Biofilm inhibitors and acne treatment agents

Abstract

This invention provides a biofilm inhibitor that is naturally derived and capable of suppressing biofilms derived from acne bacteria. [Solution] A biofilm inhibitor that suppresses biofilms derived from acne bacteria, comprising at least one selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a biofilm inhibitor that inhibits biofilms derived from acne bacteria and an acne-improving agent.

Background Art

[0002] Acne vulgaris is an inflammatory skin disease that develops when sebum accumulates in hair follicles, causing inflammation. Rashes are observed corresponding to the hair follicles mainly on the face, chest, back, etc. Acne is caused mainly by Cutibacterium acnes. Since Cutibacterium acnes is an anaerobic bacterium, it lives deep inside the hair follicles and grows using sebum accumulated in the hair follicles as a nutrient source. Hair follicles clogged with sebum like this are called comedones.

[0003] As a composition for treating acne, compositions containing adapalene or benzoyl peroxide have been proposed (see Non-Patent Documents 1 and 2). According to such a composition, it is possible to reduce (treat) the generated acne. However, the use of adapalene or benzoyl peroxide may cause side reactions such as a feeling of irritation, erythema, and dryness on the skin.

[0004] Also, in hair follicles, Cutibacterium acnes forms a biofilm by accumulating polysaccharides and the like secreted from the Cutibacterium acnes, making it difficult for drugs such as antibacterial agents to penetrate the Cutibacterium acnes inside. As a composition for destroying such a biofilm and killing Cutibacterium acnes, an acne bacteria biofilm-destroying composition containing an acne bacteria biofilm-destroying component composed of a chemically synthesized component such as isopropylmethylphenol has been proposed (see Patent Document 1). However, the use of such a chemical synthetic product may cause side reactions such as a feeling of irritation, erythema, and dryness on the skin as described above. Therefore, in order to improve acne by prevention, treatment, etc., it is desirable to use natural-derived products.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2017-1993 [Non-patent literature]

[0006] [Non-Patent Document 1] Acne vulgaris treatment agents: Adapalene® Gel, Differin® Gel 0.1%, Differin® Gel, Pharmaceutical Interview Form, Revised January 2023 (14th Edition) [Non-Patent Document 2] Acne vulgaris treatment agent: Benzoyl peroxide preparations: BEPIO® Gel 2.5%, BEPIO® Lotion 2.5%. Pharmaceutical Interview Form, revised July 2024 (13th edition). [Overview of the project] [Problems that the invention aims to solve]

[0007] In view of the above circumstances, the present invention aims to provide a biofilm inhibitor that is naturally derived and can suppress biofilms derived from acne bacteria, and an acne treatment agent that is naturally derived and can improve acne by suppressing biofilms caused by acne bacteria. [Means for solving the problem]

[0008] The characteristic configuration of the biofilm inhibitor according to the present invention, which solves the above problems, is as follows: A biofilm inhibitor that suppresses biofilms derived from acne bacteria, The active ingredient is selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract.

[0009] This biofilm inhibitor, by containing the above-mentioned specific naturally derived extract as an active ingredient, can suppress the formation of biofilms derived from Propionibacterium acnes (hereinafter also referred to as "Propionibacterium acnes-derived biofilm"). By applying such a biofilm inhibitor to the skin, the formation of Propionibacterium acnes-derived biofilms in the pores of the skin can be suppressed, thereby preventing the occurrence (including exacerbation) of inflammatory reactions caused by such Propionibacterium acnes-derived biofilms, and consequently treating or preventing acne. Furthermore, while the emergence of drug-resistant bacteria is a problem when acting on Propionibacterium acnes itself, by acting on Propionibacterium acnes-derived biofilms, it is possible to suppress inflammatory reactions regardless of whether or not drug-resistant bacteria emerge.

[0010] In the biofilm inhibitor according to the present invention, Preferably, it contains at least one selected from the group consisting of water, ethanol, and butylene glycol.

[0011] According to this biofilm inhibitor, by containing at least one selected from the group consisting of water, ethanol, and butylene glycol, it is possible to suppress skin damage caused by the solvent while providing an easy-to-handle dosage form such as an extract.

[0012] In the biofilm inhibitor according to the present invention, It is preferable that the concentration of the extract is adjusted to 0.1 to 10% by mass.

[0013] With this biofilm inhibitor configuration, by adjusting the concentration (content) of the extract to fall within the appropriate range described above, it is possible to directly contact the skin cells with the inhibitor, thereby further suppressing acne-derived biofilms while minimizing damage such as cytotoxicity.

