WO2012105542A1 - 皮膚外用剤 - Google Patents
皮膚外用剤 Download PDFInfo
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- WO2012105542A1 WO2012105542A1 PCT/JP2012/052118 JP2012052118W WO2012105542A1 WO 2012105542 A1 WO2012105542 A1 WO 2012105542A1 JP 2012052118 W JP2012052118 W JP 2012052118W WO 2012105542 A1 WO2012105542 A1 WO 2012105542A1
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- adenosine
- skin
- glucosyl
- acid
- glucosyl adenosine
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- 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
- A61K8/60—Sugars; Derivatives thereof
- A61K8/602—Glycosides, e.g. rutin
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- 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
- A61K8/60—Sugars; Derivatives thereof
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- 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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- 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
- A61K8/60—Sugars; Derivatives thereof
- A61K8/606—Nucleosides; Nucleotides; Nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- 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
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- 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
- A61P17/16—Emollients or protectives, e.g. against radiation
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- 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
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- 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
- A61Q19/001—Preparations for care of the lips
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- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Definitions
- the present invention relates to ⁇ -D-glucopyranosyl- (1 ⁇ 3 ′)-adenosine and / or ⁇ -D-glucopyranosyl- (1 ⁇ 5 ′)-adenosine (hereinafter “3′-glucosyladenosine” and “5′-”, respectively).
- the present invention relates to a long-lasting anti-wrinkle skin preparation, which is referred to as “glucosyl adenosine” and sometimes collectively referred to as “glucosyl adenosine”.
- the skin forms the outermost layer of the body, protects the living body from the outside world, and prevents the moisture and nutrients in the body from leaking out to the outside world, mainly the fibroblasts, Collagen (collagen) fiber, elastic fiber, proteoglycan, etc. have a complex three-dimensional structure and are composed of thick dermis (connective tissue) that brings strength, extensibility and elasticity to the skin. .
- the skin condition is maintained by the epidermis and dermis both maintaining their healthy tissue structure and physiology.
- the epidermis of the skin is divided from the dermis side into “basal layer”, “spinous layer”, “granular layer” and “keratin layer (horny layer)”, and is mainly composed of cells called keratinocytes (keratinocytes) ing.
- Keratinocytes form a monolayer as an immature cell with the ability to divide in the lowest basal layer, divide and proliferate while being pushed up to the upper layer (“keratinization"), and finally die without a nucleus It becomes a stratum corneum which is a cell, forms a stratum corneum, and becomes a plaque from the surface layer part and falls off.
- the keratinocyte proliferation, migration, differentiation, and shedding processes are maintained in a constant state by smoothly turning over at a constant cycle.
- the skin deteriorates due to various factors such as aging, drying, oxidation, sunlight (ultraviolet rays), etc., and loses moisture, wrinkles are generated, sagging occurs, tension and elasticity decrease Presents a phenomenon.
- Such skin deterioration is considered to be caused by a decrease in the amount of collagen in the dermis due to a decrease in collagen synthesis by fibroblasts in the dermis, in order to improve the decrease in the amount of collagen.
- Numerous external preparations for skin, foods and drinks, etc., in which collagen or a collagen production enhancer is blended, have been developed (see, for example, Japanese Patent No. 3495217 and Japanese Patent Application Laid-Open No. 2007-186471).
- prolactin is a peptide
- stability when formulated or used as an external preparation on the skin There are problems in terms of permeability to the basement layer.
- a novel component that has an excellent anti-wrinkle action by acting on both the epidermis and the dermis and also has a long-lasting effect and a skin external preparation using the same are still strong. It is desired.
- the present invention helps improve the tissue structure and physiological function of both the epidermis and the dermis, has an effect of improving the skin, maintains the normal state of the skin, and It is an object of the present invention to provide a novel and long-lasting anti-wrinkle skin external preparation that is stable even when applied and does not adversely affect the living body.
- the present inventor pays attention to nucleic acid-related substances in order to solve the above-mentioned problems, repeats various studies and repeats various trials and errors, and is a component originally present in the living body from the viewpoint of safety to the living body.
- Adenosine was selected and studied.
- Adenosine itself is known to have an action of enhancing collagen production in the dermis and an effect of enhancing proliferation of keratinocytes of hair papilla cells (for example, International Publication WO05 / 034902 and International Publication WO05 / 044205).
- adenosine is rapidly metabolized by an enzyme originally present in the living body, and its effect may not be sustained.
- adenosine has low solubility in a hydrophilic solvent, and it is an external preparation for skin. It has been found that it is difficult to handle in the case of blending with.
- the present inventor conducted further research to solve such problems, and as a result, D-glucose was present at the hydroxyl group at the 3′-position or 5′-position of adenosine (the 3-position or 5-position of ribose in the adenosine molecule).
- the present inventor has found that the above-mentioned glucosyl adenosine also acts on dermal fibroblasts, and has a significantly superior collagen production enhancing action than adenosine.
- the present inventor has found that the above glucosyl adenosine is more soluble in a hydrophilic solvent than adenosine.
- the present inventor has found that the glucosyl adenosine is less susceptible to cell damage than adenosine and is more stable than adenosine in vivo as described above, but is gradually degraded and the produced adenosine is rapidly metabolized. Therefore, it has been found that it is a safe substance even when applied to humans, and it is more useful than adenosine as a component of an external preparation for skin that has an anti-wrinkle effect, in terms of strength, durability, and safety. It was confirmed that the present invention was completed.
- the present invention solves the above-mentioned problems by providing a skin external preparation having a persistent anti-wrinkle action containing 3′-glucosyl adenosine and / or 5′-glucosyl adenosine as an active ingredient.
- 3′-glucosyl adenosine and 5′-glucosyl adenosine itself are, for example, Yukio Suzuki, Satoshi Uchida, “Science and Industry”, 65, 6, 265-274 (1991), Shuji Takahashi, etc. Proliferation of epidermal keratinocytes, although it is a known substance, as disclosed in “The Journal of Antibiotics”, Vol. 47, No. 1, pp.
- the topical skin preparation of the present invention comprising the glucosyl adenosine of the present invention as an active ingredient has both the proliferation and differentiation promoting action of epidermal keratinocytes and the collagen production enhancing action of dermal fibroblasts.
- the glucosyl adenosine of the present invention is more soluble in a hydrophilic solvent than adenosine, and is easier to handle than adenosine when blended in a topical skin preparation.
- the glucosyl adenosine of the present invention is more stable than adenosine and is more difficult to be degraded by enzymes than other glycosides such as ascorbic acid 2-glucoside
- the glucosyl adenosine is gradually degraded by the action of degrading enzymes present in the living body. Since it is decomposed into D-glucose and adenosine and the produced adenosine is rapidly metabolized, it is a safe substance to be applied to humans, and is superior in a sustained effect than adenosine.
- the glucosyl adenosine of the present invention has an anti-wrinkle action with excellent durability of the effect, it is particularly suitable for use as a skin external preparation applied to the skin for a relatively long time.
- the antibacterial agent having antiseptic or bacteriostatic action to be used is preferably one that has little irritation to the skin even when used continuously and does not impair the effects of the glucosyladenosine of the present invention. The effects of the present invention can be greatly exhibited.
- the glucosyl adenosine of the present invention is stable against enzymes and the like, it is hardly affected even when used in combination with other components used for external preparations for skin, especially in combination with natural components such as plant extracts. In that case, since there is little fear of being decomposed by enzymes contained therein, it is possible to produce an excellent skin external preparation that can stably exhibit the anti-wrinkle action of glucosyladenosine as well as the activities of these other components. .
- the present invention relates to a skin external preparation having a persistent anti-wrinkle action comprising 3′-glucosyl adenosine and / or 5′-glucosyl adenosine as an active ingredient.
- the external preparation for skin referred to in the present invention contains the above glucosyl adenosine, it has both the action of promoting the proliferation and differentiation of epidermal keratinocytes and the action of enhancing collagen production, and is also excellent in sustainability of the effect. It can suppress the deterioration of the skin including wrinkles and can be used to improve various skin symptoms such as rough skin, dullness, decreased tension and elasticity, and xeroderma.
- the glucosyl adenosine used as an active ingredient of the external preparation for skin of the present invention may be any of those containing one or both of 3′-glucosyl adenosine and 5′-glucosyl adenosine.
- 5′-glucosyl adenosine is excellent in stability to biological components such as enzymes existing in the living body, has high solubility in hydrophilic solvents such as water, and 3′-glucosyl adenosine. Since it is easy to exert an effect at a relatively low concentration, it is desirable to appropriately combine them according to the dosage form and purpose of the external preparation for skin.
- the external preparation for skin of the present invention comprises ⁇ -glycosyladenosine in which one or more molecules of D-glucose are ⁇ -1,4, ⁇ -1,6 and / or ⁇ -1,3 linked to glucose of the above glucosyladenosine. You may contain.
- Glucosyladenosine used in the external preparation for skin of the present invention can be prepared by an enzymatic method. Moreover, it can also prepare by a fermentation method and a synthesis method as needed. If economic efficiency is a problem, an enzymatic method using a glycosyltransferase is advantageous.
- the glucosyl adenosine used in the present invention does not necessarily have to be highly purified, there is no problem in safety such as containing impurities that adversely affect the skin, and the purity thereof is not impaired unless the intended effect is hindered. There are no particular restrictions on. Depending on the dosage form and dosage form of the external preparation for skin of the present invention, even the reaction solution itself containing an adenosine used as a production raw material itself is an unseparated composition with other substances specific to the production method It may be in the form of a partially purified product or a highly purified product. Further, highly purified 3′-glucosyl adenosine and 5′-glucosyl adenosine can be appropriately mixed and used depending on the purpose.
- the proliferation and differentiation promoting action of epidermal keratinocytes referred to in the present invention means keratinocyte division and proliferation existing in the basal layer of the epidermis, and forms spiny layer, granule layer and stratum corneum while such cells are pushed up to the upper layer. It refers to an action that promotes the whole process and maintains a healthy epidermis, either in the process of differentiating into cells and eventually reaching dead cells without nuclei.
- the collagen production enhancing action referred to in the present invention means an action of enhancing the collagen production of fibroblasts existing in the dermis.
- the dosage of the external preparation for skin of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained, and is usually divided into one to several times per day, and a predetermined amount per day is administered. do it.
- the daily dose is usually 0.001 to 100 mg per 1 cm 2 of skin as glucosyl adenosine as an active ingredient, preferably 0.05 to 10 mg, more preferably 0.01 to 1 mg. If the transdermal dose is less than 0.001 mg / cm 2 of skin, the desired effect may not be obtained, and if it exceeds 100 mg, the effect corresponding to the dose may not be obtained. .
- the glucosyl adenosine of the present invention can be used alone as an active ingredient of an external preparation for skin. However, as long as the effects of the present invention are not hindered, other skin improvement actions, collagen production enhancing actions and keratinocyte proliferation and differentiation promoting actions are exhibited. It is also optional to use any other medicinal ingredients such as whitening agents, antioxidants, anti-inflammatory agents, moisturizers, etc., as appropriate, and together with their effects, the effects of the above glucosyl adenosine It can also be used as a skin external preparation.
- the blending amount of glucosyl adenosine in the external preparation for skin of the present invention is not limited as long as the effect can be achieved depending on the form, dosage form, etc., but is usually 0.001 to 30% by mass in the total amount of the preparation. What is necessary is just to mix
- the glucosyl adenosine of the present invention may not provide an effect commensurate with the blended amount even when blended in an external preparation for skin exceeding 30% by mass, and the desired effect is obtained when it is less than 0.001% by mass. It may not be obtained.
- the external preparation for skin of the present invention promotes the proliferation and differentiation of keratinocytes present in the epidermis, improves the supply of corneocytes and promotes the turnover of the stratum corneum, thereby improving the state of the skin that has deteriorated function, It can be maintained in an appropriate state.
- the skin external preparation of the present invention enhances the expression of genes involved in ceramide synthesis by epidermal keratinocytes and collagen synthesis by dermal fibroblasts, and increases the amount of ceramide in the epidermis and the amount of collagen in the dermis to increase the elasticity of the skin. It can improve the property, tension, barrier function, etc., improve the condition of the skin that has deteriorated, and maintain it in an appropriate state.
- the external preparation for skin of the present invention improves the skin moisturizing function, and is suitable for rough skin, wrinkles, reduced skin tension and elasticity, reduced skin barrier function, dry skin, psoriasis, psoriasis, etc. Can prevent or ameliorate skin symptoms.
- the skin external preparation of the present invention is a keratinocyte proliferation and differentiation promoting agent, Not only for the production of collagen production enhancers and ceramide synthesis promoters, but also for use in the production of humectants.
- it is also optional to produce an expression enhancer for ⁇ -glucocerebrosidase (GCase) and sphingomyelinase (SMase), which are enzymes involved in ceramide synthesis.
- Antibacterial agents include benzoic acid and its salts, salicylic acid and its salts, sorbic acid and its salts, dehydroacetic acid and its salts, paraoxybenzoic acid esters including paraoxybenzoic acid alkyl esters, edetic acid, 2,4,4 '-Trichloro-2'-hydroxydiphenyl ether, 3,4,4'-trichlorocarbanilide, hexachlorophene, benzalkonium chloride, phenoxyethanol, hinokitiol, resorcinol, ethanol, 1,3-butylene glycol, photosensitizer 201 , Lactic acid, 1,2-alkanediol, gardenia extract, clara extract, sage extract, thyme extract
- the external preparation for skin of the present invention has the effect of the present invention by blending with an antibacterial agent that causes little irritation to the skin even when used continuously and does not impair the effects of the glucosyladenosine of the present invention. It can be demonstrated greatly.
- non-paraoxybenzoic acid ester components such as 1,2-alkanediol and 1,3-butylene glycol are low in skin irritation, high in preservative effect and moisturizing. To preferred.
- 1,2-alkanediol a 1,2-alkanediol having 4 to 10 carbon atoms is preferable, and in particular, 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, One or more 1,2-alkanediols selected from 2-octanediol are particularly preferred.
- blend the glucosyl adenosine of this invention and these antibacterial agents uniformly in a skin external preparation unless the effect of this invention is impaired, it is also optional to use an appropriate surfactant.
