WO2021125346A1 - Method for activating skin stem cells through mpc1 suppression, and skin stem cell activation agent - Google Patents

Method for activating skin stem cells through mpc1 suppression, and skin stem cell activation agent Download PDF

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WO2021125346A1
WO2021125346A1 PCT/JP2020/047530 JP2020047530W WO2021125346A1 WO 2021125346 A1 WO2021125346 A1 WO 2021125346A1 JP 2020047530 W JP2020047530 W JP 2020047530W WO 2021125346 A1 WO2021125346 A1 WO 2021125346A1
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extract
mpc1
skin stem
skin
stem cell
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Japanese (ja)
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俊介 入山
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株式会社 資生堂
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method of activating skin stem cells by suppressing MPC1.
  • Patent Document 1 discloses a cell senescence inhibitor for stem cells containing astaxanthin.
  • Patent Document 2 discloses a stem cell-maintaining and activating agent for mesenchymal stem cells, which contains hydroxyproline or a pharmacologically acceptable salt thereof as an active ingredient.
  • Non-Patent Document 1 stem cells in the S phase increase at night when the NADH / NAD + ratio is high and glycolysis is predominant, but decrease during the daytime when the NADH / NAD + ratio is low and the electron transport chain is predominant. It has been reported (Non-Patent Document 2).
  • Non-Patent Document 3 In addition, in hair follicle cells genetically engineered to block the pathway to the electron transport chain by deleting the function of the mitochondrial pyruvate carrier (MPC1), stem cell proliferation is activated and the hair cycle is promoted. Was shown (Non-Patent Document 3).
  • Patent Document 3 discloses a beauty composition containing a mitochondrial transfer promoter, and its use in skin fibroblasts, epidermal keratinocytes, adipose-derived mesenchymal stem cells, epidermal stem cells and dermal stem cells can be used. Are listed.
  • Patent Document 4 includes exposing keratinocytes to a stressor, detecting metabolic indicators associated with glycolysis and oxidative phosphorylation, respectively, and providing a response to the stressor. Disclose a method for identifying a test substance as a skin care activator that improves metabolism.
  • Patent Document 5 non-lethally detects metabolic indicators associated with contacting cells with the first and second test substances, glycolysis and oxidative phosphorylation, respectively, and responds to the test substance with respect to the metabolic pathway. Disclosed are methods of identifying or evaluating synergistic combinations of active ingredients for use in cosmetic compositions, including providing.
  • An object of the present invention is to provide a method for activating skin stem cells, a skin stem cell activator, and a screening method for a skin stem cell activator.
  • the present application includes the following inventions: (1) A cosmetic method that activates skin stem cells by applying an MPC1 inhibitor. (2) The beauty method according to (1), wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (3) An MPC1 inhibitor containing at least one of akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (4) A skin stem cell activator containing an MPC1 inhibitor.
  • Agent. (6) A method for screening a skin stem cell activator using the MPC1 inhibitory effect as an index. (7) Step of bringing the skin sample into contact with the candidate drug, A step of measuring the activity, amount, and / or expression level of MPC1 in a skin sample contacted with a candidate drug.
  • a step of determining the MPC1 inhibitory effect of a candidate drug from the measured MPC1 activity, amount, and / or expression level The step of selecting a skin stem cell activator based on the determined MPC1 inhibitory effect, The method according to (6).
  • an agent and a method effective for activating skin stem cells by suppressing MPC1 are provided. If skin stem cells are activated, it is effective in suppressing skin aging.
  • FIG. 1 shows the results of Experiment 1-1, in which the MCSP / B2M value when control (Cont), UK5099 (10 ⁇ M UK5099, 20 ⁇ M UK5099), and echinomycin (10nM Echinomycin, 20nM Echinomycin) were added was controlled (Cont). It is a graph showing a relative value with the result of) as 1.
  • FIG. 2 shows the results of Experiment 1-2 and is a photograph showing the expression of MCSP and DAPI when Control (Cont) and UK5099 (20 ⁇ M UK5099) were added, respectively.
  • FIG. 3 shows the results of Experiment 1-2 and is a photograph showing the expression of Integrin ⁇ 1 and DAPI when Control (Cont) and UK5099 (20 ⁇ M UK5099) were added, respectively.
  • FIG. 4 shows the results of Experiment 1-3, showing the number of Integrin ⁇ 1-positive cells when no antibody was added (Negative cont), when control was added (Cont), and when UK5099 was added (20 ⁇ M UK5099).
  • FIG. 5 shows the results of Experiment 1-4. Integrin ⁇ 6, MCSP, and DAPI in the tissue section of a three-dimensional cultured skin model in which control (Cont) and UK5099 (1 ⁇ M UK5099, 10 ⁇ M UK5099) were added and cultured for 4 days, respectively.
  • FIG. 6 shows the results of Experiments 1-5, showing the expression of Integrin ⁇ 6, MCSP, and DAPI in human skin organ culture model tissue sections cultured for 5 days with the addition of control (Cont) and UK5099 (10 ⁇ M UK5099), respectively. It is a photograph.
  • FIG. 7 shows the results of the primary screening of Experiment 2-2, and the MPC1 / B2M values when control (DMSO; Control) and each evaluation target sample (drug Nos. 1 to 124) were added, and the control results were shown. Shown as a relative value of 1.0.
  • FIG. 6 shows the results of Experiments 1-5, showing the expression of Integrin ⁇ 6, MCSP, and DAPI in human skin organ culture model tissue sections cultured for 5 days with the addition of control (Cont) and UK5099 (10 ⁇ M UK5099), respectively. It is a photograph.
  • FIG. 7 shows the results of the primary screening of Experiment 2-2, and the MPC1 / B2M values when control (DM
  • FIG. 8 shows the results of the secondary screening of Experiment 2-2, and the MPC1 / B2M values when each evaluation target sample (37 out of drug Nos. 1 to 124) selected in the primary screening was added.
  • the result of control (DMSO) is shown as a ratio (% of control) of 100.0.
  • FIG. 9 shows the results of the tertiary screening in Experiment 2-2, when the control (DMSO; Cont) and each evaluation target sample (15 out of drug Nos. 1 to 124) selected in the secondary screening were added.
  • the MPC1 / B2M value of is shown as a ratio (% of control) with the control result as 100.0.
  • FIG. 10 shows a graph in which the results of FIG. 9 were statistically processed by Dunnett's test for each of the 6 products selected in the tertiary screening of Experiment 2-2 (* P ⁇ 0.05, ** P ⁇ 0.01).
  • the present inventors have come up with the idea that skin stem cells are activated by MPC1 inhibition, and have discovered a novel substance having an MPC1 inhibitory effect.
  • akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine have a high MPC1 inhibitory effect.
  • the present invention describes MPC1 inhibitors and skin stem cell activators (hereinafter, these may be collectively referred to as "agents of the present invention"), screening methods thereof, and skin stem cells by applying them.
  • agents of the present invention provide a method of activating.
  • the method of the present invention may be for the purpose of cosmetology and may not be treated by a doctor or healthcare professional.
  • MPC1 is a protein that forms a heterodimer with MPC2 and forms a mitochondrial pyruvate carrier, which is a transporter that delivers pyruvate into the mitochondria.
  • MPC1 inhibition refers to reducing and / or inhibiting the action, production, and / or translation of MPC1 and / or reducing the activity, amount, and / or expression of MPC1.
  • the function of MPC1 is, for example, the function of MPC1 to deliver pyruvic acid into mitochondria.
  • MPC1 suppression results in, for example, the activity, amount, and / or expression level of MPC1 when the agent of the present invention is applied, as compared to the state (control) where the agent of the present invention is not applied.
  • Decreasing with a statistically significant difference eg Dunnett's test with a significance level of 5%, or, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% Above, it can mean that it is reduced by 60% or more, 70% or more, 80% or more, 90% or more, or 100%.
  • the gene expression level of MPC1 may be measured by using a quantitative PCR method as in the examples, or the protein expression level may be measured by staining with an antibody, and a commercially available kit may be used. , Or can be obtained by the method described in documents such as Patent Document 3, but the present invention is not limited to these, and any known technique can be used.
  • activation of skin stem cells refers to promotion of stem cell proliferation in skin cells such as keratinocytes and fibroblasts.
  • Stem cell proliferation can be determined by counting the number of stem cells, measuring the expression level of stem cell markers such as integrin ⁇ 1, and the like. If skin stem cells are activated, it can be expected to promote turnover, rejuvenate the skin, improve the texture of the skin, and recover from unfavorable skin conditions such as acne and pigmentation at an early stage.
  • the MPC1 inhibitor of the present invention may consist of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine, or may be contained as an active ingredient.
  • the substance is not limited to these as long as it can suppress MPC1.
  • the agent of the present invention may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.
  • the agent of the present invention may combine the above active ingredient with one or more other ingredients such as excipients, carriers and / or diluents.
  • Akebia quinata is a deciduous shrub belonging to the genus Akebia in the family Lardizabalaceae.
  • Akevi has a hyaluronic acid production promoting effect, a collagen production promoting effect, and an MMP inhibiting effect, and has a wrinkle formation preventing / improving effect, a lipase inhibiting activity, a blood triglyceride concentration increasing inhibitory activity, or an anti-obesity activity.
  • Japanese Patent Laid-Open No. 2015-17048, Japanese Patent Application Laid-Open No. 2010-265182, etc. has been reported (Japanese Patent Laid-Open No. 2015-17048, Japanese Patent Application Laid-Open No. 2010-265182, etc.).
  • an extract of akebi stem is preferable, but since the active ingredient is also contained in the fruit, peel, seed, leaf, flower, root and the like of akebi, any of these Or one or more extracts can also be used.
  • Kuromame (Glycine max'Kuromame') is a soybean variety that is an annual plant of the legume family, and each variety such as “Tamba black” and “Wachi black” can be used. It has been reported that it is rich in polyphenols such as anthocyanins, and that black soybean-derived components have anti-obesity, anti-inflammatory, antioxidant, glucose intolerant, and other effects (Japanese Patent Laid-Open No. 2015-140298). etc).
  • the black soybean extract is preferably an extract of black soybean legume, but since the active ingredient is also contained in the pod, leaves, stems, flowers, roots, etc. of black soybean, one or more of these are extracted. You can also use things.
