WO2019123553A1 - Actinomyces culture useful for skin - Google Patents

Abstract

Provided are a method for producing an actinomyces culture broth, said method comprising subjecting a culture broth of an actinomyces to simple purification involving at least an adsorption treatment to thereby give a decolored and deodorized actinomyces culture broth, and one or more novel uses of the actinomyces culture broth as an active ingredient of cosmetics, said uses being selected from the group consisting of: (i) an effect of inhibiting elastase activity; (ii) an effect of inhibiting hyaluronidase activity; (iii) an effect of inhibiting Maillard reaction; (iv) an effect of promoting the expression of S100A7A, CD248 and/or ITGA11 genes; and (v) an effect of promoting damage recovery.

Description

Actinomycete culture useful for the skin

The present invention relates to a novel use of actinomycete metabolites as an active ingredient of cosmetics. Specifically, the present invention relates to a method for producing an actinomycete culture solution useful as a component of cosmetics, and a cosmetic composition containing the obtained actinomycete culture solution, or use of the actinomycete culture solution in cosmetic treatment On the way.

Actinomycetes are microorganisms known to produce secondary metabolites having a wide variety of structures as well as physiological activities.

Also in the composition for external use on skin such as cosmetics, for the purpose of imparting a predetermined physiological activity, blending of a component derived from a microorganism such as actinomycete is performed.

Japanese Patent Application Laid-Open No. 7-10736 describes a cosmetic having a skin-whitening effect, which contains a fermented liquid obtained by removing bacterial cells from a Streptomyces actinomycete culture liquid.

JP-A-2011-201884 describes that a compound recovered from an actinomycete culture solution is compounded in a medicine or a cosmetic. The compound is said to be a compound having melanocortin receptor regulatory activity.

Japanese Patent Application Laid-Open No. 2010-173938 describes an emulsion-type cosmetic containing a fermented converted material obtained by causing actinomycetes and lactic acid bacteria to act on a plant extract.

JP-A 2008-255079 relates to metabolism and fermentation of microorganisms (actinomycetes, bacilli, yeast) by culture using an organic medium containing volcanic sedimentary soil, rice bran, corn powder, chitosan powder, germ, molasses, etc. A process for producing cosmetic ingredients that utilizes the degradability is described. The obtained cosmetic contains chitinase which is a metabolic component of actinomycetes.

JP 10-120551 describes cosmetic compositions containing a neuropeptide Y-antagonist component obtained by fermentation of actinomycetes. The neuropeptide Y-antagonist component is said to be usable as a modulator of lipolysis / lipid biosynthesis in the skin.

JP-A-2015-224245 describes that a culture of actinomycetes (specific strains of the genera Streptomyces and Promicromonospora) isolated from plants exhibited the involucrin production promoting action. Since involucrin is a protein involved in the maturation of the stratum corneum, its promotion of production is considered to be expected to improve the barrier function of the skin.

JP 7-10736 JP 2011-201884 Unexamined-Japanese-Patent No. 2010-173938 Patent document 1: JP 2008-255079 JP-A-10-120551 JP 2015-224245

It is an object of the present invention to provide further new uses for actinomycete culture solutions.

The present inventors have found that a liquid obtained by processing a culture solution containing a metabolite of Promicromonospora spp. Actinomycetes or a lyophilizate thereof has various effects useful as an active ingredient of cosmetics. Completed the invention. Furthermore, according to the present invention, an actinomycete culture solution excellent in formulationability (palatability) to cosmetics, having reduced mutagenicity, reduced mutagenicity and reduced color taste and odor specific to actinomycete culture solution. It also provides a method of making The actinomycete culture solution of the present invention is decolorized and deodorized by performing simple purification including at least adsorption treatment on a culture solution of actinomycetes after cultivation (that is, fermentation solution of actinomycetes: fermentation broth). Or a lyophilizate of the culture solution.