- the external preparation for skin of the present invention is used together with glucosyl adenosine, usually in a form in which one or more kinds of other optional components used in cosmetics, quasi-drugs, pharmaceuticals and the like are appropriately blended as necessary. can do.
- other optional components used together with glucosyl adenosine include, for example, whitening agents, antioxidants, anti-inflammatory agents, moisturizers, ultraviolet absorbers, emulsifiers, thickeners and the like. The following components can be mentioned.
- the whitening agent examples include L-ascorbic acid or a derivative thereof, arbutin, kojic acid, ellagic acid, placental extract, hydroquinone glycoside, tranexamic acid or a derivative thereof, alkylresorcinol such as resorcin, 4-n-butylresorcinol and the like. And resorcins and derivatives thereof, such as these salts, glutathione, cyanobacteria extract, licorice extract, oxon extract, ginkgo biloba extract, age extract, magnolia extract, peonies extract, gardenia extract, sage extract, toki extract, iris extract And plant extracts such as Acacia yak extract.
- L-ascorbic acid 2-glucoside and tranexamic acid are desirable because they have an excellent effect of enhancing the physiological activity of glucosyladenosine.
- Anti-acid oxidation agent for example, butylhydroxytoluene, tocopherol, phytic, vitamin A such as retinol and its derivatives, vitamin B 6 hydrochloride, vitamin B 6 tripalmitate, vitamin B 6 dioctanoate, vitamin B 2 and its Derivatives, vitamin B 12 such as vitamin B 12 , vitamin B 15 and derivatives thereof, vitamin C such as ascorbic acid and derivatives thereof, vitamin E such as ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, vitamin E acetate, Examples of extracts of plants having antioxidant activity such as vitamin D, glutathione, rutin, hesperidin, naringin and its derivatives, hypericum perforatum extract, ages extract, ougon extract, oolong tea, tea, green tea and other tea extracts, matsutake extract It is done.
- vitamin A such as retinol and its derivatives
- vitamin B 6 hydrochloride vitamin B 6
- Anti-inflammatory agents include allantoin or derivatives thereof, glycyrrhetin or its derivatives glycyrrhetinic acid, glycyrrhizic acid, glycyrrhetinic acid allantoin, glycyrrhetinic acid glycerin, glycyrrhetinic acid stearyl, stearic acid glycyrrhetinic acid, 3-succinyloxyglycyrrhetinic acid disodium, glycyrrhizic acid Dipotassium, monoammonium glycyrrhizinate, pantothenic acid and its derivatives, vitamin E and its derivatives, L-ascorbic acid, L-ascorbic acid-2-glucoside, ascorbic acid-tocopherol phosphate diester, L-ascorbic acid sulfate, di- Ascorbyl palmitate, ascor
- humectant examples include polyethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, hexylene glycol, xylitol, sorbitol, maltitol, chondroitin sulfate, hyaluronic acid, mucoitin sulfate, caronic acid, atelocollagen, cholesteryl-12 -Hydroxystearate, sodium lactate, bile salt, dl-pyrrolidone carboxylate, short-chain soluble collagen, diglycerin (EO) PO adduct, Izayoi rose extract, yarrow extract, merilot extract, aloe extract, assembly Extract, Clara extract, Loofah extract, Maronnier extract, Yukinoshita extract, Thyme extract, Tokyo extract, Lily extract, Gardenia extract, Fennel extract, Odorikosou extract, Peppa Extracts of plants such as mint extract, etc. tranexa
- UV absorber examples include paraaminobenzoic acid (hereinafter abbreviated as “PABA”), PABA monoglycerin ester, N, N-dipropoxy PABA ethyl ester, N, N-diethoxy PABA ethyl ester, N, N-dimethyl PABA.
- PABA paraaminobenzoic acid
- Benzoic acid UV absorbers such as ethyl ester, N, N-dimethyl PABA butyl ester, N, N-dimethyl PABA methyl ester, anthranilic acid UV absorbers such as homomenthyl-N-acetylanthranilate, amyl salicylate, menthyl Salicylic acid-based UV absorbers such as salicylate, homomenthyl salicylate, octyl salicylate, phenyl salicylate, benzyl salicylate, p-isopropanol phenyl salicylate, octylcinnamate, ethyl-4-isopro Rucinnamate, methyl-2,5-diisopropylcinnamate, ethyl-2,4-diisopropylcinnamate, methyl-2,4-diisopropylcinnamate, propyl-p-methoxycinnamate, isoprop
- the ultraviolet scattering agent examples include powders of titanium oxide, fine particle titanium oxide, zinc oxide, fine particle zinc oxide, iron oxide, fine particle iron oxide, cerium oxide and the like.
- these ultraviolet scattering agents needle-like, spindle-like, spherical and granular powders are usually used.
- a fine particle powder having a particle size of 01 ⁇ m or less is preferred.
- Silicone treatment such as methyl hydrogen polysiloxane and silane coupling agent; metal soap treatment; UV scattering agent that has been hydrophobized by fluorine treatment such as perfluoroalkyl phosphate diethanolamine salt and perfluoroalkyl silane, dextrin fatty acid ester treatment, etc. Is also preferable.
- liquid fats examples include avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, rice germ oil, sasanca oil, castor oil Oil, linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, snail oil, Japanese kiri oil, jojoba oil, triglycerin and the like.
- solid fat examples include cacao butter, palm oil, horse fat, hydrogenated palm oil, palm oil, beef tallow, sheep fat, hydrogenated beef tallow, palm kernel oil, pork fat, beef bone fat, owl kernel oil, hydrogenated oil, cattle Leg fats, moles, hydrogenated castor oil and the like.
- waxes examples include beeswax, candelilla wax, cotton wax, carnauba wax, bayberry wax, ibota wax, whale wax, montan wax, nuka wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugar cane wax, lanolin fatty acid isopropyl, hexyl laurate, and reduced lanolin.
- hydrocarbon oil examples include liquid paraffin, ozokerite, squalane, pristane, paraffin, ceresin, squalene, petrolatum, microcrystalline wax, polyethylene wax, and Fischer Tropus wax.
- higher fatty acids examples include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, undecylenic acid, toluic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the like. Is mentioned.
- higher alcohols examples include linear alcohols (eg, lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol); branched chain alcohols (eg, monostearyl glycerin ether (batyl alcohol) ), 2-decyltetradecinol, lanolin alcohol, cholesterol, phytosterol, hexyl decanol, octyldodecanol and the like.
- linear alcohols eg, lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol
- branched chain alcohols eg, monostearyl glycerin ether (batyl alcohol)
- 2-decyltetradecinol lanolin alcohol
- cholesterol phytosterol
- phytosterol hexyl decanol
- Synthetic ester oils include isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyl decyl dimethyloctanoate, cetyl lactate, myristyl lactate Lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, N-alkyl glycol monoisostearate, neopentyl glycol dicaprate, apple Acid diisostearyl, di-2-heptylundecanoic acid glycerin, tri-2-ethylhexanoic acid
- silicone oil examples include linear polysiloxanes (for example, dimethylpolysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane, etc.); cyclic polysiloxanes (for example, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexyl).
- silicone resins that form a three-dimensional network structure various modified polysiloxanes (amino-modified polysiloxane, polyether-modified polysiloxane, alkyl-modified polysiloxane, fluorine-modified polysiloxane, etc.) It is done.
- lower alcohols such as ethanol
- acyl sarcosine acids for example, sodium lauroyl sarcosine
- organic acids such as citric acid, malic acid, tartaric acid, and lactic acid
- vitamins such as vitamin H, pantothenic acid, and pantethine
- nicotinamide, Saponins such as benzyl nicotinate, ⁇ -oryzanol, allantoin, glycyrrhizic acid (salt), glycyrrhetinic acid and its derivatives, hinokitiol, bisabolol, eucalptone, thymol, inositol, saikosaponin, carrot saponin, hechimasaponin, muclodisaponin
- Various drugs such as pantothenyl ethyl ether, ethinyl estradiol, cephalanthin, placenta extract, borage
- metal sequestering agents such as disodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives , Rutin, hesperidin, naringin and its derivatives and other polyphenols, licorice extract, grabrizine, hot water extract of fire thorn fruit, carnitine, coenzyme Q 10 , various herbal medicines, tocopherol acetate, hydroxydecenoic acid, glycyrrhizic acid And derivatives thereof or salts thereof, glucose, fructose, mannose, sucrose, ⁇ , ⁇ -trehalose, carbohydrate derivatives of ⁇ , ⁇ -trehalose, cyclic tetrasaccharide, dextrin, cyclodextrin, branched dextrin, starch,
- the skin external preparation of the present invention includes cosmetics, pharmaceuticals, and quasi drugs, and the dosage forms thereof are aqueous solutions, solubilization systems, emulsification systems, oil liquid systems, gel systems, paste systems, ointment systems, aerosol systems, water -Includes any two-layer system, water-oil-powder three-layer system. Moreover, what was carry
- quasi-drugs that are applied to or pasted on the skin and mucous membranes, such as ointments, patches, and film preparations, and used directly in contact with the skin when used in the oral cavity, such as mouth fresheners. This includes drugs and quasi drugs.
- glucosyl adenosine which is an active ingredient of the external preparation for skin of the present invention, should not be limited to those produced by a specific method and means as described above, but it is economical. If it is a problem, an enzymatic method using a glycosyltransferase is advantageous.
- a solution containing starch and adenosine is added to cyclomaltodextrin / glucanotransferase (CGTase), ⁇ -glucosidase, amylase, ⁇ -isomaltosylglucosaccharide producing enzyme (see, for example, pamphlet of WO02 / 10361), isomaltosyl.
- CCTase cyclomaltodextrin / glucanotransferase
- ⁇ -glucosidase ⁇ -glucosidase
- amylase ⁇ -isomaltosylglucosaccharide producing enzyme
- isomaltosyl isomaltosyl.
- Transferase see, for example, International Publication WO01 / 90338), ⁇ -glucosyltransferase (International Publication WO2008 / 136331), and hydrolyzing starch and catalyzing the transfer of glycosyl groups to produce cyclodextrin It can be produced in large quantities and at low cost by an enzymatic method using an enzyme having transglycosylation activity such as ⁇ -amylase (international publication WO2008 / 136331 pamphlet) that acts on pullulan to produce panose.
- ⁇ -glucosyltransferase disclosed in WO 2008/136331 pamphlet is used, 5′-glucosyl adenosine having high water solubility can be obtained in high yield.
- glucosyl adenosine having a higher proportion of 3′-glucosyl adenosine can be produced in a higher yield than when ⁇ -glucosyltransferase is used.
- One or two or more molecules of D-glucose is added to the hydroxyl group at the 3′-position or 5′-position of adenosine by allowing ⁇ -glucosyltransferase or CGTase having glycosyltransferase activity to act on a solution containing starch and adenosine.
- 3′-glucosyladenosine and 5′-glucosyladenosine in which one molecule of D-glucose is bonded to the 3′-position or 5′-hydroxyl group, and the 3′-position or 5′-position hydroxyl group 2 'or more D-glucose bound, for example 3'-glycosyl adenosine such as 3'-maltosyl adenosine or 3'-maltotriosyl adenosine, or for example 5'-maltosyl adenosine or 5'-malto A 5'-glycosyl adenosine such as triosyladenosine is produced.
- 3'-glycosyl adenosine such as 3'-maltosyl adenosine or 3'-maltotriosyl adenosine
- ⁇ -glucosyltransferase and CGTase are usually 1 to 2,000 units per gram of starch, preferably 1 to 2,000 units, preferably in an aqueous solution in which starch and adenosine are dissolved so that the starch concentration is 1 to 40 w / v%.
- the solution is added at a rate of 1 to 1,000 units, and the solution is allowed to react for 6 hours or more, preferably about 12 to 96 hours while maintaining the pH at about 3 to 10 and the temperature of 30 to 70 ° C.
- the mass ratio of starch and adenosine in the solution is usually set in the range of 1: 2 to 20: 1, preferably 1: 1 to 10: 1 in terms of anhydride.
- the starch content exceeds this range, the sugar transfer to adenosine proceeds efficiently, but the production rate of adenosine is limited by the initial concentration of adenosine and remains at a low level.
- the ratio of adenosine is larger than the above range, a large amount of unreacted adenosine remains, which is not preferable for industrial production. Therefore, it was determined to be the best within the above ratio range.
- isoamylase When isoamylase is used as a starch debranching enzyme in addition to ⁇ -glucosyltransferase and CGTase, isoamylase is allowed to coexist with ⁇ -glucosyltransferase and CGTase in a solution containing adenosine and starch. It is desirable to act on the starch in a wet state. The amount added depends on the optimum temperature and pH of the isoamylase, but it is usually 200 to 2,500 units per gram of starch, and it is desirable to react at 55 ° C. or lower. Moreover, what is necessary is just to use according to isoamylase also when using a pullulanase as a starch debranching enzyme.
- glucosyl adenosine-containing solutions may be used as they are as raw materials for the external preparation for skin of the present invention, and may be used by concentrating as necessary. Further, an appropriate method such as vacuum drying or spray drying is used. May be dried and pulverized if necessary.
- starch debranching enzyme such as isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41) can be advantageously used in combination.
- isoamylase is particularly preferable because it is easy to handle in terms of enzyme activity, substrate specificity, and the like.
- starch include potato starch, sweet potato starch, tapioca starch, corn starch, and wheat starch.
- starches such as dextrin
- a partially decomposed product of these starches such as dextrin
- starchy substances such as amylose.
- the glucoamylase used for cleaving two or more D-glucose chains bonded to the hydroxyl group at the 3′-position and / or the 5′-position of adenosine may be a commercial product regardless of its source and purity.
- a commercially available glucoamylase agent an enzyme agent derived from a microorganism belonging to the genus Rhizopus marketed by Nagase ChemteX Corporation under the trade name “GlucoTeam # 20000”, or Amano Enzyme Inc. under the trade name “Gluczyme”.
- Enzymes derived from microorganisms of the genus Aspergillus or Rhizopus can be used.
- ⁇ Experiment 1 Preparation of glucosyladenosine with ⁇ -glucosyltransferase>
- ⁇ Experiment 1-1 Preparation of ⁇ -Glucosyltransferase>
- yeast partial extract trade name “Paindex # 4” manufactured by Matsutani Chemical Industry Co., Ltd.