  • Peony (Paeonia lactiflora) is a perennial plant of the Peony family. Peony has been reported to have an epidermal thickening inhibitory effect, a VEGF production promoting effect, an IGF-1 production promoting effect, an HGF production promoting effect, a BMP-2 production promoting effect, and the like (Japanese Patent Laid-Open No. 2009-11252, JP2012). -121856 Gazette, etc.).
  • the syrup extract the extract of syrup root is preferable, but since the active ingredient is also contained in the leaves, stems, flowers, fruits, pericarps, seeds, etc. of syrup, one or more of these are one or more. Extracts can also be used.
  • Tea is an evergreen tree belonging to the genus Camellia of the family Camellia, and various varieties such as tea plant (Camellia sinensis var. Sinnsis) and assamcha (Camellia sinensis var. Assamica) can be used.
  • tea plant Camellia sinensis var. Sinnsis
  • assamcha Camellia sinensis var. Assamica
  • Uji tea or the like can be used. It is known that tea contains abundant polyphenols such as catechin, and has lipid metabolism improving action, antioxidant action, anticancer action, blood pressure increase suppressing action and the like.
  • the tea extract is preferably an extract of tea leaves or stems, but since the active ingredient is also contained in tea nuts, flowers, roots, seeds, etc., one or more of these extracts. Can also be used.
  • Jojoba (Simmondsia chinansis) is an evergreen shrub belonging to the family Caryophyllales jojoba. Jojoba is known to have a moisturizing effect, an emollient effect, an anti-inflammatory effect, a collagenase inhibitory effect, an elastase inhibitory effect, a hyaluronidase inhibitory effect, and the like (Japanese Patent Laid-Open No. 2003-48846, JP-A-2003-048812, JP-A-2003-048812). 2003-03464 No. 4). Jojoba leaf extract is an extract of jojoba leaves, but since jojoba seeds, fruits, flowers, roots, etc. also contain active ingredients, one or more of these extracts should be used. It can also be used.
  • Ergothioneine is an amino acid having the following structure and a molecular weight of 229.3, and is known to have antioxidant, anti-inflammatory, elastase-inhibiting, tyrosinase-inhibiting, etc. (Japanese Patent Laid-Open No. 2019-149972). etc). Ergothioneine may be chemically synthesized, or it may be used in the form of an extract of such a natural product because it is also contained in microorganisms such as basidiomycetes, animals and plants.
  • the above-mentioned various extracts may be commercially available as cosmetic raw materials or health food materials, or may be obtained by a conventional method.
  • the extraction method is not particularly limited, and examples thereof include an extraction method using a solvent and an extraction method by hydrolysis.
  • the raw material When performing extraction using a solvent, the raw material is immersed or heated at room temperature or heated with an extraction solvent at room temperature or heated and refluxed, and then when extraction by hydrolysis is performed, for example, chemical treatment with alkali, acid, etc., heat. It can be obtained by performing arbitrary hydrolysis treatment such as physical treatment with pressure or the like, biological treatment with an enzyme or the like, and then filtering and concentrating.
  • the raw material can be used as it is, but if it is crushed into granules or powder and used for extraction, the active ingredient can be extracted in a short time with high extraction efficiency under mild conditions.
  • the extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent.
  • the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.
  • any solvent can be used as long as it is usually used for extraction.
  • an aqueous solvent such as water, physiological saline, a phosphate buffer, a borate buffer, or an organic solvent such as ethanol can be used.
  • Alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination.
  • the active ingredient is extracted and dissolved in a solvent or hydrolyzed.
  • the solvent or hydrolyzate containing the extract may be used as it is, or may be used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. If necessary, it may be concentrated by freeze-drying or diluted with an arbitrary solvent before use. Further, the solvent or the hydrolyzate may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
  • the squeezed liquid obtained by squeezing the raw material also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.
  • the present application also provides a composition containing the agent of the present invention.
  • the composition of the present invention may be a cosmetic composition or a food composition.
  • the composition of the present invention may be, for example, a composition that activates skin stem cells through an MPC1 inhibitory action.
  • the subject to which the agent or composition of the present invention is applied may be a subject that requires activation of skin stem cells from an objective or subjective viewpoint such as delayed skin turnover, skin aging, and pigmentation. , It may be a subject who desires prophylactic activation of skin stem cells.
  • the agent or composition of the present invention can be applied by any route such as external administration or oral administration, but it is preferably blended with an external preparation for skin that can be directly applied to the skin.
  • an external preparation for skin for example, liquid, emulsion, cream, solid, sheet, spray, gel, foam, powder and the like can be arbitrarily selected.
  • It may be a cosmetic composition such as a milky lotion, a cream, a beauty essence, a lotion, a facial mask, or a facial cleanser.
  • oral administration for example, tablets, supplements, beverages, powders and the like can be arbitrarily selected.
  • any compounding ingredients used in compositions such as cosmetics and pharmaceuticals can be appropriately blended as necessary, as long as the effects are not impaired.
  • optional compounding ingredients include excipients, carriers, diluents, oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, and ultraviolet absorbers.
  • examples include agents, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and the like. For example, it may contain other medicinal ingredients that promote the activation of skin stem cells.
  • the frequency of administration is once every four weeks, once every two weeks, once a week, once every three days, once every two days, once a day, twice a day, three times a day. It can be arbitrarily selected, such as 4 times a day, 5 times a day, and each administration, but the present invention is not limited to these.
  • the possible forms of the agent or composition of the present invention are not limited to the above-mentioned dosage forms and forms.
  • the amount of the active ingredient such as akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine in the agent or composition of the present invention depends on the type, purpose, form, usage, etc. It can be decided as appropriate.
  • the blending amounts of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine are 0.0001 to 100% by weight, 0.0001 to 0.0001 to the total weight of the agent or composition of the present invention. It can be 90% by weight, 0.001 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.1 to 0.5% by weight, etc., but is not limited as long as the effect of the present invention is exhibited.
  • the present application also provides a screening method for a skin stem cell activator using the MPC1 inhibitory action as an index.
  • the screening method of the present invention is a step of contacting a skin sample with a candidate drug; a step of measuring the activity, amount, and / or expression level of MPC1 in the skin sample contacted with the candidate drug; And / or a step of determining the MPC1 inhibitory action of the candidate drug from the expression level; a step of selecting a skin stem cell activator based on the determined MPC1 inhibitory action; may be included.
  • it becomes possible to screen whether or not a candidate drug has a skin stem cell activating effect and it becomes possible to develop products and propose new skin care.
  • the skin sample may be a skin sample after collection, for example, a skin sample in an ex vivo state after being collected from an animal such as a human, or a cultured skin cell, for example, a cultured keratinocyte or a cultured fibroblast. It may be in vitro, such as a cell. Alternatively, it may be an artificial skin sample such as a three-dimensional skin model. Skin samples are not limited as long as the MPC1 inhibitory effect can be measured.
  • the present application also provides akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine that activate skin stem cells through MPC1 inhibition.
  • Experiment 1 Effect of MPC1 inhibition on stem cells
  • Experiment 1-1 Analysis of MCSP gene expression by PCR method
  • the epidermis was used using UK5099, which is known as an MPC1 inhibitor.
  • the expression of the stem cell marker Melanoma-associated chondroitin sulfate proteoglycan (MCSP) was analyzed.
  • MCSP Melanoma-associated chondroitin sulfate proteoglycan
  • Equinomycin is an inhibitor of HIF-1 ⁇ that regulates mitochondrial activity like MPC1, but differs in that it is a substance that inhibits the conversion of pyruvate to acetyl-CoA in mitochondria.
  • Echinomycin (Abcam Echinomycin: product number ab144247) is prepared by preparing a stock solution diluted with DMSO to a concentration of 10 ⁇ M and 20 ⁇ M in advance, and added to the medium at a dilution of 1000 times to obtain a final concentration of 10 nM and 20 nM. did. The mRNA was recovered 24 hours after the addition of these substances.
  • the gene expression levels of MCSP and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used are as follows.
  • B2M forward 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
  • Experiment 1-2 Protein expression analysis of MCSP and Integrin ⁇ 1 by cell staining Furthermore, not only MCSP but also another stem cell marker, Integrin ⁇ 1, was stained and analyzed. Subcultured epidermal keratinized cells were added to 20 ⁇ M in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 to obtain 1.0 ⁇ 10 4 cells / well / 0.5. The cells were seeded in mL in a 4-well chamber slide (Falcon, 354114) and cultured for 48 hours. 48 hours after the addition, the cells were reacted with 4% PFA for 10 minutes and fixed.
  • the reaction was carried out with 0.01% Triton-X100 / PBS for 15 minutes to partially lyse the cell membrane. After blocking with 12% BSA / PBS, it was reacted overnight at 4 ° C with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 1 integrin antibody (Santa Cruz, sc-13590, mouse mAb) as primary antibodies. .. The next day, the cells were washed 3 times with PBS for 5 minutes and then reacted with Alexa 488-anti-mouse IgG antibody as a secondary antibody at room temperature for 1 hour.
  • anti-MCSP antibody Micropore, MAB2029, mouse mAb
  • anti- ⁇ 1 integrin antibody Santa Cruz, sc-13590, mouse mAb
  • Experiment 1-3 FACS analysis of Integrin ⁇ 1-positive cells
  • FACS analysis of the number of Integrin ⁇ 1-positive cells was performed.
  • Subcultured epidermal keratinized cells were added to 20 ⁇ M in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 and cultured for 48 hours.
  • These cells were stripped with Trypsin and a cell suspension was prepared at a concentration of 1.0 ⁇ 10 6 cells / 500 uL. After removing the supernatant by centrifugation, blocking was performed with 0.5% BSA / PBS on ice for 10 minutes.
  • IgG antibody and Pacific blue-labeled anti- ⁇ 1 integrin antibody (BioLegend, 313620) were added to the cell suspension, reacted on ice for 1 hour, centrifuged 3 times with 0.1% BSA / PBS, and cell sorter. FACS analysis was performed on SH800. In addition, no antibody was added to the negative control (Negative cont), and the same treatment was performed.
  • Sections were prepared with a thickness of 3 ⁇ m using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
  • DAPI Vectashield, H-1200
  • Figure 5 shows the results of adding 1 ⁇ M and 5 ⁇ M UK5099 and controls, respectively.
  • the addition of UK5099 increased MCSP and Integrin ⁇ 1-expressing cells, which was consistent with the results of Experiments 1-1, 1-2, and 1-3 using the three-dimensional skin model.
