Agents for improving the skin's natural microbiome

Abstract

The objective is to provide agents for improving the skin microbiome for use in humans and other organisms. [Solution] An agent containing proteoglycans for improving the skin's natural microbiota.

Description

Technical Field

[0001] The present invention relates to, for example, an agent for improving the skin resident flora for use in humans and the like.

Background Art

[0002] Various bacteria exist on human skin, and it has been suggested that this skin resident flora is related to skin homeostasis and diseases. For example, in atopic dermatitis, it is said that Staphylococcus aureus has colonized in the rash area of about 90% of patients, and the association between this bacterium and the exacerbation of the rash has also been pointed out. On the other hand, Staphylococcus epidermidis is known as an epidermal resident bacterium and is said to enhance resistance to pathogenic bacteria by promoting the expression of antibacterial peptides and the like. Therefore, it is important to maintain the number of skin-beneficial bacteria and at the same time suppress the number of harmful bacteria on human skin (Patent Document 1).

[0003]

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The problem to be solved by the present invention is to provide an agent for improving the skin resident flora for use in humans and the like.

Means for Solving the Problems

[0006] Therefore, the inventor of the present invention was searching for a substance that promotes the growth of Staphylococcus epidermidis in vitro, and found that proteoglycan promotes the growth, and thus completed the present invention. • Item 1: An agent containing proteoglycans for improving the skin's natural microbiota. • Item 2: The agent described in Item 1, which has the effect of increasing the proportion of Staphylococcus epidermidis among the commensal bacteria of the skin. • Item 3: The agent according to item 1 or 2, wherein the proteoglycan is a proteoglycan derived from salmon nasal cartilage. • Item 4: The agent according to item 1 or 2, wherein the proteoglycan is a plant-derived proteoglycan. • Item 5: A composition for external use on the skin, comprising the agent described in any one of items 1 to 4. • Item 6: An oral composition containing the agent described in any one of items 1 to 4. [Effects of the Invention]

[0007] The present invention makes it possible to provide agents for improving the skin microbiota for use in humans and other organisms. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the main disaccharide structures of each chondroitin sulfate. [Figure 2] Figure 2 shows the results of HPLC analysis of the composition of Production Example 1. [Modes for carrying out the invention]

[0009] The following provides a detailed explanation of this disclosure, including examples. Unless otherwise specified, each disclosure may refer to the explanations of other disclosures.

[0010] <Definition> (derived from) In the specification of this application, the phrase "derived from" is used with the intention of encompassing (1) to (3) below. (1) It is purified, (2) Being isolated, and / or (3) Modification [this includes low molecular weight treatment and high molecular weight treatment (polymerization)] or alteration.

[0011] (Proteoglycan) "Proteoglycan" refers to a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also called "polysaccharide" or "sugar chain") are covalently bonded. Proteoglycans exist, for example, as extracellular matrix in skin, organs, and cartilage. Glycosaminoglycans are generally known as long-chain sugar chains that do not have a branched structure. Examples of the aforementioned proteoglycans include aggrecan, versican, decorin, testican, breakican, biglycan, serglycin, syndecan, perlecan, dystroglycan, agrin, claustrin, glypican, lumican, keratocan, and neurocan. These proteoglycans can be classified, for example, into chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, or keratan sulfate proteoglycans depending on the type of GAG bound to the protein.

[0012] Examples of the aforementioned GAGs include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the aforementioned chondroitin include O-type glycans whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine, and iO-type glycans whose main structure is a disaccharide structure of iduronic acid and acetylgalactosamine (hereinafter also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO," respectively). The aforementioned chondroitin sulfate has a structure in which a sulfate group is added to a glycan in which the disaccharides of glucuronic acid and acetylgalactosamine are repeated. Examples of the aforementioned chondroitin sulfate include chondroitin sulfate A (type A), which has a disaccharide structure of glucuronic acid and acetylgalactosamine 4-sulfate as its main disaccharide structure, iduronic acid and acetylgalactosamine Examples include chondroitin sulfate iA (iA type), which has a disaccharide structure with samin-4 sulfate as its main disaccharide structure; chondroitin sulfate C (C type), which has a disaccharide structure with glucuronic acid and acetylgalactosamine-6 ​​sulfate as its main disaccharide structure; and chondroitin sulfate iC (iC type), which has a disaccharide structure with iduronic acid and acetylgalactosamine-6 ​​sulfate as its main disaccharide structure. Each chondroitin sulfate has, for example, the disaccharide structure shown in Figure 1 as its main disaccharide structure. In Figure 1, the sulfate group (sulfo group) is bonded to a hydrogen atom, but this disclosure is not limited thereto, and the sulfate group of the GAG ​​may, for example, have a hydrogen atom removed and be ionized, or it may form a salt.