[0014] Another characteristic configuration of the acne-improving agent according to the present invention, which solves the above problem, is: The active ingredient is selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract.

[0015] As described above, this acne treatment agent contains the aforementioned specific naturally derived extract as an active ingredient, thereby suppressing the formation of acne bacteria-derived biofilms on the skin. By applying this acne treatment agent to the skin, the formation of acne bacteria-derived biofilms in the pores of the skin can be suppressed, thus preventing the occurrence (including exacerbation) of inflammatory reactions caused by such acne bacteria-derived biofilms, and consequently treating and / or preventing acne. Furthermore, while the emergence of drug-resistant bacteria becomes a problem when acting on acne bacteria themselves, by acting on acne bacteria-derived biofilms, it is possible to suppress inflammatory reactions regardless of whether drug-resistant bacteria emerge or not.

[0016] In the acne treatment agent according to the present invention, Preferably, it contains at least one selected from the group consisting of water, ethanol, and butylene glycol.

[0017] According to this acne treatment agent, by containing at least one selected from the group consisting of water, ethanol, and butylene glycol, it is possible to create an easy-to-handle dosage form such as an extract while suppressing skin damage caused by the solvent.

[0018] In the acne treatment agent according to the present invention, It is preferable that the concentration of the extract is adjusted to 0.1 to 10% by mass.

[0019] According to the acne-improving agent of this composition, by adjusting the concentration (content) of the extract to be within the above appropriate range and directly contacting the skin cells, it is possible to further suppress the biofilm derived from acne bacteria while suppressing damage such as cytotoxicity, so that acne can be further improved.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 is a graph showing the confirmation results of the ability to form biofilm derived from acne bacteria. [Figure 2] Figure 2 is a graph showing a partial extract of the results of the screening experiment of the plant extract capable of suppressing the biofilm derived from acne bacteria.

Modes for Carrying Out the Invention

[0021] The inventors of this invention have conducted diligent research and have found the following: As mentioned above, acne occurs when pores become clogged with sebum and comedones are formed. In the pores, Propionibacterium acnes secretes polysaccharides, and these secreted polysaccharides accumulate to cover the Propionibacterium acnes, forming a biofilm. Propionibacterium acnes proliferates in large numbers within this biofilm, and these Propionibacterium acnes are protected from the outside by the biofilm. Here, it is believed that the Propionibacterium acnes biofilm binds to the TLR2 receptor of human keratinocytes and may induce inflammatory cytokines, etc. (Rong Zeng et al, miR-146a Inhibits Biofilm-Derived Cutibacterium acnes-Induced Inflammatory Reactions in Human Keratinocytes, Journal of Investigative Dermatology (2019), Volume 139). Therefore, it is thought that it is not the acne bacteria itself, but rather the biofilm derived from acne bacteria that causes acne inflammation. Therefore, if the formation of acne bacteria-derived biofilm can be suppressed, it may be possible to suppress the inflammatory response caused by it. Focusing on this point, the inventors conducted diligent research and found that the skin of patients suffering from acne has a higher concentration of acne bacteria with a higher biofilm-forming ability than that of people without acne. Then, in order to search for an active ingredient that can suppress the formation of acne bacteria-derived biofilm from naturally derived products with low skin irritation, they screened extracts from 165 types of plants and found that extracts of perilla, burdock, ginseng, Roman chamomile, houttuynia cordata, licorice, and sage significantly suppressed the formation of acne bacteria-derived biofilm, and that this could treat and / or prevent acne, thus completing the present invention.

[0022] The biofilm inhibitor and acne treatment agent of the present invention will be described below. However, the present invention is not intended to be limited to the configurations described in the embodiments below. In this specification, when a numerical range is indicated by "~", it means that the numerical values ​​enclosing "~" are included as the upper and lower limits, and the numerical range may be formed by appropriately combining the upper and lower limits. In this specification, "biofilm inhibition" means inhibiting the formation of biofilm. "Acne treatment" means treating acne and / or preventing acne.

[0023] [Biofilm inhibitors] The biofilm inhibitor suppresses biofilms derived from acne bacteria. The biofilm inhibitor contains at least one active ingredient selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract. The biofilm inhibitor can suppress the formation of acne bacteria-derived biofilms in the pores of human skin. The biofilm inhibitor is preferably a liquid extract, but may contain only the above-mentioned specific extracts.