  • Sections were prepared with a thickness of 3 ⁇ m using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti- ⁇ 6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
  • DAPI Vectashield, H-1200
  • Figure 6 shows the results of adding 10 ⁇ M UK5099 and control respectively.
  • the addition of UK5099 increased MCSP and Integrin ⁇ 1-expressing cells, which is consistent with the results of Experiments 1-1, 1-2, 1-3, and 1-4 using ex vivo human skin samples. It was.
  • Experiment 2 Screening of substances with MCP1 inhibitory effect
  • Experiment 1 suggested that inhibition of MCP1 activates stem cells. Therefore, we screened substances that have an MCP1 inhibitory effect.
  • Experiment 2-1 Sample preparation The following samples were used for evaluation of the MPC1 inhibitory effect.
  • a total of 124 types of candidate samples were prepared, including naturally derived components such as extracts of animals and plants and synthetic components.
  • Experiment 2-2 Evaluation of MCP1 inhibitory effect
  • normal human fetal-derived epidermal keratinocytes were cultured, and each drug was added 24 hours after seeding.
  • Candidate samples are prepared by preliminarily making 10% in DMSO and added to the medium at 1000-fold dilution in the primary and secondary screenings to a final concentration of 0.01%. The concentrations were adjusted to 0.001%, 0.01%, and 0.1%.
  • As a positive control UK5099 was added to 20 ⁇ M in the same manner as in Experiment 1.
  • DMSO was added as a negative control at a 1000-fold dilution. The mRNA was recovered 24 hours after the addition of each drug.
  • the gene expression levels of MPC1 and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan).
  • the primers used are as follows.
  • B2M forward 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
  • the result of the primary screening is shown in FIG. Thirty-seven products (substances below the line shown in FIG. 7) were selected from 124 products as candidates for substances having an MPC1 inhibitory effect.
  • the result of the secondary screening is shown in FIG. Thirty-seven to fifteen substances (dark gray substances shown in FIG. 8) were selected as candidates for substances having an MPC1 inhibitory effect in a concentration-dependent manner.
  • the result of the tertiary screening is shown in FIG. From 15 products as candidates for substances that have a reproducible and concentration-dependent MPC1 inhibitory effect, 6 products: akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine (Fig. 9). (Dark gray substance shown in) was selected.
  • Fig. 10 shows a graph in which the results of Fig. 9 were statistically processed by Dunnett's test for each of the 6 selected products. As shown in FIG. 10, all the substances exerted an MPC1 inhibitory effect with a statistically significant difference in a concentration-dependent manner.
  • skin stem cells are activated by MCP1 inhibition, and akebi extract, black bean extract, sycamore extract, tea extract, jojoba leaf extract, and ergothioneine have an MCP1 inhibitory effect. I understood it. If skin stem cells are activated by the MCP1 inhibitor of the present invention, it is effective in suppressing skin aging.

Abstract

Provided are a method for activating skin stem cells, a skin stem cell activation agent, and a screening method for skin stem cell activation agents.　The application of an MPC1 suppressant is effective in activating skin stem cells. Examples of such MPC1 suppressants include agents that include at least an akebia extract, a black bean extract, a peony extract, a tea extract, a jojoba leaf extract, or ergothioneine as an active ingredient. Moreover, skin stem cell activation agents can be screened using MPC1 suppression action as an index.

Description

ＭＰＣ１抑制により皮膚幹細胞を活性化する方法および皮膚幹細胞活性化剤Method of activating skin stem cells by suppressing MPC1 and skin stem cell activator
　本発明は、MPC1抑制により皮膚幹細胞を活性化する方法に関する。 The present invention relates to a method of activating skin stem cells by suppressing MPC1.
　皮膚幹細胞を活性化するために様々な方策が検討されている。例えば、特許文献１は、アスタキサンチンを含有する幹細胞の細胞老化抑制剤を開示する。特許文献２は、ヒドロキシプロリン又は薬理学的に許容されるその塩を有効成分として含有する、間葉系幹細胞の幹細胞性維持及び賦活化剤を開示する。 Various measures are being studied to activate skin stem cells. For example, Patent Document 1 discloses a cell senescence inhibitor for stem cells containing astaxanthin. Patent Document 2 discloses a stem cell-maintaining and activating agent for mesenchymal stem cells, which contains hydroxyproline or a pharmacologically acceptable salt thereof as an active ingredient.
　分化細胞は、解糖系に加え電子伝達系を使いATPを産生させるため、活性酸素が細胞内に蓄積する。一方で、幹細胞は解糖系のみでATP産生を行うため、活性酸素は発生せず、細胞が守られる（非特許文献１）。ネズミ表皮では、NADH/NAD+比が高く解糖系が優勢な夜間にはS期にある幹細胞が増加するが、NADH/NAD+比が低く電子伝達系が優勢な昼間には減少するというサイクルを繰り返すことが報告されている(非特許文献２）。また、ミトコンドリアピルビン酸キャリア（mitochondrial pyruvate carrier (MPC1)）の機能を欠失させ電子伝達系への経路をブロックするよう遺伝子操作した毛包細胞では幹細胞の増殖が活性化されヘアサイクルが促進されことが示された（非特許文献３）。 Differentiated cells use an electron transport chain in addition to glycolysis to produce ATP, so active oxygen accumulates inside the cells. On the other hand, since stem cells produce ATP only in glycolysis, active oxygen is not generated and the cells are protected (Non-Patent Document 1). In the murine epidermis, stem cells in the S phase increase at night when the NADH / NAD + ratio is high and glycolysis is predominant, but decrease during the daytime when the NADH / NAD + ratio is low and the electron transport chain is predominant. It has been reported (Non-Patent Document 2). In addition, in hair follicle cells genetically engineered to block the pathway to the electron transport chain by deleting the function of the mitochondrial pyruvate carrier (MPC1), stem cell proliferation is activated and the hair cycle is promoted. Was shown (Non-Patent Document 3).
　特許文献３には、ミトコンドリアトランスファー促進剤を含有する美容組成物を開示し、係る組成物の皮膚線維芽細胞、表皮角化細胞、脂肪由来間葉系幹細胞、表皮幹細胞及び真皮幹細胞への使用が記載されている。特許文献４は、角化細胞をストレッサーに暴露すること、解糖及び酸化的リン酸化のそれぞれと関連付けられる代謝指標を検出し、ストレッサーに対するそれぞれの応答を提供すること等を含む、角化細胞の代謝を改善するスキンケア活性剤として被検物質を特定する方法を開示する。特許文献５は、細胞を第１および第２被験物質と接触させること、解糖及び酸化的リン酸化のそれぞれと関連付けられる代謝指標を非致死的に検出して、被験物質に対する代謝経路に関する応答を提供すること等を含む、化粧品組成物に使用するための有効成分の相乗作用する組み合わせを特定又は評価する方法を開示する。 Patent Document 3 discloses a beauty composition containing a mitochondrial transfer promoter, and its use in skin fibroblasts, epidermal keratinocytes, adipose-derived mesenchymal stem cells, epidermal stem cells and dermal stem cells can be used. Are listed. Patent Document 4 includes exposing keratinocytes to a stressor, detecting metabolic indicators associated with glycolysis and oxidative phosphorylation, respectively, and providing a response to the stressor. Disclose a method for identifying a test substance as a skin care activator that improves metabolism. Patent Document 5 non-lethally detects metabolic indicators associated with contacting cells with the first and second test substances, glycolysis and oxidative phosphorylation, respectively, and responds to the test substance with respect to the metabolic pathway. Disclosed are methods of identifying or evaluating synergistic combinations of active ingredients for use in cosmetic compositions, including providing.
特開2018-52879号公報Japanese Unexamined Patent Publication No. 2018-52879 特開2019-26617号公報Japanese Unexamined Patent Publication No. 2019-26617 特開2018-123130号公報Japanese Unexamined Patent Publication No. 2018-123130 特表2015-534644号公報Special Table 2015-534644 特表2015-531881号公報Special Table 2015-531881
　本発明の課題は、皮膚幹細胞を活性化する方法、皮膚幹細胞活性化剤、及び皮膚幹細胞活性化剤のスクリーニング方法を提供することにある。 An object of the present invention is to provide a method for activating skin stem cells, a skin stem cell activator, and a screening method for a skin stem cell activator.
　本発明者らは、鋭意検討の結果、MPC1抑制により電子伝達系への経路を阻害することで皮膚幹細胞を活性化することに想到し、本発明を為すに至った。 As a result of diligent studies, the present inventors have come up with the idea of activating skin stem cells by inhibiting the pathway to the electron transport chain by suppressing MPC1, and have come to the present invention.
　本願は下記の発明を包含する：
（１）MPC1抑制剤の適用により皮膚幹細胞を活性化する美容方法。
（２）前記MPC1抑制剤が、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含む（１）に記載の美容方法。
（３）アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含むMPC1抑制剤。
（４）MPC1抑制剤を含む、皮膚幹細胞活性化剤。
（５）前記MPC1抑制剤が、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含む（４）に記載の皮膚幹細胞活性化剤。
（６）MPC1抑制作用を指標とする、皮膚幹細胞活性化剤のスクリーニング方法。
（７）皮膚試料を候補薬剤に接触させる工程、
　候補薬剤に接触させた皮膚試料におけるMPC1の活性、量、及び／又は発現量を測定する工程、
　測定したMPC1の活性、量、及び／又は発現量から候補薬剤のMPC1抑制作用を決定する工程、
　決定したMPC1抑制作用に基づき、皮膚幹細胞活性化剤を選択する工程、
　を含む（６）に記載の方法。 The present application includes the following inventions:
(1) A cosmetic method that activates skin stem cells by applying an MPC1 inhibitor.
(2) The beauty method according to (1), wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.
(3) An MPC1 inhibitor containing at least one of akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.
(4) A skin stem cell activator containing an MPC1 inhibitor.
(5) The skin stem cell activity according to (4), wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. Agent.
(6) A method for screening a skin stem cell activator using the MPC1 inhibitory effect as an index.
(7) Step of bringing the skin sample into contact with the candidate drug,
A step of measuring the activity, amount, and / or expression level of MPC1 in a skin sample contacted with a candidate drug.
A step of determining the MPC1 inhibitory effect of a candidate drug from the measured MPC1 activity, amount, and / or expression level,
The step of selecting a skin stem cell activator based on the determined MPC1 inhibitory effect,
The method according to (6).
