Composition for oral care

Abstract

[Problem] To produce: a product that is for care of the oral cavity and that can suppress adhesion of bacteria roaming in the oral cavity to the teeth, by using no antibacterial components or by using an antibacterial component at a low concentration; and a material or the like to be contained in such a product. [Solution] This composition for oral cavity care contains a Codiaceae algae extract. The composition contains, as an active component, a protein fraction of a molecular weight of 5000 or more included in the Codiaceae algae extract.

Description

Cross-reference

[0001] This application claims priority under Japanese Patent Application No. 2021-20876, filed in Japan on 12 February 2021, and Japanese Patent Application No. 2021-140997, filed in Japan on 31 August 2021, all contents of said said application are incorporated herein by reference in their entirety. Furthermore, all contents of all patents, patent applications, and documents cited herein are also incorporated herein by reference in their entirety. [Technical Field]

[0002] The present invention relates to an oral care composition containing a protein fraction having a specific molecular weight obtained from an extract of a type of algae belonging to the family Myrrhaceae as an active ingredient. [Background technology]

[0003] Periodontal disease is an inflammatory disease that begins when a type of pathogenic bacteria in the oral cavity, known as periodontal disease-causing bacteria, settles in the mouth. If oral hygiene is insufficient, plaque (oral bacteria and their metabolites) adheres to the gum line, settles, and proliferates. Neutrophils and macrophages infiltrate in response to this foreign substance, causing inflammation. If oral hygiene is performed early through brushing, this inflammation can be improved. However, if plaque accumulation is left untreated, the inflammation will spread, and once periodontal pockets are formed, the plaque accumulated there becomes difficult to remove by brushing alone. Therefore, suppressing plaque, that is, killing or stabilizing pathogenic bacteria in the oral cavity, is considered a useful means of preventing and improving periodontal disease.

[0004] In recent years, dental plaque has been viewed as a biofilm, and it has become clear that bacteria in oral biofilms (dental plaque) differ significantly from free-floating bacteria in their protein expression patterns and drug resistance, and that drugs effective against free-floating bacteria are not effective against bacteria that make up biofilms.

[0005] Numerous antimicrobial agents, such as cationic antimicrobial agents like cetylpyridinium chloride, benzethonium chloride, and chlorhexidine, as well as nonionic antimicrobial agents like triclosan, have been incorporated into oral compositions as effective sterilization methods. However, due to the drug resistance mechanisms of biofilm bacteria, these antimicrobial agents alone do not provide sufficient biofilm suppression. While improvement techniques involving the combined use of other components have been proposed to enhance the bactericidal power of these antimicrobial agents, none have yielded significant results due to factors such as low drug penetration into biofilms.

[0006] In contrast, Patent Document 1 discloses that mushroom-derived lectin ABA, which recognizes glycans having the terminal structures of Galβ1-3GalNAc and GlcNAc, has the effect of suppressing the adhesion and growth of oral bacteria to plaque or biofilm in the oral cavity. Furthermore, it has been reported that lectins contained in the algal extracts of Milaceae algae, which bind to glycans that compete with GalNAc, can prevent tooth decay by suppressing the adhesion and growth of oral bacteria, particularly Streptococcus mutans (see, for example, Patent Document 2). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 5558362 [Patent Document 2] Patent No. 5925431 [Non-patent literature]

[0008] [Non-Patent Document 1] Dental Medicine Research 30(1):9-14,2010 [Non-Patent Document 2] Showa Dental Journal 22:214-219, 2002 [Overview of the project] [Problems that the invention aims to solve]

[0009] When using oral care products such as mouthwash that contain antibacterial ingredients, side effects such as diarrhea may occur, and these side effects usually disappear when antibiotic use is discontinued. In addition, antibacterial ingredients may reduce non-pathogenic bacteria that are already established in the body, potentially giving other pathogens an opportunity to infect. Furthermore, there are concerns that bacteria resistant to antibacterial ingredients may develop. Therefore, there is a strong desire to provide oral care products and materials that can be incorporated into such products that can serve as alternatives to the use of antibacterial ingredients (including reducing the amount of antibacterial ingredients).

[0010] The problems that this invention aims to solve include creating oral care products and materials contained in oral care products that can suppress the adhesion of bacteria floating in the oral cavity to teeth without using antibacterial components or by using antibacterial components at a low concentration. [Means for solving the problem]

[0011] The present invention was made to solve the above problems, and includes, for example, an oral care product containing a protein fraction having a specific molecular weight contained in an extract of Mircea algae, and a material to be included in said product (oral care material).

[0012] In other words, the present invention includes the following embodiments. [1] An oral care composition containing an extract of a myrrh algae as an active ingredient. An oral care composition containing a protein fraction with a molecular weight of 5000 or more contained in the myrrh algae extract as an active ingredient. This protein fraction preferably has a molecular weight of 5000 or more and less than 50000, and more preferably has a molecular weight of 5000 or more and less than 30000. Alternatively, it may be a protein present in a high molecular weight fraction with a molecular weight of 50000 or more. [2] The oral care composition according to [1] further comprising an antibacterial component. [3] The oral care composition according to [1] or [2], wherein the antibacterial component is one or more selected from the group consisting of cetylpyridinium chloride (CPC), chlorhexidine, benzalkonium chloride, benzethonium chloride, decamethonium chloride, chlorhexidine gluconate, protamine, dodecyldiaminoethyl glycine, triclosan, 3-methyl-4-isopropylmethylphenol (IPMP), thymol, carvacrol, farnesol, bisabolol, cineole, hinokitiol, sodium lauroyl sarcosinate, and l-menthol. [4] The oral care composition according to any one of [1] to [3], wherein the active ingredient is contained in an amount of 0.001 to 5% by mass based on the total amount of the composition in terms of the extract of the Myrionectaceae algae. [5] The oral care composition according to any one of [2] to [4], wherein the antibacterial component is contained in an amount of 0.0001 to 1% by mass based on the total amount of the composition.

