Coaggregation inhibitor and coaggregation inhibition method

Abstract

To provide an oral preparation that is useful for preventing and improving periodontal disease-related diseases by inhibiting the coaggregation of periodontal disease-related bacteria on the tooth surface and thereby preventing the adhesion and colonization of periodontal disease-related bacteria on the tooth surface.SOLUTION: Provided is a coaggregation inhibitor for periodontal disease-related bacteria containing Equisetum arvense extract as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a coaggregation inhibitor and a coaggregation inhibition method. [Background technology]

[0002] Periodontal disease is an oral infection that develops when pathogenic bacteria adhere to and colonize the tooth surface. The mechanism by which periodontal disease-related bacteria colonize the tooth surface is as follows: first, early colonizing bacteria such as Streptococcus oralis, Streptococcus sanguis, Streptococcus gordonii, and Actinomyces naeslundii adhere to the enamel surface, which is covered with a thin film of saliva (pellicle). As these early colonizing bacteria grow, they coaggregate with each other and initiate the formation of dental plaque. As the plaque matures, the microbial flora transitions from facultative anaerobes to obligate anaerobes, and obligate anaerobes such as Fusobacterium nucleatum coaggregate with the initial colonizing bacteria. It is believed that periodontal disease-related bacteria such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis coaggregate with Fusobacterium nucleatum and colonize the plaque.

[0003] Coaggregation is caused by nonspecific electrostatic interactions between bacteria, lectin-receptor type interactions, adhesion through adhesive polysaccharide synthesis, nonspecific hydrophobic interactions, etc., and plays a particularly important role in the adhesion of pathogenic bacteria to tooth surfaces.

[0004] In order to prevent the colonization and proliferation of pathogenic bacteria that cause dental caries and periodontal disease in dental plaque, it is effective to inhibit the coaggregation of bacteria, and various means for inhibiting the coaggregation of bacteria have been studied. For example, Patent Document 1 discloses an oral bacterial coaggregation inhibitor that contains erythritol. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-8579 A Summary of the Invention [Problem to be solved by the invention]

[0006] Since erythritol is a type of sugar alcohol and is generally known as a sweetener, oral compositions containing erythritol as a coaggregation inhibitor have a sweet taste, which makes them difficult to use for users who do not like sweet things. In addition, since sweeteners are reminiscent of dental caries, there is a concern that when used as a coaggregation inhibitor for periodontal disease-related bacteria, it may give users an unfavorable impression.

[0007] Therefore, an objective of the present invention is to provide an oral preparation that prevents the adhesion and settlement of periodontal disease-related bacteria on tooth surfaces by inhibiting coaggregation of periodontal disease-related bacteria on the tooth surface, thereby being useful for the prevention and amelioration of periodontal disease-related diseases. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors discovered that horsetail extract has the effect of inhibiting coaggregation of periodontal disease-related bacteria, and thus completed the present invention.

[0009] That is, the present invention is achieved by the following (1) to (3). (1) An inhibitor of coaggregation of periodontal disease-related bacteria containing horsetail extract as an active ingredient. (2) The coaggregation inhibitor according to (1) above, wherein the periodontal disease-associated bacterium is Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans. (3) A method of suppressing coaggregation of periodontal disease-related bacteria by applying an oral composition containing horsetail extract as an active ingredient into the oral cavity. Effect of the Invention

[0010] The coaggregation inhibitor of periodontal disease-related bacteria containing the horsetail extract of the present invention as an active ingredient can inhibit coaggregation of periodontal disease-related bacteria, so by applying an oral composition containing horsetail extract as an active ingredient to the oral cavity, the formation of dental plaque can be inhibited and tooth decay and periodontal disease can be prevented. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the absorbance at OD 600 nm measured in Test Example 1 for Control Example 1, Examples 1 to 4, and Comparative Examples 1 to 3. [Diagram 2] 1 is a photographic diagram of Control Example 1, Examples 1 to 4, and Comparative Examples 1 to 3 of Test Example 1. [Diagram 3] 1 is a graph showing the absorbance at OD 600 nm measured for Examples 5 to 10 in Test Example 2, Control Example 1 in Test Example 1, and Comparative Examples 1 to 3. [Figure 4] 1 is a graph showing the absorbance at OD 600 nm measured in Test Example 3 for Control Example 2, Examples 11 to 13, and Comparative Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, the embodiments of the present invention will be described in more detail.