[0013] The PG derived from salmon nasal cartilage is PG obtained by extraction from salmon nasal cartilage. Here, salmon refers to fish belonging to the genus Oncorhynchus, for example, but preferably salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of efficiently regulating the immune response. The proteoglycan contained in the agent or composition of this embodiment is prepared, for example, by the method described in the publication (Japanese Patent Publication No. 6317053). Furthermore, the proteoglycan content in a composition (1g) containing the composition of this embodiment (for example, a composition for topical application to the skin, a composition for oral use) is preferably at a lower limit of 0.1 μg / g or more, more preferably 100 μg / g or more, and even more preferably 1 mg / g or more, from the viewpoint of effectively exhibiting the desired effect.

[0014] The proteoglycans used in this invention, such as PG derived from salmon nasal cartilage, can be produced by methods described in, for example, Japanese Patent Publications (Japanese Patent No. 6875701, Japanese Patent No. 6317053, and Japanese Patent No. 7295572).

[0015] Commercially available PG products include, for example, PG derived from salmon nasal cartilage, such as proteoglycan IPC (Ichimaru Pharcos) and proteoglycan F (Ichimaru Pharcos).

[0016] (Plant-derived proteoglycans) The "plant-derived proteoglycan" used in the present invention is obtained through processes such as extraction from parts of plants of the Combretaceae family (including sap such as ghatti gum). Plants of the Combretaceae family (Quisqualis indica L.) are distributed in southern China, Southeast Asia, etc., and are evergreen vine-like woody plants cultivated for medicinal and ornamental purposes. In the early growth stage, it is shrubby, but afterwards, it grows to about 10 m while twining around other objects. Ghatti gum is the dried and solidified sap secreted from the wounds of the trunk of Anogeissus latifolia Wallich of the Combretaceae family, and is also called Indian Gum. The main component of ghatti gum is a water-soluble polysaccharide composed of arabinose, galactose, mannose, xylose, and glucuronic acid as constituent sugars, and naturally exists mainly as Ca, Mg, and K salts. Ghatti gum usually dissolves in water up to about 30% by mass at room temperature or higher temperature conditions. Ghatti gum contains about 3% protein and forms a "polysaccharide-protein complex" similar to gum arabic. The "plant-derived proteoglycan" in the present invention also includes arabinogalactan-protein (AGP). Arabinogalactan-protein (AGP) is a proteoglycan that is ubiquitously distributed in plant tissues and is mainly localized in the cell wall (extracellular matrix). AGP generally has a core protein rich in hydroxyproline (Hyp) to which arabinogalactan (AG) sugar chains rich in galactose (Gal) and L-arabinose (L-Ara) are bound. There are also many types of core proteins, and the structures of the sugar chains are complex and rich in diversity.

[0017] The plant-derived proteoglycan used in the present invention can be produced by the method described in the publication (International Publication WO2023074491).

[0018] (Manufacturing method of the plant-derived proteoglycan (Production Example 1) used in the following examples) As follows, the plant-derived proteoglycan (Production Example 1) used in the following examples was produced as described in the examples of the publication (International Publication WO2023074491).

[0019] 1. Dissolution step Gattigum (2.5 kg) was dissolved in 20 times the amount of purified water (50 kg).

[0020] 2. Ultrafiltration process A solution for use in the ultrafiltration process was prepared by adding purified water to the filtrate obtained by passing it through a membrane filter with a pore size of 0.45 μm. Ultrafiltration was performed on this prepared solution to remove solutions with a molecular weight of 50,000 or less, and the solution with a molecular weight of over 50,000 (39 kg) was recovered.

[0021] 3.Filtration process The solution (39 kg) with a molecular weight exceeding 50,000 recovered in step 2 was passed through a membrane filter with a pore size of 0.45 μm, and the resulting filtrate was collected.

