JP2014043441A - Composition for oral cavity and candida biofilm formation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for oral cavity and a candida biofilm formation inhibitor which are effective for prevention and suppression of intraoral mucous and oral candidiasis with inflammation generated on a tongue, caused by a biofilm formed by form conversion from a yeast type to a mycelial type of candida present in an oral cavity or by proliferation of mycelial type candida and have high safety.SOLUTION: A composition for oral cavity and the candida biofilm formation inhibitor contain geraniol and menthol, where the geraniol and the menthol have a mass standard compounding ratio (geraniol/menthol) of 3/97 to 95/5.

Description

  The present invention relates to an oral composition and a Candida biofilm formation inhibitor.

  Oral candidiasis is one of the oral mucosal diseases and is caused by Candida albicans, an opportunistic infection. Major clinical symptoms include white moss and redness, pain and discomfort in the tongue, and abnormal taste. Opportunistic infectious bacteria are attenuated microorganisms that are harmless to healthy people or non-pathogenic microorganisms, but exhibit pathogenicity against susceptible hosts with reduced immunity. Local and systemic factors associated with oral candidiasis include reduced saliva, denture wear, long-term administration of steroids, abuse of antibacterial drugs, chemotherapy and radiation therapy, reduced nutritional status, HIV (human immunodeficiency virus) ) There are infections, endocrine disorders, etc., which are caused by a decrease in host resistance and fungal replacement.

  Candida has the characteristic of dimorphism that reversibly transforms the spherical yeast type and the filamentous mycelium type, and the transformation from the yeast type to the mycelium type is biofilm formation, tissue invasion, etc. It is thought to be a cause of pathogenicity. Oral candidiasis high-risk individuals (susceptible hosts) are prone to microbial replacement, and the oral mucosa is often fragile due to decreased salivary secretion or the like. Therefore, for effective prevention of oral candidiasis, in addition to controlling infection with Candida, it does not disturb the bacterial flora balance of oral resident bacteria and may be less irritating to mucosal cells (living body) Desired. As one of the means, inhibition of morphological conversion of Candida to mycelium and biofilm formation is considered to be an effective preventive method that can specifically suppress the pathogenicity of Candida.

  Geraniol is a linear monoterpene alcohol contained in essential oils of plants such as rose, palmarosa, and geranium, and has been reported to show effects on Candida and biofilm formation (patents) Reference 1 and 2). However, Patent Document 1 does not specifically show that geraniol is effective in preventing the onset of oral candidiasis and suppressing progression. Further, Patent Document 2 shows that biofilm formation has an inhibitory effect on various bacteria, but the concentration is 0.1%, which is an inappropriate concentration for the oral cavity, and the inhibitory effect is also 50%. The level is not satisfactory.

On the other hand, in oral compositions, the combined use of geraniol and menthol has been suggested (see Patent Documents 3 to 5). Patent Document 3 contains a glycoside having a bitter taste such as geraniol and menthol, and N-substituted-p-menthane-3-carboxamides, and has a different taste and odor due to menthol or essential oils containing the same. Masked oral compositions are disclosed.
Patent Document 4 discloses a composition for oral cavity that contains geraniol and menthol, has a degree of irritation and bitterness that is not accidentally swallowed by the user, and is excellent in use feeling. Patent Document 5 discloses a composition for oral cavity containing a base having sweetness and a terpene alcohol-based bitter substance such as geraniol or menthol, masked with sweetness and having a refreshing aftertaste.

  However, Patent Documents 3 to 5 do not describe examples in which geraniol and menthol are actually used in combination. Patent Documents 3 to 5 do not describe the effect of suppressing oral candidiasis of menthol and the synergistic improvement of the effect of suppressing oral candidiasis by the combined use of geraniol and menthol.

JP 2000-290176 A JP 2007-091706 A JP-A-8-310930 JP 2000-026260 A JP 2001-064136 A

  The objective of this invention is effective in prevention and control of oral candidiasis, and is providing the composition for oral cavity and Candida bacteria biofilm formation inhibitor with high safety | security.

  As mentioned above, it has been reported that some plant essential oils have an inhibitory effect on the morphological conversion of Candida albicans to the mycelium type, but no compound having a sufficiently satisfactory inhibitory effect has been obtained. . In the research process for solving the above-mentioned problems, the present inventors have the highest activity of geraniol contained in rose essential oil at a high concentration among the currently known compounds to suppress the above-mentioned form conversion. I found out. Furthermore, as a result of repeated research by the present inventors, the combination of geraniol with geraniol suppresses the growth of the above-mentioned morphological conversion or mycelium-type Candida, and synergistically suppresses the formation of biofilm. As a result, the present invention has been completed.

That is, the present invention provides the following oral composition.
(1) A composition for oral cavity containing geraniol and menthol and having a mass ratio of geraniol and menthol of 3/97 to 95/5.
(2) The composition for oral cavity of said (1) whose compounding ratio of the mass reference | standard of geraniol and menthol is 5 / 95-95 / 5.
(3) The oral composition according to (1) or (2), wherein the geraniol content is 0.001% by mass or more and less than 1% by mass of the total amount of the oral composition.