[0024] <Biofilm derived from Propionibacterium acnes> A biofilm derived from acne bacteria is a film formed when polysaccharides and other substances secreted by acne bacteria accumulate and surround the bacteria. Within this biofilm, acne bacteria proliferate in large numbers, and these bacteria are protected from external elements. Furthermore, as described above, acne bacteria biofilms trigger inflammatory responses in hair follicles. Therefore, suppressing the formation of acne bacteria biofilms can suppress inflammatory responses caused by them.

[0025] <Perilla extract> Perilla frutescens (L.) Britton var. crispa (Benth.) W. Deane is a perennial herbaceous plant belonging to the genus Perilla in the family Lamiaceae, cultivated throughout Japan. Any part of the plant can be used for extraction, including the leaves, branches, stems, flowers, buds, seeds, roots, or the entire plant, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from acne bacteria.

[0026] <Burdock extract> Burdock (Arctium lappa) is a perennial herb belonging to the genus Arctium in the Asteraceae family, native to the Eurasian continent. Any part of the plant can be used for extraction, including the leaves, stems, flowers, buds, seeds, roots, or the entire plant, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from Propionibacterium acnes.

[0027] <Ginseng extract> Panax ginseng (also known as Korean ginseng, medicinal ginseng) is a perennial plant belonging to the genus Panax in the family Araliaceae, and is the source plant for the herbal medicine "ginseng" (Ginseng Radix). Any part of the plant can be used for extraction, including the leaves, branches, stems, flowers, buds, seeds, roots, or the entire plant, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from Propionibacterium acnes.

[0028] <Anthropocene chamomile extract> Roman chamomile (Anthemis nobilis) is a perennial plant belonging to the genus Anthemis in the family Asteraceae. Any part of the plant can be used for extraction, including the leaves, branches, stems, flowers, buds, seeds, roots, or the entire plant, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from acne bacteria.

[0029] <Houttuynia cordata extract> Houttuynia cordata is a perennial herb belonging to the genus Houttuynia in the family Saururaceae. Any part of the plant—leaves, stems, roots, flowers, or the entire plant—can be used for extraction, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from Propionibacterium acnes.

[0030] <Licorice extract> Glycyrrhiza is a perennial herb belonging to the genus Glycyrrhiza in the family Fabaceae. Any part of the plant can be used for extraction, including the leaves, branches, stems, flowers, buds, seeds, roots, or the entire plant. It is preferable to use the part that can more effectively suppress the formation of biofilms derived from acne bacteria.

[0031] <Sage extract> Sage (Salvia officinalis) is a perennial herb or evergreen shrub belonging to the genus Salvia in the Lamiaceae family. Any part of the plant can be used for extraction, including the leaves, branches, stems, flowers, buds, seeds, roots, or the entire plant, but it is preferable to use the part that can more effectively suppress the formation of biofilms derived from acne bacteria.

[0032] The above-mentioned extracts of perilla, burdock, ginseng, Roman chamomile, houttuynia cordata, licorice, and sage can be obtained by finely chopping or grinding the parts of each plant to make coarse powder, and then extracting them with an extraction solvent such as water, ethanol (including anhydrous ethanol), butylene glycol, or a mixture thereof.

[0033] In addition to the specific extracts that are the active ingredients, biofilm inhibitors may contain water or organic solvents such as ethanol to dissolve or dilute them. That is, in addition to the specific extracts, biofilm inhibitors may contain at least one selected from the group consisting of water, ethanol, and butylene glycol. The extraction solvents described above may be used as is as the solvent to be included in the biofilm inhibitor. By including at least one selected from the group consisting of water, ethanol, and butylene glycol in the biofilm inhibitor, it is possible to make it into an easy-to-handle dosage form such as an extract while suppressing skin damage caused by the solvent.

[0034] <Concentration (content) of the extract> When the biofilm inhibitor is an extract, the concentration of the specific extract in the biofilm inhibitor can be appropriately set to a concentration capable of suppressing the formation of acne bacteria-derived biofilms. For example, the concentration of the specific extract in the biofilm inhibitor is preferably adjusted to 0.1 to 10% by mass, more preferably to 0.1 to 5% by mass, and even more preferably to 0.5 to 3% by mass. By adjusting the concentration (content) of the specific extract to fall within the above appropriate range, it is possible to further suppress the formation of acne bacteria-derived biofilms while minimizing damage such as cytotoxicity by directly contacting skin cells.