　本発明によれば、MPC1抑制により皮膚幹細胞を活性化に有効な剤および方法が提供される。皮膚幹細胞が活性化されれば、皮膚の老化抑制に有効である。 According to the present invention, an agent and a method effective for activating skin stem cells by suppressing MPC1 are provided. If skin stem cells are activated, it is effective in suppressing skin aging.
図１は、実験1-1の結果であり、コントロール(Cont)、UK5099（10μM UK5099、20μM UK5099）、エキノマイシン（10nM Echinomycin、20nM Echinomycin）をそれぞれ添加した場合のMCSP/B2M値をコントロール(Cont)の結果を1とした相対値で示すグラフである。FIG. 1 shows the results of Experiment 1-1, in which the MCSP / B2M value when control (Cont), UK5099 (10 μM UK5099, 20 μM UK5099), and echinomycin (10nM Echinomycin, 20nM Echinomycin) were added was controlled (Cont). It is a graph showing a relative value with the result of) as 1. 図２は、実験1-2の結果であり、コントロール(Cont)、UK5099（20μM UK5099）をそれぞれ添加した場合のMCSP及びDAPIの発現を示す写真である。FIG. 2 shows the results of Experiment 1-2 and is a photograph showing the expression of MCSP and DAPI when Control (Cont) and UK5099 (20 μM UK5099) were added, respectively. 図３は、実験1-2の結果であり、コントロール（Cont）、UK5099（20μM UK5099）をそれぞれ添加した場合のIntegrin β1及びDAPIの発現を示す写真である。FIG. 3 shows the results of Experiment 1-2 and is a photograph showing the expression of Integrin β1 and DAPI when Control (Cont) and UK5099 (20 μM UK5099) were added, respectively. 図４は、実験1-3の結果であり、抗体を添加しない場合(Negative cont）、コントロールを添加した場合(Cont)、UK5099を添加した場合（20μM UK5099）のIntegrinβ１陽性細胞数を示す。FIG. 4 shows the results of Experiment 1-3, showing the number of Integrin β1-positive cells when no antibody was added (Negative cont), when control was added (Cont), and when UK5099 was added (20 μM UK5099). 図５は、実験1-4の結果であり、コントロール（Cont）、UK5099（1μM UK5099，10μM UK5099）をそれぞれ添加し4日間培養した三次元培養皮膚モデルの組織切片におけるIntegrin α6、MCSP、及びDAPIの発現を示す写真である。FIG. 5 shows the results of Experiment 1-4. Integrin α6, MCSP, and DAPI in the tissue section of a three-dimensional cultured skin model in which control (Cont) and UK5099 (1 μM UK5099, 10 μM UK5099) were added and cultured for 4 days, respectively. It is a photograph showing the expression of. 図６は、実験1-5の結果であり、コントロール（Cont）、UK5099（10μM UK5099）をそれぞれ添加し5日間培養したヒト皮膚器官培養モデル組織切片におけるIntegrin α6、MCSP、及びDAPIの発現を示す写真である。FIG. 6 shows the results of Experiments 1-5, showing the expression of Integrin α6, MCSP, and DAPI in human skin organ culture model tissue sections cultured for 5 days with the addition of control (Cont) and UK5099 (10 μM UK5099), respectively. It is a photograph. 図７は、実験2-2の一次スクリーニングの結果であり、コントロール(DMSO；Control)、各評価対象試料（薬剤No.1～124）を添加した場合のMPC1/B2M値を、コントロールの結果を1.0とした相対値で示す。FIG. 7 shows the results of the primary screening of Experiment 2-2, and the MPC1 / B2M values when control (DMSO; Control) and each evaluation target sample (drug Nos. 1 to 124) were added, and the control results were shown. Shown as a relative value of 1.0. 図８は、実験2-2の二次スクリーニングの結果であり、一次スクリーニングで選定された各評価対象試料（薬剤No.1～124のうち37品）を添加した場合のMPC1/B2M値を、コントロール(DMSO)の結果を100.0とした割合(% of control)で示す。FIG. 8 shows the results of the secondary screening of Experiment 2-2, and the MPC1 / B2M values when each evaluation target sample (37 out of drug Nos. 1 to 124) selected in the primary screening was added. The result of control (DMSO) is shown as a ratio (% of control) of 100.0. 図９は、実験2-2の三次スクリーニングの結果であり、コントロール(DMSO；Cont)、二次スクリーニングで選定された各評価対象試料（薬剤No.1～124のうち15品）を添加した場合のMPC1/B2M値を、コントロールの結果を100.0とした割合(% of control)で示す。FIG. 9 shows the results of the tertiary screening in Experiment 2-2, when the control (DMSO; Cont) and each evaluation target sample (15 out of drug Nos. 1 to 124) selected in the secondary screening were added. The MPC1 / B2M value of is shown as a ratio (% of control) with the control result as 100.0. 図１０は、図９の結果を、実験2-2の三次スクリーニングで選定した６品それぞれについてDunnett's testにより統計処理を施したグラフを示す（*P＜0.05，**P＜0.01）。FIG. 10 shows a graph in which the results of FIG. 9 were statistically processed by Dunnett's test for each of the 6 products selected in the tertiary screening of Experiment 2-2 (* P <0.05, ** P <0.01).
　本発明者らは、MPC1抑制により皮膚幹細胞が活性化されることに想到し、MPC1抑制作用を有する新規物質を発見した。とりわけ、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインには高いMPC1抑制作用があることを発見した。 The present inventors have come up with the idea that skin stem cells are activated by MPC1 inhibition, and have discovered a novel substance having an MPC1 inhibitory effect. In particular, we found that akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine have a high MPC1 inhibitory effect.
　本発明はかかる知見に基づき、MPC1抑制剤および皮膚幹細胞活性化剤（以降これらを総称して「本発明の剤」という場合がある。）、そのスクリーニング方法、並びにそれらを適用することにより皮膚幹細胞を活性化する方法を提供する。本発明の方法は、美容を目的とする方法の場合があり、医師や医療従事者による治療ではないことがある。 Based on such findings, the present invention describes MPC1 inhibitors and skin stem cell activators (hereinafter, these may be collectively referred to as "agents of the present invention"), screening methods thereof, and skin stem cells by applying them. Provide a method of activating. The method of the present invention may be for the purpose of cosmetology and may not be treated by a doctor or healthcare professional.
　MPC1は、MPC2とヘテロダイマーを形成し、ミトコンドリア内部へとピルビン酸を送達するトランスポーターであるミトコンドリアピルビン酸キャリアを形成するタンパク質である。MPC1抑制とは、MPC1の働き、産生、及び／又は翻訳を低減及び／又は阻害すること、並びに／或いは、MPC1の活性、量、及び／又は発現量を減少させることを指す。MPC1の働きとしては、例えば、MPC1がミトコンドリア内部へピルビン酸を送達する機能が挙げられる。ある実施形態では、MPC1抑制は、本発明の剤を付与していない状態（コントロール）に比べて、本発明の剤を付与した場合に、MPC1の活性、量、及び／又は発現量が、例えば有意水準を5%とした統計学的有意差（例えばDunnettの検定）をもって減少していること、あるいは、例えば5％以上、10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、又は100％減少していることを意味し得る。 MPC1 is a protein that forms a heterodimer with MPC2 and forms a mitochondrial pyruvate carrier, which is a transporter that delivers pyruvate into the mitochondria. MPC1 inhibition refers to reducing and / or inhibiting the action, production, and / or translation of MPC1 and / or reducing the activity, amount, and / or expression of MPC1. The function of MPC1 is, for example, the function of MPC1 to deliver pyruvic acid into mitochondria. In certain embodiments, MPC1 suppression results in, for example, the activity, amount, and / or expression level of MPC1 when the agent of the present invention is applied, as compared to the state (control) where the agent of the present invention is not applied. Decreasing with a statistically significant difference (eg Dunnett's test) with a significance level of 5%, or, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% Above, it can mean that it is reduced by 60% or more, 70% or more, 80% or more, 90% or more, or 100%.
　MPC1の発現量は、例えば、実施例のように定量PCR法を用いて遺伝子発現量を測定してもよく、抗体を用いて染色することによりタンパク質発現量を測定してもよく、市販のキットを用いて測定してもよく、あるいは特許文献３等の文献に記載の方法により求めることができるがこれらに限定されず、任意の公知技術が使用できる。 For the expression level of MPC1, for example, the gene expression level may be measured by using a quantitative PCR method as in the examples, or the protein expression level may be measured by staining with an antibody, and a commercially available kit may be used. , Or can be obtained by the method described in documents such as Patent Document 3, but the present invention is not limited to these, and any known technique can be used.
　本明細書において、皮膚幹細胞の活性化とは、角化細胞や線維芽細胞といった皮膚の細胞における幹細胞の増殖が促進することを指す。幹細胞の増殖は、幹細胞数の計数、integrin β1等の幹細胞マーカーの発現量の測定、等によって決定できる。皮膚幹細胞が活性化されれば、ターンオーバーの促進、肌の若返り、肌のキメの改善、ニキビや色素沈着といった好ましくない肌状態からの早期回復、等が期待できる。 As used herein, activation of skin stem cells refers to promotion of stem cell proliferation in skin cells such as keratinocytes and fibroblasts. Stem cell proliferation can be determined by counting the number of stem cells, measuring the expression level of stem cell markers such as integrin β1, and the like. If skin stem cells are activated, it can be expected to promote turnover, rejuvenate the skin, improve the texture of the skin, and recover from unfavorable skin conditions such as acne and pigmentation at an early stage.
　本発明のMPC1抑制剤は、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及び/又はエルゴチオネインからなってもよく、又は有効成分として含有してもよい。しかしながら、MPC1を抑制することができる物質であればこれらに限定されない。 The MPC1 inhibitor of the present invention may consist of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine, or may be contained as an active ingredient. However, the substance is not limited to these as long as it can suppress MPC1.
　本発明の剤は、上記の有効成分の何れか1種を単独で含有してもよく、2種類以上を任意の組み合わせ及び比率で含有してもよい。 The agent of the present invention may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.
　本発明の剤は、上記の有効成分を、1種又は2種以上の他の成分、例えば賦形剤、担体及び/又は希釈剤等と組み合わせてもよい。 The agent of the present invention may combine the above active ingredient with one or more other ingredients such as excipients, carriers and / or diluents.