The present invention also provides the following Candida biofilm formation inhibitor.
(4) A Candida biofilm formation inhibitor containing geraniol and menthol and having a geraniol / menthol mass ratio (geraniol / menthol) of 3/97 to 95/5.
(5) The Candida biofilm formation inhibitor of the above (4), wherein the mass-based blending ratio of geraniol and menthol (geraniol / menthol) is 5/95 to 95/5.

  The composition for oral cavity of the present invention is effective in preventing the onset and progression of oral candidiasis and has high safety. The Candida bacterium biofilm formation inhibitor of the present invention has an effect of suppressing biofilm formation by suppressing Candida morphological conversion to mycelium type or growth of mycelium type Candida.

It is a graph which shows the biofilm formation rate (%) in presence of the Candida biofilm formation inhibitor containing geraniol and menthol at each density | concentration. It is a graph which shows the relationship between the density | concentration of geraniol or menthol, and a biofilm formation rate (%). It is a graph which shows the growth inhibitory effect with respect to yeast type Candida of geraniol or menthol. It is a graph which shows the onset suppression effect of the oral candidiasis by the composition for oral cavity of this invention. It is a photograph which shows the external appearance of the tongue of the ICR mouse | mouth of each group of the test preparation X process group (a), the test preparation Y process group (b), and the infection only process group (c) in the test example 4. FIG.

[Oral composition]
The composition for oral cavity of the present invention contains geraniol and menthol, and the mass ratio of geraniol and menthol is 3/97 to 95/5, preferably 5/95 to 95/5, more preferably 15 / 85 to 95/5, more preferably 20/80 to 95/5. Due to such characteristics, the composition for oral cavity of the present invention is effective in preventing the onset of oral candidiasis and suppressing the progression thereof.

Geraniol is a linear monoterpene alcohol represented by the chemical formula: C ₁₀ H ₁₈ O contained in essential oils of plants such as rose, palmarosa, and geranium, and has a fragrance resembling the scent of roses. Geraniol is widely used as a fragrance for oral compositions, various cosmetics, foods and the like, and is known as a highly safe substance. Furthermore, geraniol has been reported to have an effect on Candida bacteria and a biofilm formation inhibitory effect on various bacteria, but it is not at a sufficiently satisfactory level.

  1-Menthol (2-isopropyl-5-methylcyclohexanol) is a cyclic monoterpene that is abundant in peppermint plants such as peppermint and mint oil, and has been added to foods and pharmaceuticals for a long time. It is also frequently used in cosmetic compositions and cosmetics. Menthol, like geraniol, is highly safe.

  The present inventors have found that menthol is effective in preventing onset of oral candidiasis and suppressing progression. However, even if menthol is used alone, its effect is not at a sufficiently satisfactory level, like geraniol. In addition, when menthol is used at a high concentration, the effect of suppressing the growth of Candida fungi in the mycelium type is increased, but since menthol has strong irritation properties, an oral composition containing a high concentration of menthol Makes it hard to take in the mouth.

  In the composition for oral cavity of the present invention, geraniol and menthol are mixed in a mass-based mixing ratio of 3/97 to 95/5, preferably 5/95 to 95/5, more preferably 15/85 to 95/5, More preferably, it is used so as to be 20/80 to 95/5. In this way, by using geraniol and menthol together at a predetermined blending ratio, the geraniol single use effect, the menthol single use effect, and the combined effect of both of them (additive effect) are even higher. An effect (synergistic effect) is obtained.

  When the blending ratio of geraniol is less than 3 (or when the blending ratio of menthol exceeds 97), only an additive effect combining the effects of single use of both can be obtained. Moreover, when the blending ratio of geraniol exceeds 95 (or when the blending ratio of menthol is less than 5), only an additive effect combining the effects of using both alone can be obtained.

  Moreover, in this invention, content of geraniol and menthol in an oral composition can be reduced by using geraniol and menthol by the predetermined | prescribed compounding ratio mentioned above. Thereby, the composition for oral cavity which is effective for the onset prevention and progression suppression of oral candidiasis, has low irritation, and can be easily taken into the oral cavity is obtained. Moreover, since both geraniol and menthol have high safety, a highly safe oral composition can be obtained.

  The content of geraniol in the oral composition of the present invention is not particularly limited, but preferably from the viewpoint of ease of administration of the oral composition into the oral cavity, reduced efficacy due to dilution with saliva, etc. 0.001% by mass or more and less than 1% by mass of the total amount of the composition for use, and more preferably 0.005% by mass to 0.1% by mass of the total amount of the composition for oral cavity.

  When the content of geraniol is less than 0.001% by mass, the effect of preventing or suppressing the progression of oral composition against oral candidiasis is inferior even if the blending ratio of geraniol and menthol is within the predetermined range. May be sufficient. On the other hand, when the content of geraniol is 1% by mass or more, although the effect of preventing onset and suppressing progression of oral candidiasis can be obtained, both geraniol and menthol have a strong aroma. There is a risk that the property becomes too strong and it becomes difficult to take the oral composition into the oral cavity.

  The oral composition of the present invention can be formulated into various forms. The preparation form of the oral composition of the present invention is not particularly limited, and examples thereof include toothpastes, pastes (cream-like lumber), liquid dentifrices, mouthwashes, oral moisturizers, liquids, gels, Pasta agents, ointments, sprays, gums and the like can be mentioned. Among these, liquid or liquid forms such as mouthwashes, liquid dentifrices, sprays, liquids, soft pastes (cream-form preparations) are preferable, and mouthwashes, liquid dentifrices, sprays, liquids and the like are more preferable. A mouthwash is more preferable.