[0035] Biofilm inhibitors, by containing the above-mentioned specific naturally derived extracts as active ingredients, can suppress the formation of biofilms derived from Propionibacterium acnes. By applying such biofilm inhibitors to the skin, the formation of biofilms by Propionibacterium acnes in the pores of the skin can be suppressed, thereby preventing the occurrence (including exacerbation) of inflammatory reactions caused by such Propionibacterium acnes-derived biofilms, and consequently treating or preventing acne. Furthermore, while the emergence of drug-resistant bacteria is a problem when acting on Propionibacterium acnes itself, by acting on the Propionibacterium acnes-derived biofilm, it is possible to suppress inflammatory reactions regardless of whether drug-resistant bacteria emerge. In addition, by destroying the Propionibacterium acnes-derived biofilm, it becomes possible to bring antibacterial components into contact with the Propionibacterium acnes inside.

[0036] Furthermore, biofilm inhibitors can also be used in vitro to suppress the formation of acne bacteria-derived biofilms by applying them to pores in the skin, thereby contributing to basic research on skin tissue.

[0037] [Acne treatment] The acne treatment contains at least one active ingredient selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract. The acne treatment is preferably in the form of a liquid extract, but may contain only the above-mentioned specific extracts.

[0038] Acne treatment agents can treat and / or prevent acne by suppressing the formation of acne bacteria-derived biofilms in the pores of the skin. In this respect, acne treatment agents can be said to be the application of the aforementioned biofilm inhibitors to acne treatment agents. Since the composition of acne treatment agents is the same as that of biofilm inhibitors, a detailed explanation will be omitted.

[0039] As described above, acne treatment agents can suppress the formation of acne bacteria-derived biofilms by containing the specific naturally derived extracts mentioned above as active ingredients. By applying such acne treatment agents to the skin, the formation of acne bacteria-derived biofilms in the pores of the skin can be suppressed, thereby preventing the occurrence (including exacerbation) of inflammatory reactions caused by such acne bacteria-derived biofilms, and consequently treating and / or preventing acne. Furthermore, while the emergence of drug-resistant bacteria is a problem when acting on acne bacteria themselves, acting on acne bacteria-derived biofilms makes it possible to suppress inflammatory reactions regardless of whether drug-resistant bacteria emerge. In addition, by destroying the acne bacteria-derived biofilm, it becomes possible to bring the antibacterial components into contact with the acne bacteria inside.

[0040] Furthermore, acne-improving agents can also be used in in vitro methods to improve acne by contacting skin cells, thereby allowing them to be used in basic experiments on skin tissue.

[0041] [Application form] The biofilm inhibitors and acne-improving agents described above can be in the form of cosmetics, pharmaceuticals, quasi-drugs, experimental reagents, etc., containing them as active ingredients.

[0042] When formulated as cosmetics, quasi-drugs, pharmaceuticals, etc., the above-mentioned specific extract can be incorporated into the above-mentioned forms by, for example, incorporating the above-mentioned biofilm inhibitor or the above-mentioned acne-improving agent. In addition, when formulated as cosmetics, quasi-drugs, pharmaceuticals, etc., the above-mentioned biofilm inhibitor and acne-improving agent, as extracts, can be appropriately combined with conventionally known ingredients used in cosmetics, quasi-drugs, and pharmaceuticals (moisturizers, solubilizers, emulsifiers, oils, drugs, thickeners, stabilizers, preservatives, etc.) to the extent that the effects of containing the above-mentioned specific extract are not hindered.

[0043] The amounts of biofilm inhibitors and acne-improving agents in these forms can be appropriately set so that the concentration of the specific extract is sufficient to suppress biofilm formation on the skin and treat or prevent acne. In this case, the concentration of the specific extract may be appropriately adjusted by appropriately removing the solvent contained in the biofilm inhibitor and acne-improving agent as extracts by reducing pressure, drying, etc. Furthermore, the amount used (applied amount) in a single application to the skin in the above forms can be appropriately adjusted so that it is sufficient to treat or prevent acne. Therefore, the concentration of the specific extract in the above forms can be appropriately adjusted according to the amount used in a single application. Furthermore, when used in the form of experimental reagents, the amount of biofilm inhibitor and acne-improving agent in the above forms can be appropriately adjusted so that, for example, the concentration of the specific extract after addition to cells is preferably 0.01 to 10 mg / mL, more preferably 0.05 to 5 mg / mL, and even more preferably 0.1 to 1 mg / mL.