　アケビ（Akebia quinata）は、アケビ科アケビ属に属する落葉低木である。アケビは、ヒアルロン酸生成促進効果、コラーゲン生成促進効果及びＭＭＰ阻害効果を有し、しわ形成防止・改善効果や、リパーゼ阻害活性、血中中性脂肪濃度上昇抑制活性、あるいは抗肥満活性を有することが報告されている（特開2015-17048号公報、特開2010-265182号公報等）。本発明に用いられるアケビ抽出物としては、アケビの茎の抽出物が好ましいが、アケビの果実、果皮、種子、葉、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Akebia quinata is a deciduous shrub belonging to the genus Akebia in the family Lardizabalaceae. Akevi has a hyaluronic acid production promoting effect, a collagen production promoting effect, and an MMP inhibiting effect, and has a wrinkle formation preventing / improving effect, a lipase inhibiting activity, a blood triglyceride concentration increasing inhibitory activity, or an anti-obesity activity. Has been reported (Japanese Patent Laid-Open No. 2015-17048, Japanese Patent Application Laid-Open No. 2010-265182, etc.). As the akebi extract used in the present invention, an extract of akebi stem is preferable, but since the active ingredient is also contained in the fruit, peel, seed, leaf, flower, root and the like of akebi, any of these Or one or more extracts can also be used.
　黒豆（Glycine max 'Kuromame'）は、マメ科の一年草であるダイズの品種であり、「丹波黒」、「和知黒」等の各品種を用いることができる。アントシアニン等のポリフェノールを豊富に含んでいること、黒豆由来成分には抗肥満作用、抗炎症作用、抗酸化作用、耐糖能改善作用等があることが報告されている（特開2015-140298号公報等）。黒豆抽出物は、黒豆の豆果の抽出物が好ましいが、黒豆の鞘、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Kuromame (Glycine max'Kuromame') is a soybean variety that is an annual plant of the legume family, and each variety such as "Tamba black" and "Wachi black" can be used. It has been reported that it is rich in polyphenols such as anthocyanins, and that black soybean-derived components have anti-obesity, anti-inflammatory, antioxidant, glucose intolerant, and other effects (Japanese Patent Laid-Open No. 2015-140298). etc). The black soybean extract is preferably an extract of black soybean legume, but since the active ingredient is also contained in the pod, leaves, stems, flowers, roots, etc. of black soybean, one or more of these are extracted. You can also use things.
　シャクヤク（Paeonia lactiflora）は、ボタン科の多年草である。シャクヤクは、表皮肥厚抑制作用、ＶＥＧＦ産生促進作用、ＩＧＦ－１産生促進作用、ＨＧＦ産生促進作用、及びＢＭＰ－２産生促進作用等が報告されている（特開201９-11252号公報、特開2012-121856号公報等）。シャクヤク抽出物は、シャクヤクの根の抽出物が好ましいが、シャクヤクの葉、茎、花、果実、果皮、種子等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Peony (Paeonia lactiflora) is a perennial plant of the Peony family. Peony has been reported to have an epidermal thickening inhibitory effect, a VEGF production promoting effect, an IGF-1 production promoting effect, an HGF production promoting effect, a BMP-2 production promoting effect, and the like (Japanese Patent Laid-Open No. 2009-11252, JP2012). -121856 Gazette, etc.). As the syrup extract, the extract of syrup root is preferable, but since the active ingredient is also contained in the leaves, stems, flowers, fruits, pericarps, seeds, etc. of syrup, one or more of these are one or more. Extracts can also be used.
　茶（Camellia sinensis）は、ツバキ科ツバキ属の常緑樹であり、チャノキ(Camellia sinensis var. sinensis)、アッサムチャ(Camellia sinensis var. assamica)等の各品種を用いることができる。例えば、宇治茶等を使用できる。茶にはカテキン等のポリフェノールを豊富に含んでいること、脂質代謝改善作用、抗酸化作用、抗ガン作用、血圧上昇抑制作用等があることが知られている。茶抽出物は、茶の葉や茎の抽出物が好ましいが、茶の実、花、根、種子等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Tea (Camellia sinensis) is an evergreen tree belonging to the genus Camellia of the family Camellia, and various varieties such as tea plant (Camellia sinensis var. Sinnsis) and assamcha (Camellia sinensis var. Assamica) can be used. For example, Uji tea or the like can be used. It is known that tea contains abundant polyphenols such as catechin, and has lipid metabolism improving action, antioxidant action, anticancer action, blood pressure increase suppressing action and the like. The tea extract is preferably an extract of tea leaves or stems, but since the active ingredient is also contained in tea nuts, flowers, roots, seeds, etc., one or more of these extracts. Can also be used.
　ホホバ(Simmondsia chinensis)は、ナデシコ目ホホバ科に属する常緑低木である。ホホバには、保湿作用、エモリエント作用、抗炎症作用、コラゲナーゼ阻害効果、エラスターゼ阻害効果、ヒアルロニダーゼ阻害効果等が知られている（特開2003-48846号公報、特開2003-048812号公報、特開2003-034644号公報等）。ホホバ葉抽出物は、ホホバの葉の抽出物であるが、ホホバの種子、実、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。 Jojoba (Simmondsia chinansis) is an evergreen shrub belonging to the family Caryophyllales jojoba. Jojoba is known to have a moisturizing effect, an emollient effect, an anti-inflammatory effect, a collagenase inhibitory effect, an elastase inhibitory effect, a hyaluronidase inhibitory effect, and the like (Japanese Patent Laid-Open No. 2003-48846, JP-A-2003-048812, JP-A-2003-048812). 2003-03464 No. 4). Jojoba leaf extract is an extract of jojoba leaves, but since jojoba seeds, fruits, flowers, roots, etc. also contain active ingredients, one or more of these extracts should be used. It can also be used.
　エルゴチオネイン（L-Ergothioneine）は以下の構造を有する分子量229.3のアミノ酸であり、抗酸化、抗炎症作用、エラスターゼ阻害作用、チロシナーゼ阻害作用等を有することが知られている（特開2019-149972号公報等）。エルゴチオネインは、化学的に合成してもよく、あるいは、担子菌類等の微生物や動植物等にも含まれているため、このような天然物の抽出物等の形態で用いてもよい。
Figure JPOXMLDOC01-appb-C000001
 Ergothioneine is an amino acid having the following structure and a molecular weight of 229.3, and is known to have antioxidant, anti-inflammatory, elastase-inhibiting, tyrosinase-inhibiting, etc. (Japanese Patent Laid-Open No. 2019-149972). etc). Ergothioneine may be chemically synthesized, or it may be used in the form of an extract of such a natural product because it is also contained in microorganisms such as basidiomycetes, animals and plants.
Figure JPOXMLDOC01-appb-C000001
　上述の各種抽出物は、化粧品原料や健康食品材料として市販のものを使用してもよく、常法により得てもよい。抽出方法は特に限定されるものではないが、溶媒を用いた抽出法や加水分解による抽出法が挙げられる。溶媒を用いた抽出を行う際には、原料を抽出溶媒とともに常温又は加熱して浸漬または加熱還流した後、加水分解による抽出を行う際には、例えば、アルカリ、酸等による化学的処理、熱、圧力等による物理学的処理、酵素等による生物学的処理等、任意の加水分解処理を行った後、濾過し、濃縮して得ることができる。原料をそのまま使用することもできるが、顆粒状や粉末状に粉砕して抽出に供した方が、穏和な条件で短時間に高い抽出効率で有効成分の抽出を行うことができる。抽出温度は特に限定されるものではなく、粉砕物の粒径や溶媒の種類等に応じて適宜設定すればよい。通常は、室温から溶媒の沸点までの範囲内で設定される。また、抽出時間も特に限定されるものではなく、粉砕物の粒径、溶媒の種類、抽出温度等に応じて適宜設定すればよい。さらに、抽出時には、撹拌を行ってもよいし、撹拌せず静置してもよいし、超音波を加えてもよい。 The above-mentioned various extracts may be commercially available as cosmetic raw materials or health food materials, or may be obtained by a conventional method. The extraction method is not particularly limited, and examples thereof include an extraction method using a solvent and an extraction method by hydrolysis. When performing extraction using a solvent, the raw material is immersed or heated at room temperature or heated with an extraction solvent at room temperature or heated and refluxed, and then when extraction by hydrolysis is performed, for example, chemical treatment with alkali, acid, etc., heat. It can be obtained by performing arbitrary hydrolysis treatment such as physical treatment with pressure or the like, biological treatment with an enzyme or the like, and then filtering and concentrating. The raw material can be used as it is, but if it is crushed into granules or powder and used for extraction, the active ingredient can be extracted in a short time with high extraction efficiency under mild conditions. The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.
　抽出溶媒としては、通常抽出に用いられる溶媒であれば任意に用いることができ、例えば、水性溶媒、例えば水、生理食塩水、リン酸緩衝液、ホウ酸緩衝液、あるいは有機溶媒、例えばエタノール、プロピレングリコール、1、3-ブチレングリコール、グリセリン等のアルコール類、含水アルコール類、クロロホルム、ジクロルエタン、四塩化炭素、アセトン、酢酸エチル、ヘキサン等を、それぞれ単独あるいは組み合わせて用いることができる。 As the extraction solvent, any solvent can be used as long as it is usually used for extraction. For example, an aqueous solvent such as water, physiological saline, a phosphate buffer, a borate buffer, or an organic solvent such as ethanol can be used. Alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination.
　このような抽出操作により、有効成分が抽出され、溶媒に溶け込む又は加水分解される。抽出物を含む溶媒又は加水分解物は、そのまま使用してもよいが、滅菌、洗浄、濾過、脱色、脱臭等の慣用の精製処理を加えてから使用してもよい。また、必要により凍結乾燥などにより濃縮あるいは任意の溶媒で希釈してから使用してもよい。さらに、溶媒又は加水分解物を全て揮発させて固体状（乾燥物）としてから使用してもよいし、該乾燥物を任意の溶媒に再溶解してから使用してもよい。 By such an extraction operation, the active ingredient is extracted and dissolved in a solvent or hydrolyzed. The solvent or hydrolyzate containing the extract may be used as it is, or may be used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. If necessary, it may be concentrated by freeze-drying or diluted with an arbitrary solvent before use. Further, the solvent or the hydrolyzate may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
　また、原料を圧搾することにより得られる圧搾液にも抽出物と同様の有効成分が含まれているので、抽出物の代わりに圧搾液を使用することもできる。 Further, since the squeezed liquid obtained by squeezing the raw material also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.
