Proliferation inhibitor for bacteria, cosmetic, quasi-drug and pharmaceutical composition comprising same, and method for inhibiting proliferation of bacteria

Abstract

The present disclosure relates to a proliferation inhibitor which is a proliferation inhibitor of at least one type of bacterium selected from the group consisting of the Gardneria genus and the Atopobium genus, and which contains a glycosylglycerol glycoside therein, and to a method for producing a proliferation inhibitor of at least one type of bacterium selected from the group consisting of the Gardneria genus and the Atopobium genus.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a proliferation inhibitor of at least one bacterium selected from the genus Gardnerella and the genus Atopobium, a cosmetic, quasi-drug, and pharmaceutical composition containing the proliferation inhibitor, and a method for inhibiting proliferation of bacteria. BACKGROUND

[0002] Various resident bacteria exist in the vagina of healthy adult women, of which the genus Lactobacillus accounts for 75 to 95%. The Lactobacillus genus (Lactobacillus dudlei) that is resident in the vagina produces lactic acid, keeping the pH in the vagina acidic at 4.5 or less, thereby preventing the invasion of miscellaneous bacteria (non-patent document 1).

[0003] As the Lactobacillus dudlei, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus jensenii, Lactobacillus casei, Lactobacillus vaginalis, Lactobacillus crispatus, Lactobacillus iners, etc. are known, and it has been reported that Lactobacillus crispatus and Lactobacillus iners among them are dominant bacteria in all four ethnic groups (whites, blacks, Hispanics, and Asians) (non-patent document 2).

[0004] Vaginitis (vaginal disease) is a disease concept in which abnormal leukorrhea is the chief complaint, and representative examples thereof are bacterial vaginosis (BV), vulvovaginal candidiasis, and trichomoniasis. The pathogenic microorganism of vulvovaginal candidiasis is Candida genus fungi, and the pathogenic microorganism of trichomoniasis is Trichomonas vaginalis, but bacterial vaginosis is caused by the collapse of the resident flora, and is not caused by a specific pathogenic microorganism.

[0005] In bacterial vaginosis, the Lactobacillus dudlei decreases, and various aerobic bacteria and / or anaerobic bacteria abnormally proliferate. Although more than half of patients with bacterial vaginosis have no symptoms, the main symptoms of bacterial vaginosis include leukorrhea with malodor, grayish-white to white leukorrhea, burning sensation when urinating, itching around the vagina, etc., and it is considered that bacterial vaginosis increases the risk of pelvic inflammatory disease, miscarriage, premature birth, postpartum endometritis, etc.

[0006] In bacterial vaginosis, as an overproliferated aerobic bacterium, Streptococcus agalactiae, Escherichia coli, Gardnella vaginalis, etc. are known, and as an overproliferated anaerobic bacterium, Anaerocossus spp., Atopobium vaginae of the genus Atopobium, Peptostreptococcus spp., Finegoldia spp., Micromonas spp., Peptoniphilus spp., etc. are known.

[0007] As a medicament having a bacterium proliferation inhibitory effect mainly against Gardnella vaginalis, metronidazole is known, and is widely used for the treatment of bacterial vaginosis (non-patent literature 1).

[0008] However, it is known that bacteria of the genus Atopobium are resistant to metronidazole (patent literature 1), and the effect of metronidazole is insufficient.

[0009] On the other hand, glycerol glucoside is a sugar derivative having a structure in which glucose and glycerol are combined, and has been used as a material for cosmetics.

[0010] It is reported that a-D-pyranoglycuroglucosyl glycerol, which is one of glycerol glucosides, has no skin irritation, and is very excellent in safety, and has antibacterial properties against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus epidermidis, and Corynebacterium minutissimum (patent literature 2).

[0011] However, the antibacterial property in Patent Literature 2 is evaluated based on the number of viable bacteria after bacteria are inoculated into a-D-glucopyranosyl glycerol, exposed for 24 hours or 48 hours, the collected sample is cultured in a culture medium, and the number of bacteria is counted. Patent Literature 2 does not clarify the effect of a-D-glucopyranosyl glycerol on bacteria in proliferation. In addition, it is unknown how a-D-glucopyranosyl glycerol affects other bacteria, particularly bacteria of the genus Lactobacillus, the genus Gardnerella, and the genus Atopobium.

