JP7219033B2 - M. Skin external composition containing aerilata as an active ingredient - Google Patents

Description

The present invention is based on M.S. The present invention relates to an external skin composition containing aerilata as an active ingredient.

The skin is roughly divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. The epidermis, which is the outermost layer, is further classified into four layers, the stratum corneum, the stratum granulosum, the stratum spinosum and the stratum basale, and the main cells constituting these layers are called epidermal keratinocytes.

The skin plays an important role in preventing external stimuli, foreign matter from entering the body, and excessive moisture release, and this is called a barrier function. A decrease in barrier function is known to cause rough skin, itching, and inflammation. Therefore, improvement or preservation of the barrier function is one of the important themes in the development of cosmetics (for example, Patent Documents 1 and 2).

Barrier function consists of the interaction of a plurality of defense components, and among them, intercellular tight junctions have recently been considered to be particularly important elements and have attracted attention (for example, Non-Patent Document 1).

Intercellular tight junctions present in the granular layer adjacent to the stratum corneum, that is, intercellular adhesion structures, have the function of separating the inside and outside of the living body in the stratum corneum, and are recognized to play a major role in the barrier function. (Patent Document 3).

A TER value (Transepithelial electrical resistance) is used as an index of the barrier function, and a technique for enhancing and improving the barrier function by increasing the TER value has been proposed (Non-Patent Document 1).

Adhesion proteins involved in the TER value include TJP1 (Tight Junction Protein 1), CLD1 (Claudin 1), OCL (Occludin), ZO1 (Zonula Occludens Protein 1), and the like (Non-Patent Document 2).

By the way, in recent years, there has been proposed a cosmetic containing fungal cells, which have a beneficial effect on the human body, as an active ingredient (for example, Patent Document 4).

JP 2018-104322 A JP 2017-002013 A JP 2018-090631 A JP-A-09-020638

The Journal of Nutrition, 2011 Jan;141(1):87-94 Frontiers in Immunology, December 2015, Volume 6, Article 612

An object of the present invention is to provide a novel technique for improving the barrier function and promoting the formation of a tight junction structure.

As a result of diligent research efforts, the present inventors have found that bacteria belonging to the genus Massilia have the effect of improving the barrier function and promoting the formation of tight junction structures, and have completed the present invention.

That is, the present invention for solving the above problems is a composition for external use on the skin containing Massilia bacterium as an active ingredient.
The composition for external use on skin of the present invention has an effect of improving barrier function and/or promoting formation of tight junction structure.

In a preferred embodiment of the present invention, the Massilia bacterium is Massilia aerilata.
The external composition for skin of the present invention in the form containing Massilia errata as an active ingredient is excellent in improving the barrier function and/or promoting the formation of tight junction structures.

In a preferred embodiment of the present invention, the composition for external use on skin is used for improving barrier function and/or promoting tight junction structure formation.

In a preferred embodiment of the present invention, the external composition for skin is used for prevention or improvement of one or more selected from dry skin, rough skin, wrinkles, pigmentation, and slackness.

The present invention also relates to a barrier function-improving and/or tight junction structure formation promoting agent containing Massilia bacterium as an active ingredient.

In a preferred form of the invention, the Massilia bacterium is Massilia aerilata.

Moreover, in a preferred embodiment of the present invention, it is used for prevention or improvement of one or more selected from dry skin, rough skin, wrinkles, pigmentation and saggy skin.

The present invention has a barrier function-enhancing action and/or a tight junction structure formation-promoting action, more specifically, an action for preventing or improving dry skin, rough skin, wrinkles, pigmentation, sagging, and the like.

4 is a bar graph showing measurement results of TER values, which are indicators of barrier function. It is an image captured by a confocal microscope in which formation of a tight junction structure is visualized by an immunostaining method.

Preferred embodiments of the present invention will now be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.

As the bacterium belonging to the genus Massilia, which is an active ingredient in the present invention, any bacterium belonging to the genus Massilia can be used without particular limitation, including Massilia aerilata, Massilia aurea, and Massilia timonae. , Massilia plicata, Massilia dura, Massilia albidiflava, Massilia lutea, and the like.

Among them, it is more preferable to use Massilia aerilata as an active ingredient.

Bacteria of the genus Massilia can be used by isolating from samples collected from various environments reported as habitats of bacteria of the genus Massilia, or commercially available strains and deposited strains can be used.

