass (matrix-assisted desorption ionization-time-of-flight measurement) method using a time-of-flight mass spectrometer. After subtracting the molecular weight of H ₂ O (about 18) from the obtained molecular weight, the degree of polymerization is calculated by dividing by the total value (293.32) of the amount of arginine residues (156.18) and the amount of histidine residues (137.14). To do. The residue amount is a value obtained by subtracting the H ₂ O molecular weight from the amino acid molecule.

  The polyarginyl histidine sequence is analyzed by an automated Edman degradation analyzer (Applied Biosystems, model 492). This reveals that the polyarginyl histidine contained in the oral composition of the present invention has a structure in which arginine and histidine are alternately linked.

  The polyarginyl histidine represented by the general formula (2) or (3) is produced by a known method such as a chemical synthesis method, a biochemical method, or a microbial fermentation known in the art. As a method for producing polyarginyl histidine contained in the composition for oral cavity of the present invention at low cost, fermentation production of microorganisms is most preferable.

As a chemical synthesis method of the polyarginyl histidine, for example, developed by RBMerrifield in 1963, the C-terminal carboxyl group of the peptide is covalently bonded to the solid support, and the amino acid is sequentially bonded in the N-terminal direction to the peptide. , ΑNH ₂ protected amino acids and N-terminal protected peptides with protected side chains, coupling of α-carboxy protected amino acids and C-terminal protected peptides with a condensing agent such as carbodiimide, reverse reaction of proteolytic enzyme And a method of polymerizing N-carboxyanhydride of side chain protected arginine and side chain protected histidine, and the like.

  Examples of the biochemical production method of the polyarginyl histidine include an intracellular or cell-free protein synthesis system using DNA or RNA encoding polyarginyl histidine, depending on the functions of transcription and translation systems provided in the organism. And a method of producing it by genetic engineering.

  Examples of the method for producing polyarginyl histidine by microbial fermentation include the method shown in Patent Document 2 (WO2004 / 014944 A1). Below, the example of the manufacturing method by microbial fermentation is shown.

  In the production of the polyarginyl histidine used in the present invention, preferably an epichloroe strain is used. A preferred microorganism is Epichloe kibiensis strain E18 (FERM P-18923) or a variant thereof. The Epichloe kibiensis E18 strain (FERM P-18923) (hereinafter referred to as “E18 strain”) is 1st, 1st East, Tsukuba City, Ibaraki Prefecture. Research Institute Deposited at the Patent Biological Deposit Center (Microbiological labeling: Epichloe kibiensis E18). With the E18 strain as a parent strain, a derivative strain that enhances the productivity of polyarginyl histidine can be obtained by mutant induction or recombinant gene technology. Derivative strains include those that have been artificially mutagenized and those obtained by screening.

A medium for producing a polyarginyl histidine included in the present invention, for example, a medium such as E18 strain, is appropriately selected according to the properties of the microorganism and can be obtained from a commercial product, but should be prepared by a method known to those skilled in the art. You can also. A complete medium, a synthetic medium, or a semi-synthetic medium having an appropriate liquid or solid composition can be used, but a liquid medium is suitable because of ease of operation. The medium may be any medium as long as it contains a carbon source, nitrogen source, inorganic salt and other nutrients as general components. Examples of the carbon source include glucose, galactose, fructose, glycerol, starch, and the like, and the content is preferably 0.1 to 10% (w / v). Examples of the nitrogen source include yeast extract, peptone, casein hydrolyzate, organic compounds such as amino acids, and inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and sodium nitrate. The content is 0.1 to 5% ( w / v) is preferred. If desired, other nutrient sources (such as those that give inorganic salts, phosphate ions, potassium ions, sodium ions, magnesium ions, zinc ions, iron ions, manganese ions, nickel ions, sulfate ions, etc.), vitamins ( For example, vitamin B ₁ ) and antibiotics (eg, ampicillin, tetracycline, kanamycin, etc.) may be added.

Culturing can be performed by shaking culture, stirring culture, or the like under aerobic conditions. The culture temperature is about 25-40 ° C., and the pH of the medium is 2.0-8.0, preferably 3.0-8.0, more preferably about 5.0. The culture period is usually 1 day to 14 days, but the culture can be continued for a longer period.
The above derivative strain (mutant strain) derived from E18 strain as a parent strain can also be cultured in the same manner.