Advantages of the Invention

[0013] According to the oral care composition of the present invention, it is possible to suppress the adhesion of bacteria floating in the oral cavity to teeth without using an antibacterial component or by reducing its concentration.

Brief Description of the Drawings

[0014] [Figure 1] Figure 1 shows the results of a bacterial adsorption test for Myrionectaceae extract (ML), cetylpyridinium chloride (CPC), and their mixture (CPC + ML). [Figure 2] Figure 2 shows the results of a bacterial adsorption test for Myrionectaceae extract (ML), benzalkonium chloride, and their mixture (benzalkonium chloride + ML). [Figure 3] Figure 3 shows the results of a bacterial adsorption test for Myrionectaceae extract (ML), chlorhexidine, and their mixture (chlorhexidine + ML). [Figure 4] Figure 4 shows the results of a bacterial adsorption test for Myrionectaceae extract (ML), IPMP, and their mixture (IPMP + ML). [Figure 5]Figure 5 shows the results of the bacterial adsorption test for the four fractions (molecular weight 50,000 or more, 30,000 or more but less than 50,000, 5,000 or more but less than 30,000, and less than 5,000) that were fractionated by molecular weight in Example 2. [Figure 6] Figure 6 shows the results of the bacterial adsorption test for the three fractions (molecular weight less than 5000, 5000 to less than 30000, and 30000 to less than 50000) that were fractionated by molecular weight in Example 2. [Figure 7] Figure 7 shows the results of bacterial adsorption tests when CPC was added as an antibacterial agent to the four fractions (molecular weight 50,000 or more, 30,000 or more but less than 50,000, 5,000 or more but less than 30,000, and less than 5,000) that were fractionated by molecular weight in Example 2. [Figure 8] Figure 8 shows the results of a bacterial adsorption test using mill extract and a commercially available mouthwash, which investigated the combined effects of the mill extract and the mouthwash. [Modes for carrying out the invention]

[0015] Next, embodiments of the present invention will be described with reference to the drawings. It should be noted that the embodiments described below are not intended to limit the invention as defined in the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

[0016] (Oral care composition) In this specification, "oral care composition" refers to, for example, the following: • Products that, in their normal use, are not intended to be swallowed for the purpose of systemic administration of a specific therapeutic agent, but rather are held in the oral cavity for a sufficient time to come into contact with substantially all tooth surfaces and / or oral tissues for the purpose of oral activity (oral care products). • The materials contained in the product. These materials contain an extract from the family Myrrhizaceae algae.

[0017] Oral care products may be in the form of mouthwash, mouth rinses, toothpastes, toothpastes, gels, or solutions. Oral care products may also be incorporated into floss, strips, or films for direct application or adhesion to the oral surface, or into devices or applicators such as toothbrushes or rotary applicators. Such applicators may be disposable or reusable.

[0018] An oral care composition in one embodiment of the present invention contains an extract of a myrrh-family algae or a protein fraction therefrom with a molecular weight of 5000 or more as an active ingredient. The details thereof will be described below.

[0019] (Extract from algae of the family Myrrhaceae) The extract of the family Codium algae used in one embodiment of the present invention (hereinafter referred to as "the extract of this embodiment") is extracted from algae belonging to the family Codium, which belongs to the order Codium (sometimes classified as Bryopsidales in recent classifications) of the class Chlorophyceae. These algae include Codium fragile, Codium tomentosum, Codium minus, Codium spongiosum, Codium subtubulosum, Codium intricatum, Codium adhaerens, Codium arabicum, Codium coactum, Codium barbatum, Codium contractum, Codium latum, and Codium It may belong to the genus Codium, which includes, but is not limited to, *Codium cylindricum*.

[0020] The method for recovering the extract in this embodiment may be any method, provided that it does not substantially impair the activity of inhibiting or suppressing the binding of oral bacteria to biomolecules in saliva.

[0021] The extract in this embodiment may be collected from any tissue of the algae. The algae may be washed, then crushed with a homogenizer or the like, or freeze-dried into a powder, or pulverized with a grinder before being subjected to extraction. The aqueous solution used for extraction may include, but is not limited to, an aqueous solution containing NaCl or other salts, such as physiological saline, a mixture of acetone or other water-soluble organic solvents and / or alcohol with water, and purified water or deionized water, but may also include other aqueous solvents well known to those skilled in the art. Here, the alcohol includes, but is not limited to, ethanol, ethylene glycol, butylene glycol, and glycerin. The aqueous solvent may have an acidic or alkaline pH. To adjust the pH of the aqueous solvent, adipic acid, citric acid, gluconic acid, succinic acid, acetic acid, tartaric acid, lactic acid, fumaric acid, malic acid, phosphoric acid, potassium carbonate, sodium carbonate, sodium bicarbonate, and potassium or sodium salts of phosphates may be used, but is not limited to these. Furthermore, to maintain a constant pH, the solution may include, but is not limited to, Tris-HCl buffer, Hepes buffer, phosphate buffer, acetate buffer, citrate buffer, and glycine-HCl buffer. The extraction method may be appropriately selected from immersion extraction, pressure extraction, supercritical or subsupercritical extraction, or a combination thereof. The extraction conditions may be any conditions, provided that the activity of inhibiting or suppressing the binding of oral bacteria to biomolecules in saliva is not substantially impaired, but the extraction time is preferably from 10 minutes to 24 hours, and the extraction temperature may be 4°C or higher, preferably room temperature or higher, and more preferably 15°C or higher.

[0022] (Active ingredients) Next, the extract of this embodiment can be used as is, or purified or concentrated to obtain an active ingredient for an oral care composition. In a preferred embodiment, this active ingredient is present in a protein fraction with a molecular weight of 5000 or more. More preferably, it is in a fraction with a molecular weight of 5000 or more but less than 50000, and even more preferably in a fraction with a molecular weight of 5000 or more but less than 30000.

