WO2012115247A1 - Stratum corneum peeling accelerator - Google Patents

Stratum corneum peeling accelerator Download PDF

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Publication number
WO2012115247A1
WO2012115247A1 PCT/JP2012/054638 JP2012054638W WO2012115247A1 WO 2012115247 A1 WO2012115247 A1 WO 2012115247A1 JP 2012054638 W JP2012054638 W JP 2012054638W WO 2012115247 A1 WO2012115247 A1 WO 2012115247A1
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extract
stratum corneum
mesotrypsin
corneum peeling
ectoine
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PCT/JP2012/054638
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French (fr)
Japanese (ja)
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利彦 日比野
章子 山田
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株式会社資生堂
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/738—Rosa (rose)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
    • 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
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9771—Ginkgophyta, e.g. Ginkgoaceae [Ginkgo family]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/20—Chemical, physico-chemical or functional or structural properties of the composition as a whole
    • A61K2800/28—Rubbing or scrubbing compositions; Peeling or abrasive compositions; Containing exfoliants

Definitions

  • the present invention provides a mesotrypsin expression promoter selected from the group consisting of ginkgo biloba extract, saxifrage extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine, and a stratum corneum peeling promoter containing the same.
  • the outermost layer of the epidermis the stratum corneum (SC) consists of keratinized cells (keratinocytes) and a continuous extracellular lipid layer, forming a physiochemical barrier to effectively protect the body from environmental disasters.
  • SCs are created continuously through terminal differentiation of keratinocytes.
  • cell shedding occurs and the SC is maintained at a constant thickness.
  • individual keratinocytes or small aggregates detach from the skin surface. This process is undetectable in healthy people, but in some pathological conditions there is an imbalance between SC creation and ablation. The result is a visible excess scale. This is seen in inflammatory skin diseases such as psoriasis.
  • Non-patent Document 1 Lundstrom and Egelrud have reported that chymotrypsin-like serine protease exists in SC and is involved in detachment.
  • This enzyme was purified from human sole SC as a stratum corneum chymotrypsin enzyme, and a cDNA encoding the stratum corneum chymotrypsin enzyme was isolated from a human keratinocyte cDNA library (Non-patent document 2; Non-patent document). 3).
  • the present inventor has shown that not only chymotrypsin enzyme but also trypsin-like serine protease is involved in the SC exfoliation process other than the sole.
  • KLK7 stratum corneum chymotrypsin enzyme
  • KLK5 trypsin-like enzyme
  • KLK5 and KLK7 have a very important function in peeling.
  • trypsinogens are called cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen based on their equipotential points. According to recent studies, these trypsinogens are encoded by different genes PRSS1, PRSS2, and PRSS3 (Non-patent Document 8). Trypsinogen 1 which is a PRSS1 gene product and trypsinogen 2 which is a PRSS2 gene product are major digestive pancreatic proteases. PRSS3 is a mesotrypsinogen gene and at least two splicing variants are generated (trypsinogen 3 and 4).
  • trypsinogens have the very highly conserved enteropeptidase-activated putative cleavage site sequence DDDDK-1 present in pancreatic trypsinogen from various species (Light and Janska, 1989) . Trypsinogen is expressed not only in the pancreas but also in other tissues, including epithelial cells of various tissues and the human brain. Interestingly, mesotrypsin differs from the other two trypsins in substrate specificity and inhibitor sensitivity. Mesotrypsin does not cleave protein substrates very much.
  • the inventor performed cDNA cloning of the trypsinogen gene from a human keratinocyte cDNA library, resulting in the isolation of two splicing isoforms of the mesotrypsinogen gene PRSS3, one of which is identical to trypsinogen 4 (brain trypsinogen), and The other was different only in the exon encoding the N-terminal region and was named trypsinogen 5 (Non-Patent Document 9).
  • Both isoforms have the activation sequence DDDDK-1.
  • DDDDK-1 is hardly cleaved by trypsin itself because of the presence of mainly negatively charged residues at the cleavage site.
  • enteropeptidase is highly specific for the DDDDK-1 sequence (Non-Patent Document 10).
  • the catalytic efficiency of enteropeptidase for bovine trypsinogen is 34,000 times that for bovine trypsin.
  • enteropeptidase is the only enzyme that physiologically converts trypsinogen to trypsin. It has also been found that enteropeptidase is present exclusively in the granular layer of the epidermis. The expression and location of these trypsinogens and their activating enzyme enteropeptidase is consistent with the view that mesotrypsin is involved in terminal differentiation of keratinocytes.
  • LEKTI serine protease inhibitor lympho-epithelial-casal-type 5 inhibitor
  • SPINK5-deficient mice show hyperproliferation of epidermal protease and show symptoms similar to Netherton syndrome through excessive degradation of desmoglen-1.
  • Multiple epidermal kallikreins may be involved in stratum corneum detachment through desmoglen 1 cleavage, and they are regulated by LEKTI.
  • stratum corneum detachment occurs when KLK acts on a desmosome that exhibits cell adhesion and decomposes it, and when LEKTI suppresses the action of KLK, stratum corneum detachment is also suppressed.
  • LEKTI suppresses the action of KLK
  • mesotrypsin was thought to promote stratum corneum peeling only by degrading LEKTI.
  • An object of the present invention is to provide a new stratum corneum peeling accelerator.
  • the stratum corneum peeling accelerator literally promotes the peeling of the stratum corneum, thereby exhibiting an effect of improving rough skin.