- yeast extract Product name “Polypeptone”, manufactured by Nippon Pharmaceutical Co., Ltd.
- yeast extract trade name “Yeast Extract S”, manufactured by Nippon Pharmaceutical Co., Ltd.
- 0.1 w / v% dipotassium phosphate 0 0.1 w / v%, monosodium phosphate dihydrate 0.06 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, manganese sul
- the culture supernatant was collected by centrifugation, added with ammonium sulfate so as to be 80% saturated, and allowed to stand at 4 ° C. for 24 hours for salting out.
- the salted-out product was collected by centrifugation, dissolved by adding 20 mM acetate buffer (pH 6.0), dialyzed against the same buffer, and concentrated to prepare a concentrated crude enzyme solution.
- the ⁇ -glucosyltransferase activity of this concentrated crude enzyme solution was about 1300 units / ml.
- an amylase activity of about 140 units / ml was also observed in this concentrated enzyme solution.
- the activity of the ⁇ -glucosyltransferase is measured as follows. Maltose is dissolved in 20 mM acetate buffer (pH 6.0) to a final concentration of 1 w / v% to make a substrate. After adding 0.5 ml of the enzyme solution to 5 ml of this substrate solution and reacting at 40 ° C. for 30 minutes, 0.5 ml of the reaction solution and 5 ml of 20 mM acetate buffer (pH 7.0) are mixed, and the mixture is then added in boiling water. After stopping the reaction by heating for 10 minutes, the amount of glucose in the reaction solution is measured by a glucose oxidase method according to a conventional method, and the amount of glucose produced by the reaction is calculated.
- One unit of activity of ⁇ -glucosyltransferase is defined as the amount of enzyme that produces 1 micromole of glucose per minute under the above conditions.
- the activity of the mixed amylase was measured as follows. That is, short-chain amylose (trade name “Amylose EX-I”, sold by Hayashibara Biochemical Laboratories, Inc., average polymerization degree 17) is 50 mM acetate buffer containing 1 mM calcium chloride so that the final concentration is 1 w / v%. Dissolve in (pH 6.0) to obtain a substrate solution, add 0.2 ml of the enzyme solution to 2 ml of the substrate solution, and perform an enzyme reaction at 35 ° C.
- reaction solution for 30 minutes, and 0.2 ml of the reaction solution into 8 ml of 0.02N sulfuric acid solution. After mixing and stopping the reaction, 0.2 ml of 0.1N iodine solution is added, and after maintaining at 25 ° C. for 15 minutes, the absorbance at 660 nm is measured. Separately, the reaction solution at 0 hour reaction is measured in the same manner, and the decrease in iodine coloration per reaction time is measured.
- One unit of amylase activity was defined as 10 times the amount of enzyme that reduced the absorbance (iodine coloration) at 660 nm of 20 mg of short-chain amylose by 10% under the above conditions.
- glucoamylase Nagase Seikagaku Corporation, trade name “XL-4”, 3,800 units / ml
- 1,000 units per unit were added and reacted at 50 ° C. for 24 hours and treated at 100 ° C. for 10 minutes to stop the enzyme reaction.
- the column was washed with 20 volume% ethanol solution (5 times the volume) and 20 volume% ethanol solution (3 times the wet resin volume) and eluted with 40 volume% ethanol solution.
- the eluate was fractionated by 50 ml, and fractions having an ultraviolet absorption (260 nm) of 0.15 or more were collected.
- HPLC high performance liquid column chromatography
- Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) disclosed in JP-A-50-63189 (JP-B-53-27791) Tc-91 strain (Accession No. FERM BP- 11273, deposited at the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba City 1-1-1 Tsukuba City, Ibaraki Prefecture; transferred to the International Deposit of Deposit No. FERM P-2225) CGTase (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) was added at 1,000 units per gram of dextrin, reacted at 50 ° C. for 24 hours, then heated at 100 ° C.
- the column was washed with 20 volume% ethanol solution (6 times the wet resin volume) and eluted with 40 volume% ethanol solution (16 times the wet resin volume).
- the eluate was fractionated by 50 ml, and fractions having an ultraviolet absorption (260 nm) of 0.15 or more were collected.
- the collected fraction was concentrated and then subjected to preparative HPLC using an ODS column to collect the main peak of the glucosyladenosine-containing fraction.
- the collected fraction was adsorbed on an activated carbon column for desalting, eluted with 40% by volume ethanol, and concentrated with a rotary evaporator to collect the eluted fraction of glucosyladenosine.
- Preparative HPLC was the same as in Experiment 1-2, except that 20 mM acetic acid-ammonium acetate buffer (pH 3.5) / methanol 82/18 (volume / volume) was used as the mobile phase, and the column temperature was 40 ° C. Then, about 1.6 g of a preparation containing 99% by mass or more of glucosyl adenosine in terms of anhydride was prepared under the same conditions as the fractionation of glucosyl adenosine.
- the main peak of glucosyl adenosine eluted from preparative HPLC using an ODS column was eluted at a different retention time from the main peak of glucosyl adenosine eluted in Experiment 1-2. Further, it was confirmed in advance by measurement of molecular weight by mass spectrum (MS) analysis by a conventional method that glucosyl adenosine was contained in such a main peak.
- MS mass spectrum
- glucoseladenosine 100 mg of 5′-glucosyladenosine or 3′-glucosyladenosine (hereinafter, both compounds may be collectively referred to as “glucosyladenosine”) are dissolved in 500 ⁇ l of pure water at room temperature (25 ° C.). It was. Moreover, 5 mg of adenosine was dissolved in 500 ⁇ l of pure water at room temperature (25 ° C.). After standing in a temperature-controlled room at 25 ° C. for 1 day, the supernatant from which the undissolved precipitate was removed by centrifugation was subjected to ultrafiltration (trade name “Ultrafree 0.5 Biomax-5 Membrane”, sold by Japan Millipore).
- adenosine is only 0.8 w / v% soluble in water at room temperature (25 ° C.), whereas 3′-glucosyladenosine dissolves in water up to 2 w / v%.
- the solubility in water also increased.
- 5′-glucosyl adenosine was dissolved in water at a temperature exceeding 16.1 w / v% at room temperature, and the solubility in water was significantly increased as compared with adenosine.
- adenosine only 0.4 w / v% of adenosine is dissolved in water at 4 ° C., whereas 3′-glucosyl adenosine is dissolved up to 2.0 w / v%, and the solubility in water is higher than that of adenosine. . Furthermore, 5′-glucosyl adenosine was dissolved in water at 4 ° C. in excess of 15.7 w / v%, and the solubility in water was significantly increased as compared with adenosine.
- 3′-glucosyl adenosine and 5′-glucosyl adenosine can be prepared, for example, in the form of a solution when used for the manufacture of a skin external preparation using a hydrophilic solvent, and there is no risk of insolubilization and precipitation during the preparation. It shows that the processability in producing a homogeneous skin external preparation is improved.
- ⁇ Experiment 4 Persistence of glucosyl adenosine>
- ⁇ Experiment 4-1 Resistance to adenosine deaminase> It has been confirmed that adenosine is rapidly degraded into inosine and hypoxanthine by adenosine deaminase (ADA) originally present in the living body. Therefore, in order to investigate the possibility that the effect of glucosyl adenosine, which is a glycoside of adenosine, can be exerted continuously, cell lysate of human fetal normal fibroblasts is used as a source of enzymes involved in adenosine metabolism.
- ADA adenosine deaminase
- FCS Dulbecco's minimum essential medium containing 10% by volume fetal bovine serum
- DMEM Human fetal normal fibroblasts
- NHDF cells Human fetal normal fibroblasts (sold by Kurabo Industries, hereinafter referred to as “NHDF cells”) diluted in 1 in a 150 cm 2 culture flask (Corning) at 1.5 ⁇ 10 6 cells / 20 ml / flask. Cultured for days.
- the suspension was treated with a 0.05 mass% trypsin / EDTA solution (trade name “Trypsin-EDTA” sold by Gibco) and the cell suspension was recovered from the flask. After centrifuging the cell suspension and removing the supernatant, 10 mM Tris-HCl (pH 7.5) buffer solution (protease inhibitor cocktail (Complete, EDTA-free, tablets, manufactured by Roche)) dissolved in the cell pellet ( Lysis buffer solution) was added, and NHDF cells were suspended to 4.0 ⁇ 10 6 c / ml.
- Tris-HCl pH 7.5
- prote inhibitor cocktail Complete, EDTA-free, tablets, manufactured by Roche
- the suspension was sonicated (BRANSON SINIFIER CELL DISPTOR 185), and the cells were crushed (3 times, 30 seconds ⁇ 2 times each), then centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected.
- Cellulase was prepared. 5 ml of 5′-glucosyl adenosine prepared in Experiment 1-2 or 3 ml of 3′-glucosyl adenosine prepared in Experiment 2 were dissolved to a concentration of 5 mM, and 0.1 ml of the above-mentioned cell lysate was added. The mixture was stirred and reacted at 37 ° C.
- adenosine was rapidly degraded by the enzyme contained in the cell lysate and completely disappeared after 7 hours of reaction. More than 90% of 5′-glucosyladenosine remained even after 24 hours of reaction. More than 80% of 3′-glucosyladenosine remained after 7 hours of reaction, and 17% remained after 24 hours. As a result, adenosine is degraded in a short period of time in vivo and its function is easily lost, whereas 3′-glucosyladenosine and 5′-glucosyladenosine are produced by adenosine deaminase (ADA) and ⁇ -glucosidase.
- ADA adenosine deaminase
- EPI100 assay medium (trade name “EPI assay medium” sold by Kurabo Industries Co., Ltd.) 0.33 ml / well was added to a 12-well plate (Becton Dickinson, trade name “12-well multiwell plate”), and a three-dimensional skin model EPI200 ( The skin model cup sold by Kurabo Industries Co., Ltd. (trade name “EPI-200 kit”) was transferred to the well.
- 6-well plate (Becton Dickinson) supplemented with 0.9 ml / well of DMEM medium (Nissui Pharmaceutical Co., Ltd., trade name “Dulbecco's Modified Eagle Medium“ Nissui ””) after culturing at 37 ° C. with 5% CO 2 .
- the skin model cup was transferred to the company name, “6 well multi-well plate”.
- 5′-glucosyl adenosine prepared by the method of Experiment 1-2, 3′-glucosyl adenosine or adenosine prepared by the method of Experiment 2 were dissolved in 30% ethanol to a final concentration of 0.1%, and obtained. 0.1 ml of the solution was added into the skin model cup.
- profilaggrin a marker of granule layer cells
- profilaggrin a marker of granule layer cells
- Western blotting using a specific antibody against this marker protein. Expression levels were compared to evaluate the effect of glucosyl adenosine on keratinocyte differentiation.
- the increase in profilagrin means that the differentiation of keratinocytes in the basal layer was promoted and the cells in the granule layer increased.
- EDGS Cell growth additive
- EpiLife medium Cell growth additive (EDGS: EpiLife Defined Growth Supplement) containing EpiLife medium (Caskade Biologies Inc. sold)
- EpiLife medium NHEK cells suspended in a 5 ⁇ 10 5 cells /1.5ml/well on a 6-well plate (Sold by Becton Dickinson, trade name "FALCON MULTI-WELL TM 6WELL”) coated with collagen (sold by Nitta Gelatin Inc., trade name "Cellmatrix Type IV”)
- the cells were seeded and cultured in an incubator for 2 days at 37 ° C.
- culture was performed for 8 days while changing the medium with a medium containing the same components as those initially added.
- the culture solution in each well is removed by aspiration, washed with PBS, extraction buffer (extraction buffer) is added at 100 ⁇ l / well, the cells are detached from the well using a cell scraper, and a 1.5 ml microtube (Eppendorf) Collected in sales and product name "Eppendorf tube”).
- the collected cells were mixed and suspended with a vortex mixer, then left on ice for 30 minutes, centrifuged (15,000 ⁇ g, 10 minutes, 4 ° C.), and the supernatant was collected to prepare a sample for analysis. .
- SDS sodium dodecyl sulfate
- EDTA 1 mM edetic acid
- PMSF phenylmethylsulfuride fluoride
- leupeptin 20 ⁇ M leupeptin
- 0.1 ⁇ M aprotinin 20 mM HCl buffer
- This blocking nitrocellulose membrane was treated with 50 mM TBS (50 mM Tris-HCl (pH 7.4) containing 200 mM NaCl) containing 10% by volume of Block Ace of an anti-profilagri antibody (sold by Santa Cruz, trade name “Flaggrin (AKH1) Antibody”). ) After immersing in a solution diluted 100-fold with a buffer (hereinafter referred to as “TBS buffer”) at room temperature for 1 hour, the excess antibody was removed by washing with 50 mM TBS buffer containing 0.05 vol% Tween 20 It was. The nitrocellulose membrane was immersed in an HRP-labeled anti-mouse IgG rabbit polyclonal antibody (sold by DAKO) for 2 hours at room temperature.
- TBS buffer a buffer
- the membrane was washed with 50 mM TBS buffer containing 0.05% by volume of Tween 20 for 30 minutes, and then the color reaction was carried out using a commercially available Western blotting detection kit (sold by GE Healthcare Biosciences, Inc., trade name “ECL Western blotting detection reagent and Hyperfilm ECL ").
- ECL Western blotting detection reagent and Hyperfilm ECL As a control, the same treatment was performed except that an anti- ⁇ -actin antibody was used instead of the anti-profilagri antibody.
- the density (intensity) of each band is measured with a densitometer (trade name “Image Master 1D”, sold by Amersham Pharmacia Biotech) on the developed nitrocellulose membrane, and ⁇ -actin is used as an internal standard.
- profilaggrin was divided by the value of ⁇ -actin band intensity to obtain the expression level of profilaggrin.
- the expression level of profilagrin in control 2 cultured only in EpiLife medium is taken as 1, and the relative value of the expression level of profilagrin when cultured with 5′-glucosyladenosine, 3′-glucosyladenosine or adenosine added is determined. As shown in FIG.
- both 5′-glucosyl adenosine and 3′-glucosyl adenosine have a keratinocyte differentiation promoting action for differentiating keratinocytes in the epidermal basal layer into cells of the granule layer, and are components of an external preparation for skin having an anti-wrinkle effect It is shown that it is suitable as.