[0023] The purification and concentration of algal extracts from the family Myrrhidae may be performed using techniques including, but not limited to, centrifugation, salting out, dialysis, vacuum concentration, ultrafiltration, gel filtration, ion exchange chromatography, and affinity chromatography, either individually or in combination of two or more. After purification or concentration, the algal extract may be dried under vacuum or diluted with a solvent before use, as needed.

[0024] One candidate for the active ingredient of the present invention is a lectin that binds to glycans that compete with GalNAc (see Patent Document 2). However, this active ingredient does not necessarily bind to all glycans that have GalNAc at their terminus. Furthermore, the second or third sugar residue from the terminus may bind only to glycans with a specific structure, but this binding may be inhibited in the presence of GalNAc.

[0025] In this specification, lectins refer to proteins other than antibodies, T cell receptors, Toll-like receptors, and other immune proteins involved in the animal immune system, which have the ability to specifically bind to sugar chains. Typically, lectins can aggregate red blood cells and other animal cells. According to Kanji Hori (Chemistry and Biology, 32:586-594, (1994)), lectins derived from Mircea algae share the common characteristic of binding to sugar chains that compete with GalNac. While not bound by any theory, the active ingredient of the present invention is thought to bind to GalNAc present on the surface of a membrane formed by polysaccharides contained in the biopolymers of saliva, thereby inhibiting the adhesion of oral bacteria to it.

[0026] The active ingredient of the present invention may be present at any concentration, provided that its activity in suppressing or inhibiting the binding of oral bacteria to biomolecules in saliva is not substantially impaired. Preferably, it is present at 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, based on the total amount of the oral care composition in terms of the extract of the family Myrrhizaceae algae. Furthermore, the upper limit is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, and even more preferably 2% by mass or less.

[0027] The protein concentration in the active ingredient of the present invention may be any concentration, provided that it does not substantially impair the activity of inhibiting or suppressing the binding of oral bacteria to biomolecules in saliva, but it is preferably 0.5 μg / mL or higher, and more preferably 5 μg / mL or higher.

[0028] (Antibacterial ingredient) The oral care composition of this embodiment preferably contains a predetermined amount of an antibacterial component. The antibacterial component is a component that has antibacterial activity. Antibacterial activity includes, for example, sterilization activity, bactericidal activity, disinfection activity, bacteriostatic activity, antibacterial activity, antimicrobial activity, preservative activity, antimicrobial activity, and antifungal activity. The antibacterial agent is an agent that contains an antibacterial component. Examples of antibacterial agents include bactericides, antibacterial agents, antifungal agents, antifungal agents, preservatives, deodorants, and insecticides, and among these, microbial control agents such as bactericides, antibacterial agents, antifungal agents, and antifungal agents, as well as deodorants, can be suitably used. As the antibacterial component contained in the oral care composition of this embodiment, known compounds having the above-mentioned activity may be appropriately selected and used.

[0029] Known antimicrobial agents include, for example, organically synthesized antimicrobial agents, natural product antimicrobial agents, and inorganic antimicrobial agents. Examples of antimicrobial components contained in these antimicrobial agents include cetylpyridinium chloride (CPC), chlorhexidine, benzalkonium chloride, benzethonium chloride, depotassium chloride, chlorhexidine gluconate, protamine, dodecyldiaminoethylglycine, triclosan, 3-methyl-4-isopropylmethylphenol (IPMP), thymol, carvacrol, farnesol, bisabolol, cineole, hinokitiol, sodium lauroyl sarcosinate, l-menthol, and the like. These antimicrobial agents may be used individually or in combination of two or more. The concentration of these antimicrobial components is appropriately determined for each component, taking into account the purpose of use and antimicrobial activity. For example, in the case of cationic antimicrobial agents, the concentration can be used in the range of 0.00001 to 0.5% by mass, preferably in the range of 0.0001 to 0.01% by mass. When using an acidic antibacterial agent, it is used in a range of 0.01 to 0.5% by mass. In the case of polyols with 5 to 6 carbon atoms, a range of 0.1 to 5.0% by mass is preferred, and 1.0 to 4.0% by mass is more preferred.

[0030] The manufacturing and marketing approval standards for oral external preparations claiming efficacy or effects related to medicinal toothpastes (Pharmaceutical and Food Safety Bureau Director-General, Ministry of Health, Labour and Welfare, Notification No. 0325-37, March 25, 2015) specify the types, specifications, and quantities of active ingredients that can be used for each efficacy or effect. For example, it is stated that the concentration of chlorhexidine hydrochloride, an antibacterial ingredient that can be used for the prevention of periodontitis (pyorrhea), should be 0.001 to 0.05% by mass. Furthermore, it is stated that the concentration of cetylpyridinium chloride, benzalkonium chloride, and isopropylmethylphenol, which can be used for the prevention of gingivitis, should be 0.01 to 0.05% by mass (see Appendix 1 of the Manufacturing and Marketing Approval Standards (Pharmaceutical and Food Safety Bureau Director-General, Ministry of Health, Labour and Welfare, Notification No. 0325-37, March 25, 2015)). The oral care composition of this embodiment, by containing these antibacterial agents in the specified specifications and quantities described herein, enhances the effect of suppressing or inhibiting the binding of oral bacteria to biomolecules in saliva, while simultaneously exhibiting bactericidal or bacteriostatic effects against oral bacteria. Alternatively, the above antibacterial agents may be included in a reduced amount (concentration) below the amount described herein. In this case as well, it is possible to enhance the effect of suppressing or inhibiting the binding of oral bacteria to biomolecules in saliva while reducing the side effects of the antibacterial agents.

[0031] While the synergistic effect between the algal extract and the antibacterial agent is not bound by any particular theory, since ionic antibacterial agents have a greater synergistic effect than nonionic antibacterial agents, it is thought that the active ingredient of the present invention and the antibacterial agent form some kind of complex, thereby further enhancing lectin activity.