  The composition for oral cavity of this invention can contain various additives as an arbitrary component in the range which does not impair the effect of this invention according to a formulation form etc., for example. The additive is not particularly limited as long as it is an additive conventionally used in oral compositions. For example, surfactants, fragrances, thickeners, sweeteners, wetting agents, preservatives, colorants, Examples include pH adjusters, medicinal components, bases, and abrasives. An additive can be used individually by 1 type or in combination of 2 or more types.

Examples of the surfactant include nonionic surfactants, anionic surfactants, and amphoteric surfactants.
Specific examples of the nonionic surfactant include sugar fatty acid esters such as sucrose fatty acid ester, maltose fatty acid ester, and lactose fatty acid ester, fatty acid alkanolamides, sorbitan fatty acid ester, fatty acid monoglyceride, and polyoxyethylene addition coefficient of 4 to 15, polyoxyethylene alkyl ether having an alkyl group having 10 to 18 carbon atoms, polyoxyethylene addition coefficient of 10 to 18, polyoxyethylene alkyl phenyl ether having an alkyl group having 9 carbon atoms, diethyl sebacate, poly Oxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyethylene glycol fatty acid ester, polyester Renranorin, polyethylene sterols, polyethylene lanolin alcohols, alkyl glucosides, polyoxyethylene polyoxypropylene block copolymers, and the like.

  Specific examples of the anionic surfactant include, for example, sulfate ester salts such as sodium lauryl sulfate and polyoxyethylene lauryl ether sodium sulfate, sodium lauryl sulfosuccinate, sulfosuccinate such as sodium polyoxyethylene lauryl ether sulfosuccinate, and cocoyl sarcosine. Examples thereof include acyl amino acid salts such as sodium and sodium lauroylmethylalanine, and cocoylmethyl taurine sodium.

Specific examples of zwitterionic surfactants include, for example, betaine acetate type activators such as lauryl dimethylaminoacetic acid betaine, coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine, and N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine. Examples include imidazoline type activators such as sodium and amino acid type activators such as N-lauryldiaminoethylglycine.
Surfactant can be used individually by 1 type or in combination of 2 or more types.

  As the fragrance, for example, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronell, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineol, linalool, ethyl linalool, Examples include thymol, spearmint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, perilla oil, winter green oil, clove oil, eucalyptus oil, and pimento oil. A fragrance | flavor can be used individually by 1 type or in combination of 2 or more types.

  Examples of the thickener include carboxymethyl cellulose sodium, carboxymethyl ethyl cellulose salt, cellulose compounds such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microbial polymer compounds such as xanthan gum, gellan gum, tragacanth gum, karaya gum, arabiya gum, carrageenan , Natural polymer compounds such as dextrin or natural rubbers, synthetic polymer compounds such as polyvinyl alcohol and polyvinylpyrrolidone, and cationic thickening such as O- [2-hydroxy-3- (trimethylammonio) propyl] hydroxyethylcellulose chloride Agents and the like. A thickener can be used individually by 1 type or in combination of 2 or more types.

  Examples of the sweetening agent include saccharin, saccharin sodium, acesulfame potassium, stevia extract, stevioside, neohesperidyl dihydrochalcone, glycyrrhizin, perilartin, thaumatin, aspartylphenylalanine methyl ester, methoxycinnamic aldehyde, palatinose, palatinit, erythritol, Examples include maltitol, xylitol, and lactitol. A sweetener can be used individually by 1 type or in combination of 2 or more types.

  Examples of the wetting agent include glycerin, ethylene glycol, propylene glycol, sorbit, polyethylene glycol, and polypropylene glycol. Wetting agents can be used alone or in combination of two or more.

  Examples of the preservative include parabens such as methylparaben and propylparaben, and benzoates such as sodium benzoate. A preservative can be used individually by 1 type or in combination of 2 or more types.

  Examples of the colorant include legal pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral pigments such as ultramarine and enhanced ultramarine, and titanium oxide. A coloring agent can be used individually by 1 type or in combination of 2 or more types.

  Examples of the pH adjuster include citric acid, phosphoric acid, malic acid, gluconic acid, maleic acid, aspartic acid, succinic acid, glucuronic acid, fumaric acid, glutamic acid, adipic acid, and salts thereof, hydrochloric acid, hydroxylated Sodium, potassium hydroxide, sodium silicate, etc. are mentioned. A pH adjuster can be used individually by 1 type or in combination of 2 or more types. The pH of the composition for oral cavity of the present invention is not particularly limited as long as it is in a range that can be used in the oral cavity, but is usually pH 5.5 to 9.5, preferably pH 5.8 to 8.0, more preferably. Is 6.0-7.5.