- 5′-glucosyl adenosine was compared with 3′-glucosyl adenosine, the addition of 5′-glucosyl adenosine showed a stronger effect of enhancing the expression of profilagrin protein.
- NHDF human fetal normal fibroblasts
- FCS Dulbecco's minimum essential medium
- the total amount was transferred to a 96 microwell plate (Bekton Decktonson, trade name “FALCON MICROTEST TM 96”), and the absorbance meter (Molecular Devices, trade name “Vmax microplate reader”) was used. Absorbance (A560-A650) was measured. Using the same method, the amount of collagen in each well was determined based on a calibration curve for color development using a color-developing reagent for collagen quantification prepared using type I collagen (sold by KOKEN), and is also shown in Table 6.
- glucosyl adenosine was confirmed to have an effect of promoting the differentiation of epidermal keratinocytes effective for skin improvement.
- a real-time PCR analysis method was used, and the differentiation marker gene of keratinocytes was used as an index.
- a test for examining the change in the expression level was performed as follows. Furthermore, in order to investigate the effect of glucosyl adenosine on ceramide synthesis, which has an important role in moisture retention and barrier function of human skin and is effective for skin improvement, the expression level of genes involved in ceramide synthesis is also examined. We also examined.
- profilagrin late marker
- involucrin early differentiation marker
- GCase ⁇ -glucocerebrosidase
- Sphingomyelinase Sphingomyelinase
- CypB cyclophilin B
- each well was washed twice with PBS (K-), 0.2 ml / well of a 0.025 mass% trypsin / EDTA solution was added, and the cells were detached from the plate by allowing to stand at room temperature for 5 minutes. . Further, 1 ml / well of neutralization solution (Cylex-treated PBS containing 0.5% by volume FCS) was added, and the cell suspension was added to a 1.5 ml microtube while pipetting (trade name “Eppendorf tube”, sold by Eppendorf). ). The tube was centrifuged at 5,000 rpm for 5 minutes, and the cell pellet obtained by removing the supernatant was subjected to total RNA extraction. As a control, cells cultured by adding only EpiLife medium were treated in the same manner. The test used 3 wells each for control, glucosyl adenosine or adenosine.
- RNA extraction from these cell pellets was performed using commercially available RNA extraction kits (both sold by GIAGEN, cell disruption: trade name “QIA shredder”, RNA recovery: trade name “RNeasy Mini kit”). Use and operation followed the instructions. On the way, DNase (Qiagen sales) treatment was added. Next, a cDNA template was synthesized using a commercially available reverse transcriptase (trade name “M-MLVRT”, sold by Gibco) according to the attached instructions. Total RNA was mixed with oligo dT random primer (Gibco Co., Ltd.) and dNTPs (GE Healthcare Co., Ltd., trade name “DNA polymerization Mix”), treated at 70 ° C.
- reverse transcriptase and dithiothreitol were added and incubated at 25 ° C. for 10 minutes, 42 ° C. for 50 minutes, and then 70 ° C. for 15 minutes.
- the base sequences (Forward and Reverse primers) necessary for PCR analysis of the internal standard and the gene to be analyzed are designed from the GENBANK mRNA sequence using "primer3 software" (freeware), or known base sequences
- the primer was prepared at the request of a commissioned synthesis company (Sigma Aldrich Co., Ltd.).
- Table 7 also shows the analysis target genes used in this experiment, the genes used as internal standards, and the base sequences used as primers for PCR reactions of these genes.
- the expression level of each gene to be analyzed was quantified by a commercially available PCR quantitative analysis system (trade name “Light Cycler 480” sold by Roche Diagnostics).
- PCR reaction of each cDNA template a commercially available PCR kit (trade name “SYBR Green I Master kit” sold by Roche Diagnostics) was used, and the reaction solution was first treated at 95 ° C. based on the attached instructions. After 5 minutes of heat denaturation, PCR reaction (95 ° C. for 10 seconds, 60 ° C. for 10 seconds, 72 ° C. for 15 seconds was repeated for 45 cycles) was performed. Using a DNA encoding cyclophilin B as an internal standard, the relative expression level of each gene to be analyzed was calculated. Table 8 shows the average value of the three wells of the relative expression level of each analysis target gene, assuming that the average value of the expression level of each analysis target gene in the control is 1.00.
- NHEK cells when added with adenosine, were cultured with profilagrin (PFG), involucrin (INV), ⁇ -glucocerebrosidase (GCase) and sphingomyelinase (SMase) genes.
- the expression levels were 1.41, 1.15, 1.37, and 1.35, respectively, and no significant enhancement was observed for any gene compared to the control expression level.
- glucosyl adenosine also acts at the gene level for the synthesis of ceramides, such as ⁇ -glucocerebrosidase and sphingomyelinase, which play an important role in skin moisturizing and barrier functions. It shows that adenosine can be used as an expression enhancer for ⁇ -glucocerebrosidase and sphingomyelinase, and further as a synthesis accelerator for ceramide.
- ⁇ Experiment 8 Effect on keratinocyte growth factor>
- glucosyl adenosine was confirmed to have an effect of promoting keratinocyte differentiation, so the effect on keratinocyte proliferation was also examined.
- the effects of 5'-glucosyl adenosine and 3'-glucosyl adenosine on keratinocyte growth factor (KGF) expression were compared in normal human skin fibroblasts (NHDF).
- DMEM medium (trade name “Dulbecco Modified Eagle Medium“ Nissui ””) containing 10% FBS (JRS, trade name “Fetal Bovine Serum (FBS) Australian Origin”) Inoculated at 3 ⁇ 10 5 cells / ml / well in a 12-well plate (trade name “12-well multi-well plate” sold by Becton Dickinson) and cultured at 37 ° C. for 1 day with 5% CO 2 .
- DMEM medium trade name “Dulbecco Modified Eagle Medium“ Nissui ”
- FBS Fetal Bovine Serum
- 5′-glucosyl adenosine obtained by the method of Experiment 1-2 or 3′-glucosyl adenosine obtained by the method of Experiment 2 were adjusted to final concentrations of 0.15 and 1.5 mM, respectively.
- a control supplemented with 1% FCS-containing DMEM medium without glucosyladenosine was used.
- the culture supernatant was aspirated and washed twice with PBS ( ⁇ ), and the cells were subjected to RNA extraction.
- RNA extraction was performed using a commercially available RNA extraction kit (Qiagen, trade name “QIA shredder + RNeasy Mini kit”). The operation followed the instructions attached to the kit.
- RNA extraction kit Qiagen, trade name “QIA shredder + RNeasy Mini kit”.
- Super Script VILO cDNA synthesis kit (Invitrogen's product name, “Super Script VILO cDNA synthesis kit”) was used, and RNA 10.5 ⁇ l, 5 ⁇ VILO reaction mix 3 ⁇ l, 10 ⁇ enzyme mix 1.5 ⁇ l in total were mixed. 15 ⁇ l) was performed under conditions of treatment at 25 ° C. for 10 minutes, 42 ° C. for 60 minutes, and then 85 ° C. for 5 minutes.
- LightCycler 480 SYBR Green I Master (Roche Diagnostics, trade name “Light Cycler 480 SYBR Green I Master” (mu) 10 ⁇ l, MasterMix 0.6 ⁇ m, Ml0.6p ) 0.6 ⁇ l and 3.0 ⁇ l of template were mixed (12.0 ⁇ l in total), and 45 cycles were performed under the conditions of holding at 95 ° C. for 10 seconds, 60 ° C. for 10 seconds, and then 72 ° C. for 15 seconds.
- the KGF expression level of each sample was calculated from the standard curve and corrected with the expression level of the internal standard (18s rRNA). The relative value when the expression level in the control was 1.0 was determined, and the expression level of each sample was evaluated.
- the primers shown in Table 9 were used. The results are shown in Table 10.
- 5'-glucosyl adenosine has a strong effect on keratinocyte differentiation and 3'-glucosyl adenosine has a strong promoting effect on keratinocyte proliferation. Therefore, when used as an external skin preparation, both glucosyl adenosines can be used in combination as appropriate. It is preferable to do this.
- EPI100 assay medium (trade name “EPI assay medium” sold by Kurabo Industries Co., Ltd.) added to a 24-well plate (Becton Dickinson, trade name “24-well multiwell plate”) is added to the 3D skin model EPI200 ( A skin model cup sold by Kurabo Industries, trade name “EPI-200 kit”) was transferred to the well. After culturing at 37 ° C. for 5 hours with 5% CO 2 , add 0.9 ml / well of EPI100 assay medium to a new 6-well plate (trade name “6-well multi-well plate” sold by Becton Dickinson), and remove the skin model cup. Moved.
- 5′-glucosyl adenosine obtained by the method of Experiment 1-2, 3′-glucosyl adenosine obtained by the method of Experiment 2 or adenosine was dissolved in 30% ethanol to a final concentration of 5%. .1 ml was added into the skin model cup, and after 18 hours, the permeate (medium in the well) was collected. A control was added with glucosyl adenosine and 30% ethanol not containing adenosine.
- the lactose dehydrogenase (LDH) activity released from the skin model was measured using a cytotoxicity detection kit (trade name “Cytotoxicity detection kit (LDH)” sold by Roche), and the LDH activity of the control was measured.
- the relative LDH activity was determined as 1.0, and the cell damage of each sample was evaluated. The results are shown in Table 11.
- the glucosyl adenosine of the present invention is gradually degraded by ⁇ -glucosidase, and the produced adenosine is rapidly metabolized. Therefore, even if it is degraded to glucose and adenosine by ⁇ -glucosidase, cell damage is caused. It is thought that it does not accumulate as adenosine that may occur.
- Test samples 1 and 2 were prepared by dissolving 3′-glucosyladenosine obtained by the method of Experiment 2 or 5′-glucosyladenosine obtained by the method of Experiment 1-2 in purified water so as to have the following concentrations. Purified water was used as a control.
- Test sample 1 aqueous solution containing 3'-glucosyl adenosine 2 w / v%
- Test sample 2 aqueous solution containing 5'-glucosyl adenosine 2 w / v%
- Control purified water only
- test sample 1 and the control were applied to 10 people per group (Experimental group 1). Of the 10 subjects, 5 randomly selected subjects applied the control to the inside of the left upper arm, applied test sample 1 to the inside of the upper right arm, and the remaining 5 subjects to the inside of the left upper arm. Sample 1 was applied and a control was applied to the inside of the upper right arm. 15 ⁇ l of each of the test sample 1 and the control was subjected to occlusion application for 24 hours by a conventional method using a fin chamber (Finland, manufactured by Epitest Ltd.).
- the remaining 10 subjects in 1 group were subjected to a 24 hour occlusion application test under the same conditions as test sample 1 except that 15 ⁇ l of test sample 2 was used instead of test sample 1 (experimental group 2).
- the fin chamber was removed 24 hours after the start of application, and the state of the application site at 1 hour and 24 hours after removal was visually confirmed.
- the state of the skin at the sample application site was determined according to the following criteria, and the number of subjects is shown in Table 12.
- bathing, showering, and extreme exercise are prohibited until the fin chamber is removed, and extreme exercise is prohibited for 24 hours after removal, as well as stimulating acts on the sample application site such as friction with a towel. Banned.
- enzyme solution ( ⁇ -glucosyltransferase) prepared by diluting the concentrated crude enzyme solution of ⁇ -glucosyltransferase with 50 mM acetate buffer (pH 6.0) so that the activity of ⁇ -glucosyltransferase is 500 units / ml.
- the enzyme was added at 20 units per 1 g of dextrin), mixed with stirring, and reacted at 50 ° C. for 24 hours.
- the column was washed with 20 volume% ethanol solution (5 times the volume) and 20 volume% ethanol solution (3 times the wet resin volume) and eluted with 40 volume% ethanol solution.
- the eluate was fractionated by 50 ml, and fractions having an ultraviolet absorption (260 nm) of 0.15 or more were collected.
- This product contains approximately 85% by mass of 5′-glucosyl adenosine, approximately 10% by mass of 3′-glucosyl adenosine, approximately 0.3% by mass of nigerosyl adenosine, and approximately 2% by mass of adenosine in terms of anhydride. It was.
- This product is an appropriate carrier, excipient, and stabilizer as it is as an active ingredient of a topical skin preparation with a long-lasting anti-wrinkle effect to help maintain and improve the tissue structure and physiological functions of the epidermis and dermis. , Buffers, pH adjusters, solvents, and the like, or any pharmaceuticals, quasi-drugs, cosmetics, etc., in which these are added.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- the compound was determined to be a compound in which bimolecular glucose was bound to adenosine. Furthermore, using the collected fraction, the substance contained in the collected fraction is one molecule of nigerose at the 5′-position hydroxyl group of adenosine based on the chemical shift values of carbon and proton (see Table 13) by MNR analysis under the following conditions. Identified as 5′-nigerosyl adenosine ( ⁇ -D-glucopyranosyl- (1 ⁇ 3) - ⁇ -D-glucopyranosyl- (1 ⁇ 5 ′)-adenosine; 5′- ⁇ -nigerosyl adenosine) did.
- a buffer, a pH adjuster, a solvent, etc. can be used as a preparation to which an optional auxiliary agent is added.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- CGTase manufactured by Hayashibara Biochemical Laboratories, Inc.
- Geobacillus stearothermophilus Tc-91 strain Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center accession number FERM BP-11273
- glucoamylase sold by Nagase ChemteX Corporation, trade name “Glucoteam # 20000”, 20, 000 units / g
- the reaction solution was heated at 100 ° C. for 10 minutes and then centrifuged (11,500 rpm), and 800 ml of the supernatant was collected.
- the column was washed with 7 volume) and 20 volume% ethanol solution (6 volumes of wet resin volume) and eluted with 40 volume% ethanol solution (16 volumes of wet resin volume).
- the eluate was fractionated by 50 ml, and fractions with ultraviolet absorption (260 nm) were collected. About half of the collected fraction was subjected to membrane filtration (pore size 0.22 ⁇ m) and dried under reduced pressure to obtain about 4 g of glucosyladenosine-containing powder.
- This product contained about 26% by mass of 5′-glucosyl adenosine and about 52% by mass of 3′-glucosyl adenosine and about 21% by mass of adenosine in terms of anhydride.