[0032] Alternatively, it is thought that the active ingredient of the present invention inhibits the interaction between saliva-derived biopolymers, particularly glycans containing galactose at their termini, and oral bacteria. This may be due to the coexistence of the antibacterial component altering the cell membrane structure of oral bacteria, thereby reducing the binding affinity of lectin-like substances derived from oral bacteria that are present on the membrane surface.

[0033] In any case, since the inhibitory or suppressive activity of the Myrrhizae algae extract in inhibiting the binding of oral bacteria to biomolecules in saliva is enhanced in the presence of extremely low concentrations of antimicrobial components, it may be possible to use it as a means to solve various problems caused by biofilms formed by microorganisms. For example, in the medical field, it has been pointed out that biofilms formed on the surface of catheters can cause serious infections, but it is also possible to suppress the formation of biofilms on the surface of medical devices by treating them with a composition containing Myrrhizae algae extract and antimicrobial components.

[0034] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % in the numerical values ​​indicating the amount of each component added means mass % (w / v%). [Examples]

[0035] [Example 1] Preparation of algal extract (Mil extract) Codium fragile (Codium fragile) thallus was used as the raw material. This raw material was purchased from a cultivator as fresh, undried seaweed. The purchased fresh seaweed was dried and pulverized according to a prescribed method and used as the raw material. 50 g of the raw material was added to 10 times the volume of Dulbecco phosphate-buffered saline (pH 7.3, D5652, Sigma-Aldrich, Ca and Mg-free, hereinafter referred to as "PBS"), and allowed to stand at room temperature for 30 minutes to prepare a Codium fragile extract. The extract was centrifuged at 37000 × g for 15 minutes, and the supernatant was collected. The protein concentration of the extract was measured using a BCA protein assay kit (Thermo Fisher Scientific Co., Ltd.). A 2-fold dilution series of the extract with PBS was prepared and used in experiments to evaluate its effect on the adhesion and growth of Streptococcus mutans.

[0036] [Example 2] Fractionation of Mill extract Ten mL of the mill extract prepared in Example 1 was sequentially treated with vivaspin 20 (Sartorius) with molecular weight cutoffs of 50k, 30k, and 5k, respectively, to obtain four fractions with molecular weights of less than 5000, 5000 to less than 30000, 30000 to less than 50000, and 50000 or more. The protein concentrations of these four fractions were measured using a BCA protein assay kit (Thermo Fisher Scientific Co., Ltd.), diluted with PBS to the desired concentration, and subjected to the following bacterial adsorption tests.

[0037] [Test Example 1] Bacterial Adsorption Test <Preparation of bacterial suspension> Streptococcus mutans (NBRC13955) was purchased from the National Institute of Technology and Evaluation (NITE). After culturing in Todd-Hewitt Broth at 37°C overnight (approximately 16 hours), the absorbance OD was measured at 660 nm. 660 We used organisms that had grown until the absorbance reached 0.8-1.4. Based on the measured absorbance, we performed OD analysis using PBS. 660 The bacterial suspension used in the following tests was prepared by diluting it to a concentration of 0.125.

[0038] <Preparation of evaluation samples> The protein concentration of the mill extract prepared in Example 1 was 765 μg / mL. This was diluted with PBS to a concentration of 1% and used in the following tests. Each antibacterial agent used in this study was prepared at twice the concentration of the test, as it was mixed in a 1:1 ratio with the mill extract, its specified molecular weight fraction, or PBS.

[0039] To prepare the CPC solution, 0.2 g of CPC was dissolved in 10 mL of ethanol solution to create a storage solution (2% CPC). This was then diluted 20-fold with purified water to prepare a 0.1% CPC (5% ethanol) solution. This was further diluted with 5% ethanol solution to prepare 0.05% CPC (5% ethanol) and 0.02% CPC (5% ethanol) solutions, respectively.

[0040] A benzalkonium chloride solution was prepared by dissolving 0.2 g of benzalkonium chloride in 10 mL of ethanol solution to make a storage solution (2% benzalkonium chloride). This was then diluted five times with ethanol to prepare a 0.4% benzalkonium chloride solution in ethanol. This was further diluted twentyfold with purified water to prepare a 0.02% benzalkonium chloride (5% ethanol) solution. This was then further diluted twice with 5% ethanol to obtain a 0.01% benzalkonium chloride (5% ethanol) solution, and then diluted twice again with 5% ethanol to obtain a 0.005% benzalkonium chloride (5% ethanol) solution.

[0041] A 20% aqueous solution of chlorhexidine was used as the stock solution and diluted 20-fold with ethanol and purified water to obtain a 0.1% chlorhexidine (5% ethanol) solution. This was further diluted 5-fold with a 5% ethanol solution to obtain a 0.02% chlorhexidine (5% ethanol) solution, which was then further diluted 10-fold with 5% ethanol to prepare a 0.002% chlorhexidine (5% ethanol) solution.

[0042] To prepare the IPMP solution, 0.4 g of IPMP was dissolved in 10 mL of ethanol solution to create a storage solution (4% IPMP). This was then diluted 20-fold with ethanol and purified water to obtain a 0.2% IPMP (20% ethanol) solution. This was further diluted 2-fold with 20% ethanol to obtain a 0.1% IPMP (20% ethanol) solution, and then diluted again with 20% ethanol to prepare a 0.04% IPMP (20% ethanol) solution.

[0043] <Bacterial adsorption test> Saliva was collected at least two hours after brushing teeth. Stimulated saliva secreted by chewing Parafilm was collected and centrifuged at 4°C and 2000×g for 30 minutes. The supernatant was aspirated and filtered through a cellulose mixed ester (0.1 μm, 90 mm) membrane filter, and the saliva was diluted to an appropriate concentration using PBS.