As the deposited strain, Massilia aerilata KACC 12505 (hereinafter referred to as M. erilata KACC 12505) can be used.

M. In 2009, Errata KACC 12505 was registered at the Patent Organism Depositary Center, National Institute of Technology and Evaluation (zip code: 305-8566, address: Tsukuba Center Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture), Budapest Treaty and has been assigned accession number NBRC 106432.

M. Errata KACC 12505 can be cultured by conventional methods. For example, using R2A agar medium, it is possible to culture under aerobic conditions at a temperature of 5 to 35° C. (preferably 30° C.) and a pH of 5 to 9 (preferably pH 6 to 8).

The Massilia bacterium, which is the active ingredient of the composition for external use on skin of the present invention, has an effect of improving the barrier function and/or promoting the formation of tight junction structures.
In addition, it is particularly useful for preventing or improving skin conditions caused by decreased barrier function and/or decreased ability to form tight junction structures.
Dry skin, rough skin, wrinkles, pigmentation, slackness and the like can be mentioned as skin conditions caused by a decrease in barrier function and/or a decrease in ability to form tight junction structures.
Bacteria belonging to the genus Massilia are effective in preventing or improving the aforementioned skin conditions.

As described above, the genus Massilia bacterium exerts an effective effect on the skin, and is therefore preferably provided in the form of a composition for external use on the skin.

The amount of cells of the genus Massillia in the external composition for skin of the present invention varies depending on the dosage form of the external composition for skin, but is preferably 50 CFU/mL or more, more preferably 5×10 ² to 5×10 ⁵ CFU. /mL can be used as a guideline. In addition, CFU means a colony forming unit: Colony Forming Unit.

Embodiments of the composition for external use on the skin of the present invention are not particularly limited, and suitable examples include cosmetics, quasi-drugs, and pharmaceuticals.
As the dosage form, any of commonly known lotion dosage form, emulsion dosage form, essence dosage form, cream dosage form and powder-containing dosage form can be used.

When it is provided in the form of a composition for external use on the skin, its composition is not particularly limited, and it may contain commonly used optional ingredients as long as the effects of the present invention are not impaired. Examples of such optional ingredients include macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, and hydrogenated coconut oil. , hydrogenated oil, Japanese wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, ibota wax, lanolin, reduced lanolin, hard lanolin, jojoba wax, oils and waxes; liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, vaseline , Hydrocarbons such as microcrystalline wax; Higher fatty acids such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, undecylenic acid; Cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol , octyldodecanol, myristyl alcohol, cetostearyl alcohol, etc.; , ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glyceryl di-2-heptylundecanoate, glyceryl tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, triisostearate Oil agents such as synthetic ester oils such as methylolpropane and pentane erythritol of tetra-2-ethylhexanoate; anions such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and triethanolamine ether of alkyl sulfates Surfactants; cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide; imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salts, etc.), betaine surfactants (alkylbetaine, amidobetaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyl taurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.) , glycerin fatty acids (glyceryl monostearate, etc.), propylene glycol fatty acid esters (monostearate acid propylene glycol, etc.), hydrogenated castor oil derivatives, glycerin alkyl ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.) ), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkylphenyl ether (POE nonylphenyl ether, etc.), pluronic type, POE/POP alkyl ethers (POE/POP 2-decyltetradecyl ether, etc.), tetronics, POE castor oil/hardened castor oil derivatives (POE castor oil, POE hardened castor oil, etc.), nonionic surfactants such as sucrose fatty acid esters and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, erythritol, sorbitol, xylitol, maltitol, propylene glycol and 2,4-hexanediol Moisturizing ingredients such as sodium pyrrolidonecarboxylate, lactic acid, sodium lactate; para-aminobenzoic acid UV absorber; anthranilic acid UV absorber; salicylic acid UV absorber; cinnamic acid UV absorber; benzophenone UV absorber; Sugar-based UV absorbers; UV absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane, and the like are preferably exemplified.

Alternatively, the Massilia bacterium may be provided in the form of an oral composition.
Oral composition forms include the form of food compositions or pharmaceutical compositions.

Food compositions include general foods such as confectionery, bread, and noodles, and food groups with the purpose of promoting health in the form of drink preparations, granules, powders, capsules, and tablets (for example, food for specified health use, etc.). and other embodiments can be suitably exemplified.
Preferred examples of pharmaceutical compositions include embodiments such as granules, powders, capsules, and orally administered pharmaceuticals in the form of tablets. Particularly preferably, it is in the form of a food composition.