- This product is an appropriate carrier, excipient, and stabilizer as it is as an active ingredient of a topical skin preparation with a long-lasting anti-wrinkle effect to help maintain and improve the tissue structure and physiological functions of the epidermis and dermis. , Buffers, pH adjusters, solvents, and the like, or any pharmaceuticals, quasi-drugs, cosmetics, etc., in which these are added.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- Example 3 The remaining half of the fraction eluted from the activated carbon column collected in Example 3 was subjected to preparative HPLC using an ODS column under the same conditions as in Experiment 1-2, and the 3′-glucosyl adenosine fraction and 5′- The glucosyl adenosine fractions were individually collected, desalted by activated carbon column chromatography in the same manner as in Example 3, and eluted with 40% by volume ethanol to collect the eluted glucosyl adenosine fraction.
- the collected fraction is subjected to membrane filtration (pore size 0.22 ⁇ m) and then dried under reduced pressure to obtain approximately 1.6 g of a standard product containing 3% -glucosyladenosine 99% by mass or more in terms of anhydride and 5′-glucosyladenosine. About 0.6 g of a sample containing 99% by mass or more was prepared.
- any of these products can be used as an active ingredient of a skin external preparation having a continuous anti-wrinkle action to help maintain and improve the tissue structure and physiological function of the epidermis and dermis, as they are, as appropriate carriers, excipients, It can be used as a preparation to which an optional auxiliary agent such as a stabilizer, a buffer, a pH adjuster, a solvent or the like is added, or in the form of a pharmaceutical, a quasi-drug, a cosmetic or the like containing these.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- CGTase manufactured by Hayashibara Biochemical Laboratories, Inc.
- Geobacillus stearothermophilus Tc-91 strain Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center accession number FERM BP-11273
- per gram of dextrin 1,000 units were added, reacted at 50 ° C. for 24 hours, heated at 100 ° C. for 15 minutes to inactivate CGTase, centrifuged (11,500 rpm), and 400 ml of the supernatant was recovered.
- the collected fraction was subjected to membrane filtration (pore size 0.22 ⁇ m) and dried under reduced pressure to obtain about 5 g of a powder containing glycosyl adenosine and adenosine containing glucosyl adenosine.
- This product is 3'-glucosyl adenosine, such as 3'-glucosyl adenosine, 3'-maltosyl adenosine, 3'-glycosyl adenosine such as 3'-maltosyl adenosine, and 5'-glucosyl adenosine, 5'-maltosyl.
- the total amount of 5′-glycosyl adenosine such as adenosine and 5′-maltotriosyladenosine was about 58% by mass and adenosine was about 37% by mass.
- about 30% by mass of the glycosyl adenosine in this product was glycosylated at the 5 ′ position.
- This product is an appropriate carrier, excipient, and stabilizer as it is as an active ingredient of a topical skin preparation with a long-lasting anti-wrinkle effect to help maintain and improve the tissue structure and physiological functions of the epidermis and dermis.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- ⁇ Glucosyladenosine-containing composition > 2 parts by mass of ⁇ , ⁇ -trehalose was added to 1 part by mass of any of the glucosyl adenosine-containing powders prepared by the methods of Examples 1 to 5, and mixed uniformly to prepare a glucosyl adenosine-containing composition.
- This product can be used as an active ingredient of an external preparation for skin having a long-lasting anti-wrinkle action to help maintain and improve the tissue structure and physiological function of the epidermis and dermis.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- Glucosyladenosine-containing composition Syrup containing saccharide derivatives of ⁇ , ⁇ -trehalose (sales by Hayashibara Biochemical Laboratories, Inc., trade name “Tornale”) is added to any 2 parts by mass of the glucosyladenosine-containing powder prepared by the methods of Examples 1 to 5. 3 parts by weight of spray dried powder (prepared by Hayashibara Biochemical Laboratories Co., Ltd.) was added and mixed homogeneously to prepare a glucosyladenosine-containing composition.
- This product can be used as an active ingredient of an external preparation for skin having a long-lasting anti-wrinkle action to help maintain and improve the tissue structure and physiological function of the epidermis and dermis.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- Glucosyladenosine-containing composition > Ascorbic acid 2-glucoside (sold by Hayashibara Biochemical Laboratories, Inc., trade name “AA2G”), edetic acid 2 with respect to any 1 part by mass of the glucosyladenosine-containing powder prepared by the methods of Examples 1 to 5 0.1 parts by weight of sodium was added and mixed homogeneously to prepare a glucosyladenosine-containing composition.
- This product can be used as an active ingredient of an external preparation for skin having a long-lasting anti-wrinkle action to help maintain and improve the tissue structure and physiological function of the epidermis and dermis.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- glucosyladenosine-containing composition With respect to 20 parts by mass of purified water, any 1 part by mass of the glucosyl adenosine-containing powder prepared by the method of Examples 1 to 5, 2 parts by mass of ⁇ -glucosyl hesperidin or ⁇ -glucosyl rutin, cyclonigerosyl nigerose (cyclic four Sugar: Manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) 1 part by mass was added, dissolved by stirring, and then spray-dried by a conventional method to prepare a glucosyladenosine-containing composition.
- This product can be used as an active ingredient of an external preparation for skin having a long-lasting anti-wrinkle action to help maintain and improve the tissue structure and physiological function of the epidermis and dermis.
- the product is optionally used as a keratinocyte differentiation and growth promoter, collagen production enhancer, ceramide synthesis promoter or moisturizer.
- Example 1 Cream external skin preparation> Compounding ingredients (mass%) (1) Propylene glycol 5 (2) Beeswax 5 (3) Cetyl alcohol 4 (4) Reduced lanolin 5 (5) Squalane 35 (6) Glyceride stearate 2 (7) Polyoxyethylene (20 mol) Sorbitan monolaurate 2 (8) 3′-glucosyladenosine prepared in Example 4 or 5'-Glucosyladenosine 1 (9) Preservative Appropriate amount (10) Fragrance Appropriate amount Purified water is added to make the total amount 100% by mass.
- the above-mentioned cream (3′-glucosyl adenosine formulation: Cream 1) was applied to the rough skin area 3 times a day (morning, noon, evening) for 1 month for 10 people per group.
- the above-mentioned cream (5′-glucosyl adenosine combination: cream 2) was applied to the rough skin area 3 times a day (morning, noon, evening) for 1 month for 10 people per group.
- cream (Cream 3) with the same formulation as above except that 1% by weight of adenosine was added instead of glucosyl adenosine in the cream above, 3 times a day (morning, noon, evening) ) Applied daily for 1 month.
- cream (Cream 4) with the same composition as above except that it does not contain glucosyl adenosine or adenosine, 3 times a day (morning, noon, evening) every day for 1 month.
- the moisture content of the skin was measured by the following method, and the wrinkle state was evaluated as a wrinkle score to determine the rough skin state.
- the rough skin state means that the moisture content of the skin after application is higher and the wrinkle score is lower as compared to before applying the test sample. ⁇ Measurement method of skin moisture content> On the day before the cream was applied and the day after the end of the cloth, the moisture content and wrinkle score of the skin where the cream was applied were measured.