[0022] 4.Lyophilization The filtrate obtained from step 3 was freeze-dried to obtain a dried product (1.4 kg of composition containing proteoglycans derived from Gadigum).

[0023] This dried material was dissolved at a rate of 10g in 100mL of water at 20°C. The resulting solution was clear.

[0024] (3) Determination of protein content by BCA Protein Assay Quantitative analysis of the protein content using BCA Protein Assay confirmed that the composition contains approximately 2% protein.

[0025] (4) Analysis of amino acids using an automated amino acid analyzer Amino acid analysis confirmed that the protein in the composition contained approximately 0.5% hydroxyproline (Hyp). Hydroxyproline is a linking site in the sugar chain of plant-derived proteoglycans.

[0026] (5) HPLC analysis HPLC analysis was performed on the composition of Production Example 1. Approximately 1 g of the sample obtained from the composition of Production Example 1 (dried) was accurately weighed, and phosphate buffer (pH 6.8) was added to make exactly 10 mL to prepare the sample solution. After passing each sample through a 0.45 μm membrane filter, HPLC was performed under the following operating conditions, and the molecular weight was determined from the calibration curve below.

[0027] Furthermore, the molecular weight of the peak top was determined from a calibration curve created using Shodex STANDARD P-82 (manufactured by Showa Denko Corporation) as a molecular weight marker. A calibration curve (standard pullulan) was created for molecular weights in the range of 5,000 to 800,000.

[0028] Operating conditions Analyzer: HPLC analyzer Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation) Column: Gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation) Column temperature: 40℃ Sample injection volume: 50 μL Mobile phase: Phosphate buffer (pH 6.8) Flow rate: 0.5mL / min

[0029] Figure 2 shows the HPLC results. In the results shown in Figure 1, the molecular weight of the peak top, calculated from the column retention time, was between 300,000 and 400,000.

[0030] (Improves skin flora) In the present invention, the term "improvement of the skin microbiota" refers to maintaining the skin microbiota present in healthy skin and providing a barrier function that prevents the invasion of pathogenic bacteria from the outside. When the balance of the skin microbiota is disrupted, excessive proliferation of resident bacteria and invasion and proliferation of other harmful bacteria occur, inducing various skin symptoms. Staphylococcus epidermidis, one of the skin microbiota, has an antagonistic effect against pathogenic bacteria, such as Staphylococcus aureus and Pseudomonas aeruginosa, and can inhibit the proliferation of such harmful bacteria. Therefore, the effect of promoting the growth of Staphylococcus epidermidis is considered to be one function as a balance regulator of the skin microbiota. Alternatively, it may mean that the growth of harmful Staphylococcus aureus and other bacteria is suppressed without affecting the growth of Staphylococcus epidermidis and other bacteria. In the present invention, improvement of the skin microbiota includes, for example, increasing the proportion of Staphylococcus epidermidis and / or decreasing the proportion of Staphylococcus aureus in the skin microbiota.

[0031] (Composition for external use on the skin) In the present invention, compositions for external use on the skin, such as cosmetics, topical pharmaceuticals, and quasi-drugs, are prepared in the form of general topical skin preparations and put into practical use, for example, using a suitable pharmaceutical carrier that is pharmaceutically acceptable.Examples of such formulation carriers include humectants such as glycerin, petrolatum, urea, hyaluronic acid, and heparin; PABA derivatives (para-aminobenzoic acid, Escalol 507, etc.), cinnamic acid derivatives (Neo-Heliopan, Parsol MCX, Sunguard B, etc.), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsol DAM, etc.), heterocyclic derivatives (Tinuvin derivatives, etc.), and ultraviolet absorbers / scatterers such as titanium dioxide; disodium edetate, etc. Metal sequestering agents such as trisodium decodium, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, and gluconic acid; sebum inhibitors such as salicylic acid, sulfur, caffeine, and tannins; disinfectants such as benzalkonium chloride, benzethonium chloride, and chlorhexidine gluconate; and antibacterial agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and its salts, glycyrrhizic acid derivatives, and glycyrrhetinic acid. Inflammatory agents; Vitamins such as Vitamin A, Vitamin B group (B1, B2, B6, B12, B15), Folic acid, Nicotinic acid derivatives, Pantothenic acid derivatives, Biotin, Vitamin C, Vitamin D group (D2, D3), Vitamin E, Ubiquinone derivatives, Vitamin K (K1, K2, K3, K4); Amino acids and their derivatives such as Aspartic acid, Glutamic acid, Alanine, Lysine, Glycine, Glutamine, Serine, Cysteine, Cystine, Tyrosine, Proline, Arginine, Pyrrolidone carboxylic acid; Retinol, Tocopherol acetate, Magnesium ascorbate phosphate Whitening agents such as sodium, ascorbic acid glucoside, arbutin, kojic acid, ellagic acid, and placental extract; antioxidants such as butylhydroxytoluene, butylhydroxyanisole, and propyl gallate; astringents such as zinc chloride, zinc sulfate, zinc carbohydrate, zinc oxide, and aluminum potassium sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, and lactitol; and specified plant extracts, as well as oily components, surfactants, thickeners, alcohols, powder components, and pigments.These can be supplied by performing processing according to the type and form of product to be added (for example, a combination of processes such as crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc., as arbitrarily selected from various materials).