[0012] Prior Art Documents

[0013] Patent Literature

[0014] Patent Literature 1: Japanese Patent Laid-Open No. 2012-524117

[0015] Patent Literature 2: Japanese Patent Laid-Open No. 2004-331577

[0016] Non-Patent Literature

[0017] Non-Patent Literature 1: Bacterial Vaginosis, Sexually Transmitted Infections Diagnosis and Treatment Guidelines (Bacterial Vaginosis, Sexually Transmitted Infections Diagnosis and Treatment Guidelines) 2008, Japanese Society of Sexually Transmitted Infections, pp. 77-80, November 30, 2008

[0018] Non-Patent Literature 2: "Vaginal microbiome of reproductive-age women", Jacques Ravel et al., Proc Natl Acad Sci U S A (2011), 108 (Suppl 1); 4680-4687 SUMMARY

[0019] PROBLEMS TO BE SOLVED BY THE INVENTION

[0020] Based on the above background, the present inventors have found that if the number of bacteria of the genus Gardnerella and the genus Atopobium can be reduced without reducing the number of Doderlein's bacilli, it is helpful to maintain the health of a site where at least one bacterium selected from the genus Gardnerella and the genus Atopobium is present or suspected to be present, for example, the private parts, and to prevent or treat infections.

[0021] Thus, the technical problem to be solved by the present disclosure is to provide a bacterial proliferation inhibitor that inhibits the proliferation of at least one bacterium selected from the group consisting of Gardnerella and Atopobium without inhibiting the proliferation of Doderlein's bacillus, a preparation containing the proliferation inhibitor, and a method for inhibiting the proliferation of bacteria.

[0022] Technical solution to solve the technical problem

[0023] The present inventors have conducted intensive studies in order to solve the above-described technical problem. As a result, they have found that the above-described technical problem can be solved by having the following configuration, thereby completing the present disclosure.

[0024] The present disclosure relates to, for example, [1] to [7] below.

[0025] [1] A proliferation inhibitor that is a proliferation inhibitor of at least one bacterium selected from the group consisting of Gardnerella and Atopobium, wherein glycerol glucoside is contained.

[0026] [2] The proliferation inhibitor according to [1], wherein the bacterium is at least one selected from the group consisting of Gardnerella vaginalis and Atopobium vaginae.

[0027] [3] The proliferation inhibitor according to [1] or [2], which is used as a cleaning agent or an external agent.

[0028] [4] A cosmetic or quasi-drug, wherein the proliferation inhibitor according to [1] or [2] is contained.

[0029] [5] A pharmaceutical composition for the treatment or prevention of a disease in which Gardnerella vaginalis or Atopobium vaginae is involved, wherein the proliferation inhibitor according to [2] is contained.

[0030] [6] The pharmaceutical composition according to [5], wherein the disease is bacterial vaginosis.

[0031] [7] A method for inhibiting the proliferation of at least one bacterium selected from the group consisting of Gardnerella and Atopobium, which comprises a step of administering glycerol glucoside to a subject.

[0032] Effects of the invention

[0033] According to the present disclosure, it is possible to provide a bacterial proliferation inhibitor that inhibits the proliferation of at least one bacterium selected from the group consisting of Gardnerella and Atopobium without inhibiting the proliferation of Doderlein's bacillus, a preparation containing the proliferation inhibitor, and a method for inhibiting the proliferation of bacteria.

[0034] Glycerol glucoside was not confirmed to be toxic in a primary skin irritation test (rabbit), a repeated skin irritation test, a skin sensitization test (guinea pig intracutaneous maximization method), a phototoxicity test (guinea pig), a photosensitization test, an eye irritation test (rabbit), a mutagenicity test (in vitro), and a human patch test, and thus is highly safe. Therefore, according to the present disclosure, it is possible to inhibit the proliferation of at least one bacterium selected from the group consisting of Gardnerella and Atopobium without worrying about safety and side effects. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a graph showing the measured values (average of n = 3) of turbidity in the bacterial proliferation test.

[0036] Figure 2 is a graph showing the relative values (average of n = 3) of turbidity in the bacterial proliferation test with respect to the control. DETAILED DESCRIPTION

[0037] Next, the present application will be specifically described.

[0038] Unless otherwise specifically stated, the notation "A to B" with respect to a numerical range means A or more and B or less. In addition, % means mass %.

[0039] <glycerol glucoside>

[0040] In the present specification, glycerol glucoside, also referred to as GG, means a compound in which glycerol is combined with one molecule or more of glucose through an α- or β-glycosidic bond.

[0041] As the glycerol glucoside, for example, compounds represented by formula (1) to formula (5) can be exemplified, and one kind of these compounds can be used alone, or two or more kinds thereof can be used.

[0042] [Chemical Formula 1]

[0043]

[0044] [Chemical Formula 2]

[0045]

[0046] The compound represented by formula (1) is 1-O-(α- or β-) D-(mono or poly) glucopyranosyl glycerol. This compound is also sometimes referred to as 1-O-(α- or β-) D-dihydroxypropyl (mono or poly) glucopyranoside.