- the moisture content of the skin was measured using a moisture content measuring device (trade name “SKICON-200EX”, sold by IBS), and the average value of the moisture content of 10 subjects who applied each cream is shown in Table 15.
- cream (cream 1 or 2) containing glucosyl adenosine was applied, the water content and wrinkle score of the skin after application were significantly improved and rough skin were improved compared to before application.
- cream (cream 3) containing adenosine instead of glucosyl adenosine and cream (cream 4) containing no glucosyl adenosine or adenosine were applied after applying to the moisture content and wrinkle score of the skin. No significant improvement was observed compared to before, and rough skin did not improve.
- ⁇ Formulation Example 2 Composition in solution form> Compounding ingredients (mass%) (1) Glycerin 3 (2) Propylene glycol 4 (3) Ethanol 8 (4) Polyoxyethylene (20 mol) oleic alcohol 0.5 (5) Any one of glucosyl adenosine-containing powders prepared by the methods of Examples 1 to 5 0.5 Or any one of the glucosyl adenosine-containing compositions prepared by the methods of Examples 6 to 8 3 (6) magnolia extract 2 (7) Citric acid 0.01 (8) Sodium citrate 0.1 (9) 1,2-pentanediol 0.1 (10) Fragrance 0.05 Purified water is added to make the total amount 100 mass%.
- ⁇ Composition Example 3 External preparation for skin in pack form> Compounding ingredients (mass%) (1) Polyvinyl alcohol 15 (2) Polyethylene glycol 3 (3) Propylene glycol 7 (4) Ethanol 10 (5) Matsutake extract 1 (6) Any one of glucosyl adenosine-containing powders prepared by the methods of Examples 1 to 5 0.1 Or any one of the glucosyl adenosine-containing compositions prepared by the methods of Examples 6 to 8 0.5 (7) 1,2-pentanediol 0.1 (8) Fragrance Appropriate amount Purified water is added to make the total amount 100% by mass.
- ⁇ Formulation Example 4 Lip Balm> A lip balm was prepared according to a conventional method with the following composition. Compounding ingredients (mass%) (1) Fatty acid dextrin ester 8.0 (2) Beeswaw 4.0 (3) Microcrystalline wax 3.0 (4) Capric acid triglyceride 15.0 (5) Diglyceryl triisostearate 20.0 (6) Diisostearyl malate 32.0 (7) Polybutene 10.0 (8) Tocopherol acetate 2.0 (9) 5′-glucosyladenosine 2.0 prepared by the method of Example 2 (10) Licorice extract 0.1 (11) 1,2-pentanediol 0.1 (12) Perfume appropriate amount
- the various skin external preparations of these formulation examples can continuously exert an anti-wrinkle action and improve the skin condition in each typical usage mode.
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Abstract
Description
<実験1-1:α-グルコシル転移酵素の調製>
国際公開WO2008/136331号パンフレットの実験5の方法により、澱粉部分分解物(商品名『パインデックス#4』、松谷化学工業株式会社製造)1.5w/v(質量/容積)%、酵母抽出物(商品名『ポリペプトン』、日本製薬株式会社製造)0.5w/v%、酵母抽出物(商品名『酵母エキスS』、日本製薬株式会社製造)0.1w/v%、リン酸二カリウム0.1w/v%、リン酸一ナトリウム・2水和物0.06w/v%、硫酸マグネシウム・7水和物0.05w/v%、硫酸マンガン・5水和物0.001w/v%、硫酸第一鉄・7水和物0.001w/v%及び水からなる液体培地に、バチルス・サーキュランス PP710株(受託番号FERM BP-10771として茨城県つくば市東1-1-1中央第6所在の独立行政法人産業技術総合研究所、特許生物寄託センターに寄託されている)を接種し、ファーメンターで約24時間培養した。培養後、遠心分離して培養液上清を回収し、80%飽和となるように硫安を添加して4℃、24時間放置することにより塩析した。塩析物を遠心分離して回収し、20mM酢酸緩衝液(pH6.0)を加えて溶解した後、同緩衝液に対して透析し、膜濃縮することにより濃縮粗酵素液を調製した。本濃縮粗酵素液のα-グルコシル転移酵素活性は約1300単位/mlであった。また、本濃縮酵素液には約140単位/mlのアミラーゼ活性も認められた。
アデノシン(和光純薬株式会社販売、試薬特級)の濃度が10質量%となるように0.3Nの塩酸で溶解した。このアデノシンの10質量%溶液40mlとデキストリン(松谷化学株式会社販売、商品名『パインデックス#1』、固形分約92.3質量%)12gとを、2mMのCaCl2を含む50mM酢酸緩衝液(pH6.0)336mlに加え撹拌混合し、実験1-1で調製した濃縮粗酵素液を50mM酢酸緩衝液(pH6.0)でα-グルコシル転移酵素の活性が20単位/mlとなるように希釈した酵素液9mlを加え、撹拌混合し、50℃で24時間反応を行った。反応終了後の反応液に1N塩酸を加えpH3.5に調整後、グルコアミラーゼ(ナガセ生化学工業社販売、商品名『XL-4』、3,800単位/ml)を、デキストリンの固形分1g当たり1,000単位加え50℃で24時間反応させ、100℃で10分間処理し、酵素反応を停止した。この酵素反応を停止した反応液を、活性炭カラム(100mm×φ41mm)に、流速SV=3(5ml/分)で通液することによりグルコシルアデノシンをカラムに吸着させた後、脱イオン水(湿潤樹脂容積の5倍量)及び20容積%エタノール溶液(湿潤樹脂容積の3倍量)でカラムを洗浄し、40容積%エタノール溶液で溶出した。溶出液を50mlずつ分画し、紫外吸収(260nm)が0.15以上の画分を回収した。回収した画分をロータリーエバポレーターで濃縮し、ODSカラムを用いた分取用高速液体カラムクロマトグラフィー(HPLC)に供し、グルコシルアデノシンの溶出画分のメインピークを回収した。ODSカラムを用いたHPLCによる分取を2回に分けて実施し、固形物換算でグルコシルアデノシンを99質量%以上含有する標品を、合計で約0.8g調製した。なお、分取用HPLCは以下の条件で行った。
装置:SHIMADZU Shodex RI-102(検出器),LC-10AD(ポンプ),SIL-10ADvp(オートサンプラー)、C-R7Aplus(記録計)
カラム:ODSカラム(YMC社販売、商品名『YMC-Pack R&D ODS-A』、φ20mm×250mm)
移動相 : 20mM酢酸-酢酸アンモニウム緩衝液(pH3.5)/メタノール 84/16(容積/容積)
検出: RI
流速 :3.0ml/分
カラム温度 35℃
無水物換算で、アデノシン(東京化成工業株式会社販売、試薬特級)の濃度が1w/v%、デキストリン(松谷化学株式会社販売、商品名『パインデックス#1』)の濃度が10w/v%となるように、10mM酢酸ナトリウム溶液(pH5.5)に添加し、50℃に加温し、撹拌し完全に溶解させた。特開昭50-63189号公報(特公昭53-27791号公報)に開示されたジオバチルス・ステアロサーモフィラス(旧分類ではバチルス・ステアロサーモフィラス) Tc-91株(受託番号FERM BP-11273として茨城県つくば市東1-1-1中央第6所在の独立行政法人産業技術総合研究所、特許生物寄託センターに寄託されている;受託番号FERM P-2225を国際寄託に移管したもの)由来のCGTase(株式会社林原生物化学研究所製造)をデキストリン1g当たり1,000単位添加し、50℃で24時間反応後、100℃で15分間加熱しCGTaseを失活させた後、グルコアミラーゼ(ナガセケムテックス株式会社販売、商品名『グルコチーム#20000』、20,000単位/g)をデキストリン1g当たり2,600単位加え、50℃で24時間反応させた。この反応液を100℃で10分間加熱した後、遠心(11,500rpm)し、上清を回収した。この上清を、活性炭カラム150ml(120mm×φ41mm)に、流速SV=3(5ml/分)で通液することによりグルコシルアデノシンをカラムに吸着させた後、脱イオン水(湿潤樹脂容積の7倍量)及び20容積%エタノール溶液(湿潤樹脂容積の6倍量)でカラムを洗浄し、40容積%エタノール溶液で溶出した(湿潤樹脂容積の16倍量)。溶出液を50mlずつ分画し、紫外吸収(260nm)が0.15以上の画分を回収した。回収した画分を濃縮後、ODSカラムを用いた分取用HPLCに供し、グルコシルアデノシン含有画分のメインピークを回収した。この回収した画分を、活性炭カラムに吸着させて脱塩し、40容積%エタノールにより溶出し、ロータリーエバポレーターで濃縮し、グルコシルアデノシンの溶出画分を回収した。分取用HPLCは、移動相 に20mM酢酸-酢酸アンモニウム緩衝液(pH3.5)/メタノール82/18(容積/容積)を用い、カラム温度を40℃とした以外は前記実験1-2と同様にグルコシルアデノシンの分取と同じ条件で行い、無水物換算でグルコシルアデノシンを99質量%以上含有する標品を約1.6g調製した。ちなみに、ODSカラムを用いた分取用HPLCから溶出されたグルコシルアデノシンのメインピークは、実験1-2で溶出されたグルコシルアデノシンのメインピークとは異なるリテンションタイムに溶出された。また、斯かるメインピークにグルコシルアデノシンが含まれていることは、予め、常法によるマススペクトラム(MS)分析による分子量の測定でも確認した。
<NMR分析条件>
装置:日本電子株式会社販売、商品名『JNM-AL300』、1H:300.4MHz、13C:75.45MHz
溶媒:重水(0.6ml)
内部標準:3-トリメチルシリル-1-プロパンスルフォン酸ナトリウム塩(TPS)
積算回数:1H-NMR 128回、13C-NMR 4,000回、DEPT 400回、H-H COSY 32回、H-C COSY 36回
試料量:20mg
本発明のグルコシルアデノシンの基本的な性質として、水に対する溶解性をアデノシンと比較した。実験1-2で調製した5´-グルコシルアデノシン及び実験2で調製した3´-グルコシルアデノシンの水に対する溶解度を以下のようにして求め、アデノシンの溶解度と比較した。すなわち、500μlの純水に100mgの5´-グルコシルアデノシン又は3´-グルコシルアデノシン(以下、両方の化合物を総称し「グルコシルアデノシン」という場合がある。)を、各々室温(25℃)で溶解させた。また、500μlの純水に5mgのアデノシンを室温(25℃)で溶解させた。25℃の恒温室に1日静置後、遠心分離により溶け残った沈殿を除去した上清を、限外濾過(日本ミリポア社販売、商品名『ウルトラフリー 0.5 Biomax-5 Membrane』)後、移動相で希釈し、さらに膜濾過(日本ミリポア社販売、商品名『Millex-LH』、ポアサイズ0.45μm)した。これを下記条件のHPLCに供し、紫外線検出器によるクロマトグラムに出現したピークの面積からグルコシルアデノシン及びアデノシン量をそれぞれ求め、濃度を計算して、これを室温(25℃)での最大溶解濃度とした。同じサンプルを4℃の冷室に1ヶ月間静置した後、再度遠心分離し、同様に膜濾過処理した上清中のグルコシルアデノシン及びアデノシン量をそれぞれ同様に測定し、これを4℃での最大溶解濃度とした。その結果を表2に示す。
<HPLC分析条件>
装置:SHIMADZU UV-VIS DETECTOR SPD-10AV(検出器),LC-10AT(ポンプ),C-R8A(記録計),SIL-20AC(オートサンプラー)
カラム :ODSカラム(YMC社販売、商品名『YMC-Pack ODS-A』、φ4.6mm×250mm)
移動相 : 0.1容積%酢酸水溶液/メタノール=96/4(容積/容積)
検出波長 :260nm
流速 :1.0ml/分
注入量: 20μl
カラム温度: 40℃
<実験4-1:アデノシンデアミナーゼに対する耐性>
アデノシンは生体内にもともと存在するアデノシンデアミナーゼ(ADA)によって速やかにイノシン、ヒポキサンチンへと分解されることが確認されている。そこで、アデノシンの配糖体であるグルコシルアデノシンについて、その効果を持続して発揮できる可能性について調べるため、アデノシン代謝に関与する酵素の給源としてヒト胎児正常線維芽細胞のセルライゼート(cell lysate)を用い、5´-グルコシルアデノシン及び3´-グルコシルアデノシンが生体内で分解されるかどうかを確認する試験を実施した。10容積%ウシ胎児血清(以下、「FCS」と略記する)含有ダルベッコのミニマムエッセンシャルメディウム(日水製薬株式会社販売、商品名『ダルベッコ変法イーグル培地「ニッスイ」』、以下、DMEMと略記する)で希釈したヒト胎児正常線維芽細胞(クラボウ社販売、以下「NHDF細胞」という)を、150cm2培養フラスコ(コーニング社製)に、1.5×106細胞/20ml/フラスコで播種し、4日間培養した。培養上清を除去後、0.05質量%トリプシン/EDTA溶液(ギブコ社販売、商品名『Trypsin-EDTA』)で処理し、細胞懸濁液をフラスコから回収した。細胞懸濁液を遠心し、上清を除去後、細胞のペレットに、プロテアーゼインヒビターカクテル(Complete,EDTA-free、tablets、ロシュ社製)を溶解した10mM Tris-HCl(pH7.5)緩衝液(Lysis緩衝液)を加え、NHDF細胞を4.0×106c/mlとなるように懸濁した。懸濁液を、超音波処理(BRANSON SINIFIER CELL DISROPTOR 185)し、細胞を破砕(3回、各30秒×2回)した後、15,000rpm、5分間遠心分離し、上清を回収し、セルライゼートを調製した。実験1-2で調製した5´-グルコシルアデノシン又は実験2で調製した3´-グルコシルアデノシンを濃度5mMとなるよう溶解したリン酸緩衝生理食塩水(PBS)0.1mlと上記セルライゼート0.9mlを混合、撹拌し、37℃で反応させた。反応開始時(0時間)、反応開始1、7及び24時間の反応液を回収し、実験3と同じ方法で反応液を処理し、HPLC法によりアデノシン又はグルコシルアデノシン量を定量した。各々の化合物の反応開始時の濃度を100%とし、各反応時間における化合物の反応液中の濃度の相対値を計算し、アデノシン或いはグルコシルアデノシンの残存率として表3に示す。
<実験4-2:3次元皮膚モデル中での残存性>