　また、本願は、本発明の剤を含む組成物も提供する。本発明の組成物は、化粧品組成物又は食品組成物であってもよい。本発明の組成物は、例えば、MPC1抑制作用を介して皮膚幹細胞を活性化する組成物であってもよい。 The present application also provides a composition containing the agent of the present invention. The composition of the present invention may be a cosmetic composition or a food composition. The composition of the present invention may be, for example, a composition that activates skin stem cells through an MPC1 inhibitory action.
　本発明の剤又は組成物を適用する対象は、例えば、肌のターンオーバーの滞り、皮膚老化、色素沈着といった客観的又は主観的な観点から皮膚幹細胞の活性化が必要である対象であっても、予防的に皮膚幹細胞の活性化を希望する対象であってもよい。 The subject to which the agent or composition of the present invention is applied may be a subject that requires activation of skin stem cells from an objective or subjective viewpoint such as delayed skin turnover, skin aging, and pigmentation. , It may be a subject who desires prophylactic activation of skin stem cells.
　本発明の剤又は組成物は、外用投与または経口投与など任意の経路により適用できるが、皮膚に直接適用することができる皮膚外用剤に配合することが好ましい。外用投与の形態としては、例えば、液状、乳液状、クリーム状、固形状、シート状、スプレー状、ゲル状、泡状、パウダー状等任意に選択することができる。乳液、クリーム、美容液、ローション、パック、洗顔料等の化粧品組成物であってもよい。経口投与の形態としては、例えば、錠剤、サプリメント、飲料、粉末など任意に選択することができる。本発明の剤又は組成物は、その効果を損なわない範囲で、化粧品や医薬品等の組成物に用いられる任意配合成分を、必要に応じて適宜配合することができる。前記任意配合成分としては、例えば、賦形剤、担体、希釈剤、油分、界面活性剤、粉末、色材、水、アルコール類、増粘剤、キレート剤、シリコーン類、酸化防止剤、紫外線吸収剤、保湿剤、香料、各種薬効成分、防腐剤、pH調整剤、中和剤などが挙げられる。例えば、皮膚幹細胞の活性化を促進する他の薬効成分などが含まれていてもよい。 The agent or composition of the present invention can be applied by any route such as external administration or oral administration, but it is preferably blended with an external preparation for skin that can be directly applied to the skin. As the form of external administration, for example, liquid, emulsion, cream, solid, sheet, spray, gel, foam, powder and the like can be arbitrarily selected. It may be a cosmetic composition such as a milky lotion, a cream, a beauty essence, a lotion, a facial mask, or a facial cleanser. As the form of oral administration, for example, tablets, supplements, beverages, powders and the like can be arbitrarily selected. In the agent or composition of the present invention, any compounding ingredients used in compositions such as cosmetics and pharmaceuticals can be appropriately blended as necessary, as long as the effects are not impaired. Examples of the optional compounding ingredients include excipients, carriers, diluents, oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, and ultraviolet absorbers. Examples include agents, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and the like. For example, it may contain other medicinal ingredients that promote the activation of skin stem cells.
　また、投与頻度は、４週間に１回、２週間に１回、１週間に１回、３日に１回、２日に１回、１日１回、１日２回、１日３回、１日４回、１日５回、都度投与等任意に選択できるがこれらに限定されない。 The frequency of administration is once every four weeks, once every two weeks, once a week, once every three days, once every two days, once a day, twice a day, three times a day. It can be arbitrarily selected, such as 4 times a day, 5 times a day, and each administration, but the present invention is not limited to these.
　しかしながら、本発明の剤や組成物の採り得る形態は、上述の剤型や形態に限定されるものではない。 However, the possible forms of the agent or composition of the present invention are not limited to the above-mentioned dosage forms and forms.
　本発明の剤又は組成物におけるアケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネイン等の有効成分の配合量は、種類、目的、形態、利用方法などに応じて、適宜決めることができる。例えば、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及び／又はエルゴチオネインの配合量は、本発明剤又は組成物の総重量当たり0.0001～100重量％、0.0001～90重量％、0.001～50重量％、0.01～5重量％、0.01～1重量％、0.1～0.5重量％、等とすることができるが、本発明の効果が発揮されれば限定されない。 The amount of the active ingredient such as akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine in the agent or composition of the present invention depends on the type, purpose, form, usage, etc. It can be decided as appropriate. For example, the blending amounts of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine are 0.0001 to 100% by weight, 0.0001 to 0.0001 to the total weight of the agent or composition of the present invention. It can be 90% by weight, 0.001 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.1 to 0.5% by weight, etc., but is not limited as long as the effect of the present invention is exhibited.
　さらに、本願は、MPC1抑制作用を指標とする、皮膚幹細胞活性化剤のスクリーニング方法も提供する。本発明のスクリーニング方法は、皮膚試料を候補薬剤に接触させる工程；候補薬剤に接触させた皮膚試料におけるMPC1の活性、量、及び／又は発現量を測定する工程；測定したMPC1の活性、量、及び／又は発現量から候補薬剤のMPC1抑制作用を決定する工程；決定したMPC1抑制作用に基づき、皮膚幹細胞活性化剤を選択する工程；を含んでもよい。本発明の方法により，候補薬剤が皮膚幹細胞活性化作用を有するか否かについてのスクリーニングが可能となり，製品開発や新たな肌ケアの提案が可能になる。 Furthermore, the present application also provides a screening method for a skin stem cell activator using the MPC1 inhibitory action as an index. The screening method of the present invention is a step of contacting a skin sample with a candidate drug; a step of measuring the activity, amount, and / or expression level of MPC1 in the skin sample contacted with the candidate drug; And / or a step of determining the MPC1 inhibitory action of the candidate drug from the expression level; a step of selecting a skin stem cell activator based on the determined MPC1 inhibitory action; may be included. According to the method of the present invention, it becomes possible to screen whether or not a candidate drug has a skin stem cell activating effect, and it becomes possible to develop products and propose new skin care.
　皮膚試料は、採取後の皮膚試料、例えば、ヒト等の動物から採取された後のex vivoの状態の皮膚試料であってもよいし、培養皮膚細胞、例えば、培養角化細胞又は培養線維芽細胞といったin vitroの状態であってもよい。あるいは、三次元皮膚モデルなどの人工皮膚試料であってもよい。皮膚試料は、MPC1抑制作用を測定することができれば限定されない。 The skin sample may be a skin sample after collection, for example, a skin sample in an ex vivo state after being collected from an animal such as a human, or a cultured skin cell, for example, a cultured keratinocyte or a cultured fibroblast. It may be in vitro, such as a cell. Alternatively, it may be an artificial skin sample such as a three-dimensional skin model. Skin samples are not limited as long as the MPC1 inhibitory effect can be measured.
　また、本願は、MPC1抑制を介して、皮膚幹細胞を活性化するアケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及び／又はエルゴチオネインも提供する。 The present application also provides akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and / or ergothioneine that activate skin stem cells through MPC1 inhibition.
　次に実施例によって本発明をさらに詳細に説明する。なお、本発明はこれにより限定されるものではない。 Next, the present invention will be described in more detail by way of examples. The present invention is not limited thereto.
　実験１:MPC1抑制による幹細胞への影響
　実験1-1:PCR法によるMCSPの遺伝子発現解析
　MPC1を阻害することによる幹細胞への影響を調べるために、MPC1阻害剤として公知のUK5099を用いて、表皮幹細胞マーカーであるMelanoma-associated chondroitin sulfate proteoglycan (MCSP)の発現を解析した。 Experiment 1: Effect of MPC1 inhibition on stem cells Experiment 1-1: Analysis of MCSP gene expression by PCR method In order to investigate the effect of inhibiting MPC1 on stem cells, the epidermis was used using UK5099, which is known as an MPC1 inhibitor. The expression of the stem cell marker Melanoma-associated chondroitin sulfate proteoglycan (MCSP) was analyzed.
　細胞培養
　正常ヒト胎児由来表皮角化細胞（ScienCell社、Cat No.2120）を表皮角化細胞培養培地（クラボウ社、KK-2150S）にて培養した。継代後に2.5×105 cells/well/2mLになるように6well plateへ播種し、24時間後にUK5099、コントロール、エキノマイシンをそれぞれ添加した。エキノマイシンは、MPC1と同様にミトコンドリア活性を制御するHIF-1αの阻害剤であるがミトコンドリア内におけるピルビン酸からアセチルCoAへの変換阻害を行う物質である点で異なる。ミトコンドリア内へのピルビン酸送達を阻害する経路と、それとも、ミトコンドリア内におけるピルビン酸からアセチルCoAへの変換を阻害する経路のいずれが、表皮角化細胞の幹細胞を活性化するのかを検討するためにエキノマイシンを使用した。UK5099（メルク社製：カタログ番号504817）は、あらかじめ10mM及び20mMの濃度になるようにDMSOで希釈したストック溶液を作成し、1000倍希釈で培地に添加することで最終濃度10μM及び20μMとした。コントロールはDMSOを1000倍希釈で添加した。エキノマイシン（Abcam社製Echinomycin：製品番号ab144247）は、あらかじめ10μM及び20μMの濃度になるようにDMSOで希釈したストック溶液を作成し、1000倍希釈で培地に添加することで最終濃度10nM及び20nMとした。これらの物質を添加24時間後にmRNAを回収した。 Cell culture Normal human fetal-derived epidermal keratinocytes (ScienCell, Cat No. 2120) were cultured in epidermal keratinocyte culture medium (Krabou, KK-2150S). After passage, seeding was performed on a 6-well plate so as to have 2.5 × 10 5 cells / well / 2 mL, and UK5099, control, and equinomycin were added 24 hours later, respectively. Equinomycin is an inhibitor of HIF-1α that regulates mitochondrial activity like MPC1, but differs in that it is a substance that inhibits the conversion of pyruvate to acetyl-CoA in mitochondria. To investigate whether the pathway that inhibits the delivery of pyruvate into mitochondria or the pathway that inhibits the conversion of pyruvate to acetyl-CoA in mitochondria activates the stem cells of epidermal keratinocytes. Equinomycin was used. UK5099 (Merck Co., Ltd .: Catalog No. 504817) prepared a stock solution diluted with DMSO in advance so as to have a concentration of 10 mM and 20 mM, and added it to the medium at a 1000-fold dilution to obtain a final concentration of 10 μM and 20 μM. Control added DMSO at 1000-fold dilution. Echinomycin (Abcam Echinomycin: product number ab144247) is prepared by preparing a stock solution diluted with DMSO to a concentration of 10 μM and 20 μM in advance, and added to the medium at a dilution of 1000 times to obtain a final concentration of 10 nM and 20 nM. did. The mRNA was recovered 24 hours after the addition of these substances.