  Examples of medicinal ingredients include cationic fungicides such as cetylpyridinium chloride, chlorhexidine hydrochloride, chlorhexidine gluconate, benzethonium chloride, and benzalkonium chloride; amphoteric fungicides such as dodecyldiaminoethylglycine; non-amphoteric agents such as isopropylmethylphenol and triclosan Ion fungicides; Enzymes such as dextranase, amylase, protease, mutanase, lysozyme, lytic enzyme (lytechenzyme); anti-inflammatory agents that are glycyrrhizinate such as glycyrrhetinic acid, dipotassium glycyrrhizinate; tocopherol nicotinate, tocopherol acetate, etc. Blood circulation promoters; antiplasmin agents such as tranexamic acid and epsilon aminocaproic acid; bleeding improvers such as ascorbic acid; tissue repair agents such as allantoin; fluorine such as sodium fluoride Remineralising agent compounds and the like; plant extract extracted with a water-soluble solvent, chlorophyll, sodium chloride, Karopeputaido, zinc chloride, hinokitiol, or the like. Medicinal components can be used alone or in combination of two or more.

  Examples of the base include alcohol, silicon, apatite, white petrolatum, paraffin, liquid paraffin, micro crostalin wax, squalane and the like. A base can be used individually by 1 type, or can be used in combination of 2 or more type.

  As an abrasive, for example, calcium carbonate, magnesium carbonate, dicalcium phosphate, tricalcium phosphate, magnesium phosphate, silica, zeolite, sodium metaphosphate, aluminum hydroxide, magnesium hydroxide, calcium pyrophosphate, bengara, calcium sulfate, anhydrous Silica etc. are mentioned. An abrasive | polishing agent can be used individually by 1 type or in combination of 2 or more types.

  The composition for oral cavity of the present invention can be obtained, for example, by mixing predetermined amounts of geraniol and menthol with one or more of the above additives depending on the form of the preparation. In addition, the oral composition of the present invention can be obtained by dissolving a predetermined amount of geraniol and menthol in water and mixing the obtained aqueous solution with one or more of the above additives as necessary. Can be obtained.

  Although the usage method of the composition for oral cavity of this invention is not specifically limited, For example, usage methods, such as gargle, intraoral mucosa, sticking or application | coating to the tongue surface, a gum, etc., and brushing using a toothbrush, are mentioned.

[Candida biofilm formation inhibitor]
The Candida biofilm formation inhibitor of the present invention (hereinafter sometimes referred to as “the biofilm formation inhibitor of the present invention”) is a morphological change from the yeast type to the mycelium type or the growth of mycelium type Candida bacteria. And the amount of biofilm formed of Candida cells and their secretions can be significantly reduced. Due to such effects, the biofilm formation inhibitor of the present invention is considered to be effective in preventing the onset, suppressing the progression and treating the oral candidiasis caused by Candida in the oral cavity.

  Examples of Candida bacteria effective for the biofilm formation inhibitor of the present invention include Candida ciferrii, Candida famata, Candida lambica, Candida lipolytica, Candida norvegensis, Candida rugosa, Candida viswanathii, Candida zeylanoides, Candida albicans, Candida albicans, tropical Candida tropida・ Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida kefyr, Candida giriel guilli mondii), Candida dubliniensis and the like. Among these Candida bacteria, it is particularly effective against Candida albicans.

  In the biofilm formation inhibitor of the present invention, geraniol and menthol are mixed in a mass ratio (geraniol / menthol) of 3/97 to 95/5, preferably 5/95 to 95/5, more preferably 15/85 to 95/5, more preferably 20/80 to 95/5. By containing geraniol and menthol in the above blending ratio, the effect on oral candidiasis is further increased than the additive effect obtained by combining the effects of geraniol alone and menthol alone. Outside the range of the blending ratio, only the additive effects described above can be obtained, and the onset prevention and treatment effects for oral candidiasis are insufficient.

  The content of geraniol in the biofilm formation inhibitor of the present invention is not particularly limited, but from the viewpoint of efficacy against oral candidiasis, ease of formulation and administration, reduced efficacy due to dilution with saliva, etc. It is 0.001 mass% or more of film formation inhibitor whole quantity, and less than 1 mass%, More preferably, it is 0.005 mass%-0.1 mass% of biofilm formation inhibitor whole quantity. When the content of geraniol is less than 0.001% by mass, even if the blending ratio of geraniol and menthol is within the above-mentioned predetermined range, the onset prevention and progression inhibition efficacy for oral candidiasis tends to decrease. On the other hand, when the content of geraniol is 1% by mass or more, efficacy against oral candidiasis increases, but formulation and administration tend to be difficult.

  The content of menthol in the biofilm formation inhibitor of the present invention may be appropriately selected according to the content of geraniol so that the blending ratio of geraniol and menthol is within a predetermined range.

  The biofilm formation inhibitor of the present invention is administered, for example, by oral administration. The dosage of the biofilm formation inhibitor of the present invention is appropriately selected according to age, sex, symptoms, administration route, number of administrations, formulation, etc. For example, in the case of an adult (60 kg), geraniol and menthol per day The total amount is preferably 0.1 mg to 1000 mg, more preferably 1 mg to 100 mg. Administration may be, for example, once or divided into 2 to 4 times per day.

  The formulation form of the biofilm formation inhibitor of the present invention is not particularly limited. For example, tablets, powders, fine granules, granules, capsules, powders, pills, troches, chewable tablets, orally disintegrating tablets, Examples include solid preparations such as film preparations, solutions such as solutions, suspensions, emulsions, and syrups, and patch preparations. In the formulation, pharmacologically acceptable carriers, excipients, diluents, disintegrants, extenders, solubilizers and the like are used. Furthermore, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, buffers, antioxidants, and bacteria inhibitors may be used.