The main components of the present invention are as follows.
(1) An actinomycete culture solution (ferment filtrate of actininobacteria), the following (i) to (v):
(i) Elastase inhibitory activity,
(ii) Hyaluronidase activity inhibitory activity,
(iii) Maillard reaction inhibitory action
(iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
(v) A cosmetic composition as an active ingredient for one or more actions selected from the group consisting of damage recovery promoting actions.
(2) A cosmetic composition comprising an actinomycete culture solution as an active ingredient for two or more actions selected from the group consisting of (i) to (v).
(3-1) Elastase activity inhibitor, hyaluronidase activity inhibitor, Maillard reaction inhibitor, S100A7A, CD248, and / or ITGA11 gene expression promoter, or damage recovery promoter, or actinomycete culture solution The cosmetic composition containing as an agent.
(3-2) The cosmetic composition which is a cosmetic which is applied to the skin and used.
(4) The cosmetic composition having anti-wrinkle effect, enhancement of firm feeling, anti-inflammatory, moisturizing, anti-glycation and anti-staining action.
(5) Use method of the actinomycete culture solution, which comprises the following (i) to (iii):
(i) anti-wrinkle and / or elasticity improving effect,
(ii) anti-inflammatory and / or moisturizing effects,
(iii) A method of using the culture solution for cosmetic treatment to obtain one or more effects selected from the group consisting of antiglycation and / or anti-staining effects.
(6) The actinomycete is cultured, and the obtained culture solution is subjected to adsorption treatment using an adsorbent, and then subjected to a filtration treatment, thereby reducing the color and odor peculiar to the actinomycete culture solution, and the cosmetic The manufacturing method of the actinomycete culture solution which improved the compounding property or palatability to it.
(7) An actinomycete is cultured, and the obtained culture solution is subjected to adsorption treatment using an adsorbent and then subjected to a filtration treatment to obtain a decolorized and deodorized actinomycete culture solution, an actinic radiation Methods for purification of fungal cultures.
(8) The method for producing the actinomycete culture or the method for purifying the culture solution, using an adsorbent capable of removing an odorous component while maintaining the usefulness of the culture solution.
(9) The above-mentioned method wherein an actinomycete culture solution or an actinomycete culture having no mutagenicity and low skin irritation is obtained.
(10) An actinomycete is cultured, the obtained culture solution is subjected to adsorption treatment, and then filtration treatment is performed to obtain an actinomycete culture solution, which is not mutagenic and is skin irritant A method for producing an actinomycete culture solution, which is low.
(11) (a) Inoculating the actinomycete in a liquid medium containing a carbon source and a nitrogen source and aerobically culturing it at room temperature for 1 to 7 days to obtain a seed solution;
(B) adding a seed solution to a liquid medium having the same composition as the primary culture, and aerobically culturing it at 20-30 ° C. for 3-10 days to obtain a secondary culture solution;
(C) An adsorbent is mixed with the secondary culture solution, shaken at room temperature for about 1 day to perform adsorption treatment, and then the adsorbent is removed to obtain a decolorized / deodorized culture solution; ,
And (d) filtering the decolorized / deodorized culture solution using one or more filter devices, and a method of producing an active ingredient of cosmetics,
The active ingredient is
(i) Elastase inhibitory activity,
(ii) Hyaluronidase activity inhibitory activity,
(iii) Maillard reaction inhibitory action
(iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
(v) A manufacturing method which exerts one or more actions selected from the group consisting of damage recovery promoting actions.
(12) Furthermore, (e) Physiological activity of the culture solution after filtration (preferably, (i) Elastase activity inhibition activity; (ii) Hyaluronidase activity inhibition activity; (iii) Maillard reaction inhibition activity; Evaluating the expression promoting action of the CD248 and / or ITGA11 gene; (v) one or more actions selected from the damage recovery promoting action;
A method of producing an active ingredient of a cosmetic, including:
(13) The composition or method as described above, wherein the actinomycete is an actinomycete isolated from a plant, particularly an actinomycete of the genus Promicromonospora.
(14) Promicromonospora Ferment Filtrate, the following (i) to (v):
(i) Elastase inhibitory activity,
(ii) Hyaluronidase activity inhibitory activity,
(iii) Maillard reaction inhibitory action
(iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
(v) A cosmetic composition as an active ingredient for one or more actions selected from the group consisting of damage recovery promoting actions.
(15) The cosmetic composition as described above, wherein the culture solution of Promicromonospora Ferment Filtrate is derived from the culture solution of Promicromonospora strain K12-1001.

According to the present invention, a simple and low-cost method for purifying an actinomycete culture is provided which includes adsorption treatment for deodorization and decolorization and filtration treatment for sterilization. The purified product has multiple effects useful for the skin and can be safely incorporated into skin cosmetics at high loading rates.

It is a migration result of a Maillard reaction product when making a sample act at 0.25, 0.5, 0.75, 1.0, and 1.5 mg / mL. It is a microscope observation image of the culture | cultivation human epidermal keratinocyte injury site | part before and after a test substance addition.

Processing of Actinomycete Culture Solution The actinomycete culture solution of the present invention can be obtained by processing a culture solution (fermentation solution) of actinomycetes containing metabolites of actinomycetes. In the present invention, the culture solution (fermentation solution) of actinomycetes includes any of a culture solution containing culture cells and a culture supernatant. The culture solution of actinomycetes containing the cultured cells contains a secondary culture solution described later. The culture supernatant is obtained by removing the cells by centrifuging or filtering the culture solution after culture.

In a certain embodiment, the actinomycete used in the present invention is preferably a plant-derived actinomycete.

In a certain embodiment, the actinomycete used in the present invention is Promicromonospora sp. K12-1001 (NITE P-01848) having a phototogosis source. This strain is described in JP-A-2015-224245. In addition, this strain has been deposited as an NITE P-01848 (K12-1001) on April 24, 2014 at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary (NPMD).

In the present invention, the culture solution can be obtained by aerobic liquid culture of actinomycetes. Although the medium composition and the method of culture can be referred to the section of Examples in the present specification, they are merely exemplary and do not limit the present invention.

The medium may be a liquid medium containing a carbon source and a nitrogen source, and optionally, an inorganic salt, and a medium having a general composition known to those skilled in the art can be used. Examples of carbon sources are starch, glucose, peptone, calcium carbonate, sucrose, cellulose, glycerol, solubilized starch, examples of nitrogen sources are V8 juice, corn steep powder, oatmeal, far media, meat extract, yeast extract Examples of defatted wheat germ, dried broth, and inorganic salts include dipotassium hydrogen phosphate, magnesium sulfate, sodium metasilicate and magnesium chloride. Furthermore, if necessary, hot spring water, hot spring crystals, seawater, sea salt, iron sulfate, zinc sulfate, copper sulfate, cobalt chloride and the like may be added to the medium. The pH of the culture medium is preferably 6.5 to 8.0.