[0032] Furthermore, by combining the various cosmetic and pharmaceutical effects of each known raw material, it is possible to create a product that offers not only the skin microbiome improvement effect targeted by this invention, but also multifunctional effects.

[0033] Specific examples of topical skin preparations include cosmetic creams, lotions, toners, face masks, skin milks (emulsifiers), gels, powders, lip balms, lipsticks, under-makeup products, foundations, sunscreens, bath products, body washes, body rinses, soaps, cleansing foams, ointments, patches, gels, aerosols, and the like.

[0034] (Beauty method) Another embodiment of the present invention relates, for example, to a beauty method using the above-described composition for external skin application. The beauty method of this embodiment is characterized by improving the skin's resident microbiota and skin condition by applying the above-described composition for external skin application to a subject. The application method is, for example, to apply it to the skin of a desired area of ​​the subject. Application to the skin may be performed, for example, once or multiple times a day. This can be appropriately selected depending on the type of composition. Generally, when applied as a composition prepared as a lotion, emulsion, gel, cream, or ointment, it is preferable to apply it to the skin, such as the face, about once or twice a day. The composition for external skin application of the present invention can also be used as a cosmetic, for example, and can be applied to lotions, emulsions, serums, creams, liquid foundations, powder foundations, lipsticks, etc. In this embodiment, the beauty method includes not only methods performed personally, but also those provided as a cosmetic formulation tailored to the customer when providing beauty-related products, and provided by cosmetic salespeople or estheticians other than doctors. It also includes those provided as a method of use for beauty-related products in product instructions (package inserts, etc.).

[0035] The cosmetic method of this embodiment can be used, for example, to improve skin conditions where improvement of the skin's resident microbiota is desired. For example, it can be used to impart skin smoothness to individuals prone to skin inflammation.

[0036] (Oral composition) The oral compositions according to the present invention include, for example, foods and beverages (including functional foods, foods for specified health uses, supplements, etc.), pharmaceuticals, and the like.

[0037] For example, if the oral composition is a food or beverage, the form of the food or beverage may include various foods and beverages such as bread, cakes, noodles, confectionery, jellies, frozen foods, ice cream, dairy products, and beverages, as well as forms similar to those of the oral formulations described above (tablets, capsules, syrups, etc.). Foods in various forms can be prepared by using the active ingredient of the present invention alone, or by combining it with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, humectants, thickeners, etc., as appropriate.

[0038] For example, if the oral composition is a pharmaceutical, it is generally easy to assemble a convenient daily dosing regimen that can be adjusted according to the degree of discomfort, but the form of the pharmaceutical may be, for example, a solid or a liquid. Examples of the solid form include powders, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules. For example, in the case of a powder, the carrier is generally a finely ground solid that is a mixture with the finely ground active ingredient. For example, in the case of a tablet, the active ingredient is generally mixed in an appropriate proportion with a carrier having the required binding ability and molded into the desired shape and size. Suitable carriers may, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point waxes, cocoa butter, etc. The pharmaceutical product may also contain, as necessary, excipients, stabilizers, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, antiseptics, etc., to the extent that it does not impair the desired effect.