[0013] The coaggregation inhibitor of periodontal disease-associated bacteria of the present invention (hereinafter also simply referred to as "coaggregation inhibitor") contains horsetail extract as an active ingredient. Horsetail extract is an extract (plant extract) obtained from Equisetum arvense, a plant of the Equisetum genus in the Equisetaceae family, which is a type of herbal medicine.

[0014] The horsetail extract may be obtained by extracting the whole plant, stems and / or leaves of the horsetail, but it is preferable to obtain it by extracting the whole plant of the horsetail, and a commercially available product may be used. Methods for obtaining the horsetail extract by extraction include solvent extraction, supercritical extraction, steam distillation, etc.

[0015] Examples of extraction solvents used in the solvent extraction treatment include water and alcohols. Examples of alcohols include methanol, ethanol, propylene glycol, 1,3-butylene glycol, or mixtures thereof, or a 1% urea-containing ethanol solution, or a 1,3-butylene glycol solution containing 1% urea. It is preferable to use an extract obtained by extraction with these solvents, and it is particularly preferable to use an extract obtained by extraction with water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, or a mixture thereof.

[0016] The obtained extract may be used as it is, or may be dried before use. If necessary, the obtained extract may be purified, concentrated, or the like. Examples of the purification process include filtration, or adsorption, decolorization, and separation using an ion exchange resin or an activated carbon column. Examples of the concentration process include conventional methods such as an evaporator. These may be further dried, such as freeze-dried, or may be powdered according to a conventional method. If necessary, the extract thus obtained may be dissolved in water, ethanol, or the like before use.

[0017] Commercially available products of horsetail extract include, for example, "Horsetail Extract BG" (manufactured by Maruzen Pharmaceutical Co., Ltd.), "Horsetail Extract" (manufactured by Maruzen Pharmaceutical Co., Ltd.), and "Sugina Extract" (manufactured by Koei Kogyo Co., Ltd.).

[0018] One type of horsetail extract may be used alone, or two or more types of horsetail extracts extracted from different parts or by different extraction methods may be used in combination.

[0019] The horsetail extract is preferably contained in the coagulation inhibitor at a content of 0.00001 w / v% or more in terms of solid content. When the content of the horsetail extract in the coagulation inhibitor is 0.00001 w / v% or more in terms of solid content, the desired effect of the present invention can be obtained when the coagulation inhibitor is applied to the oral cavity. The content of the horsetail extract in terms of solid content is preferably 0.0001 w / v% or more, more preferably 0.001 w / v% or more, and more preferably 1 w / v% or less, more preferably 0.1 w / v% or less, and even more preferably 0.05 w / v% or less, in terms of solid content, in the coagulation inhibitor.

[0020] The coagulation inhibitor of the present invention may contain other optional components that are applied to the oral cavity, as long as the effects of the present invention are not impaired. Examples of the optional components include solvents, bactericides, anti-inflammatory agents, tartar prevention agents, pH adjusters, preservatives, surfactants, colorants, and flavors.

[0021] Examples of the solvent include water such as purified water and ion-exchanged water; lower monohydric alcohols such as ethanol; and polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol.

[0022] Examples of the disinfectant include quaternary ammonium salts such as cetylpyridinium chloride, benzethonium chloride, and benzalkonium chloride, hinokitiol, triclosan, isopropylmethylphenol, thymol, and alkyldiaminoethylglycine hydrochloride solution.