For culture, actinomycetes are inoculated into a culture medium and aerobically cultured at 20 ° C. to 40 ° C., usually for 3 to 10 days. Actinomycetes can be grown in the culture medium by shaking culture, stationary culture, industrial culture in a tank, culture in a solid medium such as agar medium, or the like. The culture temperature is preferably 20 ° C to 30 ° C, particularly preferably 24 ° C to 28 ° C.

Prior to culture in a culture medium (main culture), it is also possible to inoculate the culture medium with actinomycetes precultured in a seed medium (seed medium) as an inoculum to start culture. For example, in the case of using actinomycetes refrigerated or stored frozen, actinomycetes are activated by performing pre-culture, and culture efficiency in main culture is improved. When adopting such a culture method, in the present specification, pre-culture is referred to as "primary culture" and main culture as "secondary culture". In the case of performing pre-culture, for the culture period, primary culture (pre-culture) may be performed for 1 to 7 days, and secondary culture may be performed for 3 to 10 days.

A typical example of the culture method is a primary culture step of inoculating actinomycetes in a liquid medium containing a carbon source and a nitrogen source, and culturing at about 25 ° C. for 3 days to obtain an inoculum, and the same composition as the primary culture. Adding a seed solution to the liquid medium and culturing at 20 to 30 ° C. for 7 days to obtain a secondary culture solution;

The actinomycete culture solution of the present invention is produced by performing simple purification including at least adsorption treatment on a culture solution of actinomycetes after culture. In the present invention, an adsorption treatment can be performed on a culture solution (for example, a secondary culture solution) containing cells after culture, before or after the cells are removed. The adsorption treatment is an operation of selectively separating one or more components from a culture of actinomycetes using an adsorbent. The adsorption process may employ either a batch system or a continuous system, and one skilled in the art can select an appropriate system according to the production amount. The adsorption treatment removes the color and odor specific to the actinomycete culture solution. As an adsorbent for decolorization / deodorization, for example, synthetic adsorbents such as aromatic or acrylic synthetic adsorbents are suitably used, but not limited thereto. Those skilled in the art can select and use adsorbents capable of removing odorous components while maintaining the usefulness of the culture solution.

In the present invention, an adsorption treatment is performed to obtain an actinomycete culture solution from which colors and odors which are undesirable for use as cosmetics are removed. Furthermore, it is preferable that the adsorption-treated actinomycete culture solution has no mutagenicity and low skin irritation.

As a means for the adsorption treatment, it is preferable to use a method which does not reduce the physiological activity. In addition, it is more preferable if it is a means that can be directly subjected to the adsorption treatment to the culture solution containing the cultured cells without removing the cells. For example, as described in the Example section of the present specification, the adsorbent is mixed with the secondary culture solution, and adsorption treatment is performed by shaking at room temperature (about 25 ° C.) for 1 day (about 24 hours) be able to. Thereafter, the adsorbent can be removed by an appropriate method such as filtration or centrifugation to obtain a decolorized / deodorized culture solution after adsorption purification.

Then, the culture solution after adsorption purification is filtered using one or more kinds of filter aids and / or a filter device. The filtration process can be performed by applying a general existing technology. In the present invention, the filtration process can be performed inexpensively by using an inexpensive filter aid (for example, diatomaceous earth, cellulose powder, etc.) in combination with a filter such as a horizontal filter plate type filter device, a filter press filtration device, or a cartridge filter. It is preferred to do. The final step of filtration is filter sterilization using a filter with a pore diameter of 0.2 to 0.6 μm, and typically microfiltration sterilization using a filter with a pore diameter of 0.22 μm or 0.45 μm is performed.

The adsorptive purified, sterilized actinomycete culture solution (i.e., culture filtrate) may be subjected to a lyophilization treatment thereafter.

Physiological activity of actinomycete culture solution The actinomycete culture solution of the present invention,
(i) Elastase inhibitory activity,
(ii) Hyaluronidase activity inhibitory activity,
(iii) Maillard reaction inhibitory action
(iv) S100A7A, CD248, ITGA11 gene expression promoting action
(v) exerts one or more actions selected from the group consisting of damage recovery promoting actions.

(1) Elastase Activity Inhibitory Effect on Anti-Wrinkle, Elasticity or Firmness Improving Effect The term "elastase" in the present invention refers to a protease having elastin as a typical substrate, unless otherwise specified. Elastin is a type of elastic hard protein that constitutes the connective tissue, tendon, aortic integument, cervical cord, etc. of higher animals. There is a lot of crosslinking between the peptide chains that make up this protein, which brings about elasticity.

In the dermis, elastin binds reticulated collagen and maintains the firmness of the skin with the collagen. Therefore, when elastin decreases due to aging, ultraviolet light, active oxygen, stress, elastase (elastase), etc., it causes aging such as wrinkles and sag. From this, it is expected that the component that inhibits elastase has the effect of recovering or maintaining firmness of the skin or improving the elasticity of the skin, preventing wrinkles, and as a result, keeping the skin youthful.