[0039] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % in the numerical values ​​indicating the amount of each component added means mass %. [Examples]

[0040] The following describes embodiments of the present invention. [Experiment 1]: Confirmation of the effect of adding proteoglycans on the growth of Staphylococcus epidermidis. (Experimental method) Standard Staphylococcus epidermidis strain (ATCC12228) was mixed with proteoglycan IPC (Ichimaru Falcos, dilution of proteoglycan IPC was performed with 30% 1,3-butylene glycol solution (BG solution)) diluted to various concentrations, or with Production Example 1 (dilution of the above-mentioned plant-derived proteoglycan was performed with 30% 1,3-butylene glycol solution (BG solution)) diluted to various concentrations. After incubation at 37°C for 24 hours, a portion of the mixture was seeded onto Nutribouillon No. 2 (Nissui Pharmaceutical Co., Ltd.) plates as culture medium and incubated at 37°C for 24 hours. The number of colonies formed on the plates was counted to examine the assimilation of proteoglycan IPC or Production Example 1 by Staphylococcus epidermidis.

[0041] Proteoglycan IPC was diluted with pure water to final concentrations of 0.1%, 1.00%, and 10.0%, respectively, and Production Example 1 was diluted with pure water to final concentrations of 1.00% and 10.0%, respectively. A control group (Control 2) containing neither proteoglycan IPC nor Production Example 1 was also simultaneously tested by adding BG to a final concentration of 0.3%. A control group (Control 1) containing neither proteoglycan IPC, Production Example 1, nor BG was also simultaneously tested. For Staphylococcus epidermidis, frozen samples were thawed, and the final concentration was 5 × 10⁻⁶. 6 It was diluted with water to a concentration of CFU / mL before use.

[0042] The above-mentioned samples and diluted Staphylococcus epidermidis were dispensed into 24-well microplates and incubated at 37°C for 1 day. 24 hours after the start of incubation, each sample was diluted 10 to 10,000 times with PBS(-), and 8 μL of each sample was taken and dropped onto Nutrient Broth No. 2 plates and incubated at 37°C for 24 hours. The number of colonies formed on the plates after incubation was measured and the results are shown in Table 1 below. This measurement was performed with N=3 for each group.

[0043] (Experimental results) The values ​​(measurement results) shown in Table 1 represent the average value of the number of colonies (N=3) measured in each group. Compared to the control group, both the addition of proteoglycan IPC and the addition of Production Example 1 were confirmed to promote the growth of Staphylococcus epidermidis.

[0044] [Table 1]

[0045] [Experiment 2] Collection and analysis of the skin microbiota of subjects (Experimental method) Four male subjects aged 30 to 59 years old had their left cheeks moistened with PBS (Fujifilm Wako Pure Chemical Industries, 162-19321) and rubbed 10 times vertically and horizontally with a swab (Plain Swab, Plastic Stick, Aseptic manufacture, SCC, 155C). The swab was then agitated in PBS to collect bacteria. This solution was added to 1% NB medium (Kanto Chemical, CM0067B) with or without proteoglycan IPC, and incubated at 37°C for 24 hours. A control group was also prepared (set up) with only PBS added, without any bacteria collected, and the test was conducted using this culture medium. After culturing, the growth of bacteria (aerobic bacteria, mainly Staphylococcus species (mainly Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus hominis, Staphylococcus capitis, mainly aerobic), and other species such as Corynebacterium)) was confirmed by measuring the absorbance of the culture solution at 620 nm. The absorbance measurement results are shown in Table 2.

[0046] (Experimental results) As shown in Table 2, in all four subjects, the addition of proteoglycan IPC resulted in the growth of aerobic bacteria (higher absorbance compared to the sample without proteoglycan IPC).

[0047] [Table 2]

[0048] Although embodiments of the present invention (including examples) have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and any design changes, etc., that do not depart from the spirit of the present invention are still included. [Industrial applicability]

[0049] The present invention makes it possible to provide agents for improving the skin microbiota for use in humans and other organisms.

Claims

1. An agent containing proteoglycans for improving the skin's natural microbiome.

2. The agent according to claim 1, which has the effect of increasing the proportion of Staphylococcus epidermidis among the commensal bacteria of the skin.

3. The agent according to claim 1 or 2, wherein the proteoglycan is a proteoglycan derived from salmon nasal cartilage.

4. The agent according to claim 1 or 2, wherein the proteoglycan is a plant-derived proteoglycan.

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