  If the produced polyarginyl histidine is secreted into the culture, the culture is filtered or centrifuged to isolate the crude product. Purification of the produced polyarginyl histidine is carried out from the collected culture supernatant by a method known in the art (for example, ion-exchange resin treatment method, used for purification or isolation of natural or synthetic amino acids and proteins). An activated carbon adsorption treatment method, an organic solvent precipitation method, a vacuum concentration method, a freeze-drying method, a crystallization method, and the like) can be appropriately combined. When the produced polyarginyl histidine is present in the periplasm and cytoplasm of the cultured microorganism, the cells are collected by filtration and centrifugation, and their cell walls and / or cell membranes are destroyed, for example, by ultrasonication and / or lysozyme treatment. To obtain debris (cell debris). The debris can be dissolved in an appropriate aqueous solution (for example, a buffer solution), and the product can be isolated and purified according to the above method.

The physicochemical properties of polyarginyl histidine produced by E18 strain are as follows.
(1) Only arginine and histidine are produced by hydrolysis with 6N hydrochloric acid solution.
(2) Polyarginyl histidine and its hydrolyzate are positive for Sakaguchi reaction and Pauli reaction.
(3) The bonding mode between monomers is a peptide bond between the α-position carboxyl group and the α-position amino group.
(4) The amino sequence determined by the automatic Edman degradation method is an alternating repetition of arginine and histidine with the N-terminus as arginine.
(5) In the molecular weight measurement by the MALDI-TOF Mass (matrix-assisted desorption ionization-time-of-flight measurement) method, a molecule having a molecular weight of about 1486 is a main component. In addition to this, it is a hybrid of different molecules with a regular molecular weight difference of about 293.
(6) The produced histidine shows the same Rf value (0.19) as the histidine standard in thin layer chromatography, and is positive for ninhydrin reaction and Pauli reaction.
(7) About 85% of the optical purity of the produced histidine is D-form by chromatographic analysis using an optical resolution column.

  Various chemical modifications (for example, acylation) can be performed on the functional groups released in the residues of polyarginyl histidine obtained by the above chemical synthesis method, biochemical method, microbial fermentation method and the like. . These derivatization methods are known in the art. For example, it can be changed to ornithine by alkaline hydrolysis of a guanidino group released as a side chain to an arginine residue in polyarginyl histidine. Since arginine and ornithine have different guanidino group and amino group dissociation constants, the arginine and ornithine dissociation constants are appropriately adjusted to adjust the composition ratio of arginine and ornithine, so that the polyion having a dissociation constant (pKa) of the electrolytic functional group that is optimal for the purpose of use. Arginyl histidine can be produced.

  The polyarginyl histidine contained in the composition for oral cavity of the present invention does not show remarkable growth inhibitory activity against oral microorganisms, as shown in Test Examples described later. Moreover, the polyarginyl histidine does not inhibit the enzyme activities of arginine-specific proteolytic enzyme and lysine-specific proteolytic enzyme, which are potent pathogenic factors of Porphyromonas gingivalis.

On the other hand, the polyarginyl histidine contained in the composition for oral cavity of the present invention suppresses the hemagglutination activity of Porphyromonas gingivalis, which is an oral microorganism, as shown in the following test examples. In addition, the polyarginyl histidine inhibits adhesion between Porphyromonas gingivalis and saliva-coated hydroxyapatite, and also suppresses coaggregation of Porphyromonas gingivalis and Streptococcus oralis.

The composition for oral cavity of the present invention comprises hemagglutination activity of Porphyromonas gingivalis, adhesion of Porphyromonas gingivalis and saliva-coated hydroxyapatite, and co-use of Porphyromonas gingivalis and Streptococcus oralis. Since polyarginyl histidine which suppresses aggregation is contained, periodontal disease bacteria can be prevented from adhering to oral tissues.
This effect in the present invention is due to periodontitis, gingivitis, periodontitis, periodontitis, peri-implantitis, and other periodontal diseases such as caries (cavities), stomatitis, bad breath, and prevention and treatment of diseases. The composition of the present invention is also used in these. Among these, the composition of the present invention is suitable for periodontal disease prevention and periodontal disease treatment.