[0047] The carbon atom at position 2 in formula (1) marked with an asterisk is a chiral carbon atom, and optical isomers, namely, (2R) and (2S) forms, exist.

[0048] n in formula (1) is the degree of sugar condensation, and represents an integer of 1 or more. In commercially available glyceroglucosides manufactured by known methods such as enzymatic methods and organic synthesis methods, n is usually an integer of 1 to 6, and most of them are integers of 1 to 3, and especially, a large amount of compounds in which n is 1 exist in GG compositions.

[0049] The compound represented by formula (2) is 2-O-(α- or β-) D-(mono or poly) glucopyranosyl glycerol. This compound is also sometimes referred to as 2-O-(α- or β-) D-dihydroxypropyl (mono or poly) glucopyranoside.

[0050] n in formula (2) is the degree of sugar condensation, and represents an integer of 1 or more. In commercially available glyceroglucosides manufactured by known methods such as enzymatic methods and organic synthesis methods, n is usually an integer of 1 to 6, and most of them are integers of 1 to 3, and especially, a large amount of compounds in which n is 1 exist in GG compositions.

[0051] m and n in formula (3), n and 1 in formula (4), and 1, m and n in formula (5) are the degrees of sugar condensation, and each independently represent an integer of 1 or more, and are usually integers of 1 to 6, and most of them are integers of 1 to 3, and especially, a large amount of compounds in which n, m and 1 are 1 exist in GG compositions. In formulae (3), (4) and (5), in the case where n, m and 1 are 2 or more, glucopyranosyl groups can be linked to each other by α1-4 bond, β1-4 bond, α1-6 bond, β1-6 bond or the like.

[0052] The glyceroglucoside of the present application preferably contains at least one of 1-O-D-glucopyranosyl glycerol and 2-O-D-glucopyranosyl glycerol represented by formula (1) or formula (2) in which n is 1, and more specifically, preferably contains at least one selected from 1-O-α-D-glucopyranosyl glycerol, 1-O-β-D-glucopyranosyl glycerol, 2-O-α-D-glucopyranosyl glycerol and 2-O-β-D-glucopyranosyl glycerol.

[0053] The glyceroglucoside of the present application can be one compound selected from 1-O-α-D-glucopyranosyl glycerol, 1-O-β-D-glucopyranosyl glycerol, 2-O-α-D-glucopyranosyl glycerol and 2-O-β-D-glucopyranosyl glycerol, and can contain two or more compounds.

[0054] Further, the glycerol glucosides of 1-O-α-D-glucopyranosyl glycerol, 1-O-β-D- glucopyranosyl glycerol, 2-O-α-D-glucopyranosyl glycerol and 2-O-β-D- glucopyranosyl glycerol are compounds in which glycerol is combined with one molecule of glucose, but in the present application, the glycerol glucosides can also be compounds in which glycerol is further combined with glucose, i.e., n is 2 or more, and can also be a mixture of a compound in which n is 1 and a compound in which n is 2 or more.

[0055] For example, in the case where n, m and 1 are each independently 2 or more in the formulae (1) to (5), the bonding mode of the glucopyranosyl groups to each other is not particularly limited.

[0056] Further, the wavy line in the formulae (1) to (5) each independently represents that the hydroxyl group of glycerol can react with and bond to the 1-position hydroxyl group of α-D-glucopyranosyl group (α bond), or can react with and bond to the 1-position hydroxyl group of β-D-glucopyranosyl group (β bond).

[0057] The purity of the glycerol glucosides of the present application (mass ratio of the components of formulae (1) and (2) in which n = 1 to 3, formula (3) in which n + m = 2 or 3, formula (4) in which n + 1 = 2 or 3, formula (5) in which n = m = 1 = 1, in the glycerol glucosides) is preferably 70 to 100 mass% in 100 mass% of the glycerol glucosides, the component of formulae (1) and (2) in which n = 1 is more preferably 60 to 95 mass%, and further preferably 75 to 90 mass%. Further, in the glycerol glucosides, 1-O-α-D-glucopyranosyl glycerol is preferably the main component, and 1-O-α-D-glucopyranosyl glycerol is more preferably 10 to 50 mass% in 100 mass% of the glycerol glucosides.

[0058] The glycerol glucosides can also be used as a composition (also referred to as a GG composition) in which, in addition to the compounds represented by the formulae (1) to (5), a compound other than the compounds represented by the formulae (1) to (5) is contained, such as water, an unreacted substance or a reaction by-product (an impurity) that can be mixed in when the compounds represented by the formulae (1) to (5) are produced by an organic synthesis method.