These food compositions and pharmaceutical compositions may also contain acceptable optional ingredients. In the case of a food composition, such optional ingredients include, in addition to general food materials, seasoning ingredients such as salt, sugar, sodium glutamate, sodium inosinate, and vinegar; Viscous agents, emulsifying/dispersing agents, preservatives, stabilizers, various vitamins, etc. can be suitably exemplified. Preferred examples include binders such as gum arabic and hydroxypropylcellulose, disintegrants such as croscarmellose sodium and starch, lubricants such as magnesium stearate, flavoring agents, coloring agents, and various vitamins. The composition of the present invention can be produced by treating these in accordance with conventional methods.

<Culture of strain>
Massilia aerilata, Staphylococcus epidermidis, and Staphylococcus aureus used in the test examples described later were all purchased from National Institute of Technology and Evaluation. Incidentally, Massilia Elirata is M.I. A strain of Errata KACC 12505 was purchased.

The purchased bacteria were cultured using appropriate agar or liquid media to obtain strains used in subsequent tests.

<Test Example 1: Evaluation of Effect of Improving Barrier Function (TER Value Measurement Test)>
The barrier function-improving effect of Massilia bacteria was evaluated by the following method.
First, normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries) were seeded (70,000 cells/well) in a 12-well transwell plate. After that, the seeded NHEKs were cultured for 3 days at 37° C. under 5% CO ₂ environment. After culturing for 3 days, the culture medium was replaced with a high calcium medium (1.45 mM). Suspensions of each strain were added thereto, the concentration of which was adjusted with the medium so that the final concentration was 7,500 CFU/well. A sample containing no strain was prepared as a control.

After culturing for 6 hours, transepithelial electrical resistance (TER) was measured using Millicell ERS2 (manufactured by Millipore). The results are shown in FIG. In FIG. 1, the vertical axis represents the TER value (Ω·cm ² ).

From the results of FIG. 1, Massilia erilata showed a significant increase in TER value compared to the control. In addition, Massilia erilata shows a remarkable increase in TER value compared to other strains. This result indicates that bacteria belonging to the genus Massilia have an excellent barrier function-enhancing effect.

<Test Example 2: Evaluation of Tight Junction Structure Formation Promotion Effect>
The formation of tight junction structures was visualized by the following method, and the effect of promoting the formation of tight junction structures in Massilia bacteria was evaluated.
First, normal human epidermal keratinocytes (NHEK) (manufactured by Kurabo Industries) were seeded (30,000 cells/well) in a 4-well chamber plate. After that, the seeded NHEKs were cultured for 3 days at 37° C. under 5% CO ₂ environment. After culturing for 3 days, the culture medium was replaced with a high calcium medium (1.45 mM). Suspensions of each strain were added thereto, the concentration of which was adjusted with the medium so that the final concentration was 2,300 CFU/well. A sample containing no strain was prepared as a control.

After 6 hours of culture, the medium was removed and washed once with phosphate-buffered saline (PBS). Next, in order to fix the cells, ethanol treatment was performed at 4° C. for 15 minutes, followed by acetone treatment at room temperature for 1 minute. After that, it was treated with a 0.1% Tween20/PBS solution for 5 minutes, and further treated with a 0.5% TritonX100/PBS solution for 20 minutes. Then, in order to block non-specific reactions, it was treated with a 10% Blockace/PBS solution at room temperature for 30 minutes.

Thereafter, a 100-fold diluted mouse anti-Occludin antibody (manufactured by Invitrogen) and a 200-fold diluted rabbit anti-Zonula Occludin-1 (ZO-1) antibody (manufactured by Invitrogen) were added as primary antibodies to the 4-well chamber plate. , at 4° C. overnight. After washing with 0.1% Tween 20/PBS solution, 250-fold diluted goat anti-mouse IgG antibody Alexa Fluor 488 (manufactured by Invitrogen) and 250-fold diluted goat anti-rabbit IgG antibody Alexa Fluor 555 (manufactured by Invitrogen) ) was added as a secondary antibody in a 4-well chamber plate and allowed to react at room temperature for 45 minutes. After washing, it was mounted using Fluoromount-G (manufactured by Diagnostic BioSystems). The prepared sample was observed using a confocal microscope (LSM510: manufactured by Carl Zeiss).