　定量PCR法による遺伝子発現解析
　薬剤添加24時間後の細胞からRNAの回収は、Quiagen Rneasy mini Kit（Quiagen社）を用い、cDNAの合成はSuperScript VILO cDNA Synthesis Kit（Thermo Fisher Scientific社）にて行った。MCSPおよびB2Mの遺伝子発現量は、Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan)を用いてSyber Green法にて行った。使用したプライマーは以下の通りである。
B2M forward： 5’-GTGGGATCGAGACATGTAAGCA-3’ （配列番号１）
B2M reverse： 5’-CAATCCAAATGCGGCATCT-3’ （配列番号２）
MCSP forward： 5’-CACGGCTCTGACCGACATAG-3’ （配列番号３）
MCSP reverse： 5’-CCCAGCCCTCTACGACAGT-3’ （配列番号４） Gene expression analysis by quantitative PCR method RNA was recovered from cells 24 hours after drug addition using the Quiagen Rneasy mini Kit (Quiagen), and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). .. The gene expression levels of MCSP and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used are as follows.
B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2)
MCSP forward: 5'-CACGGCTCTGACCGACATAG-3' (SEQ ID NO: 3)
MCSP reverse: 5'-CCCAGCCCTCTACGACAGT-3' (SEQ ID NO: 4)
　結果を図１に示す。UK5099を添加すると濃度依存的にMCSPの発現が増加していた。一方、エキノマイシを添加するとMCSPの発現は減少していた。これらの結果より、MCP1抑制により幹細胞が活性化することが示唆される。 The results are shown in Fig. 1. The addition of UK5099 increased the expression of MCSP in a concentration-dependent manner. On the other hand, the addition of Ekinomaishi reduced the expression of MCSP. These results suggest that MCP1 suppression activates stem cells.
　実験1-2:細胞染色によるMCSPおよびIntegrin β1のタンパク質発現解析
　更に、MCSPのみならず、別の幹細胞マーカーであるIntegrin β1について細胞染色し解析を行った。
　実験1-1と同様に継代した表皮角化細胞をUK5099を20μMとなるように添加し、対照にはUK5099で処理せず同量のDMSOを添加して1.0×104 cells/well/0.5mLで4well chamber slide（Falcon社、354114）に播種し48時間培養した。添加48時間後に細胞を4%PFAにて10分反応させ、固定した。その後0.01% Triton-X100/PBSにて15分反応させ、細胞膜を部分的に溶解させた。12% BSA/PBSでブロッキング後、1次抗体として抗MCSP抗体(Millipore, MAB2029, mouse mAb)、抗β1 integrin抗体(Santa Cruz, sc-13590, mouse mAb)にて4℃で一晩反応させた。翌日、PBSで5分で3回洗浄後、二次抗体としてAlexa488-anti-mouse IgG抗体にて室温、1時間反応させた。PBSで5分、3回洗浄後、DAPIを含む封入剤（Vectashield, H-1200）にて封入し、顕微鏡観察を行った。
　20μMのUK5099、コントロールをそれぞれ添加した結果を図２、３に示す。UK5099を添加するとMCSPおよびIntegrin β1のいずれも発現量が増加しており、実験1-1の結果と一致する結果となった。 Experiment 1-2: Protein expression analysis of MCSP and Integrin β1 by cell staining Furthermore, not only MCSP but also another stem cell marker, Integrin β1, was stained and analyzed.
Subcultured epidermal keratinized cells were added to 20 μM in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 to obtain 1.0 × 10 4 cells / well / 0.5. The cells were seeded in mL in a 4-well chamber slide (Falcon, 354114) and cultured for 48 hours. 48 hours after the addition, the cells were reacted with 4% PFA for 10 minutes and fixed. Then, the reaction was carried out with 0.01% Triton-X100 / PBS for 15 minutes to partially lyse the cell membrane. After blocking with 12% BSA / PBS, it was reacted overnight at 4 ° C with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-β1 integrin antibody (Santa Cruz, sc-13590, mouse mAb) as primary antibodies. .. The next day, the cells were washed 3 times with PBS for 5 minutes and then reacted with Alexa 488-anti-mouse IgG antibody as a secondary antibody at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
The results of adding 20 μM UK5099 and control are shown in FIGS. 2 and 3, respectively. The addition of UK5099 increased the expression levels of both MCSP and Integrin β1, which was consistent with the results of Experiment 1-1.
　実験1-3: Integrinβ１陽性細胞のFACS解析
　次に、Integrinβ１陽性細胞数のFACS解析を行った。実験1-1と同様に継代した表皮角化細胞をUK5099を20μMとなるように添加し、対照にはUK5099で処理せず同量のDMSOを添加して48時間培養した。これらの細胞を、Trypsinで剥がし、1.0×106 cells/500uLの濃度で細胞懸濁液を調整した。遠心で上清を除去後に、0.5% BSA/PBS on iceで10分ブロッキングした。遠心で上清除去後、IgG抗体、Pacific blue-標識抗β1 integrin抗体（BioLegend, 313620）を細胞懸濁液に加え氷上で1時間反応させ、0.1% BSA/PBSで3回遠心洗浄し、セルソーターSH800でFACS解析を行った。また、陰性対照（Negative cont）には抗体を添加せず、同様の処理を行った。 Experiment 1-3: FACS analysis of Integrin β1-positive cells Next, FACS analysis of the number of Integrin β1-positive cells was performed. Subcultured epidermal keratinized cells were added to 20 μM in the same manner as in Experiment 1-1, and the same amount of DMSO was added to the control without treatment with UK5099 and cultured for 48 hours. These cells were stripped with Trypsin and a cell suspension was prepared at a concentration of 1.0 × 10 6 cells / 500 uL. After removing the supernatant by centrifugation, blocking was performed with 0.5% BSA / PBS on ice for 10 minutes. After removing the supernatant by centrifugation, IgG antibody and Pacific blue-labeled anti-β1 integrin antibody (BioLegend, 313620) were added to the cell suspension, reacted on ice for 1 hour, centrifuged 3 times with 0.1% BSA / PBS, and cell sorter. FACS analysis was performed on SH800. In addition, no antibody was added to the negative control (Negative cont), and the same treatment was performed.
　結果を図４に示す。UK5099を添加するとIntegrin β1陽性細胞数が増加しており、実験1-1および実験1-2の結果と一致する結果となった。 The results are shown in Fig. 4. The addition of UK5099 increased the number of Integrin β1-positive cells, which was consistent with the results of Experiment 1-1 and Experiment 1-2.
　実験1-4；組織染色によるMCSPおよびIntegrin β1のタンパク質発現解析
次に、MatTeK社製皮膚モデルEFT-400を用いて、培地にUK5099を1μMおよび5μMとなるように添加し、対照にはUK5099で処理せず同量のDMSOを添加して2.5mL/wellで皮膚モデルを培養した。2日後に培地を交換し、培養開始から4日後に冷蔵アセトンに浸漬させ脱水固定した。その後、室温アセトン、室温の安息香酸メチルで２回、室温のキシレンで２回溶媒置換を行い、パラフィン包埋することでパラフィンブロックを作成した。ミクロトームを使い3μmの厚さで切片を作成した。キシレンを用いて脱パラフィンを行い、12% BSA/PBSでブロッキング後、1次抗体として抗MCSP抗体(Millipore, MAB2029, mouse mAb)、抗α6 integrin抗体(Santa Cruz, sc-19622, rat mAb)にて4℃で一晩反応させた。翌日、PBSで5分で3回洗浄後、二次抗体としてAlexa488-anti-mouse IgG抗体およびAlexa594-anti-rat IgG抗体にて室温、1時間反応させた。PBSで5分、3回洗浄後、DAPIを含む封入剤（Vectashield, H-1200）にて封入し、顕微鏡観察を行った。 Experiment 1-4; Protein expression analysis of MCSP and Integrin β1 by tissue staining Next, using MatTeK skin model EFT-400, UK5099 was added to the medium to 1 μM and 5 μM, and UK5099 was used as a control. The skin model was cultured at 2.5 mL / well with the same amount of DMSO added without treatment. The medium was changed 2 days later, and 4 days after the start of the culture, the medium was immersed in refrigerated acetone for dehydration and fixation. Then, the solvent was replaced twice with acetone at room temperature and methyl benzoate at room temperature, and twice with xylene at room temperature, and the paraffin was embedded to prepare a paraffin block. Sections were prepared with a thickness of 3 μm using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
　1μMおよび5μMのUK5099、コントロールをそれぞれ添加した結果を図５に示す。UK5099を添加するとMCSPおよびIntegrin β1発現細胞が増加しており、三次元皮膚モデルを使用しても実験1-1、1－2、1-3の結果と一致する結果となった。 Figure 5 shows the results of adding 1 μM and 5 μM UK5099 and controls, respectively. The addition of UK5099 increased MCSP and Integrin β1-expressing cells, which was consistent with the results of Experiments 1-1, 1-2, and 1-3 using the three-dimensional skin model.
　実験1-5；組織染色によるMCSPおよびIntegrin β1のタンパク質発現解析
次に、BioPredic社から購入した新鮮ヒト腹部皮膚を用いて、培地にUK5099を10μMとなるように添加し、対照にはUK5099で処理せず同量のDMSOを添加して3mL/wellで培養した。2日後、4日後に培地を交換し、培養開始から5日後に冷蔵アセトンに浸漬させ脱水固定した。その後、室温アセトン、室温の安息香酸メチルで２回、室温のキシレンで２回溶媒置換を行い、パラフィン包埋することでパラフィンブロックを作成した。ミクロトームを使い3μmの厚さで切片を作成した。キシレンを用いて脱パラフィンを行い、12% BSA/PBSでブロッキング後、1次抗体として抗MCSP抗体(Millipore, MAB2029, mouse mAb)、抗α6 integrin抗体(Santa Cruz, sc-19622, rat mAb)にて4℃で一晩反応させた。翌日、PBSで5分で3回洗浄後、二次抗体としてAlexa488-anti-mouse IgG抗体およびAlexa594-anti-rat IgG抗体にて室温、1時間反応させた。PBSで5分、3回洗浄後、DAPIを含む封入剤（Vectashield, H-1200）にて封入し、顕微鏡観察を行った。 Experiment 1-5; Protein expression analysis of MCSP and Integrin β1 by tissue staining Next, using fresh human abdominal skin purchased from BioPredic, UK5099 was added to the medium to a concentration of 10 μM, and the control was treated with UK5099. Instead, the same amount of DMSO was added and cultured at 3 mL / well. After 2 days and 4 days, the medium was changed, and 5 days after the start of culturing, the cells were immersed in refrigerated acetone for dehydration and fixation. Then, the solvent was replaced twice with acetone at room temperature and methyl benzoate at room temperature, and twice with xylene at room temperature, and the paraffin was embedded to prepare a paraffin block. Sections were prepared with a thickness of 3 μm using a microtome. After deparaffinization with xylene and blocking with 12% BSA / PBS, anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb) were used as primary antibodies. The reaction was carried out at 4 ° C. overnight. The next day, after washing with PBS three times for 5 minutes, the cells were reacted with Alexa 488-anti-mouse IgG antibody and Alexa 594-anti-rat IgG antibody as secondary antibodies at room temperature for 1 hour. After washing with PBS for 5 minutes and 3 times, the cells were encapsulated with a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.