  Furthermore, the biofilm formation inhibitor of the present invention can be formulated into the form of the oral composition described above, and may be further formulated into a confectionery form such as chewing gum, hard candy, soft candy, and gummy candy.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, “%” and “parts” are based on mass.

(Test Example 1)
In order to examine the effect of a biofilm formation inhibitor containing geraniol and menthol on Candida, which is a causative bacterium of oral candidiasis, the following test was performed. Specifically, the test bacteria, devices, instruments, reagents and medium used in this test example are as follows.

[Test bacteria]
C. albicans NBRC 1594. This Candida strain is available from the National Institute of Technology and Evaluation Biotechnology Center.
[Equipment / Equipment]
Fluorescent microplate reader: trade name; Gemini XPS, manufactured by Molecular Device Japan Co., Ltd. Spectrophotometer: trade name: UV-1600, manufactured by Shimadzu Corporation)
96-well plate: manufactured by Sumitomo Bakelite Co., Ltd.

[reagent]
Geraniol: manufactured by Wako Pure Chemical Industries, Ltd. l-menthol: manufactured by Nagaoka Perfume Co., Ltd. AlamarBlue (trade name): Cell staining reagent, trade name; invitrogen (trade name), Life Technologies Japan Co., Ltd. Phosphate buffered saline (hereinafter referred to as “PBS”): Dainippon Sumitomo Pharma Co., Ltd. Ethanol: Wako Pure Chemical Industries, Ltd.

[Culture medium]
YPD liquid medium: manufactured by Nippon Pecton Dickinson Co., Ltd. R10 liquid medium: prepared by dissolving 10% FBS (fetal bovine serum, manufactured by Biowest) in RPMI 1640 medium (trade name, manufactured by Sigma Aldrich Japan Co., Ltd.) .
The R10 liquid medium was used as a culture medium for mycelium-type Candida, and the YPD liquid medium was used as a culture medium for yeast-type Candida.

(1) Preparation of Candida Bacteria Solution Candida was inoculated into 10 ml of YPD liquid medium, and cultured with shaking at 28 ° C. for 24 hours. Candida was cultured in yeast form. The obtained culture solution was centrifuged at 3000 G for 5 minutes. The obtained Candida cells were washed once with PBS, then suspended in an R10 liquid medium, and the turbidity at 660 nm (OD ₆₆₀ , McFarland turbidimetric method) measured by an absorptiometer was about 1.0. To obtain a Candida fungus solution. The resulting Candida bacterium solution contained about 1.0 × 10 ⁶ Candida bacteria per ml.

(2) Preparation of test preparation liquid Geraniol and menthol were each diluted to 200 mg / ml with ethanol, and further diluted 400 times with R10 liquid medium, and adjusted so that geraniol and menthol were each 500 μg / ml. An adjustment liquid was prepared. At this time, the solvent is an R10 liquid medium containing 0.25% ethanol. Each adjustment liquid of geraniol and menthol was diluted with this solvent, and dilution liquids having respective concentrations of 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 (μg / ml) were obtained. .

  50 μl of each diluted solution of geraniol and 50 μl of each diluted solution of menthol were mixed to obtain a test preparation solution. In this test preparation solution, the concentrations of geraniol and menthol are diluted by a factor of 2, and are 50, 12.5, 6.25, 3.13, 1.56, and 0.78. For comparison, a comparative test preparation solution containing geraniol or menthol alone by mixing 50 μl of a diluted solution of geraniol with each concentration or 50 μl of a dilution solution of menthol with the above-described dilution liquid. Got.

(3) Culture 100 μl of the obtained test preparation solution and comparative test preparation solution are supplied to each well of a 96-well plate, and 10 μl of the Candida fungus solution obtained above is further added to each well. cell concentration of the mixed liquid of the formulation liquid (or comparative test Shisei agent solution) and Candida solution was adjusted to OD ₆₆₀ = 0.1. One well of the 96-well plate was supplied with 100 μl of R10 liquid medium containing ethanol at a ratio of 0.25% and 10 μl of Candida bacteria solution, and used as a negative control.

  This 96-well plate was placed in a thermostatic bath and cultivated at 37 ° C. for 90 minutes. By this culture, Candida bacteria in the mixed solution were attached to the 96-well plate, and the form was changed from the yeast type to the mycelium type. Subsequently, shaking culture was carried out at 37 ° C. for 24 hours to form a Candida biofilm.

(4) Measurement of the number of viable bacteria After discarding the culture supernatant of the culture solution in each well of the 96-well plate and washing the biofilm in each well three times with 200 μl of PBS, a 10-fold dilution of Alamar Blue Was supplied to each well in a volume of 100 μl, and a color reaction was carried out at 37 ° C. for 180 minutes. Alamar Blue becomes a redox-type pigment due to its metabolic activity and develops fluorescence, so that the fluorescence intensity increases with the amount of biofilm formed. The fluorescence intensity of the specimen after the color development reaction was measured with a fluorescence microplate reader at an excitation wavelength Ex: 560 and a fluorescence wavelength Em: 590. This test was performed three times to determine the average value of the fluorescence intensity.