The presence or absence and / or the degree of the ability to inhibit elastase activity can be confirmed and / or measured by a person skilled in the art using known methods as appropriate. For detailed conditions, reference can be made to the Examples section of this specification. In the present invention, when referring to the presence or absence and / or the degree of inhibition of elastase activity, the method described in the Examples of the present specification is used unless otherwise specified.

(2) Anti-inflammatory and moisturizing effect by inhibiting hyaluronidase activity "Hyaluronidase" is an enzyme that lowers the molecular weight of hyaluronic acid and is an enzyme that hydrolyzes the N-acetyl-D-hexosaminide bond of hyaluronic acid. Hyaluronic acid is a type of glycosaminoglycan having a repeating structure of only disaccharides of O-β-D-glucuronosil (1 → 3) -N-acetyl-β-D-glucosaminil (1 → 4) units. Hyaluronic acid is abundantly present in connective tissues such as animal joint fluid and the surface layer of the dermis, and is involved in joint lubrication, skin softening and moisturization, and the like. Hyaluronidase is present in the skin and is thought to be responsible for cellular activity in remodeling of connective tissue by degrading hyaluronan. Therefore, an inhibitor for hyaluronidase that degrades hyaluronic acid in connective tissue is expected to improve the condition of the skin in which the degradation of hyaluronic acid is enhanced. In addition, hyaluronidase is thought to be activated at the time of inflammation, destroying the structure of tissues, and enhancing the permeability of inflammatory cells. It is also involved in the release of histamine from mast cells and is thought to be involved in the development of type I allergy. Therefore, by measuring the hyaluronidase activity inhibitory effect, it is also possible to predict the anti-inflammatory action or anti-allergic action of the test substance.

The presence or absence and / or degree of the ability to inhibit hyaluronidase activity can be confirmed and / or measured by a person skilled in the art using known means as appropriate. For detailed conditions, reference can be made to the Examples section of this specification. In the present invention, when the presence or absence and / or the degree of inhibition of hyaluronidase activity is referred to, the method described in the examples of the present specification is used unless otherwise specified.

(3) Anti-glycation and dulling suppression effect by inhibition of Maillard reaction When the term "Maillard reaction" is referred to in the present invention, brown matter (melanoidin) is generated from aminocarbonyl reaction from sugar, protein, etc., unless otherwise specified. I say a reaction. The Maillard reaction is generally a non-enzymatic reaction in which an Amadori rearrangement product (Amadori compound) is formed after a reducing sugar and an amino group of a protein react to form a Schiff base. Then, through complex reactions such as oxidation, dehydration, condensation, intramolecular and intermolecular cross-linking, and the like, it gradually turns to yellow-brown, and AGEs (advanced glycosylation end products), which are Maillard reaction late stage products, are produced.

The Maillard reaction that occurs in vivo is thought to be involved in the formation of brown spots on the skin found in diabetic patients and the like. The pigment that causes brown spots contains AGE. The in vivo Maillard reaction also appears to be related to lifestyle-related diseases, diseases promoted by aging (eg, diabetes complications, Alzheimer's disease, arteriosclerosis), or glycation of the body that promotes aging. It is being made. Also, with the development of anti-aging medicine, AGE is a generic term for various structures generated from the Amadori rearrangement product through complex reactions such as oxidation, dehydration, condensation, etc. , Pyraline, pentosidine, crosulin, imidazolone, etc., and polymers of dimers or more of proteins are also known as AGEs. In the skin, it is considered that, as the Maillard reaction progresses, crosslinking, denaturation, and the like of the dermis-constituting proteins such as collagen and elastin become recognized due to cross-linking and denaturation.

The presence or absence and / or extent of the Maillard reaction suppression ability may be confirmed and / or measured by a person skilled in the art using, for example, a known means, as an indicator of inhibition of formation of Amadori rearrangement product or inhibition of AGE formation. it can. For detailed conditions, reference can be made to the Examples section of this specification. In the present invention, when referring to the presence / absence and / or degree of the Maillard reaction inhibition ability, the method described in the Examples of the present specification is used unless otherwise specified.

(4) S100A7A, CD248, ITGA11 gene expression promoting differentiation / mature promotion, cell adhesion enhancement, growth promotion
S100A7A (Homo sapiens S100 calcium binding protein A7A) encodes a calcium binding protein, and is involved in epidermal cell differentiation and maturation promotion, innate immunity, antibacterial protection, psoriatic inflammation, signal transmission and the like. The S100A7A protein, known as psoriasin, acts as a mediator and regulator in skin differentiation and disease (psoriasis).
CD248 (Homo sapiens CD248 molecule, endosialin) encodes endosialin, which is involved in cell adhesion. ITGA11 (Homo sapiens integrin, alpha 11) encodes alpha integrin 11 which is a membrane protein that binds collagen to cells. It is expressed in skeletal muscle cells and interstitial fibroblasts, and is involved in collagen binding and cell proliferation. These gene expression variations can be confirmed and measured by known techniques such as DNA microarrays.

(5) Cell activity promoting effect by damage recovery promoting action When "damage recovery" is referred to in the present invention, surrounding cells migrate and proliferate when the cell tissue is damaged in any way, unless otherwise specified. It means to repair the damaged part. In vivo, in particular, the skin epidermis is mainly composed of keratinocytes, and cells proliferated in the basal layer of the epidermis differentiate into the stratum corneum. In the epidermis, when wounds occur due to various factors, the keratinocytes around the injured part migrate and proliferate, and the wounds heal. If this damage recovery can be promoted, it acts on the recovery promotion effect and keratinocytes from damage such as cracking, aggravation, cuts and contact dermatitis, rough skin due to ultraviolet rays and dryness, etc., thus promoting cell proliferation promotion and turnover. Can also be expected to be effective.