[0044] 100 μL of human saliva prepared as described above was added to a 96-well multiplate (polystyrene, Thermo Fisher Scientific, MultiSoap), plate sealed, and incubated at 37°C for 1 hour. Then, each well was washed twice with 300 μL of PBS, 100 μL of each sample as described above was added, plate sealed, and incubated at 37°C for 1 hour. Afterward, the liquid from each well was discarded, washed twice with 300 μL of PBS, and OD (Oral Disintegration) was performed. 660 A bacterial suspension prepared to achieve a ratio of 0.125 was added to each well at a rate of 100 μL. The plate was sealed and incubated at 37°C for 16 hours. The bacterial suspension was discarded from each well of this plate, washed twice with 300 μL of PBS, and fixed at room temperature for 30 minutes with 100 μL of 0.25% glutaraldehyde solution. The solution was then discarded, and 100 μL of 0.1% crystal violet solution was added to each well and allowed to stand at room temperature for 30 minutes. The staining solution was aspirated and discarded using a pipette, and the plate, thoroughly washed with purified water, was dried in a 37°C incubator. 100 μL of 30% acetic acid solution was added to each well, and the dye was eluted using a plate shaker. The number of bacterial cells was quantified by measuring the dye concentration in the 30% acetic acid solution in each well at an absorption wavelength of 570 nm using a plate reader.

[0045] The results are shown in Figures 1 to 7. Figure 1 is a graph showing the inhibitory effect of mill extract (ML), cetylpyridinium chloride (CPC), and mixtures thereof (CPC+ML) on the adhesion of Streptococcus mutans to a saliva-coated substrate. The adhesion rate in this graph is shown as the average value of measurements repeated five times under the same conditions. A smaller adhesion rate indicates a greater inhibition of Streptococcus mutans adhesion. The Cont. group shows the measurement results for samples containing only PBS. The BSA group on the horizontal axis is the group to which 100 μg / mL of bovine serum albumin (BSA) was added as a positive control. In Figure 1, the relative values ​​of each sample are shown with the Cont. group set to 100. The numerical values ​​(adhesion rates) for each group shown in Figure 1 are shown in Table 1.

[0046] [Table 1]

[0047] As shown in Figure 1 and Table 1, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the group to which 1.0 w / v% of mill extract was added (ML) compared to the control group. Also, as shown in Figure 1 and Table 1, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the groups to which predetermined amounts of CPC and mill extract were added (CPC0.050+ML, CPC0.025+ML, CPC0.010+ML) compared to the group to which 1.0 w / v% of mill extract was added (ML).

[0048] Figure 2 shows the results of a similar test when benzalkonium chloride was used instead of CPC as the antibacterial component. In Figure 2, the relative values ​​of each sample are shown with the Cont. group set to 100. Table 2 shows the values ​​(adhesion rate) for each group shown in Figure 2.

[0049] [Table 2]

[0050] As shown in Figure 2 and Table 2, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the group to which 1.0 w / v% of the mill extract was added (ML) compared to the control group. Also, as shown in Figure 2 and Table 2, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the groups to which predetermined amounts of benzalkonium chloride and mill extract were added (benzalkonium chloride 0.010 + ML, benzalkonium chloride 0.005 + ML, benzalkonium chloride 0.003 + ML) compared to the group to which 1.0 w / v% of the mill extract was added (ML).

[0051] Figure 3 shows the results of a similar test when chlorhexidine was used instead of CPC or benzalkonium chloride as the antibacterial component. In Figure 3, the relative values ​​of each sample are shown with the Cont. group set to 100. The numerical values ​​(adhesion rates) for each group shown in Figure 3 are shown in Table 3.

[0052] [Table 3]

[0053] As shown in Figure 3 and Table 3, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the group to which 1.0 w / v% of the mill extract was added (ML) compared to the control group. Also, as shown in Figure 3 and Table 3, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the groups to which predetermined amounts of chlorhexidine and mill extract were added (chlorhexidine 0.050 + ML, chlorhexidine 0.025 + ML, chlorhexidine 0.010 + ML) compared to the group to which 1.0 w / v% of the mill extract was added (ML).

[0054] Figure 4 shows the results of similar tests when IPMP was used instead of each of the above antibacterial agents. In Figure 4, the relative values ​​of each sample are shown with the Cont. group set to 100. The numerical values ​​(adhesion rates) for each group shown in Figure 4 are shown in Table 4.

[0055] [Table 4]

[0056] As shown in Figure 4 and Table 4, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the group to which 1.0 w / v% of mill extract was added (ML) compared to the control group. Also, as shown in Figure 4 and Table 4, the adhesion rate was significantly lower (p<0.01 by Student's t-test) in the groups to which predetermined amounts of IPMP and mill extract were added (IPMP0.100+ML, IPMP0.050+ML, IPMP0.020+ML) compared to the group to which 1.0 w / v% of mill extract was added (ML).

[0057] Figure 5 shows the results of bacterial adsorption tests conducted under the same conditions as above, using samples with a protein concentration of 10 μg / mL for the four fractions of the mill extract from Example 1 (extract without fractionation) and the mill extract fractionated by molecular weight in Example 2 (molecular weight less than 5000, 5000 to less than 30000, 30000 to less than 50000, and 50000 or more). In Figure 5, data showing a significant difference (p<0.01) compared to the Control by Student's t-test are indicated by **. The values ​​(adhesion rate) for each group shown in Figure 5 are shown in Table 5.

[0058] [Table 5]

[0059] As shown in Figure 5 and Table 5, a decrease in bacterial adhesion was confirmed in the group of Mill extract (unfractionated) from Example 1, the group of Mill extract fractions with a molecular weight of 50,000 or more (50k or more), and the group of Mill extract fractions with a molecular weight of 5,000 or more but less than 30,000. Therefore, in the experiments related to Figure 5 and Table 5, the decrease in adhesion was not observed in the three groups of Mill extract fractions (molecular weight less than 5,000, 5,000 to less than 30,000, and 30,000 to less than 50,000), and the protein concentration was increased for further analysis.

[0060] To confirm the concentration dependence of the three mill extract fractions, bacterial adsorption tests were performed using higher concentration samples (diluted in a 2-fold dilution series starting from a protein concentration of 50 μg / mL). The results of this test are shown in Figure 6 and Table 6. In Figure 6, data showing a significant difference (p<0.01) compared to the control using Student's t-test are indicated by **. The values ​​(adhesion rates) for each group shown in Figure 6 are shown in Table 6.