[0023] Examples of anti-inflammatory agents include glycyrrhizic acid and derivatives thereof, tranexamic acid and derivatives thereof, aminocaproic acid and derivatives thereof, allantoin and derivatives thereof, ascorbic acid and derivatives thereof, azulene, azulene sulfonic acid and derivatives thereof, dihydrocholesterol, epidihydrocholesterol, tocopherol acetate, tocopherol nicotinate, lysozyme chloride, and the like.

[0024] Examples of anti-tartar agents include phosphates, polyphosphates, methoxyethylene maleic anhydride copolymers, zinc chloride, and zinc organic acids.

[0025] Examples of pH adjusters include sodium phosphate salts such as monosodium phosphate and disodium phosphate, sodium citrate, citric acid, glucono-δ-lactone, sodium gluconate, sodium acetate, sodium carbonate, sodium bicarbonate, sodium hydroxide, and hydrates thereof.

[0026] Examples of preservatives include paraoxybenzoic acid esters, sodium benzoate, potassium sorbate, and phenoxyethanol.

[0027] Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Specifically, examples of the nonionic surfactants include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty acid diethanolamides, fatty acid monoglycerides, polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oil, alkyl glucosides, and polyoxyethylene polyoxypropylene block copolymers. Examples of the cationic surfactants include polyoxyethylene alkylamines and fatty acid amides. Examples of the anionic surfactants include sulfate ester salts, α-olefin sulfonates, sulfosuccinates, N-acyl amino acid salts, and acylated methyl taurine salts. Examples of the amphoteric surfactants include betaine acetate amphoteric surfactants and imidazoline amphoteric surfactants.

[0028] Examples of pigments include Blue No. 1, Blue No. 2, Blue No. 201, Yellow No. 4, Yellow No. 5, Yellow No. 202(1), Yellow No. 203, Red No. 3, Red No. 102, Red No. 104, Red No. 105, Green No. 3, and Green No. 201.

[0029] Examples of fragrances include natural essential oils such as peppermint oil, spearmint oil, mint oil, eucalyptus oil, clove oil, thyme oil, rosemary oil, lemon oil, ginger oil, lime oil, cassia oil, cardamom oil, lavender oil, and cinnamon oil; fragrance components such as L-menthol, L-carvone, carvacrol, eugenol, anethole, 1,8-cineole, thymol, vanillin, pinene, 3-L-menthoxypropane-1,2-diol, and methyl salicylate; mixtures of these, natural fragrances, blended fragrances, and synthetic fragrances.

[0030] Some of the above-mentioned optional ingredients promote bacterial coaggregation when applied to the oral cavity. For example, tranexamic acid, which is an anti-inflammatory ingredient, easily causes bacteria to coaggregate, and by incorporating the coaggregation inhibitor of the present invention together, it becomes difficult for bacteria to coagulate with each other, so that the oral cavity can be kept clean and the anti-inflammatory effect of tranexamic acid is not impaired.

[0031] The coaggregation inhibitor of the present invention can inhibit coaggregation of any bacteria present in the oral cavity without any particular limitation, and can effectively inhibit coaggregation of other bacteria with initially colonizing bacteria. Periodontal disease-related bacteria that coaggregate with early colonizing bacteria include, for example, Fusobacterium nucleatum, Porphyromonas gingivalis, Tannerella (Bacteroides) forsythia, Treponema denticola, Prevotella intermedia, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Actinomyces viscosus, Aggregatibacter actinomycetemcomitans, and others. actinomycetemcomitans, Selenomonas sputigena, etc., among which coaggregation of Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans can be suitably inhibited.

[0032] The coagulation inhibitor of the present invention can be applied to the oral cavity as an oral composition. Examples of the formulation include liquid, gel, paste, cream, solid form such as tablet, powder form such as granule or fine grain, capsule, soft capsule, etc. Among them, liquid formulations are preferred because the horsetail extract can be uniformly applied to the oral cavity. Examples of liquid formulations include mouthwash (also called gargle), liquid dentifrice, mouthwash, mouth spray, etc.