The presence or absence and / or degree of the damage recovery promoting effect can be determined by a person skilled in the art using known means, as appropriate, for example, by using a cultured cell in a scratch assay (the cell where the cell was scraped). It is possible to observe and confirm the state of filling due to proliferation and migration. For detailed conditions, reference can be made to the Examples section of this specification. When the present invention refers to the presence, absence, and / or degree of the damage recovery promoting effect, the method described in the examples of the present specification is used unless otherwise specified.

Cosmetic Composition The actinomycete culture solution of the present invention can be used by mixing it with a cosmetic composition.

In the cosmetic composition of the present invention, the actinomycete culture solution comprises the following (i) to (iv):
(i) Elastase inhibitory activity,
(ii) Hyaluronidase activity inhibitory activity,
(iii) Maillard reaction inhibitory action
(iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
(v) It can be used as an active ingredient having one or more actions selected from the group consisting of damage recovery promoting actions.

The actinomycete culture solution of the present invention can be formulated according to a conventional method, and the formulated product is an expression inhibitor of elastase activity inhibitor, hyaluronidase activity inhibitor, Maillard reaction inhibitor, S100A7A, CD248, ITGA11 gene It may be used by blending it with cosmetics as a damage recovery promoter.

In the cosmetic composition of the present invention, the elastase activity inhibitor, hyaluronidase activity inhibitor, Maillard reaction inhibitor, S100A7A, CD248, ITGA11 gene expression promoter, damage recovery promoter, and other components to be blended, Any product that can usually be used in cosmetics can be used, and may be selected appropriately according to the efficacy and effect.

In the process of producing the cosmetic composition of the present invention, the method of blending the actinomycete culture solution is not particularly limited as long as the characteristics thereof are not significantly impaired. For example, you may mix | blend by mixing with another raw material in the state of aqueous solution. Alternatively, a lyophilizate obtained by lyophilization according to a conventional method may be used as it is or may be blended by mixing with other powder raw materials. From the viewpoint of preventing significant deterioration of the odor and color of the culture solution, it is preferable not to apply excessive heat when blending. From the same point of view, the aqueous solution or freeze-dried culture solution is preferably stored under refrigeration.

In the cosmetic composition of the present invention, the content of the actinomycete culture solution is not particularly limited as long as the desired effects are exhibited, but the lyophilizate obtained by the method described in the examples described later is 50 As an amount corresponding to the amount dissolved in% ethanol, usually, about 0.01 mass (w / w)% (hereinafter referred to as%) or more can be blended.

The composition or agent of the present invention may contain another naturally occurring substance in addition to the actinomycete culture solution. Examples of such substances are: Ume extract described in Japanese Patent No. 4795475, for example, Ume root extract and Mume fruit water. JP-A-2012-116761 describes water vapor distillation of plant-derived steam distilled water, for example, one or more plants selected from passion fruit, shimomikan, banana, atemoya, plume, tankan, lemon lime, ginger water. Deep ocean water described in JP-A-2000-290159. Deep sea water concentrate as described in Patent No. 5500757. A tokioki extract described in Japanese Patent No. 3454505. A seawater-derived yeast culture described in JP 2009-029788. Plum juice fermented liquid described in Japanese Patent No. 2526362. Hot water flower and / or hot water flower extract or oxidized product described in Japanese Patent No. 3967357. However, substances derived from natural products that can be contained in the composition or agent of the present invention are not limited to these, and any other substance can be used.

In the composition or agent of the present invention, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters which are components used in general external use compositions or agents, as long as the desired effects are not impaired. Amino acids, vitamins, surfactants, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, dyes, antioxidants, skin lightening agents, anti-inflammatory agents, anti-wrinkle agents It is also possible to incorporate ingredients such as skin roughening agents, drugs for acne, alkalis, chelating agents, sequestering agents and the like. Furthermore, the components such as sodium sulfate, sodium hydrogen carbonate, calcium carbonate, potassium chloride, oil components, emulsifiers, succinic acid, herbal medicines, inorganic pigments, perfumes, dyes, etc., which are components used for ordinary bath additives, should be blended. You can also.

The composition of the present invention can also be prepared into various dosage forms such as solid agents, semisolid agents, solutions and the like, depending on the purpose of use.

The form as a cosmetic is not particularly limited. For example, as a skin care cosmetic, a cleansing agent, a cleansing agent, a lotion, a cosmetic solution, a pack, a massage cream, an emulsion, a moisturizing cream, a lip balm, a foundation as a makeup cosmetic, white powder , Lipsticks, blushers, eye shadows, etc., body care cosmetics such as soaps, liquid detergents, bath additives etc., hair care cosmetics such as shampoos, rinses, hair treatments, hair styling, hair tonics, hair restorers, scalp treatments etc. it can. In the above-mentioned cosmetics, various cosmetic ingredients generally used for cosmetics can be blended suitably.

In the cosmetic composition of the present invention, the specific use and / or use (for example, efficacy / effect, amount used, frequency of use, method of use) can be displayed.