[0061] [Table 6]

[0062] As shown in Figure 6 and Table 6, in the group of fractions of mill extract with a molecular weight of 5000 or more and less than 30000, a decrease in the adhesion rate was observed in a concentration-dependent manner with respect to the protein.

[0063] Furthermore, the same bacterial adsorption tests as above were performed on the mill extract from Example 1 (extract without fractionation) and the four fractions of the mill extract fractionated by molecular weight in Example 2 (molecular weight less than 5000, 5000 to less than 30000, 30000 to less than 50000, and 50000 or more), with CPC (antimicrobial component) added to achieve a final concentration of 0.05%. Each sample of the mill extract from Example 1 and the fractionated mill extract was added to achieve a final concentration of 1.5%. The test results are shown in Figure 7 and Table 7. In Figure 7, data showing a significant difference (p<0.01) compared to the Control in Student's t-test are indicated by **. [Table 7]

[0064] As shown in Figure 7 and Table 7, a decrease in adhesion rate was observed in at least the unfractionated group, the 5k to less than 30k group, and the 30k to less than 50k group compared to the control group.

[0065] [Test Example 2] Bacterial Adsorption Test Furthermore, using the mill extract from Example 1 and a commercially available mouthwash, the effects of using the mill extract and the mouthwash together were confirmed by a bacterial adsorption test, similar to the one described in Test Example 1 above. The mill extract from Example 1 was added to a final concentration of 1.0%. In this Test Example 2, GUM Dental Rinse (Regular Type, Sunstar) was used as the commercially available mouthwash. The components of this mouthwash are listed below.

[0066] [Ingredients contained in the mouthwash] Solvent: Concentrated glycerin, ethanol / Flavoring agent: Fragrance (herb mint type), sodium saccharin / Solubilizer: POE hydrogenated castor oil / Medicinal ingredients: Cetylpyridinium chloride (bactericide CPC), dipotassium glycyrrhizate (anti-inflammatory agent GK2), benzalkonium chloride (bactericide BKC) / pH adjuster: Sodium citrate, anhydrous citric acid / Cleaning aid: Coconut oil fatty acid acyl arginine ethyl DL-PCA salt

[0067] The results of this verification are shown in Figure 8 and Table 8. In Figure 8, data showing a significant difference (p<0.01) compared to the control group in Student's t-test are indicated by **. [Table 8]

[0068] As shown in Figure 8 and Table 8, a decrease in adhesion rate was observed in at least the group to which mill extract was added and the group to which both mill extract and mouthwash were added, compared to the Control.

[0069] [Test Example 3] Confirmation of the effect of suppressing bad breath The presence or absence of the effect was confirmed using a specified mill extract, etc. The test method is described below.

[0070] <Preparing the test sample> (Preparation of the specified mill extract) To prepare a myrrh extract containing proteins with a molecular weight of 5000 or more (an extract that does not contain proteins with a molecular weight of less than 5000, hereinafter referred to as "5000 or more ML"), 10 mL of the myrrh extract prepared in Example 1 described above was prepared. Using vivaspin 20 (manufactured by Sartorius) with a fractional molecular weight of 5k, the 5000 or more ML was prepared.

[0071] Furthermore, 10 mL of the mill extract prepared in Example 1 above was prepared to create the following three samples. • Extracts from Mircea algae containing proteins with a molecular weight of 5,000 or more and less than 30,000 (extracts that do not contain proteins with molecular weights other than 5,000 or more and less than 30,000; hereinafter referred to as "5,000 or more and less than 30,000 ML"). • Extracts from Mircea algae containing proteins with a molecular weight of 30,000 or more and less than 50,000 (extracts that do not contain proteins with molecular weights other than 30,000 or more and less than 50,000; hereinafter referred to as "30,000 or more and less than 50,000 ML"). • Extracts from Mircea algae containing proteins with a molecular weight of 50,000 or more (extracts that do not contain proteins with molecular weights other than 50,000 or more; hereinafter referred to as "50,000 or more ML")

[0072] Ten mL of the mill extract prepared in Example 1 was sequentially treated with vivaspin 20 (manufactured by Sartorius) with fractional molecular weight cutoffs of 50 k, 30 k, and 5 k, respectively, to obtain three fractions: 5,000 to less than 30,000 ml, 30,000 to less than 50,000 ml, and 50,000 or more ml.

[0073] (Preparation of the specified CPC solution) The specified CPC solutions were prepared as follows: First, 0.2 g of CPC was dissolved in 10 mL of ethanol solution to prepare a preservation solution (2% CPC). This preservation solution was diluted 20 times with purified water to prepare a 0.1% CPC (5% ethanol) solution. This was further diluted with 5% ethanol solution to prepare 0.05% CPC (5% ethanol), 0.025% CPC (5% ethanol), and 0.0125% CPC (5% ethanol), respectively.

[0074] (Preparation of samples used for VSC concentration measurement (preparation of oral bacterial culture solution)) Saliva (about 2 mL) was collected from a healthy human in their 30s (one male) by a specified method. The collected saliva was centrifuged (2000×g, for 10 minutes, at 25°C (room temperature)). After this centrifugation, the supernatant was recovered. The supernatant was added to 30 mL of Todd Hewitt Broth (Becton, Dickinson and Company, 249240) and cultured at 37°C for 16 hours. The turbidity (OD660nm) of the culture solution after this culture was measured, and the culture solution was diluted with PBS so that the turbidity became 0.125. The diluted solution (oral bacterial culture solution) was used in the test.