  The biofilm formation amount (fluorescence intensity) in the negative control was taken as 100, and the biofilm formation amount at the concentration of each test preparation solution was shown as a relative value (biofilm formation rate,%) relative to the negative control. The results are shown in Table 1. In addition, the numerical value described in Table 1 is a value obtained by rounding off the third decimal place. Further, the data shown in Table 1 was graphed as shown in FIG. FIG. 1 is a graph showing the biofilm formation rate (%) in the presence of a biofilm formation inhibitor containing geraniol and menthol at various concentrations.

  In the negative control in which the concentrations of geraniol and menthol are each 0 μg / ml, almost all of Candida bacteria are present in the state of viable bacteria, so that it is considered that the amount of biofilm formation is standard. It can be seen from Table 1 that when the biofilm production amount in this negative control is 100, the biofilm production amount is smaller than 100 in a system in which a biofilm formation inhibitor containing geraniol and menthol is present. Further, it can be seen from Table 1 that the biofilm formation inhibitor in the portion surrounded by the thick line has a biofilm formation rate of less than 20% and has an excellent effect.

  From this, the biofilm formation inhibitor containing geraniol and menthol suppresses the growth of Candida in the mycelial form, suppresses the formation of biofilms that are thought to cause oral candidiasis, and oral candida It is thought to be effective in preventing the onset of symptoms, suppressing progression, and treating them.

(Test Example 2)
Two non-patent literatures (TING-CHAO CHOU, Academic, San Diego, pp. 61-102 and TING-CHAO CHOU, et al. Trends Pharmacol Sci. 1983; 4: 450-454). According to these non-patent documents, in the case of mutually exclusive drugs in which the actions of drug 1 and drug 2 have the same or similar forms, they serve as an index of the synergistic effect of the two drugs. CI _A is represented by the following formula (A).

CI _A = [(D) ₁ / (D _x ) ₁ ] + [(D) ₂ / (D _x ) ₂ ] (A)
[In the formula, (D) ₁ indicates the concentration (μg / ml) of Drug 1 at which an inhibitory effect of x% is obtained when Drug 1 and Drug 2 are used in combination. (D _x ) ₁ indicates the concentration (μg / ml) of Drug 1 at which the inhibitory effect of x% can be obtained with Drug 1 alone. (D) ₂ indicates the concentration (μg / ml) of Drug 2 that provides an x% inhibitory effect when Drug 1 and Drug 2 are used in combination. (D _x ) ₂ indicates the concentration of drug 2 (μg / ml) at which x2 inhibitory effect can be obtained with drug 2 alone. ]

In the case of mutually non-exclusive drugs in which the actions of drug 1 and drug 2 have completely independent forms, CI _{B, which} is an index of the synergistic effect of these two drugs, is It is represented by the formula (B).
CI _B = [(D) ₁ / (D _x ) ₁ ] + [(D) ₂ / (D _x ) ₂ ] + [(D) ₁ (D) ₂ / (D _x ) ₁ (D _x ) ₂ ] (B)
[Wherein (D) ₁ , (D) ₂ , (D _x ) ₁ and (D _x ) ₂ are the same as above. ]

In this test example, it is not clear whether the combined system of geraniol and menthol has a synergistic effect on the suppression of Candida biofilm formation, and whether the mechanism is exclusive or non-exclusive. Therefore, the numerical values of CI _A and CI _B were obtained from the above formulas (A) and (B) and evaluated according to the following criteria. This evaluation criterion is described in the above two non-patent documents as an evaluation criterion for the calculated values of the above formulas (A) and (B).

[Evaluation criteria]
0.1 There is a very strong synergistic effect.
0.1-0.3 There is a strong synergistic effect.
0.3-0.7 There is a synergistic effect.
0.7-0.9 Slightly synergistic.
0.9-1.1 Almost an additive effect.
1.1-1.45 Slightly antagonistic.
1.45-3.3 Has antagonism.
> 3.3 Very strong antagonism.

By the same test method as in Test Example 1, in each of geraniol alone, menthol alone, and a combination system of geraniol and menthol, each drug concentration that inhibits biofilm formation by 80% (IC ₈₀ , μg / ml) is obtained. From formula (A) and formula (B), the values of CI _A and CI _B were determined, and based on the above evaluation criteria, it was determined whether it was a synergistic effect or an additive effect. The results are shown in Table 2 below.

In the said Table 2, the example of calculation in case mass ratio of geraniol (drug 1) and menthol (drug 2) is 16: 1 is shown. When the mass ratio of geraniol to menthol is 16: 1, the concentration required for IC ₈₀ is 13.28 μg / ml as the total amount of geraniol and menthol, so (D ₈₀ ) ₁ = 13.28 × 16 ÷ 17, (D ₈₀ ) ₂ = 13.28 × 1 ÷ 17.

  Moreover, the test formulation liquid of each density | concentration of geraniol individual and the test formulation liquid of each density | concentration of menthol single were prepared like Test Example 1, and the biofilm formation rate (%) in each density | concentration was used for this. Asked. However, the concentrations of geraniol or menthol in the test preparation solution were 500, 250, 125, 62.5, 31.25, 15.63, and 7.813 (μg / ml), respectively. The biofilm formation rate here was also a relative value (biofilm formation rate,%) when the biofilm formation amount (fluorescence intensity) in a negative control not containing geraniol and menthol was defined as 100. The results are shown in FIG.