FIG. 2 shows an immunostained image of NHEK by confocal microscopy. Arrows in the images indicate locations where Occludin and ZO-1 are continuously co-stained (C-2 at the bottom of FIG. 2). In addition, arrowheads indicate sites where Occludin and ZO-1 are intermittently co-stained (C-3 and C-4 at the bottom of FIG. 2). Note that the scale bar indicates 50 μm.

NHEK to which Massilia errata was added showed strong color development between cells, and it was confirmed that Occludin and ZO-1 were largely localized in intercellular spaces (Fig. 2 top A-2 and middle B-2 ). In addition, as shown in C-2 at the bottom of FIG. 2, in the integrated image of Occludin and ZO-1, many areas were confirmed to be continuously co-stained. These results indicate that bacteria belonging to the genus Massilia have an excellent tight junction structure formation promoting effect.

<Prescription example>
(1) Preparation of Cell Concentrate A Massilia errata strain was aerobically cultured in R2A medium. Then, the grown strain was suspended in physiological saline to obtain a cell concentrate (cell concentration: about 4.6×10 ⁶ CFU/mL). The prepared bacterial cell concentrate was used as a compounding material for the formulation examples shown below. Incidentally, Massilia Elirata is M.I. A strain of Errata KACC 12505 was used.

(2) Preparation of skin lotion According to the formulation in Table 1, a skin lotion, which is an external preparation for skin of the present invention, was prepared. First, the component A shown in Table 1 was heated at room temperature, and the component B was heated to 60° C. and mixed. Thereafter, B was gradually added to A while stirring, and the mixture was stirred and cooled to obtain a lotion.

(3) Preparation of Essence According to the formulation shown in Table 2, an essence, which is an external preparation for skin of the present invention, was prepared. First, components A and B shown in Table 2 were heated to 80° C. and mixed. Thereafter, B was gradually added to A while stirring, and the mixture was stirred and cooled to obtain an essence.

(4) Preparation of Milky Lotion According to the formulation shown in Table 3, a milky lotion, which is the topical skin preparation of the present invention, was prepared. First, components A and B shown in Table 3 were heated to 80° C. and mixed. Thereafter, B was gradually added to A while stirring, and the mixture was stirred and cooled to obtain a milky lotion.

(5) Preparation of O/W Cream According to the formulation in Table 4, an O/W cream, which is an external preparation for skin of the present invention, was prepared. First, the components A and B shown in Table 4 were heated to 80° C. and mixed. Thereafter, B was gradually added to A while stirring, and the mixture was stirred and cooled to obtain an O/W cream.

(6) Preparation of W/O Cream According to the formulation in Table 5, a W/O cream, which is an external preparation for skin of the present invention, was prepared. First, components A and B shown in Table 5 were heated to 80° C. and mixed. Thereafter, A was gradually added to B while stirring, and the mixture was stirred and cooled to obtain a W/O cream.

(7) Preparation of UV Cream According to the formulation in Table 6, an O/W UV cream, which is an external preparation for skin of the present invention, was prepared. First, the components A and B shown in Table 6 were heated to 80° C. and mixed. Thereafter, B was gradually added to A while stirring, and the mixture was stirred and cooled to obtain an O/W UV cream.

The present invention can be applied to cosmetics, quasi-drugs, and pharmaceuticals.

Claims (7)

1. An external skin composition for preventing or improving one or more of wrinkles, pigmentation and sagging, comprising a bacterium of the genus Massilia as an active ingredient, by promoting tight junction structure formation . 2. The composition for external use on skin according to claim 1, wherein the Massilia bacterium is Massilia aerilata. 3. The external composition for skin according to claim 1 or 2, which improves barrier function and promotes tight junction structure formation. 4. The composition for external use on skin according to any one of claims 1 to 3, comprising 50 CFU/mL or more of the Massilia bacterium . 5. The composition for external use on skin according to any one of claims 1 to 4, which is in the form of lotion, emulsion, essence, cream or powder. A tight junction structure formation promoter for preventing or improving one or more selected from wrinkles, pigmentation, and sagging, containing a Massilia bacterium as an active ingredient. The tight junction structure formation promoter according to claim 6, wherein the Massilia bacterium is Massilia aerilata.

Images