[0059] As the impurities, glycerol and a reaction by-product derived from the glycerol, which are incorporated as a raw material for producing the glycerol glucosides, and a glucose source and a reaction by-product derived from the glucose source can be exemplified. Alcohols such as ethanol, isopropyl alcohol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, 1,3-butanediol, 1,2-pentanediol, glycerol, diglycerol, polyglycerol, and sugars such as glucose can be exemplified.

[0060] In addition, the incorporation amount of glycerin in foodstuffs, condiments, cosmetics, and the like is sometimes limited. Therefore, when these products are manufactured using the GG composition, the proportion of glycerin remaining in the GG composition with respect to the total solid components (components other than water) in the GG composition is preferably 70% by mass or less, more preferably 50% by mass or less, further preferably 45% by mass or less, particularly preferably 10% by mass or less, and most preferably 5% by mass or less. It is preferable that glycerin be not contained, and the glycerin can be 0% by mass with respect to the total solid components in the GG composition, and is usually 10% by mass or less.

[0061] The method for producing the glycerol glucoside of the present application is not particularly limited, and can be produced by a publicly known method such as an enzymatic method or an organic synthesis method. In addition, a commercially available product containing the glycerol glucoside can also be used, and for example, "COSARTE-2G (registered trademark) (manufactured by Toyo Sugar Refining Co., Ltd.)", "C-mo 2G (manufactured by Toyo Sugar Refining Co., Ltd.)", and the like can be used.

[0062] In "COSARTE-2G", the content of glycerol glucoside is about 66 to 78% by mass, and the content of glycerin is about 10% by mass or less, in 100% by mass of "COSARTE-2G".

[0063] In "C-mo 2G", the content of glycerol glucoside is about 30 to 38% by mass, and the content of glycerin is about 36 to 44% by mass, in 100% by mass of "C-mo 2G".

[0064] The glycerol glucoside has good water solubility, and therefore can be uniformly dissolved or dispersed even when added to water or a formulation having a high moisture content. In addition, the glycerol glucoside has a good moisturizing effect and low skin irritation, and therefore is suitable for use in the intimate area and the like, which are sensitive to irritation.

[0065] <proliferation inhibitor>

[0066] The proliferation inhibitor of one aspect of the present application contains glycerol glucoside, and inhibits the proliferation of at least one bacterium selected from the genus Gardnerella and the genus Atopobium.

[0067] The amount of glycerol glucoside contained in the proliferation inhibitor is not particularly limited. For example, as a lower limit of the amount of glycerol glucoside contained in the proliferation inhibitor, 20 mass%, 25 mass%, 30 mass%, 40 mass%, 45 mass%, 50 mass%, 60 mass%, 70 mass%, 80 mass% can be exemplified. Further, as an upper limit of the amount of glycerol glucoside contained in the proliferation inhibitor, 100 mass%, 99 mass%, 98 mass%, 95 mass%, 90 mass%, 85 mass% can be exemplified. The range of the amount of glycerol glucoside contained in the proliferation inhibitor can be arbitrarily set to a range arbitrarily combining the above lower limit and upper limit, for example, can be set to a range of 25 to 100 mass%, 30 to 90 mass%, 60 to 80 mass%, and the like. From the viewpoint of the proliferation inhibitory effect, the amount of glycerol glucoside contained in the proliferation inhibitor is preferably 20 mass% or more, more preferably 30 mass% or more.

[0068] The proliferation inhibitor can also contain glycerol, diglycerol, glucose, moisture, other components, as needed.

[0069] As the bacteria of the genus Gardnerella, Gardnerella vaginalis can be exemplified, and as the bacteria of the genus Atopobium, Atopobium vaginae (Fannyhessae vaginae), Atopobium rimae, Atopobium parvulum, Atopobium fossor, Atopobium minutum, Atopobium deltae can be exemplified. Among the above bacteria, at least one selected from the group consisting of Gardnerella vaginalis and Atopobium vaginae is preferred because they are the main bacteria that excessively proliferate in bacterial vaginosis.

[0070] The method for evaluating the proliferation inhibitory effect against the bacteria is not particularly limited, and a publicly known method can be used. For example, in a case where the degree of proliferation of the bacteria cultured by adding the test substance is lower than the degree of proliferation of the bacteria cultured under the same culture conditions without adding the test substance, it can be evaluated that the test substance has a proliferation inhibitory effect against the bacteria.

[0071] More specifically, as described later in the examples, when the number of bacteria cultured in the presence of the test substance is set to 100%, the test substance can be evaluated as having a proliferation inhibitory effect on bacteria when the number of bacteria cultured in the presence of the test substance is less than 100%. In this case, the number of bacteria cultured in the presence of the test substance is preferably 90% or less, more preferably 80% or less, further preferably 70% or less, and particularly preferably 60% or less, relative to the number of bacteria cultured in the absence of the test substance.