  The oral composition of the present invention is produced by blending polyarginyl histidine according to a conventional method, and the oral composition of the present invention is prepared, for example, by mixing, kneading, granulating, punching, depending on the dosage form of the preparation. Manufactured by conventional methods such as tablets, coating, sterilization, and emulsification. The blending amount of the polyarginyl histidine in the oral composition is 0.001 to 10% by weight, preferably 0.01 to 1% by weight.

  The oral composition of the present invention is not particularly limited as long as it is an oral composition, and any composition can be used as long as it is administered orally. For example, tablets, pills, granules, fine granules, powders, capsules, troches, chewables, gums and other solid preparations, emulsions, suspensions, syrups, elixirs and other liquids, gels, It can be formulated as an ointment or the like. These preparation methods can be produced by known methods. Moreover, in formulating, a suitable carrier etc. can be selected according to a dosage form, and this can be mix | blended.

  The composition for oral cavity of this invention can mix | blend arbitrary components suitably in the range which does not impair the effect of polyarginyl histidine. Optional ingredients include, for example, abrasives, binders, thickeners, wetting agents, sweeteners, flavoring agents, flavoring agents, fragrances, preservatives, pH adjusting agents, dyes, excipients, binders, lubricants. , Disintegrants, emulsifiers, emulsifying aids, non-aqueous vehicles, antioxidants, tonic solutions, suspending agents, preservatives, solubilizers, dispersants, thickeners, plasticizers, adsorbents, antioxidants, Examples include other drugs.

  The oral composition of the present invention includes, for example, various toothpastes such as toothpaste, toothpaste, liquid toothpaste, foamed toothpaste, gingival massage cream, topical application, mouthwash, gargle, mouth freshener, chewing gum and the like. It can take a form. Among these forms, toothpastes, gargles, mouthwashes, chewing gums and the like are preferable, and more preferred forms are toothpastes, mouthwashes, chewing gums.

  Below, the test example which shows the effect | action of polyarginyl histidine is described.