　10μMのUK5099、コントロールをそれぞれ添加した結果を図６に示す。UK5099を添加するとMCSPおよびIntegrin β1発現細胞が増加しており、ex vivoのヒト皮膚試料を用いても実験1-1、1-2、1-3、1-4の結果と一致する結果となった。 Figure 6 shows the results of adding 10 μM UK5099 and control respectively. The addition of UK5099 increased MCSP and Integrin β1-expressing cells, which is consistent with the results of Experiments 1-1, 1-2, 1-3, and 1-4 using ex vivo human skin samples. It was.
　実験2: MCP1抑制作用を有する物質のスクリーニング
　実験1より、MCP1を抑制すると幹細胞が活性化することが示唆された。そこで、MCP1抑制作用を有する物質のスクリーニングを行った。
　実験2-1:試料の調製
　MPC1抑制作用の評価対象試料として以下を用いた。
Figure JPOXMLDOC01-appb-T000002
 Experiment 2: Screening of substances with MCP1 inhibitory effect Experiment 1 suggested that inhibition of MCP1 activates stem cells. Therefore, we screened substances that have an MCP1 inhibitory effect.
Experiment 2-1: Sample preparation The following samples were used for evaluation of the MPC1 inhibitory effect.
Figure JPOXMLDOC01-appb-T000002
　その他動植物の抽出物といった天然由来成分や合成成分を含め、合計124種類の候補試料を調製した。 A total of 124 types of candidate samples were prepared, including naturally derived components such as extracts of animals and plants and synthetic components.
　実験2-2: MCP1抑制作用の評価
　実験1-1と同様に正常ヒト胎児由来表皮角化細胞を培養し、播種24時間後に各薬剤を添加した。候補試料は、あらかじめDMSOにて10%にしたものを一次、二次スクリーニングでは1000倍希釈で培地に加え最終濃度0.01%とし、三次スクリーニングでは10000倍、1000倍、100倍希釈で培地に加え最終濃度が0.001％、0.01％、0.1％となるようにした。陽性対照として実験1と同様の方法でUK5099を20μMとなるように添加した。陰性対照としてDMSOを1000倍希釈で添加した。各薬剤を添加24時間後にmRNAを回収した。 Experiment 2-2: Evaluation of MCP1 inhibitory effect In the same manner as in Experiment 1-1, normal human fetal-derived epidermal keratinocytes were cultured, and each drug was added 24 hours after seeding. Candidate samples are prepared by preliminarily making 10% in DMSO and added to the medium at 1000-fold dilution in the primary and secondary screenings to a final concentration of 0.01%. The concentrations were adjusted to 0.001%, 0.01%, and 0.1%. As a positive control, UK5099 was added to 20 μM in the same manner as in Experiment 1. DMSO was added as a negative control at a 1000-fold dilution. The mRNA was recovered 24 hours after the addition of each drug.
　定量PCR法による遺伝子発現解析
　薬剤添加24時間後の細胞からRNAの回収は、Quiagen Rneasy mini Kit（Quiagen社）を用い、cDNAの合成はSuperScript VILO cDNA Synthesis Kit（Thermo Fisher Scientific社）にて行った。MPC1およびB2Mの遺伝子発現量は、Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan)を用いてSyber Green法にて行った。使用したプライマーは以下の通りである。
B2M forward： 5’-GTGGGATCGAGACATGTAAGCA-3’ （配列番号１）
B2M reverse： 5’-CAATCCAAATGCGGCATCT-3’ （配列番号２）
MPC1 forward： 5’-GTGCGGAAAGCGGCGGACTA-3’ （配列番号５）
MPC1 reverse： 5’-GGCAGCAATGGGAAGACCCCA-3’ （配列番号６） Gene expression analysis by quantitative PCR method RNA was recovered from cells 24 hours after drug addition using the Quiagen Rneasy mini Kit (Quiagen), and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). .. The gene expression levels of MPC1 and B2M were measured by the Cyber Green method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used are as follows.
B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1)
B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2)
MPC1 forward: 5'-GTGCGGAAAGCGGCGGACTA-3' (SEQ ID NO: 5)
MPC1 reverse: 5'-GGCAGCAATGGGAAGACCCCA-3' (SEQ ID NO: 6)
　一次スクリーニングはN=1、薬剤濃度0.01％で行った。一次スクリーニングの結果を図７に示す。MPC1抑制作用を有する物質の候補として124品から37品（図７に示すライン以下の物質）を選定した。
　一次スクリーニングで選定された37品につき、薬剤濃度0.01％でN=3で二次スクリーニングを行った。二次スクリーニングの結果を図８に示す。濃度依存的にMPC1抑制作用を有する物質の候補として37品から15品（図８に示す濃いグレーの物質）を選定した。
　二次スクリーニングで選定された15品につき、薬剤濃度0.001％、0.01％、0.1％、各N=3で三次スクリーニングを行った。三次スクリーニングの結果を図９に示す。再現性を持って濃度依存的にMPC1抑制作用を有する物質の候補として15品からアケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの6品（図９に示す濃いグレーの物質）を選定した。 The primary screening was performed with N = 1 and a drug concentration of 0.01%. The result of the primary screening is shown in FIG. Thirty-seven products (substances below the line shown in FIG. 7) were selected from 124 products as candidates for substances having an MPC1 inhibitory effect.
The 37 products selected in the primary screening were subjected to secondary screening at a drug concentration of 0.01% and N = 3. The result of the secondary screening is shown in FIG. Thirty-seven to fifteen substances (dark gray substances shown in FIG. 8) were selected as candidates for substances having an MPC1 inhibitory effect in a concentration-dependent manner.
The 15 products selected in the secondary screening were subjected to the tertiary screening at drug concentrations of 0.001%, 0.01%, 0.1%, and N = 3 each. The result of the tertiary screening is shown in FIG. From 15 products as candidates for substances that have a reproducible and concentration-dependent MPC1 inhibitory effect, 6 products: akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine (Fig. 9). (Dark gray substance shown in) was selected.
　選定した６品それぞれについて、図９の結果にDunnett's testにより統計処理を施したグラフを図１０に示す。図１０に示すように、全ての物質が濃度依存的に統計的有意差をもってMPC1抑制作用を奏していた。 Fig. 10 shows a graph in which the results of Fig. 9 were statistically processed by Dunnett's test for each of the 6 selected products. As shown in FIG. 10, all the substances exerted an MPC1 inhibitory effect with a statistically significant difference in a concentration-dependent manner.
　以上の結果により、MCP1抑制により皮膚幹細胞が活性化されること、及び、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインには、MCP1抑制作用があることがわかった。本発明のMCP1抑制剤により皮膚幹細胞が活性化されれば、皮膚の老化抑制に有効である。 Based on the above results, skin stem cells are activated by MCP1 inhibition, and akebi extract, black bean extract, sycamore extract, tea extract, jojoba leaf extract, and ergothioneine have an MCP1 inhibitory effect. I understood it. If skin stem cells are activated by the MCP1 inhibitor of the present invention, it is effective in suppressing skin aging.

Claims (7)

  1. 　MPC1抑制剤の適用により皮膚幹細胞を活性化する美容方法。 A beauty method that activates skin stem cells by applying an MPC1 inhibitor.
  2. 　前記MPC1抑制剤が、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含む、請求項１に記載の美容方法。 The beauty method according to claim 1, wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, sardine extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.
  3. 　アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含むMPC1抑制剤。 An MPC1 inhibitor containing at least one of akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.
  4. 　MPC1抑制剤を含む、皮膚幹細胞活性化剤。 A skin stem cell activator containing an MPC1 inhibitor.
  5. 　前記MPC1抑制剤が、アケビ抽出物、黒豆抽出物、シャクヤク抽出物、茶抽出物、ホホバ葉抽出物、及びエルゴチオネインの少なくともいずれかを有効成分として含む、請求項４に記載の皮膚幹細胞活性化剤。 The skin stem cell activator according to claim 4, wherein the MPC1 inhibitor contains at least one of akebi extract, black bean extract, shakyaku extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. ..
  6. 　MPC1抑制作用を指標とする、皮膚幹細胞活性化剤のスクリーニング方法。 A screening method for skin stem cell activators using the MPC1 inhibitory effect as an index.
  7. 　皮膚細胞を候補薬剤に接触させる工程、
    　候補薬剤に接触させた皮膚細胞におけるMPC1の活性、量、及び／又は発現量を測定する工程、
    　測定したMPC1の活性、量、及び／又は発現量から候補薬剤のMPC1抑制作用を決定する工程、
    　決定したMPC1抑制作用に基づき、皮膚幹細胞活性化剤を選択する工程、
    　を含む、請求項６に記載の方法。     The process of bringing skin cells into contact with a candidate drug,
    A step of measuring the activity, amount, and / or expression level of MPC1 in skin cells contacted with a candidate drug.
    A step of determining the MPC1 inhibitory effect of a candidate drug from the measured MPC1 activity, amount, and / or expression level,
    The step of selecting a skin stem cell activator based on the determined MPC1 inhibitory effect,
    6. The method of claim 6.
PCT/JP2020/047530 2019-12-20 2020-12-18 Method for activating skin stem cells through mpc1 suppression, and skin stem cell activation agent WO2021125346A1 (en)

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KR20170025365A (en) * 2015-08-28 2017-03-08 주식회사 엘지생활건강 Composition for improving skin
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