本発明のグルコシルアデノシンの効果の持続性を判断するために、3次元皮膚環境における残存性についても調べた。12ウェルプレート(ベクトンディッキンソン社販売、商品名『12ウェルマルチウェルプレート』)にEPI100アッセイ培地(クラボウ社販売 商品名『EPIアッセイ培地』)を0.33ml/well添加し、3次元皮膚モデルEPI200(クラボウ社販売 商品名『EPI-200キット』)の皮膚モデルカップをウェルに移した。5%CO2、37℃で3時間培養後、DMEM培地(日水製薬株式会社販売、商品名『ダルベッコ変法イーグル培地「ニッスイ」』)を0.9ml/well添加した6ウェルプレート(ベクトンディッキンソン社販売、商品名『6ウェルマルチウェルプレート』)に皮膚モデルカップを移した。実験1-2の方法で調製した5´-グルコシルアデノシン、実験2の方法で調製した3´-グルコシルアデノシンまたはアデノシンを終濃度が0.1%となるよう30%エタノールに溶解し、得られた溶液の0.1mlを皮膚モデルカップ内に添加した。5%CO2、37℃で24時間培養後、サンプル透過液(ウェル中の培地)を回収し、限外濾過(日本ミリポア社販売、商品名『ウルトラフリー 0.5 Biomax-5 Membrane』)した後、移動相で希釈した。これを下記条件のHPLCに供し、別途作成した検量線により5´-グルコシルアデノシン、3´-グルコシルアデノシン及びアデノシンの量を求めた。3次元皮膚中透過後の各化合物の残存量とイノシン及びヒポキサンチンの生成量を表4に示した。
装置:HPLCポンプ Model 510、ポンプコントローラ Model 680、オートインジェクター Model 712、検出器 Model 2487、データ処理(定量計算) Empower 2 software (ミリポア社製)
カラム :ODSカラム(YMC社販売、商品名『YMC-Pack ODS AQ-303』、φ4.6mm×250mm)
移動相 :20mM酢酸-酢酸アンモニウムバッファー(pH3.5)/メタノール=92/8(容積/容積)
検出波長 :260nm
流速 :0.5ml/min
注入量 :10μl
カラム温度 :40℃
グルコシルアデノシンの抗シワ作用に関し、皮膚のケラチノサイトに及ぼす効果を調べるため、ヒトの皮膚のケラチノサイトに対する分化促進剤の評価に汎用されている正常ヒト表皮角化細胞を用い、以下のように実施した。すなわち、正常ヒト表皮角化細胞(クラボウ社販売、新生児由来、以下「NHEK細胞」という)を用い、ケラチノサイトが基底層の細胞から顆粒層の細胞に分化する場合、顆粒層の細胞に分化した際に、顆粒層の細胞に特異的に発現され、その分化マーカーとされているプロフィラグリン(profilaggrin:顆粒層の細胞のマーカー)を指標とし、このマーカー蛋白に対する特異的抗体を用いるウエスタンブロッティング法によりその発現量を比較し、ケラチノサイトの分化に対するグルコシルアデノシンの効果を評価した。本実験において、プロフィラグリンの増加は、基底層のケラチノサイトの分化が促進され、顆粒層の細胞が増加したことを意味する。
細胞増殖添加剤(EDGS:EpiLife Defined Growth Supplement)含有EpiLife培地( Caskade Biologics社販売)(以下、EDGS含有EpiLife培地を「EpiLife培地」と略記する。)に懸濁したNHEK細胞を5×105細胞/1.5ml/ウエルとなるようコラーゲン(新田ゼラチン社販売、商品名『Cellmatorix Type IV』)をコートした6ウエルプレート(ベクトンデッキンソン社販売、商品名『FALCON MULTI-WELLTM 6WELL』)に播種し、インキュベータにて、5容積%CO2存在下、37℃の条件で2日間培養した。各ウエルの培養上清を吸引除去し、実験1-2で調製した5´-グルコシルアデノシン又は実験2で調製した3´-グルコシルアデノシンを、終濃度が0.1μM、1μM又は10μMとなるように、各々EpiLife培地で希釈し1.5ml/ウエル添加した。対照1として、アデノシンを終濃度が0.1μM、1μM又は10μMとなるようにEpiLife培地で希釈し1.5ml/ウエル添加した。さらに、対照2としてEpiLife培地のみを1.5ml/ウエル添加した。以降1日おきに、最初に添加したものと同じ成分を含む培地で培地交換しながら培養を8日間行った。各ウエルの培養液を吸引除去し、PBSを加え洗浄した後、抽出緩衝液(extraction buffer)を100μl/ウエル加え、セルスクレイパーを用いて細胞をウエルから剥離し、1.5mlマイクロチューブ(エッペンドルフ社販売、商品名『エッペンドルフチューブ』)に回収した。回収した細胞を、ボルテックスミキサーにより混合、懸濁した後、氷上に30分放置し、遠心(15,000×g、10分、4℃)し、上清を回収し、分析用サンプルを調製した。なお抽出緩衝液として2質量%ソディウムドデシルサルフェイト(SDS)、1mMエデト酸(EDTA)、20μM phenylmethylsulfonyle fluoride(PMSF)、20μMロイペプチン(leupeptin)、0.1μMアプロチニン(aprotinin)を含む20mM Tris-HCl緩衝液(pH8.0)を用いた。
5容積%のジチオスレイトール(DTT)を含む2.5w/v%SDS水溶液8μlに、上記分析用サンプル12μlを加えて混合し、蛋白質量が20μg/レーンとなるようにチャージし、常法により、SDS-ポリアクリルアミドゲル電気泳動した。泳動終了後、ゲルに含まれる蛋白質を、常法により、ニトロセルロース膜に移し、非特異的な反応を抑制するためブロッキング溶液(大日本住友製薬社販売、商品名「ブロックエース」)に浸漬した。このブロッキング処理したニトロセルロース膜を、抗プロフィラグリ抗体(Santa Cruz社販売、商品名『Filaggrin(AKH1)Antibody』)を10容積%ブロックエース含む50mM TBS(200mM NaCl含有50mM Tris-HCl(pH7.4))緩衝液(以下、「TBS緩衝液」という)で100倍希釈した溶液に、室温で1時間浸漬した後、0.05容積%ツイーン20を含む50mM TBS緩衝液で洗浄し過剰の抗体を除いた。ニトロセルロース膜を、HRP標識した抗マウスIgGウサギポリクローナル抗体(DAKO社販売)溶液に、室温で2時間浸漬した。この膜を0.05容積%ツイーン20を含む50mM TBS緩衝液で30分間洗浄後、発色反応は、市販のウエスタンブロッティング検出キット(ジーイー ヘルスケア バイオサイエンス社販売、商品名「ECL western blotting detection reagent and Hyperfilm ECL」)を用いて行った。対照として、抗プロフィラグリ抗体に代えて、抗β-アクチン抗体を用いた以外は同様に処理を行った。発色後のニトロセルロース膜をデンシトメーター(アマシャム・ファルマシア・バイオテク社販売、商品名『Image Master 1D』)により、各バンドの濃さ(強度)を測定し、β-actinを内部標準とし、プロフィラグリのバンド強度をβ-actinのバンド強度の値で除しプロフィラグリンの発現量とした。EpiLife培地のみで培養した対照2のプロフィラグリンの発現量を1とし、5´-グルコシルアデノシン、3´-グルコシルアデノシン又はアデノシンを添加して培養したときのプロフィラグリンの発現量の相対値を求め表5に示す。
グルコシルアデノシンの抗シワ作用について、真皮におけるコラーゲン産生に及ぼす効果を調べるため、ヒトの皮膚のコラーゲン産生増強剤の評価に汎用されているヒト胎児正常線維芽細胞(NHDF)を用い、以下のように実施した。すなわち、ヒト胎児正常線維芽細胞(クラボウ社販売、以下「NHDF細胞」という)を、10容積%ウシ胎児血清(以下、「FCS」と略記する)含有ダルベッコのミニマムエッセンシャルメディウム(以下、DMEMと略記する)(日水製薬社販売)に懸濁し、24ウエルプレート(ベクトンデッキンソン社販売、商品名『FALCON MULTI-WELLTM 24WELL』)に1.5×105細胞/0.5ml/ウエルで播種し、インキュベータにて、5容積%2存在下、37℃の条件で1日間培養した。実験1-2で調製した5´-グルコシルアデノシン、実験2で調製した3´-グルコシルアデノシン又はアデノシンの何れかを終濃度が表6に示す濃度となるように、10容積%FCS含有DMEMで希釈し、0.5ml/ウエル添加し、インキュベータにて、5容積%CO2存在下、37℃の条件で3日間培養後、培養上清を吸引除去し、リン酸緩衝生理食塩水(PBS)で洗浄後、1M酢酸で1mg/mlに希釈したペプシン(SIGMA社販売)溶液を250μl/ウエル添加した。室温で4時間振とうした後、セルスクレイパーでウエルに付着した細胞をウエルから剥離し、ピペッティングして、ペプシン消化後の細胞懸濁液を1.5mlマイクロチューブ(エッペンドルフ社販売、商品名『エッペンドルフチューブ』)に回収した。このチューブにコラーゲン定量用発色試薬(Biocolor社販売、商品名『Sicrol Collagen Assay kit』)を500μl/チューブ添加し、ボルテックスミキサーにて混合後、室温でローテーター(TAITEC社販売、商品名『RT-50』)を用いて正確に30分間転倒混和した後、4℃で10分間遠心(15,000rpm)し、上清を除去した沈殿に、1NのNaOHを100μl/チューブ添加した後、沈殿をピペッティングにより溶かし、その全量を96マイクロウエルプレート(ベクトンデッキンソン社販売、商品名『FALCON MICROTESTTM96』)に移し、吸光度計(Molecular Devices社販売、商品名『Vmax microplate reader』)により吸光度(A560-A650)を測定した。同じ方法により、タイプIコラーゲン(KOKEN社販売)を用いて作成したコラーゲン定量用発色試薬による発色の検量線に基づき、各ウエルのコラーゲン量を求め、表6に併せて示す。
実験5において、グルコシルアデノシンが皮膚改善に有効な表皮ケラチノサイトの分化促進作用を有することが確認されたので、この作用につきさらに詳しく解析するため、リアルタイムPCR解析法を用い、ケラチノサイトの分化マーカー遺伝子を指標としてその発現量の変化を調べる試験を以下のように行った。さらに、ヒトの皮膚の保湿やバリア機能に重要な役割を有し皮膚改善に有効とされているセラミド合成に及ぼすグルコシルアデノシンの影響を調べる目的で、セラミドの合成に関与する遺伝子の発現量についても併せて検討した。すなわち、解析対象遺伝子として、ケラチノサイトの分化マーカーとしてプロフィラグリン(PFG:後期のマーカー)やインボルクリン(INV:初期の分化マーカー)を、セラミドの合成に関与する酵素であるβグルコセレブロシダーゼ(GCase)、スフィンゴミエリナーゼ(SMase)を選択し、PCR反応の際の内部標準としてサイクロフィリンB(CypB)の遺伝子を用いた(表7参照)。コラーゲンコートした6ウエルプレートにNHEK細胞を8×104細胞/1.5ml/ウエルで播種し、EpiLife培地を用い、インキュベータにて、5容積%CO2存在下、37℃の条件で2日培養し、培地を除去後、5´-グルコシルアデノシン、3´-グルコシルアデノシン又はアデノシンを含有する新しいEpiLife培地を1.5ml/ウエル添加し(グルコシルアデノシン又はアデノシンの終濃度は1μM)、1日おきにグルコシルアデノシン又はアデノシンを含有する同じ培地を用い培地交換(1.5ml/ウエル)しながら7日間培養した。培養終了後、各ウエルをPBS(K-)で2回洗浄後、0.025質量%トリプシン/EDTA溶液を0.2ml/ウエル添加し、室温で5分放置することにより細胞をプレートから剥離した。さらに、中和液(キレックス処理した0.5容積%FCS含有PBS)を1ml/ウエル添加し、ピペッティングしながら細胞懸濁液を1.5mlマイクロチューブ(エッペンドルフ社販売、商品名『エッペンドルフチューブ』)に回収した。このチューブを5,000rpm、5分間遠心し、上清を除去することにより得た細胞ペレットを、全RNA抽出に供した。対照として、EpiLife培地のみを添加し培養した細胞を同様に処理した。試験は、対照、グルコシルアデノシン又はアデノシンにつき各3ウエルを用いた。
実験5及び7において、グルコシルアデノシンはケラチノサイトの分化を促進する作用が確認されたので、ケラチノサイトの増殖に対する影響も調べた。正常ヒト皮膚線維芽細胞(NHDF)において、ケラチノサイト増殖因子(KGF)発現に対する5´-グルコシルアデノシンと3´-グルコシルアデノシンの影響を比較した。NHDFを10%FBS(JRS社販売、商品名『Fetal Bovine Serum(FBS) Australian Origin』)含有DMEM培地(日水製薬株式会社販売、商品名『ダルベッコ変法イーグル培地「ニッスイ」』)に懸濁し、12ウェルプレート(ベクトンデッキンソン社販売、商品名「12ウェルマルチウェルプレート」)に3×105cells/ml/wellで播種し、5%CO2、37℃で1日培養した。培養上清を除去後、実験1-2の方法で得た5´-グルコシルアデノシン又は実験2の方法で得た3´-グルコシルアデノシンを終濃度0.15、1.5mMとなるように、各々を1%FCS含有DMEM培地で希釈し、添加した。グルコシルアデノシンを含まない1%FCS含有DMEM培地を添加したものを対照とした。5%CO2、37℃で4時間培養後、培養上清を吸引除去し、PBS(-)で2回洗浄した後、細胞をRNA抽出に供した。
グルコシルアデノシンに関し、持続性の皮膚外用剤の成分として、皮膚に対して継続的に使用できるかどうかを調べるため、その細胞障害について調べた。グルコシルアデノシン及びアデノシンの3次元皮膚に対する影響として、細胞が障害を受けた際に放出されるラクトースデヒドロゲナーゼ(LDH)を指標とし、細胞障害の強さを調べた。24ウェルプレート(ベクトンディッキンソン社販売、商品名『24ウェルマルチウェルプレート』)にEPI100アッセイ培地(クラボウ社販売 商品名『EPIアッセイ培地』)を0.19ml/well添加し、3次元皮膚モデルEPI200(クラボウ社販売 商品名『EPI-200キット』)の皮膚モデルカップを上記ウェルに移した。5%CO2、37℃で3時間培養後、新しい6ウェルプレート(ベクトンディッキンソン社販売、商品名『6ウェルマルチウェルプレート』)にEPI100アッセイ培地を0.9ml/well添加し、皮膚モデルカップを移した。実験1-2の方法で得た5´-グルコシルアデノシン、実験2の方法で得た3´-グルコシルアデノシン、またはアデノシンを終濃度5%となるよう30%エタノールで溶解し、得られた溶液0.1mlを皮膚モデルカップ内に添加し、18時間後、透過液(ウェル内の培地)を回収した。グルコシルアデノシン及びアデノシンを含まない30%エタノールを添加したものを対照とした。回収した透過液について、皮膚モデルより放出されたラクトースデヒドロゲナーゼ(LDH)活性を細胞障害検出キット(ロシュ社販売 商品名『細胞障害検出キット(LDH)』)を用いて測定し、対照のLDH活性を1.0として相対LDH活性を求め、各試料の細胞障害を評価した。結果を表11に示した。
グルコシルアデノシンの安全性を10名のボランティアによるパッチテストにより評価した。
年齢が22乃至45歳の男女合計20名(男性10名、女性10名)を、無作為に10名ずつ(各群男性5名、女性5名)の2群に分けた。
<被験試料>
実験2の方法で得た3´-グルコシルアデノシン又は実験1-2の方法で得た5´-グルコシルアデノシンを下記濃度となるように精製水に溶解し、被験試料1及び2を調製した。対照として精製水を用いた。
被験試料1:3´-グルコシルアデノシン2w/v%を含有する水溶液
被験試料2:5´-グルコシルアデノシン2w/v%を含有する水溶液
対照:精製水のみ
1群10名には被験試料1と対照を塗布した(実験群1)。10名の被験者の中、無作為に選択した5名の被験者には、左上腕内側に対照を塗布し、右上腕内側に被験試料1を塗布し、残りの5名には左上腕内側に被験試料1を塗布し、右上腕内側に対照を塗布した。被験試料1又は対照の各々15μlを、フィンチェンバー(エピテスト(Epitest Ltd.)社製 フィンランド国)を用い、常法により24時間の閉塞塗布を実施した。残りの1群10名の被験者には、被験試料1に代えて被験試料2を15μlを用いた以外は、被験試料1と同じ条件で24時間の閉塞塗布試験を実施した(実験群2)。塗布開始24時間後にフィンチェンバーを除去し、除去後1時間と24時間の塗布部位の状態を目視により確認した。試料塗布部位の皮膚の状態を下記判定基準により判定し、その被験者数を表12に示す。なお、試験期間中、フィンチェンバーを除去するまでは、入浴、シャワー、過激な運動は禁止し、除去後24時間は過激な運動を禁止するとともに、タオル等による摩擦など、試料塗布部位に対する刺激行為を禁止した。
日本接触皮膚炎学会の「環境皮膚科学」が示すパッチテスト判定基準のうちの本邦基準(http://www.daiichiclinic.jp/derma/sesyoku/contents_03/patchtest_table_2.html)に基づき、塗布部位につき、目視により判定した。判定は、反応なし(-)、わずかな紅斑(±)、明らかな紅斑(+)、紅斑+浮腫、丘疹(++)、紅斑+浮腫・丘疹+小水疱(+++)、大水疱(++++)の6段階で評価した。