[0072] As the bacteria, bacteria in the middle of proliferation are preferably used, and bacteria in the logarithmic growth phase are more preferably used.

[0073] The method for measuring the number of bacteria is not particularly limited, and known methods can be used. For example, the number of bacteria can be measured by a direct detection method for counting the number of bacteria, such as a method for counting colonies cultured on an agar plate, a method for observing the presence or absence and size of a bacteriostatic circle formed by culturing on an agar plate, and the like, or can be measured by an indirect method for quantitatively detecting various indices caused by the presence of bacteria, such as a turbidity (absorbance) measurement using a spectrophotometer, a method for measuring the weight of bacterial bodies, a method for measuring the components or biochemical activity of bacteria, and the like.

[0074] The number of bacteria is preferably measured by an indirect method because of the convenience, and more preferably measured by a turbidity measurement using a spectrophotometer.

[0075] <Use of the Proliferation Inhibitor>

[0076] The use of the proliferation inhibitor of the present application is not particularly limited, and the proliferation inhibitor has a strong effect of inhibiting the proliferation of at least one bacterium selected from the genus Gardnerella and the genus Atopobium, and thus can be administered to an organism once or multiple times, for example, for the purpose of maintaining the health of the private parts and the like and / or for the purpose of preventing or treating infectious diseases.

[0077] The proliferation inhibitor can be used for the prevention or improvement of a disease or a symptom in which at least one bacterium selected from the genus Gardnerella and the genus Atopobium is involved, for example, for the prevention or improvement of the onset or progression of bacterial vaginosis, urinary tract infections, endometritis, acute salpingitis, postoperative infections, amniotic fluid infections, infertility, premature birth, neonatal sepsis, and the like. The disease or the symptom is preferably bacterial vaginosis.

[0078] The amount of the proliferation inhibitor to be administered is appropriately selected depending on the type or degree of a disease or a symptom in which at least one bacterium selected from the genus Gardnerella and the genus Atopobium is involved, the administration route, and the like.

[0079] The administration of the proliferation inhibitor can be appropriately selected depending on the kind or degree of the disease or symptom in which at least one bacterium selected from the genera Gardnerella and Atopobium is involved, the administration route, and the like. From the viewpoint of the effect of inhibiting proliferation, the administration is preferably performed for 3 to 80 days, more preferably for 7 days to 60 days.

[0080] The administration route of the proliferation inhibitor is not particularly limited, and a publicly known administration route can be used. The administration can be oral administration or non-oral administration. The proliferation inhibitor is preferably administered non-orally, more preferably topically, because of the ease of administration.

[0081] In the case of oral administration of the proliferation inhibitor, the proliferation inhibitor can be used as a food or drink in addition to a pharmaceutical product.

[0082] From the viewpoint of the effect of inhibiting bacterial proliferation, the proliferation inhibitor is preferably used as a cleaning agent or a topical agent.

[0083] The target site of the cleaning agent or the topical agent is a site in which at least one bacterium selected from the genera Gardnerella and Atopobium is present or is suspected to be present, and is preferably the genital area of a female or a male, more preferably the genital area of a female, and further preferably the vagina. In the present specification, the genital area of a female refers to the vulva, the vagina, and the skin around the vagina, and the genital area of a male refers to the penis, the testis, and the skin around the testis.

[0084] The topical agent refers to an agent that is applied directly to the surface of the skin, the mucosa, or the like, and can be any one of a pharmaceutical composition, a quasi-drug, and a cosmetic. The topical agent can be in the form of a solid, a semi-solid, a liquid, an emulsion, a paste, a gel, a mousse, a spray, or a two-agent system in which these are used in combination, and is not limited in terms of the form and the dosage form, as long as the agent can be applied to or permeate the skin, the mucosa, or the like.

[0085] The cleaning agent refers to an agent for cleaning the skin, the mucosa, or the like, and can be any one of a pharmaceutical composition, a quasi-drug, and a cosmetic. The cleaning agent can be in the form of a solid, a semi-solid, a liquid, an emulsion, a paste, a gel, a mousse, a spray, or a two-agent system in which these are used in combination, and is not limited in terms of the form and the dosage form.

[0086] The cleaning agent contains a surfactant, and can be a cleaning agent that is used in a foaming manner (e.g., a soap, a shower gel), or a cleaning agent that is filled in a cleaning device and sprayed to a cleaning target site.

[0087] The cleaning agent is preferably a vaginal cleaning agent. The vaginal cleaning agent is, for example, filled in a container (vaginal cleaning device) that has a nozzle that can be inserted into the vagina, sprayed into the vagina, and used to wash away blood and / or secretions that remain in or outside the vagina.