FIG. 2 is a line graph showing the relationship between the concentration of geraniol or menthol and the biofilm formation rate (%). In FIG. 2, the position where the biofilm formation rate is 20% is the drug concentration that inhibits biofilm formation by 80%. Therefore, from FIG. 2, the concentration at which geraniol inhibits biofilm formation by 80%: (D) ₁ = 25 μg / ml, and the concentration at which menthol inhibits biofilm formation by 80%: (D) ₂ = 62. 50 μg / ml. By calculating by substituting these values into equation (A) and formula (B), the value of CI _A and CI _B are obtained.

  From Table 2, it is apparent that a synergistic effect is obtained by using geraniol and menthol together in a mass ratio of geraniol to menthol in the range of 16: 1 to 1:32.

(Test Example 3)
In order to examine the effects of geraniol and menthol on yeast-type Candida that existed in suspension in the culture, the following tests were carried out. As for the test bacteria, apparatus / instrument, reagent and culture medium, test examples except that an absorption plate reader (trade name: VersaMax reader, manufactured by Molecular Devices) was used instead of the fluorescent microplate reader (Gemini XPS). The same as 1 was used.

(1) Preparation of Candida Bacterial Solution Candida was inoculated into 10 ml of YPD liquid medium, and cultured with shaking at 28 ° C. for 24 hours to grow yeast-type Candida. The obtained culture solution was centrifuged at 3000 G for 5 minutes, and the resulting Candida cells were washed once with PBS, suspended in a YPD liquid medium, and turbidity at 660 nm (OD) measured with an absorptiometer. ₆₆₀ , McFarland turbidimetry) was adjusted to approximately 1.0 to obtain a Candida bacterium solution.

(2) Test preparation solution Geraniol and menthol were each diluted to 200 mg / ml with ethanol, and further diluted 400-fold with YPD liquid medium to prepare geraniol and menthol so that each of geraniol and menthol was 500 μg / ml. Was made. At this time, the solvent becomes a YPD liquid medium containing 0.25% ethanol. Each preparation solution of geraniol and menthol was diluted with this solvent, and test preparation solutions having concentrations of 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 (μg / ml) were prepared. Obtained.

(3) Culture 100 μl of each test preparation solution of geraniol and menthol was supplied to wells of a 96-well plate, and 10 μl of the Candida fungus solution obtained above was added thereto.

  As a negative control, 100 μl of YPD liquid medium containing 0.25% ethanol was supplied to the well, and 10 μl of Candida bacteria solution was added thereto. As a blank, 100 μl of YPD liquid medium containing 0.25% ethanol was supplied to the well, and 10 μl of YPD liquid medium was added thereto. The 96-well plate was placed in a thermostatic bath and cultivated at 28 ° C. for 24 hours.

After completion of the culture, the absorbance (OD ₆₆₀ ) of the culture solution in each well was measured using an absorbance plate reader (trade name: VersaMax reader). A value obtained by subtracting the blank measurement value from each measurement value is calculated, and the results (relationship between drug concentration and absorbance) are shown in FIG. FIG. 3 is a graph showing the growth inhibitory effect of geraniol or menthol on yeast-type Candida.

  From FIG. 3, it is clear that geraniol and menthol both have a very low growth inhibitory effect on yeast-type Candida. In fact, the MIC (minimum growth inhibitory concentration) of geraniol against the yeast-type Candida was 500 μg / ml, and menthol could not find a growth inhibitory effect up to a concentration of 500 μg / ml.

Thus, since the sufficient growth inhibitory effect was not obtained with respect to the yeast-type Candida bacteria in a floating state, the effect of Candida bacteria shown in FIG. IC ₈₀ is not geraniol due to the action of Candida fungi that is necessary for biofilm formation, the transformation from yeast to mycelium, or the growth of Candida fungi that have become mycelia. It is considered that the effect was shown at a low concentration of 22.5 μg / ml for menthol and 62.5 μg / ml for menthol. The effect of the combination of geraniol and menthol in the present invention is also considered to be obtained by synergistically improving this action.

(Test Example 4)
(1) Purpose The purpose of the present invention was to examine the onset prevention effect of oral candidiasis on a test preparation using an oral candidiasis mouse model.
(2) Test animals Crj: CD1 (ICR) SPF mice (6 weeks old, female, manufactured by Charles River Japan Co., Ltd.) were used as test animals. Hereinafter, the test animal is referred to as “ICR mouse”.
(3) Test strain Candida albicans TIMMM1768 was used as a test Candida strain. This Candida strain can be obtained from Teikyo University Medical Mycology Research Center.

(4) Test preparations Test preparations X (oral moisturizers containing geraniol 0.01% by mass and menthol 0.005% by mass) and test preparations Y (geraniol 0.02) shown in Table 3 below. (Moisturizing agent for oral cavity containing 0.005% by mass and menthol) was prepared and used for the test.
Test preparation X was used in a treatment group of 9 ICR mice subjected to infection treatment described below. Hereinafter, this treatment group is referred to as “test preparation X treatment group”.
Test preparation Y was used in a treatment group of 8 ICR mice subjected to the infection treatment described below. Hereinafter, this treatment group is referred to as “sample preparation Y treatment group”.
For comparison, a treatment group of 6 ICR mice (hereinafter referred to as an “infection only treatment group”) was prepared without using any of the preparations X and Y.