[0075] <VSC Concentration Measurement> Halitosis is defined as "an offensive odor that exceeds the social recognition limit among the gases emitted through the mouth or nose". It is considered that most (more than 80%) of such halitosis is derived from the gas in the oral cavity, and the main causative substances are hydrogen sulfide (H2S), methyl mercaptan (CH3SH), and dimethyl sulfide [(CH3)2S], which are volatile sulfur compounds (VSC: Volatile Sulfur Compounds) (Ministry of Health, Labour and Welfare e-Health Net, Causes and Reality of Halitosis, URL: https: / / www.e-healthnet.mhlw.go.jp / information / teeth / h-07-001.html). Therefore, in this Test Example 3, the effect of suppressing halitosis by a predetermined mill extract or the like was confirmed by measuring the VSC concentration.

[0076] (Preparation of Plate for VSC Measurement) 100 μL of the prepared human saliva was added to a 96-well multiplate (made of polystyrene, manufactured by Thermo Fisher Scientific, Multisorp) as described in Test Example 1, the plate was sealed, and incubated at 37°C for 1 hour. Thereafter, each well was washed twice with 300 μL of PBS. In addition, including the following, preparations were made such that 5 wells were used for each group described in Table 9 below.

[0077] After this washing, 100 μL of the sample listed in Table 9 was added to each of the following test examples (Test Example 3-1, Test Example 3-2, Test Example 3-3), the plate was sealed, and incubated at 37°C for 1 hour. Then, the liquid was discarded from each well and washed twice with 300 μL of PBS.

[0078] After this washing, 100 μL of the oral bacterial culture solution was added to each well. The plate was sealed and incubated at 37°C for 16 hours. The bacterial solution was discarded from each well of the plate and washed twice with 300 μL of PBS.

[0079] After this washing, 100 μl of 3.3 mM cysteine ​​solution was added to each well, the plate was sealed, and incubated at 37°C for 1 hour.

[0080] A glass vial (AS ONE, Labran screw-cap vial, No. 3, 9 mL, 9-852-05) containing 3M phosphoric acid (250 μl, reaction stopper, Kishida Chemical, 260-61925) was prepared. To this glass vial, the solutions of each group containing the cysteine ​​and incubated (total 500 μl (= 100 μl × 5)) were added.

[0081] [Table 9]

[0082] As shown in Table 9, after preparing the samples for each group, the samples for groups 3-1, 3-2, and 3-3 were left to stand at 25°C (room temperature) for 10 minutes.

[0083] After the initial standing period, the VSC concentration in each group was measured three times using a halimeter (RH17K, Taiyo), and the average value was calculated. The average of the obtained results (numerical values) was then calculated. The calculated results are shown in Table 10.

[0084] Table 10 shows the relative values ​​(for example, below) compared to the control group, with the mean value of each group (test 3-1, test 3-2, test 3-3) set to 100. • Group 3-1 of Test 3-1: In the calculation results, the relative value compared to the 3-1 control group is shown, and "*" indicates that "p<0.05" compared to the 3-1 control group in Student's t-test. • Group 3-2 of Test 3-2: In the calculation results, the relative value compared to the 3-2 control group is shown, and "**" indicates that "p<0.05" compared to the 3-2 control group in Student's t-test. • Group 3-3 of Test 3-3: In the calculation results, the relative value compared to the 3-3 control group is shown, and "***" indicates that "p<0.05" compared to the 3-3 control group in Student's t-test.

[0085] [Table 10]

[0086] As shown in Table 10, the effectiveness of the specified mill extract in suppressing bad breath was confirmed.

[0087] [Test Example 4] Pigmentation test using apatite particles For example, aesthetics are required for dental crown prostheses used in the anterior teeth (Non-Patent Document 1, Non-Patent Document 2). Therefore, we tested whether the composition (solution) containing the mill extract and CPC prepared in Example 1 could suppress pigment deposition using apatite particles. This test was conducted as described below, based on the descriptions in Non-Patent Document 1 and Non-Patent Document 2.

[0088] <Testing Method> First, list the following reagents and other items. • Apatite particles: Bio RAD CHT Ceramic Hydroxyapatite Type I (80 μm), Cat. #158-8000, Lot. M401076 • Coffee: Commercially available canned coffee, Craft Boss Black (Suntory Foods Ltd.) • Measuring equipment for color difference measurement: CR-400, Konica Minolta, colorimeter • The saliva to be added in the following test process (saliva solution #1): was prepared as follows. Saliva from healthy adult males (in their 30s) was used. Saliva was collected by parafing gum stimulation more than two hours after brushing teeth. The collected saliva was centrifuged (4°C, 2000g, 30min), and the supernatant was collected. The supernatant was diluted with PBS to prepare a diluent. This diluent was filtered through a cellulose mixed ester (0.1μm, 90mm) membrane filter. The filtered diluent was used as the saliva to be added in the following test steps. • Oral bacterial culture solution to be added in the following test steps: Saliva (approximately 2 mL) was collected from a healthy individual (male, 30s) using a prescribed method. The collected saliva was centrifuged (2000 × g, 10 minutes, 25°C (room temperature)). After centrifugation, the supernatant was collected. 30 mL of Todd Hewitt Broth (Becton, Dickinson and Company, 249240) was added to the supernatant and incubated at 37°C for 16 hours. The turbidity (OD660 nm) of the culture solution after incubation was measured, and the culture solution was diluted with PBS until the turbidity was 0.125. This diluted solution was used as the oral bacterial culture solution to be added in the following test steps. • CPC: CPC used in Test Example 1

[0089] 100 mg of apatite particles were weighed into 1.5 mL tubes. 1 mL of PBS was added to each tube containing the particles, and the mixture was stirred at 25°C using a vortex mixer. After stirring, the mixer was spun down, and the supernatant was discarded from the tubes. After the initial disposal, 1 mL of saliva was added to the tube and mixed using a vortex mixer at 25°C. After mixing, the tube was allowed to stand at 37°C for 1 hour. After this standing period and spin-down, the supernatant was discarded from the tube. The particles in each tube were rinsed twice by adding 1 mL of PBS to each tube (rinsing by vortexing, spinning down, and discarding the supernatant).