[0088] <Preparation containing proliferation inhibitor>

[0089] The proliferation inhibitor can be administered directly to an organism, or an effective amount of the proliferation inhibitor can be administered in admixture with a pharmaceutically acceptable carrier to form a preparation.

[0090] The dosage form of the preparation is not particularly limited, and can be any one of a pharmaceutical product, a quasi-pharmaceutical product, or a cosmetic product. In other words, one aspect of the present application is a cosmetic product or a quasi-pharmaceutical product containing the proliferation inhibitor. Another aspect of the present application is a pharmaceutical composition containing the proliferation inhibitor.

[0091] The content of glycerol glucoside in the preparation can be appropriately set depending on the form and / or use of the preparation, and from the viewpoint of the effect of inhibiting bacterial proliferation, for example, 0.002% by mass or more, 0.003% by mass or more, 0.004% by mass or more, 0.005% by mass or more, 0.006% by mass or more, 30% by mass or less, 29% by mass or less, 28% by mass or less, 27% by mass or less, 26% by mass or less, 25% by mass or less, 24% by mass or less, 23% by mass or less, 22% by mass or less, 21% by mass or less, or 20% by mass or less can be exemplified.

[0092] The cosmetic product or quasi-pharmaceutical product is preferably a cosmetic product or quasi-pharmaceutical product for the private parts, and more preferably a cream, an ointment, a gel, or an emulsion for the private parts.

[0093] In the cosmetic product or quasi-pharmaceutical product, a component conventionally used in cosmetic products, skin external agents as pharmaceutical products, or cleansing agents, etc., such as an oil, a wax, a hydrocarbon oil, an ester oil, a higher alcohol, a silicone oil, a humectant, a surfactant, a water-soluble polymer, a thickening agent, a powder, a skin-protecting agent, a whitening agent, a wrinkle-improving agent, an anti-aging agent, a plant extract, a preservative, an anti-inflammatory agent, a pH adjusting agent, a metal ion blocking agent, an antioxidant, a stabilizer, a perfume, a pigment, a dye, and the like can be appropriately incorporated as needed without impairing the effects of the present application. These components can be used singly or in combination of two or more.

[0094] In addition, the cosmetic product or quasi-pharmaceutical product can also contain probiotic bacteria such as Doderlein's bacillus.

[0095] The pharmaceutical composition is preferably used for the treatment or prevention of a disease involving Gardnerella vaginalis or Atopobium vaginae.

[0096] Examples of diseases mentioned include bacterial vaginosis, urinary tract infection, endometritis, acute salpingitis, postoperative infection, amniotic fluid infection, infertility, premature birth, and neonatal sepsis, with bacterial vaginosis being preferred.

[0097] The dosage form of the pharmaceutical composition is not particularly limited and may be: solid dosage forms such as powder, granules, tablets, and capsules; or liquid dosage forms such as liquid, suspension, and emulsion.

[0098] The pharmaceutical composition is preferably a pharmaceutical composition for use in the private parts, more preferably a vaginal suppository or a cream, ointment, gel or lotion applied to the skin or mucous membrane of the private parts, and even more preferably a vaginal suppository or a cream, ointment, gel or lotion applied to the skin or mucous membrane of the female private parts.

[0099] Vaginal suppositories are external medications applied inside the vagina. They can be any dosage form, such as tablets, capsules, ointments, or gels, or they can be administered via an applicator inserted into the vagina.

[0100] The pharmaceutical composition may also contain antibiotics, antifungal agents, bactericides, disinfectants, local anesthetics, antihistamines, antipruritics, preservatives, pH adjusters, excipients, binders, disintegrants, lubricants, stabilizers, solubilizers, suspending agents, isotonic agents, buffers, antioxidants, and probiotics such as *Dudleyani*. These components may be used alone or in combination of two or more.

[0101] Examples of antibiotics include metronidazole, clindamycin, secnidazole, chloramphenicol, and nifuratel.

[0102] Examples of antifungal agents include fluconazole, butoconazole, clotrimazole, miconazole, and tiaconazole.

[0103] Examples of disinfectants include benzalkonium chloride, benzyl chloride, povidone-iodine, and isopropyl methylphenol.

[0104] Examples of local anesthetics include lidocaine, serocaine, debucaine, and ethyl aminobenzoate.

[0105] Examples of antihistamines include diphenhydramine, cetirizine, doxepin, and hydroxyzine.

[0106] As an antipruritic agent, clotrimazole can be cited as an example.

[0107] The formulation can be manufactured by adding the proliferation inhibitor according to the methods commonly used in these formulations. The proliferation inhibitor can be added at the beginning, middle or end of the manufacturing process of the formulation, and the addition method can be selected from appropriate methods such as mixing, compounding, dissolving, impregnation, dispersing, spraying, coating, etc., depending on the form of the formulation.