  • mesotrypsin not only degrades LEKTI, but surprisingly directly activates KLK7, which contributes to stratum corneum detachment. Therefore, as a result of screening candidate drugs that enhance the expression of mesotrypsin, it was found that Ginkgo biloba extract, Yukinosita extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine have an effect of enhancing mesotrypsin expression, thereby completing the present invention.
  • a stratum corneum peeling promoter comprising the mesotrypsin expression enhancer of (1).
  • a method for promoting exfoliation of stratum corneum by applying to the skin a component selected from the group consisting of Ginkgo biloba extract, Yukinoshita extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine to a subject who desires exfoliation promotion.
  • a component selected from the group consisting of ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine for promoting stratum corneum peeling.
  • ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine activate mesotrypsin, thereby exerting a stratum corneum peeling promoting effect, and as a result, rough skin is improved through peeling of the stratum corneum.
  • FIG. 5 shows that mesotrypsin has the ability to activate the precursor of KLK7 (proKLK7).
  • the mesotrypsin expression promotion effect of various crude drugs and drugs is shown.
  • the numerical value at the end of each herbal extract name represents the concentration ( ⁇ g / ml).
  • Corneal layer exfoliation promoter in one aspect, provides a stratum corneum exfoliation promoter comprising a mesotrypsin expression enhancer selected from the group consisting of ginkgo biloba extract, saxifrage extract, Isaiyo rose extract, dipotassium glycyrrhizinate and ectoine.
  • a mesotrypsin expression enhancer selected from the group consisting of ginkgo biloba extract, saxifrage extract, Isaiyo rose extract, dipotassium glycyrrhizinate and ectoine.
  • Izayoi rose extract has a ceramide synthesis promoting effect (Japanese Patent Laid-Open No. 2006-111560), a collagenase inhibitory effect (Japanese Patent Laid-Open No. 2006-241148), and the like.
  • dipotassium glycyrrhizinate and ectoin are also used for external preparations for skin, but none of the extracts is known to have a stratum corneum peeling promoting effect.
  • the above extract can be obtained by a conventional method.
  • a part or all of a plant that is the origin of the extract can be obtained by soaking or heating and refluxing together with an extraction solvent at room temperature or after heating, filtering and concentrating.
  • the extraction site may be dried.
  • the extraction solvent any solvent can be used as long as it is usually used for extraction.
  • organic solvents such as alcohols such as methanol, ethanol, propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols , Chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, or an aqueous solvent such as water, physiological saline, phosphate buffer, borate buffer, etc., either alone or in combination. it can.
  • one or more kinds selected from water, methanol, ethanol, and 1,3-butylene glycol are preferably used as the solvent.
  • the extract obtained by extraction with the above solvent can be used as it is or, for example, an extract concentrated by lyophilization or the like, and if necessary, an adsorbent method, for example, an ion exchange resin removed impurities, A polymer (eg, Amberlite XAD-2) adsorbed on a column, eluted with a desired solvent, and further concentrated can be used.
  • an adsorbent method for example, an ion exchange resin removed impurities,
  • a polymer eg, Amberlite XAD-2
  • Drugs such as Ginkgo biloba leaf extract, Yukinoshita extract, Isaiyoi rose extract, dipotassium glycyrrhizinate and ectoine show mesotrypsin expression enhancing action in a concentration-dependent manner. Therefore, from such a viewpoint, the amount of ginkgo biloba extract, saxifrage extract, Izayoi rose extract, dipotassium glycyrrhizinate and / or ectoine in the stratum corneum peeling promoter of the present invention is 0.
  • the content is 0001 to 20.0 mass%, preferably 0.0001 to 10.0 mass%.
  • the stratum corneum peeling accelerator according to the present invention can be produced according to a conventional method, and as the component constituting the stratum corneum peeling accelerator, it can be prepared by one or more of the above-mentioned extracts.
  • Ingredients used in skin preparations such as cosmetics and pharmaceuticals including quasi drugs, such as oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants UV absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers, and the like are appropriately blended as necessary.
  • the dosage form of the stratum corneum peeling accelerator of the present invention is not particularly limited, and for example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system.
  • Arbitrary dosage forms such as systems, ointments, gels, aerosols and the like can be taken.
  • the form of use is not particularly limited, and can take any form such as lotion, milky lotion, cream, essence, jelly, gel, ointment, pack, mask, foundation, and the like.
  • the stratum corneum peeling promoter of the present invention can be applied to the skin and used in a cosmetic method for preventing and / or improving rough skin.
  • the usage and dosage of the stratum corneum peeling promoter of the present invention in such a cosmetic method is not particularly limited, and is appropriately determined depending on the dosage form and the state of wrinkles on the skin to be treated.
  • Appropriate amount eg 0.1 to 1 ml per square cm 2 , can be rubbed directly into the skin, or the appropriate amount can be soaked in gauze or the like and applied to the skin.
  • RT-PCR Total RNA 500 ng isolated from human keratinocytes cultured as described above was reverse transcribed using random hexamers and Superscript II RNase H-reverse transcriptase (Gibco-BRL, Getersberg, MD) Thereafter, PCR amplification was performed using Taq DNA polymerase (Takara, Kyoto, Japan) and the following primers. 40 amplification cycles of 94 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute were performed.