[0044] The above-mentioned forms can be obtained by stirring and mixing the amount of the biofilm inhibitor or acne treatment agent containing the above-mentioned specific extract, along with other additives as needed, using conventionally known methods. [Examples]

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0046] [Confirmation of the biofilm-forming ability of Propionibacterium acnes] To determine whether there is a relationship between susceptibility to acne and the biofilm-forming ability of Propionibacterium acnes, commensal bacteria were collected from the facial skin of 19 subjects prone to acne and 13 subjects not prone to acne. Propionibacterium acnes was isolated and cultured, yielding Propionibacterium acnes from subjects prone to acne and Propionibacterium acnes from subjects not prone to acne. Each strain of Propionibacterium acnes was cultured anaerobically in 96-well plates at 37°C for 3 days, washed with physiological saline, fixed with methanol, and then stained with 0.1% by mass crystal violet solution. After staining for 10 minutes, the samples were washed with physiological saline, dried, and the dye was extracted with acetone:ethanol (volume ratio 20:80). The absorbance at a wavelength of 560 nm (OD560) was measured using an absorbance microplate reader (Emax precision microplate reader, Molecular Devices), and this was used as an indicator of biofilm formation. The higher the absorbance at the wavelength of 560 nm, the higher the biofilm formation ability is considered to be. The results are shown in Figure 1. In Figure 1, subjects prone to acne are labeled "acne," and subjects not prone to acne are labeled "normal."

[0047] As shown in Figure 1, when comparing the biofilm-forming ability of 122 strains of Propionibacterium acnes from 19 subjects prone to acne with that of 57 strains from 13 subjects less prone to acne, it was shown that the Propionibacterium acnes collected from subjects prone to acne had a higher biofilm-forming ability than the Propionibacterium acnes collected from subjects less prone to acne.

[0048] [Screening experiment] <Experimental Procedure> Using the HL056PA1 strain (BEI Resources) as the bacterium *Propionibacterium acnes*, the biofilm-inhibiting ability of each plant extract was evaluated by measuring the absorbance (OD560) at a wavelength of 560 nm, except that each extract containing extracts from 165 different plants was added so that the concentration of each extract in the total contents of each well was 2% by mass, in the same manner as described above for [Confirmation of Biofilm Formation Ability of *Propionibacterium acnes*]. The concentration of each plant extract after addition was within the range of 0.1 to 0.5 mg / mL. Figure 2 shows excerpts of the results for some of the plant extracts, including *Perilla*, *Burdock*, *Ginseng*, *Roman Chamomile*, *Houttuynia cordata*, *Licorice*, and *Sage*, out of the 165 plant extracts. Figure 2 shows the average value (n=6) of the concentration of each plant extract over 6 trials, along with the standard deviation (SD). Furthermore, the results for samples in which only physiological saline solution was added to the wells without adding acne bacteria are shown as negative controls, and the results for samples in which only a 50% by mass aqueous solution of butylene glycol (BG50%) was added to the wells containing acne bacteria are shown as positive controls.

[0049] As shown in Figure 2, extracts of perilla, burdock, ginseng, Roman chamomile, houttuynia cordata, licorice, and sage were shown to have significantly higher inhibitory activity against the formation of acne bacteria-derived biofilms compared to other plants (loquat, fermented mate tea extract, peony, honeysuckle, swertia japonica, pineapple, and jojoba). Although not shown in the figure, the results also show that these specific extracts significantly increased their inhibitory activity against acne bacteria-derived biofilms compared to the remaining extracts of the 165 plants mentioned above. [Industrial applicability]

[0050] The biofilm inhibitor and acne treatment agent of the present invention can be suitably used in cosmetics, quasi-drugs, pharmaceuticals, research and development, manufacturing, sales, and experimental reagents related thereto.

Claims

1. A biofilm inhibitor that suppresses biofilms derived from acne bacteria, A biofilm inhibitor containing at least one selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract as an active ingredient.

2. The biofilm inhibitor according to claim 1, comprising at least one selected from the group consisting of water, ethanol, and butylene glycol.

3. The biofilm inhibitor according to claim 1 or 2, wherein the concentration of the extract is adjusted to 0.1 to 10% by mass.

4. An acne treatment agent containing at least one selected from the group consisting of perilla extract, burdock extract, ginseng extract, Roman chamomile extract, Houttuynia cordata extract, licorice extract, and sage extract as an active ingredient.

5. The acne treatment agent according to claim 4, comprising at least one selected from the group consisting of water, ethanol, and butylene glycol.

6. The acne treatment agent according to claim 4 or 5, wherein the concentration of the extract is adjusted to 0.1 to 10% by mass.

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