(Sample: Production example of polyarginyl histidine (pRH))
<Binding of the first amino acid to the resin>
6 g of Fmoc-D-His (Trt) -OH (Merck Co., Ltd .: Cas No.135610-90-1) and 5 g of diisopropylethylamine (Cas No.7087-68-5) (hereinafter referred to as “DIPEA”) 20 ml of N, N-dimethylformamide (Cas No. 68-12-2) (hereinafter referred to as “DMF”) and dichloromethane (Dichloromethane: Cas No. 75-09-2) (hereinafter referred to as “ Dissolved in 50 ml). To the solution, 5 g of 2-chlorotrityl chloride resin (2-Chlorotrityl chloride resin: manufactured by Merck & Co., Inc.) (hereinafter referred to as “2-ClTrt resin”) was added and stirred at 30 ° C. for 2 hours for reaction.
After the reaction, the obtained resin was washed with about 50 ml of a solvent described later. Washing was performed with DCM: MeOH: DIPEA (17: 2: 1) 3 times, DCM 3 times, DMF 2 times, DCM 2 times, then vacuum dried over KOH, and Fmoc-D- His (Trt) -resin was obtained.
<Deprotection of α-amino-protected Fmoc group>
The total amount of the synthesized Fmoc-D-His (Trt) -resin (or Fmoc-protected peptidyl resin) was added to about 50 ml of DMF solution containing 20% by volume of piperidine (Piperidine Cas No.110-89-4), and 3% at 30 ° C. Shake for hours and drain. The same treatment was repeated 3-4 times, and finally the resin was washed with about 50 ml of DMF.
<Coupling operation>
26 g of Fmoc-L-Arg (pbf) -OH (manufactured by Merck: Cas No.154445-77-9), 14.9 g of HBTU (Cas No94790-37-1), and 1-hydroxybenzotriazole (1-Hydroxybenzotriazole: Cas No. 2592-95-2) (hereinafter referred to as “HOBt”) 5.4 g was dissolved in about 80 ml of DMF. An additional 10 g of DIPEA was added and mixed. The mixture was immediately added to the resin (His (Trt) is N-terminal) from which the α-amino group-protected Fmoc group had been deprotected, and reacted at 30 ° C. for 2 hours.
α-amino-protected Fmoc group deprotection resin (Arg (pbf) is N-terminal) When histidine is bound, 25 g of Fmoc-D-His (Trt) -OH is used and the same as above. A ring operation was performed.
<Elongation of peptide chain>
The α-amino group-protected Fmoc group deprotection and coupling operations were repeated until the desired chain length peptide was obtained.
In this test, arginine and histidine were alternately bound, and the deprotection operation and coupling operation of the α-amino group-protected Fmoc group were repeated until it became a pentamer of (arginine-histidine).
<Excision of peptide from resin>
First, the α-amino group-protected Fmoc group of the resin was deprotected to remove the N-terminal Fmoc group. The resin was then washed 5 times with about 50 ml of DMF-acetic acid (60:40) and DCM. Finally, the resin was washed 5 times with about 50 ml of methanol and dried in vacuo over KOH overnight.
To this resin, trifluoroacetic acid (hereinafter referred to as “TFA”) (Trifluoroacetic Acid: Cas No. 76-05-1): triisopropylsilane (hereinafter referred to as “TIS”) (Triisopropylsilane: Cas No. 6485-79- 6): 25 ml of a mixture of water = 95: 2.5: 2.5 (volume ratio) was added, and the mixture was stirred occasionally at room temperature and left for 24 hours. The obtained solution was subjected to suction filtration, and the filtrate was recovered. The resin was washed twice with 50 ml of the same mixture, and the filtrate was similarly collected. 2 l of ice-cooled ethyl ether was added dropwise to the collected filtrate to form a precipitate. The precipitate was collected by suction filtration, and the precipitate was further washed with a small amount of cold ethyl ether. This precipitate was vacuum-dried to obtain a crude peptide.
<Purification>
The crude peptide was dissolved in a 1% CH ₃ CN (0.1% TFA) aqueous solution at a concentration of 25 mg / ml and purified by the following semi-preparative column.
Sample: 4 ml (25 mg / ml)
Column: YMC Pack ODS-A, 20 mm ID x 250 mm
Eluent: 0.1% TFA, gradient _{CH 3 CN1% → 60% (} 80min)
Flow rate: 5ml / min
Temperature: Room temperature Detection: 220nm
The eluted part of HPLC was passed through a 30 ml column of Dowex cation exchange resin (H type), washed with water and then eluted with 1 mol / L hydrochloric acid. The eluted part was lyophilized to obtain about 48 g of pRH.

<Physical properties of polyarginyl histidine>
1. Purity test Polyarginyl histidine was analyzed with a high performance liquid chromatography apparatus (HPLC). A sample of polyarginyl histidine 0.5 μl (0.01 mg / μl) was measured under the following conditions. The peak purity at that time was 98.5%.
Column: YMC Pack ODS-A, 4.6 mm ID × 150 mm
Eluent: 0.1% TFA, gradient CH ₃ CN 1% → 60% (25 min)
Flow rate: 1 ml / min
Temperature: Room temperature Detection: 220nm

2. Amino Acid Analysis After polyarginyl histidine was hydrolyzed at 6N HCl at 110 ° C. for 22 hours, amino acids were quantified with an amino acid analyzer (Hitachi L-8800). As a result, the molar ratio was [arginine: histidine = 5.00: 5.01].
In addition, elemental analysis of polyarginyl histidine was performed. The results of elemental analysis are shown in Table 1. The theoretical value and the experimental value almost agreed.

理論値＊ C₆₀H₉₇N₃₅O₁₁・11HCｌ・6.3H₂Oからの計算値

Theoretical value * Calculated from C ₆₀ H ₉₇ N ₃₅ O ₁₁ , 11HCl, 6.3H ₂ O

3. Mass spectrometry
Mass spectrometry was performed based on the Deconvolution method. Ionization was performed by electrospray ionization (ESI-MS). The measurement result was 1485, which almost coincided with the theoretical value 1484.64.