In a certain embodiment, a cosmetic comprising the actinomycete culture solution of the present invention is
(i) anti-wrinkle and / or elasticity improving effect,
(ii) anti-inflammatory and / or moisturizing effects,
(iii) It is suitably used as a cosmetic for skin external use to claim anti-glycation and / or anti-staining effects.

Beauty method The cosmetic composition of the present invention,
(i) anti-wrinkle and / or elasticity improving effect,
(ii) anti-inflammatory and / or moisturizing effects,
(iii) It can be used for cosmetic treatment to obtain one or more effects selected from the group consisting of antiglycation and / or anti-staining effects.

Specifically, the cosmetic composition of the present invention can be topically applied to the face, neck, neckline, hand or body area when needed or desired. Preferably, it is performed as part of a treatment performed in a place such as an aesthetic salon or a beauty salon.

Hereinafter, the present invention will be specifically described by way of examples. In addition, these Examples are for demonstrating this invention, Comprising: The scope of the present invention is not limited.

<Culturing of Actinomycetes and Preparation of Specimen>
Type of Actinomycetes Promicromonospora sukumoe
Primary culture 1. Mix purified water (98.70 w / w%), yeast extract (0.50 w / w%), glucose (0.10 w / w%), calcium carbonate (0.40 w / w%), polypeptone (0.30 w / w%) .
This is used as a seed medium and used in the following procedure.
2. Autoclave the seed medium prepared in step 1 (121 ° C., 20 min).
After autoclaving, allow to cool at room temperature.
3. 1. Actinomycetes are inoculated into the seed medium prepared in step 2 for 1 ese.
4. Primary culture is performed (25 ° C., 120 rpm, 72 hours).
5. This is used as an inoculating solution and is used in the following "secondary culture" step 3.

Secondary culture 1. Mix purified water (98.70 w / w%), yeast extract (0.50 w / w%), glucose (0.10 w / w%), calcium carbonate (0.40 w / w%) and polypeptone (0.30 w / w%) Dissolve completely.
2. Autoclave the medium prepared in step 1 (121 ° C., 20 min).
3. To the secondary culture medium prepared in step 2, add 1% of the inoculum prepared in "primary culture".
4. Perform secondary culture (20-30 ° C., 7 days).
5. This is used as a secondary culture solution and used in the following "deodorization" step.

Deodorization treatment 1. A specified amount of adsorbent (Sepabeads SP850 or SP207) is weighed into the secondary culture solution and mixed.
2. The culture solution and the adsorbent mixed in step 1 are shaken and subjected to adsorption treatment (25 ° C., 120 rpm, 24 hours).
3. The culture solution prepared in step 2 is filtered with a nylon mesh (150 μm mesh) to remove the adsorbent.
4. The culture solution that has been subjected to filtration in step 3 is used in the following "processing" step.

Filtration process 1. The culture solution prepared in the "deodorization" step 4 is subjected to filter sterilization using one or more filtration devices (preferably a filter with a pore diameter of 0.2 to 0.6 μm, more preferably 0.4 to 0.5 μm).

In the following tests (1) to (4), the actinomycete culture solution after deodorization and filtration was subjected to lyophilization treatment, and a sample adjusted to 50 mg / mL with 50% EtOH was used. In addition, the yield of the actinomycete culture solution powder obtained by the lyophilization treatment was 0.3 (w / v)% = 3 mg / mL. For the tests of (5) and (6), the actinomycete culture solution after deodorization and filtration treatment is diluted with purified water or culture medium, and after the filtration treatment (deodorization treatment is performed for the test of (7) Actinomycete culture solution was used.

(1) Fibroblast-derived elastase activity inhibitory effect test Test method Normal human dermal fibroblasts were adjusted to 20 × 10 ⁴ cells / mL with DMEM containing 5% FBS, and seeded at 100 μL / well in a 96-well plate. The cells were cultured at 37 ° C. for 24 hours at 5% CO ₂ , rinsed twice with 150 μl of PBS, added with 50 μl of 0.5% Triton X-100 / dw, and allowed to stand at 4 ° C. for 24 hours to extract elastase. The extracted elastase and the synthetic substrate (Suc-Ala-Ala-Ala-pNA) and the sample were reacted and developed at 40 ° C. for 2 hours, and the absorbance at 405 nm was measured. The inhibition rate was calculated using purified water as a control and the following equation.
Elastase activity inhibition rate (%) = {(1-(A-B) / (C-D)) x 100
A: Absorbance at the time of sample addition
B: Sample addition, absorbance without enzyme
C: Absorbance at the time of addition of purified water
D: Add purified water, absorbance without enzyme

Results Elastase activity inhibitory effect was confirmed in the actinomycete culture solution.