[0033] In this specification, the term "liquid" refers to a material that has fluidity and has a viscosity of 20 mPa s or less, preferably 10 mPa s or less, at 20° C. Here, the viscosity can be measured using a commercially available rotational viscometer (for example, a B-type viscometer manufactured by Toki Sangyo Co., Ltd.).

[0034] The present invention also provides a method for inhibiting coaggregation of periodontal disease-related bacteria by applying an oral composition containing horsetail extract as an active ingredient to the oral cavity, and the coaggregation inhibitor of the present invention described above can be used as the oral composition.

[0035] The oral composition contains the horsetail extract in an amount of preferably 0.00001 w / v% or more, more preferably 0.0001 w / v% or more, and even more preferably 0.001 w / v% or more, calculated as solid content. The oral composition contains the horsetail extract in an amount of preferably 1 w / v% or less, more preferably 0.1 w / v% or less, and even more preferably 0.05 w / v% or less, calculated as solid content.

[0036] When the oral composition is a mouthwash, the method of use is to hold 5 to 25 mL of the liquid oral composition in the mouth and rinse the mouth for 15 to 60 seconds. This allows the liquid oral composition of the present invention to be sufficiently distributed in the oral cavity, thereby exerting the effect of inhibiting the coagulation of periodontal disease-related bacteria. EXAMPLES

[0037] The present invention will be further explained below with reference to the following test examples, but is not limited to these examples.

[0038] <Test Example 1: Control Example 1, Examples 1 to 4, Comparative Examples 1 to 3> (Test Method) 1. Preparation of Test Specimens Fusobacterium nucleatum (ATCC23736, hereinafter referred to as "Fn") was pre-cultured in 10 mL of BHI (Brain Heart Infusion) liquid medium under anaerobic conditions at 37°C for 24 to 48 hours to obtain an Fn pre-culture solution. Similarly, Aggregatibacter actinomycetemcomitans (ATCC33384, hereinafter referred to as "Aa") was cultured in BHI liquid medium to obtain an Aa preculture solution. The Fn preculture solution and the Aa preculture solution were transferred to a centrifuge tube, respectively, and centrifuged at 5000 rpm for 10 minutes. The supernatant was removed, and 5 mL of buffer (Tris (1 mmol / L), calcium chloride (0.1 mmol / L), magnesium chloride (0.1 mmol / L), sodium chloride (0.15 mol / L), pH 8.0) was added. The mixture was centrifuged again at 5000 rpm for 10 minutes. The supernatant was removed, and further buffer was added. The absorbance OD at 600 nm in a spectrophotometer was measured. 600 =0.5 Fn suspension and Aa suspension were obtained. Next, horsetail extract and / or tranexamic acid were poured into a 96-well round-bottom plate to give the final concentrations shown in Table 1, and 100 μL of Fn suspension and 100 μL of Aa suspension were poured thereto to prepare test samples. Horsetail extract was "Horsetail Extract BG" (extracted from the whole plant of horsetail with 50% by volume 1,3-butylene glycol solution) manufactured by Maruzen Pharmaceutical Co., Ltd., diluted with a mixture of sterilized water and butylene glycol in a mass ratio of 50:50, and added so that the concentration of horsetail extract in the sample was as shown in Table 1 in terms of solid content. Tranexamic acid was added using an aqueous tranexamic acid solution so that the final concentration of tranexamic acid in the sample was as shown in Table 1. Three wells were used for each example, and three samples were prepared. As control example 1, 4 μL of a mixture of sterilized water and butylene glycol in a mass ratio of 50:50 was poured into a plate, and 100 μL of the Fn suspension and 100 μL of the Aa suspension were poured therein to prepare test samples. Three samples were prepared for control example 1.