(Test Example 1)
Antibacterial effect of polyarginyl histidine against periodontal bacteria <microbial strain>
1. 1. Porphyromonas gingivalis ATCC 33277 strain, W50 strain Actinobacillus actinomycetemcomitans ATCC 29523, Y4 3 Prevotella intermedia ATCC 49046 shares4. 4. Prevotella nigrescens ATCC 25261 strain 5. Fusobacterium nucleatum ATCC 23726 strain, ATCC 25586 strain Treponema denticola ATCC 33520 strain <Test content 1>
The above cryopreserved strains were anaerobically used at 35 ° C for 48 hours using TSB medium supplemented with yeast extract (1 g / l), hemin (5 mg / ml) and menadione (1 mg / l), respectively. The cultured product was used as a precultured bacterial solution.
Add 100 μl of pre-cultured bacterial solution to 5 ml of TSB medium containing pRH solution (1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 500 μg / ml) The optical density (OD _{660 nm} ) was measured after 24 hours and 48 hours, respectively. OD when only pRH solution is added to the medium
The value obtained by subtracting the value of _{660 nm} was defined as the turbidity due to bacteria.
<Test result 1>
pRH was found to inhibit growth at a concentration of 100 μg / ml or higher for Prevotella nigrecens ATCC 25261 (Fig. 4), but it grew at a concentration of 500 μg / ml for other test strains. Inhibition was not seen (FIGS. 1-3, 5-9). As described above, polyarginyl histidine did not show significant growth inhibitory activity against oral microorganisms.

(Test Example 2)
Effects of polyarginyl histidine on the proteolytic enzymes of Porphyromonas gingivalis Arginine-specific proteolytic enzyme (Arg-gingipain; hereinafter referred to as “RGP”) and lysine specific Inhibition of the enzymatic activity of a proteolytic enzyme (Lys-gingipain; hereinafter referred to as “KGP”) was investigated.
<Test content 2>
pRH (final concentrations 100 μg / ml, 1000 μg / ml) and substrate (Bz-Arg-methylcoumarinamide for RGP, Boc-Val-Leu-Lys-methylcoumarinamide for KGP: final concentration, 100 μM each) Is dissolved in 800 μl of Tris-HCl buffer (pH 7.6) supplemented with NaCl (100 mM), CaCl ₂ (5 mM), and cysteine (10 mM), and 200 μl of the supernatant of Porphyromonas gingivalis culture is added, The reaction was allowed to proceed at room temperature for 15 minutes. The reaction was stopped by adding 2 mM TLCK, and the released methylcoumarin amide was measured with a spectrofluorometer (excitation wavelength: 380 nm, fluorescence wavelength: 460 nm).
Moreover, the inhibition rate was calculated | required by following formula (1).
Inhibition rate (%)
= (Blank fluorescence intensity-Sample fluorescence intensity)
÷ Blank fluorescence intensity x 100 (1)
The blank fluorescence intensity represents the fluorescence intensity when reacting without adding pRH.
The sample fluorescence intensity represents the fluorescence intensity when reacting with the addition of pRH.
<Test result 2>
pRH was 5.7% inhibition against KGP at 100 μg / ml and 23.1% inhibition at 1000 μg / ml (FIG. 10). In the case of RGP, the inhibition rate was 0.2% at 100 μg / ml and 7.7% at 1000 μg / ml (FIG. 11). pRH did not show significant inhibition of either KGP or RGP.

(Test Example 3)
Effect of polyarginyl histidine on hemagglutination activity of Porphyromonas gingivalis Since Porphyromonas gingivalis has strong hemagglutination activity, its inhibitory effect was examined.
<Test content 3>
Serial 2-fold dilutions of P. gingivalis culture supernatant and OD _{660 nm} = 2 P. gingivalis cells 90 [mu] l and the sample poly arginyl histidine was prepared (final concentration 10 μg / ml, 100 μg / ml, 500 μg / ml) Add 20 μl to a 96-well microtiter plate, add 90 μl of a test solution in which 1 ml of blood obtained from human blood is suspended in 49 ml of physiological saline, and let stand at room temperature for 2 hours. The minimum concentration at which hemagglutination activity was observed with the naked eye was determined.
<Test result 3>

  From Table 2, it can be seen that the hemagglutination activity of Porphyromonas gingivalis decreases with the concentration of polyarginyl histidine. pRH was found to inhibit the hemagglutination activity of Porphyromonas gingivalis in a concentration-dependent manner.