(2) Hyaluronidase activity inhibitory effect test test method
After mixing 12 μL of a sample prepared at a concentration with purified water and 12 μL of hyaluronidase (derived from bovine testis) in a 96-well plate and reacting at 40 ° C for 20 minutes, add 12 μL of Compound 48/80 and react at 40 ° C for 20 minutes. 12 μL of potassium hyaluronate was added and reacted at 40 ° C. for 40 minutes. Thereafter, 12 μL of NaOH was added, and the reaction was quenched by cooling with ice water for 5 minutes, and 12 μL of potassium borate solution was added, heated with boiling water for 3 minutes, and cooled with ice water for 10 minutes. 180 μL of p-DMAB was added, and after 180 μL was dispensed to a 96-well plate, reaction was performed at 40 ° C. for 20 minutes, and the absorbance at 540 nm was measured with a microplate reader. The inhibition rate was calculated using purified water as a control and the following equation.
Hyaluronidase activity inhibition rate (%) = {(1- (AB) / (CD)) × 100
A: Absorbance at the time of sample addition
B: Sample addition, absorbance without enzyme
C: Absorbance at the time of addition of purified water
D: Add purified water, absorbance without enzyme

Results: The actinomycete culture solution was confirmed to have a hyaluronidase activity inhibitory effect.

(3) Maillard reaction inhibitory effect test Test method Add 800 μL of phosphate buffer (pH 7.4), 40 μL of ribose prepared with 800 μL of phosphate buffer (pH 7.4) and 100 μL of lysozyme to a sample tube, and use purified water to make concentration 60 μL of the prepared sample was added. It filter-sterilized using a 0.45 micrometer filter in the clean bench, and made it react in a 40 degreeC incubator for 7 days. After the reaction, electrophoresis was performed by SDS-PAGE, staining was performed with 0.1% Coomassie Brilliant Blue R-250, and after decoloring, the dimer of lysozyme was visually determined. The negative control was made using purified water instead of the evaluation sample, and the appearance when aminoguanidine sulfate (AG) was added was determined as a positive control.

Results FIG. 1 shows the migration results of Maillard reaction products when the actinomycete culture solution was allowed to act at 0.25, 0.5, 0.75, 1.0 and 1.5 mg / mL, respectively. The Maillard reaction inhibitory effect was confirmed in the actinomycete culture. Moreover, the effect was high compared with the same concentration of AG.

(4) DNA Microarray Reconstruction Three-dimensional cultured skin (EFT-412) was treated with purified water and deodorized actinomycete culture solution diluted to 5% with purified water from the stratum corneum side by 100 μL, and 5%. Culture was at 37 ° C. After incubation, the cells were washed with PBS, rapidly frozen with liquid nitrogen, and the skin was obtained with biopsy punch. From the obtained skin, RNA was extracted using TRIzol and purification was performed. The total RNA amount was adjusted to the same amount, and DNA microarray was performed using Human Gene 2.0 ST Array. Genes whose expression fluctuation was doubled or halved or more were extracted and analyzed.

Results There were 230 genes associated with the skin whose expression level was more than doubled by actinomycete culture fluid action compared to media effect (Table 4). Among them, the following three genes involved in cell differentiation / maturation promoting action, psoriasis control, cell adhesion enhancement, collagen binding protein expression promotion and cell proliferation effect were confirmed and confirmed to have a broad effect .

S100A7A (Homo sapiens S100 calcium binding protein A7A) encodes a calcium binding protein, and is involved in epidermal cell differentiation and maturation promotion, innate immunity, antibacterial protection, psoriatic inflammation, signal transmission and the like. The S100A7A protein, known as psoriasin, acts as a mediator and regulator in skin differentiation and disease (psoriasis).

CD248 (Homo sapiens CD248 molecule, endosialin) encodes endosialin, which is involved in cell adhesion.

ITGA11 (Homo sapiens integrin, alpha 11) encodes alpha integrin 11 which is a membrane protein that binds collagen to cells. It is expressed in skeletal muscle cells and interstitial fibroblasts, and is involved in collagen binding and cell proliferation.

(5) Damage Recovery Promotion Test Human epidermal keratinocytes were adjusted to 20 × 10 4 cells / mL with DMEM containing 10% FBS, and seeded at 800 μL / well in a 24 well plate. After culturing for 24 hours at 37 ° C. in 5% CO 2, a part of the cultured keratinocytes was scraped (damaged) with CELL ScratcherTM, washed with PBS, and the state of damage was observed with a microscope. Thereafter, the sample diluted with 10% FBS-containing DMEM was allowed to act at 500 μL / well, and the state after 24 hours was observed with a microscope (FIG. 2). The area of the observed damaged portion was measured, and the damage recovery promotion rate based on purified water was calculated using the following equation (Table 5).
Damage recovery promotion rate (%) = (A-B) / (C-D) × 100
A: Area of damaged area before addition of sample
B: Area of damaged area 24 hours after addition of sample
C: Area of damaged area before addition of purified water (1%)
D: Damaged area 24 hours after addition of purified water (1%)

Results The recovery from damage was confirmed in the actinomycete culture solution.

(6) Patch Test Using a patch tape coated with actinomycete culture solution, an occlusion patch test was conducted for 24 hours by 20 Japanese. The concentration applied is the stock solution (100%), and one day after peeling off the patch tape using petrolatum, physiological saline, and purified water as negative targets, the state of erythema is determined according to the following Japanese standard (Taro Kawamura Others The judgment was made in light of the basic research of patch test standardization, The Nikkokukai, 80, 301-314 (1970)), and the skin irritation index was calculated using the following equation. The classification of the sample was performed based on the following table from skin irritation index (Table 5).