[0108] <Methods for inhibiting bacterial proliferation>

[0109] One aspect of the present invention is a proliferation inhibition method for at least one bacterium selected from the genera *Gardnerella* and *Atopobium*, comprising the step of administering a target glycerol glucoside.

[0110] There are no particular limitations on the method of administering glycerol glucoside to the subject, and any suitable method may be used depending on the subject. As a method of administration, non-oral administration is preferred, and topical application to the skin or mucous membranes is more preferred. Topical application to the skin or mucous membranes can be, for example, simple application, layering therapy, occlusive therapy, moisturizing therapy, etc.

[0111] The target site for administering glycerol glucoside is a site where at least one bacterium from the genera Gardnerella and Atopobium is present or is suspected to be present, preferably the female or male genital genitals, more preferably the female genital genitals, and even more preferably the vagina.

[0112] One form of the present invention is a therapeutic or preventative agent for diseases involving Gardnerella vaginalis or Atopobium vaginae, comprising glycerol glucoside.

[0113] One form of the present invention is a glycerol glucoside, which is used as a therapeutic or preventative agent for diseases involving Gardnerella vaginalis or Atopobium vaginae.

[0114] One aspect of the present invention is the use of glycerol glucoside in inhibiting the proliferation of at least one bacterium selected from the genera Gardnerella and Atopobium.

[0115] One aspect of the present invention is the use of glycerol glucoside in the manufacture of a proliferation inhibitor of at least one bacterium selected from the genera Gardnerella and Atopobium.

[0116] One aspect of the present invention is a method for treating or preventing a disease involving Gardnerella vaginalis or Atopobium vaginae, comprising administering an effective amount of glycerol glucoside to the subject.

[0117] Example

[0118] The following examples illustrate the invention in a more detailed manner, but the invention is not limited thereto.

[0119] <Example 1: Proliferation test using Gardnerella vaginalis, Atopobium vaginae, and Dudleyella>

[0120] (Test substance)

[0121] As a glycerol glucoside, C-mo 2G (manufactured by Toyo Seikan Co., Ltd.) was used. Additionally, as a positive control, penicillin-streptomycin (manufactured by Fujifilm and Kazumitsu Pure Chemicals Co., Ltd., NO. 168-23191) was used. Sterile water was used as a control.

[0122] (Bacterial cells and culture medium)

[0123] Each bacterium and culture medium was used with the bacteria and culture medium shown in Table 1 and cultured under the conditions shown in Table 1.

[0124] [Table 1]

[0125]

[0126] The GAM semi-solid puncture culture medium used is manufactured by Nissui Pharmaceutical Co., Ltd., NO. 05424.

[0127] The incubation period is the number of days in which bacterial proliferation can be visually confirmed in the control group.

[0128] Various types of Duderlein bacteria are known, but because Lactobacillus crispatus and Lactobacillus iners are the dominant vaginal bacteria in all four ethnic groups (Caucasian, Black, Hispanic, and Asian), they were used as representative Duderlein bacteria in the experiment.

[0129] As a representative bacterium of Atopobium, Atopobium vaginae was used in the experiment.

[0130] (Pre-culture)

[0131] Each frozen bacterium (with 15% (vol / vol) glycerol added to the culture medium) was inoculated into 9 mL of culture medium and cultured. After reaching the logarithmic growth phase, the culture medium was thoroughly stirred to homogenize the turbidity, and the absorbance at 600 nm was measured. The culture medium was then diluted to an absorbance (600 nm) of 0.1, and this was used as the bacterial dilution.

[0132] (Experimental Methods)

[0133] C-mo 2G was added to the culture medium to achieve final concentrations of 0.5%, 1.0%, and 2.0%. Penicillin-streptomycin was added as a positive control to achieve final concentrations of 10% in *Lactobacillus crispatus*, 1% in *Lactobacillus iners*, 1% in *Gardnerella vaginalis*, and 10% in *Atopobium vaginae*.

[0134] Add 200 μL of bacterial dilution to 9 mL of culture medium containing the test substance and begin culturing. After culturing, thoroughly stir the medium to ensure uniform turbidity, and then measure the absorbance (turbidity) at 600 nm. The experiment was conducted with n=3 points at each point. Significance of difference was tested using the Student t-test; * was p<0.05, and ** was p<0.01 relative to the control.

[0135] (result)

[0136] The results of the proliferation tests for each bacterium are as follows: Figure 1 and Figure 2 As shown.