(Test Example 4)
Effect of polyarginyl histidine on the adhesion of Porphyromonas gingivalis to saliva-coated hydroxyapatite beads Porphyromonas gingivalis is a salivary protein with high proline protein, high proline glycoprotein and staserine It has been reported to bind. Therefore, the effect of polylysine and arginyl histidine on the adhesion of Porphyromonas gingivalis to saliva-coated hydroxyapatite beads was examined.
<Test content 4>
Incubate 2 mg of hydroxyapatite beads (hereinafter referred to as “HA”) with 150 μl of human unstimulated saliva overnight at room temperature and add KCl buffer (50 mM KCl, 1 mM KH ₂ PO ₄ , 1 mM CaCl ₂ , Washed with 0.1 M MgCl ₂ ). The obtained saliva-coated hydroxyapatite beads are hereinafter referred to as “sHA”. Porphyromonas gingivalis (2 x 10 ⁸ cells) and pRH or polylysine (0.1 mg / ml, 1 mg / ml each) radiolabeled by culturing in a medium supplemented with 5 μCi / ml ³ H. ml, 10 mg / ml) was added, incubated at room temperature for 1 hour with gentle agitation, and washed with Percoll and KCl buffer to obtain a sample. Add only radiolabeled Porphyromonas gingivalis (2 x 10 ⁸ cells) to sHA, incubate for 1 hour at room temperature with gentle agitation, and wash with Percoll and KCl buffer to obtain a blank sample It was. The ³ H of Porphyromonas gingivalis bound to sHA in these samples was measured, and the inhibition rate was calculated from the following formula (2).
Inhibition rate (%)
= (Blank ³ H value-Sample ³ H value)
÷ blank ³ H value × 100 (2)
The blank ³ H value represents a ³ H measurement value of Porphyromonas gingivalis of a blank sample treated without adding pRH or polylysine.
The sample ³ H value represents the ³ H measurement value of Porphyromonas gingivalis in a sample treated with pRH or polylysine added.
<Test result 4>
It is clear that pRH inhibits the adsorption of Porphyromonas gingivalis to sHA in a concentration-dependent manner, whereas polylysine, which is a basic peptide like pRH, did not inhibit the adsorption to sHA (FIG. 12). .

(Test Example 5)
Effect of polyarginyl histidine on the adhesion of Porphyromonas gingivalis to early dental biofilm-forming bacteria Porphyromonas gingivalis adheres to normal gram-positive bacteria already established on the tooth surface ( Coaggregation) is considered important. Therefore, coaggregation between Porphyromonas gingivalis and Streptococcus oralis, which is one of the typical early dental biofilm-forming bacteria, was measured by a turbidimetric method. <Test content 5>
1. Measurement of coaggregation activity
In a reaction solution of 10 mM phosphate buffered saline (PBS) (pH 6.0), add Porphyromonas gingivalis and Streptococcus oralis (5 x 10 ⁸ cells / ml each), and use a spectrophotometer (UV-265FW Using Shimadzu Corporation), the change in OD 550 nm was continuously recorded and measured for 7.5 minutes while stirring at 37 ° C. The 7.5 minute continuous recording is a recording in which the absorbance difference for 0.5 minute before and after a certain time point, that is, each minute, is calculated and recorded continuously and automatically. The maximum change in absorbance is read from the measured values, and the value is designated as A. In addition, if only Porphyromonas gingivalis (5 x 10 ⁸ cells / ml) is added to the reaction solution of 10 mM PBS (pH 6.0) and the maximum change in absorbance when measured in the same way is B, co-aggregation The activity is calculated from the following equation (3).
Coaggregation activity = A−B (3)
2. Measurement of inhibition rate When measuring coaggregation activity, add pRH solution (final concentrations 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2.5 mg / ml), and change OD550nm to 7.5 as described above. The measurement was continuously recorded for 1 minute, and the maximum change in absorbance was read. The inhibition rate is calculated from the following equation (4), where C is the coaggregation activity when no pRH solution is added and D is the coaggregation activity when the pRH solution is added.
Inhibition rate (%) = (C−D) ÷ C × 100 (4)
<Test result 5>
The pRH solution showed 33% aggregation inhibitory activity at a concentration of 2.5 mg / ml (FIG. 13).
The maximum absorbance change (A) when both Porphyromonas gingivalis and Streptococcus oralis are added (A), and the maximum change in absorbance when only Streptococcus oralis is measured in the same way is The amount of change was 1% or less. On the other hand, the maximum amount of change in absorbance (B) measured in the same manner with the addition of Porphyromonas gingivalis was about 30% of A.