Result The actinomycete culture solution was classified as "safety".
Skin irritation index = total score / number of subjects × 100

(7) Ames test (mutagenicity test)
According to "Guidance on genotoxicity testing and interpretation of pharmaceuticals" (Medicine Review No. 0920 No. 2), Salmonella typhimurium (S. typhimurium) TA100, TA1535, TA98, TA1537 and Escherichia coli (E. coli) 2.) Using WP2 uvrA, the actinomycete culture solution was diluted by pre-incubation method using water for injection under metabolic activation and non-activation conditions.

Results No precipitation or staining on the plate by the actinomycete culture solution was observed at any dose, regardless of whether or not there was metabolic activation, both after overlaying on the plate and after incubation. As a result of observing the growth inhibition to the fungus using a stereomicroscope, no strain was observed in any strain regardless of the presence or absence of metabolic activation. Moreover, the number of revertant colonies by the actinomycete culture solution was not increased by more than twice the negative control value in any strain regardless of the presence or absence of metabolic activation, and no dose response was observed . From these, it was confirmed that the actinomycete culture solution does not have mutagenicity (gene mutagenicity).

Claims (14)

1. The actinomycete culture solution is prepared as follows (i) to (v):
   (i) Elastase inhibitory activity,
   (ii) Hyaluronidase activity inhibitory activity,
   (iii) Maillard reaction inhibitory action
   (iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
   (v) A cosmetic composition as an active ingredient for one or more actions selected from the group consisting of damage recovery promoting actions.
2. The cosmetic composition according to claim 1, wherein the actinomycete culture solution is an active ingredient for two or more actions selected from the group consisting of (i) to (v).
3. The cosmetic composition according to claim 1 or 2, which is an elastase activity inhibitor, a hyaluronidase activity inhibitor, a Maillard reaction inhibitor, an expression promoter for S100A7A, CD248, and / or ITGA11 gene, or a damage recovery promoter.
4. The cosmetic composition according to claim 1 or 2, which has anti-wrinkle effect, enhancement of firm feeling, anti-inflammatory, moisturizing, anti-glycation and anti-staining action.
5. A method of using an actinomycete culture solution, which comprises the following (i) to (iii):
   (i) anti-wrinkle and / or elasticity improving effect,
   (ii) anti-inflammatory and / or moisturizing effects,
   (iii) A method of using the culture solution for cosmetic treatment to obtain one or more effects selected from the group consisting of antiglycation and / or anti-staining effects.
6. Streptomyces actinomycetes are cultured, and the obtained culture solution is subjected to adsorption treatment using an adsorbent, followed by filtration treatment, thereby reducing the color and odor peculiar to actinomycete culture fluid, and thereby blending into cosmetics The manufacturing method of the actinomycete culture solution which improved the sex or palatability.
7. An actinomycete culture solution comprising culturing the actinomycetes, adsorbing the obtained culture solution using an adsorbent, and then filtering it to obtain a decolorized and deodorized actinomycete culture solution. Method for the purification of
8. The method according to claim 6, or the purification method according to claim 7, wherein an adsorbent capable of removing an odor component while maintaining the usefulness of the culture solution is used.
9. The method according to any one of claims 6 to 8, wherein an actinomycete culture solution or an actinomycete culture having no mutagenicity and low skin irritation is obtained.
10. Actinomycetes are cultured, the obtained culture solution is subjected to adsorption treatment, and then filtration treatment is performed to obtain an actinomycete culture solution, which has no mutagenicity and low skin irritation. A method of producing an actinomycete culture solution.
11. (A) primary culture step of inoculating actinomycetes in a liquid medium containing carbon source and nitrogen source and aerobically culturing at room temperature for 1 to 7 days to obtain a seed solution;
    (B) adding a seed solution to a liquid medium having the same composition as the primary culture, and aerobically culturing it at 20-30 ° C. for 3-10 days to obtain a secondary culture solution;
    (C) An adsorbent is mixed with the secondary culture solution, shaken at room temperature for about 1 day to perform adsorption treatment, and then the adsorbent is removed to obtain a decolorized / deodorized culture solution; ,
    And (d) filtering the decolorized / deodorized culture solution using one or more filter devices, and a method of producing an active ingredient of cosmetics,
    The active ingredient is
    (i) Elastase inhibitory activity,
    (ii) Hyaluronidase activity inhibitory activity,
    (iii) Maillard reaction inhibitory action
    (iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
    (v) A manufacturing method which exerts one or more actions selected from the group consisting of damage recovery promoting actions.
12. Furthermore, (e) physiologically active action of the culture solution after filtration (preferably, (i) elastase activity inhibiting action; (ii) hyaluronidase activity inhibiting action; (iii) Maillard reaction inhibiting action; (iv) S100A7A, CD248, And / or the expression promoting action of the ITGA11 gene; (v) evaluating one or more actions selected from the damage recovery promoting action;
    The method according to claim 11, comprising
13. The method according to any one of claims 5 to 12, wherein the actinomycete is an actinomycete of the genus Promicromonospora.
14. A culture solution of an Actinomycete of the genus Promicromonospora is as follows (i) to (v):
    (i) Elastase inhibitory activity,
    (ii) Hyaluronidase activity inhibitory activity,
    (iii) Maillard reaction inhibitory action
    (iv) S100A7A, CD248, and / or ITGA11 gene expression promoting action
    (v) A cosmetic composition as an active ingredient for one or more actions selected from the group consisting of damage recovery promoting actions.
    

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