アデノシン(和光純薬工業株式会社販売、試薬特級)の濃度が1w/v%、デキストリン(松谷化学株式会社販売、商品名『パインデックス#1』、固形分約92.3質量%)の濃度が10w/v%となるように、それぞれを、2mMのCaCl2溶液6,600mlに添加し、50℃に加温し、撹拌することにより完全に溶解した。1N HClによりpHを6.0に調整した後、実験1-1で調製したバチルス・サーキュランス PP710株(独立行政法人産業技術総合研究所、特許生物寄託センター 受託番号BP-10771)を培養することにより調製したα-グルコシル転移酵素の濃縮粗酵素液を50mM酢酸緩衝液(pH6.0)でα-グルコシル転移酵素の活性が500単位/mlとなるように希釈した酵素液30ml(α-グルコシル転移酵素をデキストリン1g当たり20単位添加)を加え、撹拌混合し、50℃で24時間反応を行った。反応終了後の反応液に1N HClを加えpH3.5に調整後、グルコアミラーゼ(ナガセ生化学工業社販売、商品名『XL-4』、3,800単位/ml)を、デキストリンの固形分1g当たり1,000単位加え50℃で24時間反応させ、100℃で10分間処理し、酵素反応を停止した。この酵素反応を停止した反応液を、活性炭カラム(100mm×φ41mm)に、流速SV=3(5ml/分)で通液することによりグルコシルアデノシンをカラムに吸着させた後、脱イオン水(湿潤樹脂容積の5倍量)及び20容積%エタノール溶液(湿潤樹脂容積の3倍量)でカラムを洗浄し、40容積%エタノール溶液で溶出した。溶出液を50mlずつ分画し、紫外吸収(260nm)が0.15以上の画分を回収した。回収した画分をロータリーエバポレーターで濃縮し、強酸性カチオン交換樹脂(オルガノ社販売、商品名『XT-1030E』(Na型)、φ4.2cm×100cm、2本)に40ml/分の流速で通液し、脱イオン水を用いて溶出し、回収した画分を実験3と同様の条件のHPLCにより分析し、無水物換算で、グルコシルアデノシンを95質量%以上含有すると計算された画分を回収した。さらにこの回収した画分を減圧乾燥することによりグルコシルアデノシン含有粉末15gを得た。本品は、無水物換算で、5´-グルコシルアデノシンを約85質量%、3´-グルコシルアデノシンを約10質量%、ニゲロシルアデノシンを約0.3質量%、アデノシンを約2質量%含有していた。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として、そのままで、適宜の担体、賦形剤、安定剤、緩衝剤、pH調整剤、溶媒等、任意の助剤等を添加した製剤として、或いは、これらを配合した医薬品、医薬部外品、化粧品などの形態で用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
<HPLC条件>
装置:SHIMADZU UV-VIS DETECTOR SPD-20A(検出器),LC-20AB(ポンプ),C-R7Aplus(記録計),SIL-20AC(オートサンプラー)
カラム:ODSカラム(YMC社販売、商品名『YMC-Pack ODS-AQ303』、φ4.6mm×250mm)
移動相 : 20mM酢酸アンモニウム緩衝液(pH3.5)/メタノール=92/8(容積/容積)
検出波長 :260nm
流速 :0.5ml/分
注入量: 20μl
カラム温度: 40℃
<NMR分析条件>
装置:日本電子株式会販売、商品名『JNM-AL300』、1H:300.4MHz、13C:75.45MHz
溶媒:重水(0.6ml)
内部標準:3-トリメチルシリル-1-プロパンスルフォン酸ナトリウム塩(TPS)
積算回数:1H-NMR 64回、13C-NMR 12,00回、DEPT 100回、H-H COSY 36回、H-C COSY 72回、HMBC 920回
試料量:36.4mg
アデノシン(東京化成工業株式会社販売、試薬特級)の濃度が1w/v%、デキストリン(松谷化学株式会社販売、商品名『パインデックス#1』、固形分約92.3質量%)の濃度が10w/v%となるように、それぞれを10mM酢酸ナトリウム溶液(pH5.5)に添加し、50℃に加温し、撹拌し完全に溶解させた。ジオバチルス・ステアロサーモフィラス Tc-91株(独立行政法人産業技術総合研究所、特許生物寄託センター 受託番号FERM BP-11273)由来のCGTase(株式会社林原生物化学研究所製造)をデキストリン1g当たり1,000単位添加し、50℃で24時間反応後、100℃で15分間加熱しCGTaseを失活させた後、グルコアミラーゼ(ナガセケムテックス株式会社販売、商品名『グルコチーム#20000』、20,000単位/g)をデキストリン1g当たり2,600単位加え、50℃で24時間反応させた。この反応液を100℃で10分間加熱した後、遠心(11,500rpm)し、上清を800ml回収した。この上清を、活性炭カラム150ml(120mm×φ41mm)に、流速SV=3(5ml/分)で通液することによりグルコシルアデノシン及びアデノシンをカラムに吸着させた後、脱イオン水(湿潤樹脂容積の7倍量)及び20容積%エタノール溶液(湿潤樹脂容積の6倍量)でカラムを洗浄し、40容積%エタノール溶液で溶出した(湿潤樹脂容積の16倍量)。溶出液を50mlずつ分画し、紫外吸収(260nm)の認められた画分を回収した。回収した画分の約半量を膜濾過(ポワサイズ0.22μm)後、減圧乾燥し、グルコシルアデノシン含有粉末約4gを得た。本品は、無水物換算で、5´-グルコシルアデノシンを約26質量%、3´-グルコシルアデノシンを約52質量%、アデノシンを約21質量%含有していた。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として、そのままで、適宜の担体、賦形剤、安定剤、緩衝剤、pH調整剤、溶媒等、任意の助剤等を添加した製剤として、或いは、これらを配合した医薬品、医薬部外品、化粧品などの形態で用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
アデノシン(東京化成工業株式会社販売、試薬特級)の濃度が1w/v%、デキストリン(松谷化学株式会社販売、商品名『パインデックス#1』、固形分約92.3質量%)の濃度が10w/v%となるように、それぞれを10mM酢酸ナトリウム溶液(pH5.5)に添加し、50℃に加温し、撹拌し完全に溶解させた。ジオバチルス・ステアロサーモフィラス Tc-91株(独立行政法人産業技術総合研究所、特許生物寄託センター 受託番号FERM BP-11273)由来のCGTase(株式会社林原生物化学研究所製造)をデキストリン1g当たり1,000単位添加し、50℃で24時間反応後、100℃で15分間加熱しCGTaseを失活させた後、遠心(11,500rpm)し、上清を400ml回収した。この上清を、活性炭カラム150ml(120mm×φ41mm)に、流速SV=3(5ml/分)で通液することによりグルコシルアデノシンを含むグリコシルアデノシン及びアデノシンをカラムに吸着させた後、脱イオン水(湿潤樹脂容積の7倍量)及び20容積%エタノール溶液(湿潤樹脂容積の6倍量)でカラムを洗浄し、40容積%エタノール溶液で溶出した(湿潤樹脂容積の16倍量)。溶出液を50mlずつ分画し、紫外吸収(260nm)の認められた画分を回収した。回収した画分を膜濾過(ポワサイズ0.22μm)後、減圧乾燥し、グルコシルアデノシンを含むグリコシルアデノシンとアデノシンとを含有する粉末約5gを得た。本品は、無水物換算で、3´-グルコシルアデノシン、3´-マルトシルアデノシンや3´-マルトトリオシルアデノシンなどの3´-グリコシルアデノシン、及び、5´-グルコシルアデノシン、5´-マルトシルアデノシンや5´-マルトトリオシルアデノシンなどの5´-グリコシルアデノシンを合計で約58質量%とアデノシンを約37質量%含有していた。また、本品中のグリコシルアデノシンの約30質量%は、その5´位が配糖化されていた。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として、そのままで、適宜の担体、賦形剤、安定剤、緩衝剤、pH調整剤、溶媒等、任意の助剤等を添加した製剤として、或いは、これらを配合した医薬品、医薬部外品、化粧品などの形態で用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
実施例1乃至5の方法により調製したグルコシルアデノシン含有粉末の何れか1質量部に対しα,α-トレハロース2質量部を添加し、均質に混合してグルコシルアデノシン含有組成物を調製した。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
実施例1乃至5の方法により調製したグルコシルアデノシン含有粉末の何れか2質量部に対しα,α-トレハロースの糖質誘導体含有シラップ(株式会社林原生物化学研究所販売、商品名『トルナーレ』)を噴霧乾燥した粉末(株式会社林原生物化学研究所調製)3質量部を添加し、均質に混合してグルコシルアデノシン含有組成物を調製した。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
実施例1乃至5の方法により調製したグルコシルアデノシン含有粉末の何れか1質量部に対しアスコルビン酸2-グルコシド(株式会社林原生物化学研究所販売、商品名『AA2G』)5質量部、エデト酸2ナトリウム0.1質量部を添加し、均質に混合してグルコシルアデノシン含有組成物を調製した。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
精製水20質量部に対し、実施例1乃至5の方法により調製したグルコシルアデノシン含有粉末の何れか1質量部、α-グルコシルヘスペリジン又はα-グルコシルルチン2質量部、シクロニゲロシルニゲロース(環状四糖:株式会社林原生物化学研究所製造)1質量部を添加し、撹拌溶解した後、常法により噴霧乾燥することによりグルコシルアデノシン含有組成物を調製した。本品は、表皮及び真皮の組織構造や生理機能の維持、改善を助けるための持続性の抗シワ作用を有する皮膚外用剤の有効成分として用いることができる。また、本品は、ケラチノサイト分化及び増殖促進剤、コラーゲン産生増強剤、セラミド合成促進剤或いは保湿剤として利用することも随意である。
配合成分 (質量%)
(1)プロピレングリコール 5
(2)ミツロウ 5
(3)セチルアルコール 4
(4)還元ラノリン 5
(5)スクワラン 35
(6)ステアリン酸グリセライド 2
(7)ポリオキシエチレン(20モル)
ソルビタンモノラウリン酸エステル 2
(8)実施例4で調製した3´-グルコシルアデノシン又は、
5´-グルコシルアデノシン 1
(9)防腐剤 適量
(10)香料 適量
精製水を加え全量を100質量%とする。
本発明のグルコシルアデノシンの皮膚外用剤における有効性を確認するため、有効成分として3´-グルコシルアデノシン又は、5´-グルコシルアデノシンを配合した上記配合例1のクリームを用い、ボランティアによる試験を実施した。すなわち、問診により慢性的な肌荒れに悩む30乃至50歳代の女性40名を被験者として選択し、無作為に10名ずつ4群に分けた。1群10名には上記クリーム(3´-グルコシルアデノシン配合:クリーム1)を、肌荒れ部分に、1日3回(朝、昼、晩)、1ヶ月間毎日塗布させた。1群10名には上記クリーム(5´-グルコシルアデノシン配合:クリーム2)を、肌荒れ部分に、1日3回(朝、昼、晩)、1ヶ月間毎日塗布させた。1群10名には、上記クリームのグルコシルアデノシンに代えてアデノシンを1質量%配合した以外は上記と同じ処方のクリーム(クリーム3)を、肌荒れ部分に、1日3回(朝、昼、晩)、1ヶ月間毎日塗布させた。残りの1群10名には、グルコシルアデノシン又はアデノシンを含まない以外は上記と同じ配合のクリーム(クリーム4)を、肌荒れ部分に、1日3回(朝、昼、晩)、1ヶ月間毎日塗布させた。下記方法により、皮膚の水分量を測定すると共に、シワの状態をシワスコアとして評価し、肌荒れの状態を判定した。なお、この方法によれば、肌荒れの状態は、被験試料を塗布する前に比べ、塗布後の皮膚の水分量が高くなり、シワスコアが低くなるほど改善したことを意味する。
<肌の水分量の測定方法>
クリームの塗前日及び布終了翌日に、クリーム塗布部位の皮膚の水分含量及びシワスコアを測定した。皮膚の水分含量は水分含量測定装置(IBS社販売、商品名『SKICON-200EX』)を用いて測定し、各クリームを塗布した被験者10名の水分含量の平均値を表15に示す。
<シワスコアの判定方法>
シワスコアの判定は、各被験者につき、判定者5名の目視により、化粧品機能評価ガイドライン(『日本香粧品学会誌』、30巻、4号、316乃至332頁(2006年)参照)に基づき、表14に示す8段階のシワスコア(0乃至7グレード)で評価した。各被験者に対する判定者5名の平均値を、その被験者のシワスコアとし、各クリームを塗布した被験者10名のシワスコアの平均値を表15に示す。
配合成分 (質量%)
(1)グリセリン 3
(2)プロピレングリコール 4
(3)エタノール 8
(4)ポリオキシエチレン(20モル)オレインアルコール 0.5
(5)実施例1乃至5の方法により調製したグルコシル
アデノシン含有粉末の何れか1種 0.5
又は実施例6乃至8の方法で調製したグルコシル
アデノシン含有組成物の何れか1種 3
(6)モクレンエキス 2
(7)クエン酸 0.01
(8)クエン酸ナトリウム 0.1
(9)1,2-ペンタンジオール 0.1
(10)香料 0.05
精製水を加え全量を100質量%とする。
配合成分 (質量%)
(1)ポリビニルアルコール 15
(2)ポリエチレングリコール 3
(3)プロピレングリコール 7
(4)エタノール 10
(5)マツタケエキス 1
(6)実施例1乃至5の方法により調製したグルコシル
アデノシン含有粉末の何れか1種 0.1
又は実施例6乃至8の方法で調製したグルコシル
アデノシン含有組成物の何れか1種 0.5
(7)1,2-ペンタンジオール 0.1
(8)香料 適量
精製水を加え全量を100質量%とする。
下記の配合により、常法に従ってリップクリームを調製した。
配合成分 (質量%)
(1)脂肪酸デキストリンエステル 8.0
(2)ミツロウ 4.0
(3)マイクロクリスタリンワックス 3.0
(4)カプリン酸トリグリセリド 15.0
(5)トリイソステアリン酸ジグリセリル 20.0
(6)リンゴ酸ジイソステアリル 32.0
(7)ポリブテン 10.0
(8)酢酸トコフェロール 2.0
(9)実施例2の方法で調整した5´-グルコシルアデノシン 2.0
(10)カンゾウエキス 0.1
(11)1,2-ペンタンジオール 0.1
(12)香料 適量
配合成分 (質量%)
(1)トリオクタレイン 51.3
(2)α,α-トレハロースの糖質誘導体含有シラップ
(株式会社林原商事販売、商品名「ハローデックス」) 16.4
(3)モノミリスチン酸ポリグリセリル-10 5.2
(4)モノステアリン酸ポリグリセリル-10 1.75
(5)アスコルビン酸2-グルコシド 1
(6)カンゾウエキス 0.1
(7)ヒアルロン酸 0.25
(8)実施例1乃至5の方法により調製したグルコシル
アデノシン含有粉末の何れか1種 0.2
又は実施例6乃至8にの方法で調製したグルコシル
アデノシン含有組成物の何れか1種 1.0
(9)1,2-ペンタンジオール 0.1
(10)香料 適量
精製水を加え全量を100質量%とする。
配合成分 (質量%)
(1)酢酸ナトリウム 1.0
(2)リン酸水素カルシウム 4.0
(3)グリセリン 10.0
(4)ハッカ油 0.5
(5)緑茶エキス 0.6
(6)L-アスコルビン酸-2-グルコシド 2.0
(7)1,2-ヘキサンジオール 0.1
(7)ワセリン 49.0
(8)木ロウ 10.0
(9)ラノリ 10.0
(10)ゴマ油 10.5
Claims (5)
- α-D-グルコピラノシル-(1→5´)-アデノシン及び/又はα-D-グルコピラノシル-(1→3´)-アデノシンを有効成分として含有する持続性の抗シワ作用を有する皮膚外用剤。
- α-D-グルコピラノシル-(1→5´)-アデノシン及び/又はα-D-グルコピラノシル-(1→3´)-アデノシンとともに、抗菌剤として1,3ブチレングリコール又は1,2-アルカンジオールから選ばれる成分を含有することを特徴とする請求項1記載の皮膚外用剤。
- さらに、美白剤、抗酸化剤、抗炎症剤及び保湿剤から選ばれる成分を含有することを特徴とする請求項1又は2記載の皮膚外用剤。
- 美白剤、抗酸化剤、抗炎症剤及び保湿剤から選ばれる成分が植物の抽出物である請求項3記載の皮膚外用剤。
- 澱粉質とアデノシンとを含む溶液に、α-グルコシル転移酵素又はシクロマルトデキストリン・グルカノトランスフェラーゼを作用させる工程と、生成したα-D-グルコピラノシル-(1→5´)-アデノシン又はα-D-グルコピラノシル-(1→3´)-アデノシンを採取する工程とを含んでなる、α-D-グルコピラノシル-(1→5´)-アデノシン及び/又はα-D-グルコピラノシル-(1→3´)-アデノシンの製造方法。
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US13/983,231 US9492368B2 (en) | 2011-02-01 | 2012-01-31 | External preparation for skin |
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JPWO2012105542A1 (ja) | 2014-07-03 |
US20130309188A1 (en) | 2013-11-21 |
CN103501757B (zh) | 2016-01-20 |
EP2671565B1 (en) | 2016-09-21 |
US9492368B2 (en) | 2016-11-15 |
CN103501757A (zh) | 2014-01-08 |
EP2671565A1 (en) | 2013-12-11 |
JP5913138B2 (ja) | 2016-04-27 |
KR101967615B1 (ko) | 2019-04-10 |
EP2671565A4 (en) | 2015-08-19 |
KR20140004196A (ko) | 2014-01-10 |
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