It is a figure which shows the influence of polyarginyl histidine on the growth of Porphyromonas gingivalis ATCC 33277 strain. It is a figure which shows the influence of polyarginyl histidine on growth of a Porphyromonas gingivalis W50 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Prevotella intermedia ATCC 49046 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Prevotella nigrecens ATCC 25261 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Actinobacillus actinomisetemcomitans ATCC 29523 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Actinobacillus actinomisetemcomitans Y4 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Fusobacterium nucleatum ATCC 23726 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Fusobacterium nucleatum ATCC 25586 strain. It is a figure which shows the influence of polyarginyl histidine on the growth of Treponema denticola ATCC 33520 strain. It is a figure which shows the influence of polyarginyl histidine on the KGP activity of Porphyromonas gingivalis ATCC 33277 strain (mean +/- SD, n = 3). It is a figure which shows the influence of polyarginyl histidine on the RGP activity of Porphyromonas gingivalis ATCC 33277 (mean +/- SD, n = 3). It is a figure which shows the influence of polylysine and polyarginyl histidine on adhesion to the saliva coat hydroxyapatite beads of Porphyromonas gingivalis ATCC 33277 strain (mean ± SD, n = 5). It is a figure which shows the influence of polyarginyl histidine on the co-aggregation of Porphyromonas gingivalis ATCC 33277 strain and Streptococcus oralis ATCC 9811 strain.

Claims (15)

  An oral composition containing a peptide in which arginine and histidine are alternately bound. 2. The composition for oral cavity according to claim 1, wherein the peptide has a structure represented by the following formula (I) or (II).

(Arg represents arginine, His represents histidine. R ₁ represents hydrogen, sugar, acyl, biotinyl, thiol, phenol, or indole, and R ₂ represents a hydroxyl group, sugar, acyl, biotinyl, thiol, phenol, or indole. And n represents an integer of 2 or more.)
The composition for oral cavity according to claim 1 or 2, wherein the peptide has a structure represented by the following formula (I).

  4. The ratio of D-form arginine and L-form arginine of the arginine is D-form arginine: L-form arginine = 10: 90 to 0: 100, according to claim 1, Oral composition.   The composition for oral cavity according to any one of claims 1 to 4, wherein the arginine is L-form arginine.   The ratio of D-form histidine and L-form histidine of the histidine is D-form histidine: L-form histidine = 100: 0 to 70:30, according to any one of claims 1 to 5, Oral composition.   The composition for oral cavity according to any one of claims 1 to 6, wherein the histidine is D-form histidine.   The composition for oral cavity according to any one of claims 1 to 7, wherein the peptide is a 1-20 mer of (arginine-histidine).   The composition for oral cavity according to any one of claims 1 to 8, wherein the peptide is a pentamer of (arginine-histidine).   The composition for oral cavity according to any one of claims 1 to 9, wherein the peptide is a peptide produced by fermentation production of a microorganism.   The composition for oral cavity according to any one of claims 1 to 10, wherein the peptide content is 0.001 to 10% by weight.   The composition for oral cavity according to any one of claims 1 to 11, wherein the content of the peptide is 0.01 to 1% by weight.   It is characterized by inhibiting the hemagglutination activity of Porphyromonas gingivalis, adhesion of Porphyromonas gingivalis and saliva-coated hydroxyapatite, and coaggregation of Porphyromonas gingivalis and Streptococcus oralis The composition for oral cavity as described in any one of Claims 1-12.   It is an object for periodontal disease prevention, The composition for oral cavity as described in any one of Claims 1-13 characterized by the above-mentioned.   It is an object for periodontal disease treatment, The composition for oral cavity as described in any one of Claims 1-13 characterized by the above-mentioned.

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