[0090] To each of the following groups (Group 4-1, Group 4-2, Group 4-3), 1 mL of the sample described in Table 11 was added to the tube, and the mixture was stirred at 25°C using a vortex mixer. After stirring, the mixture was allowed to stand at 37°C for 1 hour. After standing and spin-down, the supernatant was discarded from the tube.

[0091] [Table 11]

[0092] After discarding the supernatant, the particles in each tube were rinsed twice by adding 1 mL of PBS to each tube (rinsing by vortexing, spinning down, and discarding the supernatant).

[0093] To the tubes, 1 mL of the "oral bacterial culture solution" was added to groups 4-2 and 4-3 as described above, and the mixture was stirred using a vortex mixer. The "oral bacterial culture solution" was not added to group 4-1 as described above. After stirring, the mixtures were allowed to stand at 37°C for 1 hour. After standing and spin-down, the supernatant was discarded from the tubes. The particles in each tube were rinsed twice by adding 1 mL of PBS to each tube (rinsing by vortexing, spinning down, and discarding the supernatant).

[0094] 1 mL of coffee was added to the tube and mixed using a vortex mixer. After mixing, the mixture was allowed to stand at 37°C for 1 hour. After standing and spin-down, the supernatant was discarded from the tube. The particles contained in the tubes were rinsed three times by adding 1 mL of purified water to each tube (rinsing in the following steps: vortexing, spinning down, and discarding the supernatant).

[0095] After the three rinses, the particles were transferred from the tube to a petri dish for each group. After the transfer to the petri dish, the particles were dried at 50 °C for 2 hours. After the drying, color difference measurements were performed for each group using the measuring instrument. The background color during color measurement was white, and the particles were placed on a standard white plate for measurement. The particles before adding coffee were color-measured, and the obtained color measurement values were used as reference values.

[0096] For color representation, L * a * b * Color system (CIE1976 L * a * b * uniform color space) was used. The L * value represents brightness, and the larger the numerical value from 0 to 100, the brighter it becomes. The color tone is represented by a * b * and when both a * b * are 0, it becomes achromatic. The more a * is in the positive direction, the stronger the redness, the more in the negative direction, the stronger the greenness, and also the more b * is in the positive direction, the stronger the yellowness, the more in the negative direction, the stronger the blueness. Note that the value of ΔE* (delta-E-star), which is used to represent the difference in color, is obtained by calculating how far apart the straight-line distance between two colors in this color space is.

[0097] <Test Results> The results of the color difference measurement are shown in Table 12 below. In Table 12, the average values calculated from the values measured three times for each group are described. The "*" described in Table 12 indicates "p < 0.01" compared to Group 4-3 in the Student's T-test. In the "oral bacterial culture solution", in Group 4-2 (the group added with mill extract, etc.) compared to Group 4-3, good results (especially the results of the L * value (brightness)) were obtained.

Table 12

[0098] [Test Example 5] Confirmation of the effectiveness of toothpaste containing mill extract. The effect of toothpaste containing the mill extract prepared in Example 1 was confirmed when used in the human oral cavity. The effect was confirmed in a study involving three healthy adult males (in their 30s).

[0099] Toothpaste containing the mill extract prepared in Example 1 of Formulation Examples 1 to 3, as shown in Table 13, was prepared. As Comparative Example 1, toothpaste without the mill extract, as shown in Table 13, was prepared. The effects were confirmed by brushing teeth with each of the toothpastes from Formulation Examples 1 to 3 and Comparative Example 1, as shown in Table 13.

[0100] [Table 13]

[0101] When Comparative Example 1 was used, all three participants felt refreshed immediately after brushing their teeth, but after a certain period of time (approximately 3 hours), they felt a sticky sensation in their mouths. On the other hand, when prescription examples (prescription examples 1-3) were used, all three participants felt refreshed immediately after brushing their teeth, and unlike when Comparative Example 1 was used, they did not feel a sticky sensation in their mouths after a certain period of time (approximately 3 hours).

[0102] [Test Example 6] Confirmation of the effectiveness of a mouthwash containing millet extract. The effect of using a mouthwash containing the mill extract prepared in Example 1 in the human oral cavity was confirmed. The effect was confirmed in a study involving three healthy adult males (in their 30s).

[0103] Mouthwashes containing the mill extract prepared in Example 1 of Formulation Examples 4 to 6, as shown in Table 14, were prepared. As Comparative Example 2, a mouthwash without the mill extract, as shown in Table 14, was prepared. The effects of the mouthwashes from Formulation Examples 4 to 6 and the Comparative Example, as shown in Table 14, were confirmed by performing mouthwashes.

[0104] [Table 14]

[0105] When Comparative Example 2 was used, all three participants felt a refreshing sensation immediately after using the mouthwash, but after a certain period of time (approximately 3 hours), they felt a sticky sensation in their mouths. On the other hand, when prescription examples (prescription examples 4-6) were used, all three participants felt a refreshing sensation immediately after using the mouthwash, and unlike when Comparative Example 2 was used, they did not feel a sticky sensation in their mouths after a certain period of time (approximately 3 hours). [Industrial applicability]

[0106] It has been confirmed that the composition of the present invention can suppress or inhibit the binding of oral bacteria to biomolecules in saliva. Therefore, the composition of the present invention may be used as a cosmetic or quasi-drug for oral care.

Claims

1. It contains an extract from the family Myrrhizaceae algae and an antibacterial component. The above-mentioned extract of the family Myrrhaceae contains a protein with a molecular weight of 5000 or more as an active ingredient. The aforementioned antibacterial component is one or more selected from the group consisting of cetylpyridinium chloride (CPC), chlorhexidine, and benzalkonium chloride, in an oral care composition.

2. The oral care composition according to claim 1, wherein the active ingredient is contained in an amount of 0.001 to 5% by mass of the total amount of the composition, calculated on the basis of the extract of the family Myrrhaceae algae.

3. The oral care composition according to claim 1 or 2, wherein the antibacterial component is contained in an amount of 0.0001 to 1% by mass relative to the total amount of the composition.

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