[0137] Figure 1 In the middle, the vertical axis shows the measured values ​​of absorbance (600nm) (average value of n=3). Figure 2 In the figure, the vertical axis represents the relative growth rate when the average absorbance (600 nm) of the control is set to 100%, which is the relative value of bacterial growth relative to the control.

[0138] In the positive control, the proliferation of all bacteria, including Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis, and Atopobium vaginae, was strongly inhibited.

[0139] The addition of glycerol glucoside resulted in almost no change in the number of *Lactobacillus crispatus*, while the number of *Lactobacillus iners* increased compared to the control. Furthermore, the addition of glycerol glucoside significantly inhibited the proliferation of *Gardnerella vaginalis* and *Atopobium vaginae* in a concentration-dependent manner.

[0140] That is, it can be seen that glycerol glucoside inhibited the proliferation of Gardnerella vaginalis and Atopobium vaginae without inhibiting the proliferation of Lactobacillus cristataus and Lactobacillus iners, which are Dudleyan bacteria.

[0141] <Reference Example 1: Proliferation assay using Propionibacterium acnes, Streptococcus mutans, and Staphylococcus aureus>

[0142] (Test substance)

[0143] As a glycerol glucoside, COSARTE-2G (manufactured by Toyo Glycosaccharide Co., Ltd.) was used, diluted with deionized water to achieve a COSARTE-2G content of 40% by mass. Additionally, as a positive control, BD (Sensi-Disc Tetracycline 30 Drug Sensitivity Kit, manufactured by BD Corporation, Japan) was used. As a control, glycerol (manufactured by Nacalai Tesque Co., Ltd.) was used, diluted with deionized water to achieve a content of 40% by mass.

[0144] (Acne bacteria)

[0145] As a representative strain of acne bacteria, Propionibacterium acnes (JCM6425) was used. The acne bacteria strain was added to GAM liquid medium and pre-cultured in anaerobic gas-generating bags at 35°C under anaerobic conditions.

[0146] (Caries-causing bacteria)

[0147] As a representative cariogenic bacterium, *Streptococcus mutans* (JCM5705) was used. The cariogenic strain was added to BHI liquid medium and pre-cultured at 35°C.

[0148] (Staphylococcus aureus)

[0149] As a representative strain of Staphylococcus aureus, Staphylococcus aureus (NBRC 13276) was used. The Staphylococcus aureus strain was added to BHI liquid medium and pre-cultured at 37°C.

[0150] (Experimental Methods)

[0151] GAM agar plates and BHI agar plates were prepared. Pre-cultured acne-causing bacteria, cariogenic bacteria, and Staphylococcus aureus strains in the logarithmic growth phase were cultured at a concentration of 1×10⁻⁶. 7 50 μL of the suspension was taken and spread onto agar plates using a spreader. The cells were suspended in liquid culture medium at a concentration of 100 cells / mL.

[0152] After sterilizing each test substance by filtration through a membrane filter, 50 μL of the solution was transferred to a sterilized paper plate. The paper plate was then placed on an agar plate under aseptic conditions and incubated for 48 hours under conditions suitable for each strain.

[0153] The presence and size of the inhibition zone around the paper plate can be used to determine the effect of inhibiting bacterial growth.

[0154] (result)

[0155] When using BD (positive control), large inhibition zones were observed in Propionibacterium acnes, Streptococcus mutans, and Staphylococcus aureus, indicating that the proliferation of these bacteria was strongly inhibited.

[0156] When glycerol glucoside was used, no inhibition zones were observed in *Propionibacterium acnes*, *Streptococcus mutans*, and *Staphylococcus aureus*. That is, glycerol glucoside did not inhibit the proliferation of *Propionibacterium acnes*, *Streptococcus mutans*, or *Staphylococcus aureus*. The results for glycerol used as a control were the same as those for glycerol glucoside.

Claims

1. A proliferation inhibitor, which is a proliferation inhibitor of at least one bacterium selected from the genera Gardnerella and Atopobium, wherein it contains a glycerol glucoside.

2. The proliferation inhibitor as described in claim 1, wherein, The bacteria are selected from at least one of Gardnerella vaginalis and Atopobium vaginae.

3. The proliferation inhibitor as described in claim 1 or 2, which is used as a cleansing agent or a topical agent.

4. A cosmetic or quasi-pharmaceutical comprising the proliferation inhibitor of claim 1 or 2.

5. A pharmaceutical composition for the treatment or prevention of diseases involving Gardnerella vaginalis or Atopobium vaginae, comprising the proliferation inhibitor of claim 2.

6. The pharmaceutical composition of claim 5, wherein, The disease in question is bacterial vaginosis.

7. A method for inhibiting the proliferation of at least one bacterium selected from the genera *Gardnerella* and *Atopobium*, comprising the step of administering a target glycerol glucoside.

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