[0039] 2. Assessment of Coaggregation The plate prepared above was mixed for 15 seconds using a benchtop mixer (TAIYO MICRO MIXER manufactured by Taiyo Chemical Industry Co., Ltd.) and then left to stand for 15 seconds, with this cycle repeated for 5 minutes. After leaving the plate to stand for 5 minutes, the plate was manually mixed by holding the side of the plate in the hand and moving the plate in small circles for 20 seconds, and then left to stand at room temperature (20-25°C). After 16 hours, the plate was stirred again by hand, allowed to stand at room temperature, and after confirming that aggregation of Control Example 1 could be visually confirmed (5 to 9 hours after stirring again), the absorbance OD of each well at 600 nm was measured using a plate reader ("MULTISKAN GO" manufactured by Thermo Scientific). 600 was measured, and the average value of three specimens was calculated for each case.

[0040] Compared to control 1, OD 600 The coagulation inhibitory effect was judged to be present when the OD was reduced by 0.005 or more. More specifically, the coagulation inhibitory effect was evaluated by comparing the OD with that of Control Example 1. 600 A decrease of 0.005 or more but less than 0.020 is indicated as "〇", a decrease of 0.020 or more is indicated as "◎", and a decrease of less than 0.005 or OD 600 The case where the value was larger than that of Control Example 1 was evaluated as "x".

[0041] The results are shown in Table 1 and Figures 1 and 2.

[0042] [Table 1]

[0043] <Test Example 2: Examples 5 to 10> Coagulation was evaluated in the same manner as in Test Example 1, except that the final concentrations of the horsetail extract and tranexamic acid were set as shown in Table 2. The results are shown in Table 2 and Figure 3. In addition, Table 2 and Figure 3 also show the results of Control Example 1 and Comparative Examples 1 to 3 of Test Example 1 for comparison.

[0044] [Table 2]

[0045] From Test Example 1 and Test Example 2, Control Example 1 had an absorbance OD 600 The coaggregation of Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans was observed with a value of 0.209. Furthermore, in Comparative Examples 1 to 3 containing tranexamic acid, coaggregation was promoted compared to Control Example 1, and aggregates were precipitated at the bottom of the plate as shown in Figure 2. In contrast, Examples 1 to 3, which contained the horsetail extract, had a lower absorbance OD than Control Example 1. 600 was significantly smaller, indicating that the horsetail extract inhibited the coaggregation of Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Furthermore, a comparison between Comparative Example 1 and Examples 4-5, a comparison between Comparative Example 2 and Examples 6-7, and a comparison between Comparative Example 3 and Examples 8-10 revealed that the inclusion of the horsetail extract inhibited bacterial coaggregation even when tranexamic acid was added.

[0046] <Test Example 3: Control Example 2, Examples 11 to 13, Comparative Example 4> Test Example 1 was repeated, except that dipotassium glycyrrhizinate was used instead of tranexamic acid, and the horsetail extract and dipotassium glycyrrhizinate were adjusted to the final concentrations shown in Table 3. Dipotassium glycyrrhizinate was added in an aqueous solution of dipotassium glycyrrhizinate so that the concentration of dipotassium glycyrrhizinate in the sample, converted into glycyrrhizinic acid, was as shown in Table 3.

[0047] The results are shown in Table 3 and FIG.

[0048] [Table 3]

[0049] From Test Example 3, the absorbance OD of Control Example 2 and Comparative Example 4 containing dipotassium glycyrrhizinate was 600 The absorbance OD was almost the same, indicating that dipotassium glycyrrhizinate had no effect on bacterial coagulation. Examples 11 to 13, which contained dipotassium glycyrrhizinate and horsetail extract, were higher than those of Control Example 2 and Comparative Example 4. 600 It was also found from Test Example 3 that the horsetail extract inhibited bacterial coaggregation.

Claims

1. An inhibitor of coaggregation of periodontal disease-related bacteria containing horsetail extract as an active ingredient.

2. The coaggregation inhibitor according to claim 1, wherein the periodontal disease-associated bacterium is Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

3. A method for inhibiting coaggregation of periodontal disease-related bacteria by applying an oral composition containing horsetail extract as an active ingredient into the oral cavity.

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