(5) Test method The day before inoculation with C. albicans TIMM1768, ICR mice were injected subcutaneously with 100 mg / kg of prednisolone. From the day before inoculation, ICR mice were allowed to freely ingest tap water containing 4.13 mg / ml of tetracycline hydrochloride.
2. Next, 20 mg / kg of chlorpromazine hydrochloride was intramuscularly injected, and a suspension of C. albicans TIMM1768 was applied with a cotton swab into the oral cavity of a rested ICR mouse, and C. albicans TIMM1768 was inoculated. As the suspension, C. albicans TIMM1768 suspended in RPMI 1640 medium containing 2.5% fetal calf serum (FCS) to 2 × 10 ⁸ cells / ml was used.
3. After 5 minutes, 3 hours, 24 hours, and 27 hours after C. albicans TIMM1768 inoculation, 50 μl of each test preparation was administered dropwise to the dorsal tongue of the oral cavity of ICR mice using a stomach tube.
4). In the infection-only treatment group, prednisolone, tetracycline and chlorpromazine were administered, then C. albicans TIMM1768 was inoculated in the same manner as described above, and reared within the test period.
5. After ICR mice were euthanized 2 days after inoculation, the appearance of the tongue of each ICR mouse in the test preparation X treatment group, the test preparation Y treatment group, and the infection only treatment group was evaluated according to the following evaluation score. In addition, this evaluation score is an evaluation score of Takakura et al. (Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamaguchi H, Abe S.; Microbiol Immunol. 2003; 47 (5): 321-6. " No. 3,860775, etc.) The results are shown in Table 4 and FIGS.

(6) Evaluation score 0 point: No abnormality is observed.
1 point: Red inflammation or white moss-like white foreign matter is observed in a range of less than 5% of the tongue surface.
2 points: White moss-like white foreign matter is observed in the range of 5% or more and less than 90% of the tongue surface. No abnormal surface structure such as depression or bulge is observed on the tongue surface.
3 points: White moss-like white foreign matter is observed in the range of 5% or more and less than 90% of the tongue surface. Furthermore, abnormalities in the surface structure such as depressions or bumps are observed on the tongue surface.
4 points: White moss-like foreign matter is observed in a range of 90% or more of the tongue surface. When the white moss is removed, abnormalities in the surface structure such as depressions or bumps are observed on the tongue surface.

(7) Evaluation results (onset suppression effect of oral candidiasis by the test preparation)
The evaluation scores for each treatment group were all expressed as mean ± standard deviation (mean ± SD). Student's t-test was used for the comparison test. In all cases, a risk rate of 5% or less (p <0.05) was considered statistically significant.
First, the evaluation results are as shown in Table 4 below. FIG. 4 is a graph showing the evaluation results shown in Table 4 and shows the effect of suppressing the onset of oral candidiasis by the test preparation. FIG. 5 is a photograph showing the appearance of a typical tongue of an ICR mouse in each group of the test preparation X treatment group (a), the test preparation Y treatment group (b), and the infection-only treatment group (c). It is.

From Table 4 and FIG. 4, the infection-only treatment group has an evaluation score of 3.0 ± 0.63, whereas the test preparation X treatment group has an evaluation score of 1.7 (rounded to one decimal place) ± 0 .50, test preparation Y treatment group has an evaluation score of 1.0 ± 0.76, which is significantly lower than the infection-only treatment group, and both groups have a significant preventive effect on the development of oral candidiasis (P <0.05, t-test). Further, also from FIG. 5, in the ICR mice of the infection-only treatment group (c), white moss-like white foreign substances derived from the test Candida were observed over a wide range, whereas the test preparation X treatment group (a ) And test preparation Y treatment group (b) ICR mice have a significantly smaller range of white moss-like white foreign substances derived from the test Candida fungus, and the test preparation X treatment group (a) and the test preparation Y It was clear that there was a significant difference in the development of oral candidiasis between the treatment group (b) and the infection-only treatment group (c).
From the above, it was proved that prevention of oral candidiasis can be realized by combining geraniol and menthol at a predetermined blending ratio and applying the composition as an oral composition.

(Formulation examples 1 to 8)
Each formulation shown in Tables 5 to 12 was used in the proportions shown in Tables 5 to 12, and each formulation of Formulation Examples 1 to 8 was obtained. In addition, about the mouthwash of the formulation examples 2 and 3, pH was adjusted to the range of 5.5-7.0 using the pH adjuster.

Claims (5)

  A composition for oral cavity containing geraniol and menthol and having a mass ratio of geraniol and menthol (geraniol / menthol) of 3/97 to 95/5.   The composition for oral cavity according to claim 1, wherein a mixing ratio (geraniol / menthol) based on mass of geraniol and menthol is 5/95 to 95/5.   The oral composition according to claim 1 or 2, wherein the content of geraniol is 0.001% by mass or more and less than 1% by mass of the total amount of the oral composition.   Candida biofilm formation inhibitor containing geraniol and menthol and having a geraniol / menthol mass ratio (geraniol / menthol) of 3/97 to 95/5.   The Candida biofilm formation inhibitor according to claim 4, wherein a mass-based blending ratio of geraniol and menthol (geraniol / menthol) is 5/95 to 95/5.