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Abstract

Provided are a mesotrypsin expression enhancer selected from the group consisting of Gingko biloba extract, Saxifraga stolonifera extract, Rosa roxburghii extract, dipotassium glycyrrhizate, and ectoine, and a stratum corneum peeling accelerator including the mesotrypsin expression enhancer.

Description

角層剥離促進剤Stratum corneum peeling accelerator
　本発明は、イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム、エクトインから成る群から選ばれるメソトリプシン発現促進剤及びそれを含む角層剥離促進剤を提供する。 The present invention provides a mesotrypsin expression promoter selected from the group consisting of ginkgo biloba extract, saxifrage extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine, and a stratum corneum peeling promoter containing the same.
　表皮の最外層である角層（ＳＣ）は、角化した細胞（角質細胞）と、連続的な細胞外脂質層とからなり、生理化学的障壁を形成して環境災害から身体を効果的に保護している。ＳＣは、ケラチノサイトの最終分化を通じて継続的に作り出される。それと同時に細胞シェディング（剥離）も起こってＳＣが一定の厚さに維持される。正常な剥離では、個々の角質細胞すなわち小さな凝集体が皮膚表面から剥がれる。このプロセスは、健康な人では感知できないが、ある病的状態ではＳＣの創出と剥離の間に不均衡が存在する。その結果、目に見える過剰な鱗屑となる。これは、乾癬などの炎症性皮膚疾患で見られる。 The outermost layer of the epidermis, the stratum corneum (SC), consists of keratinized cells (keratinocytes) and a continuous extracellular lipid layer, forming a physiochemical barrier to effectively protect the body from environmental disasters. Protect. SCs are created continuously through terminal differentiation of keratinocytes. At the same time, cell shedding (detachment) occurs and the SC is maintained at a constant thickness. With normal detachment, individual keratinocytes or small aggregates detach from the skin surface. This process is undetectable in healthy people, but in some pathological conditions there is an imbalance between SC creation and ablation. The result is a visible excess scale. This is seen in inflammatory skin diseases such as psoriasis.
　LundstromとEgelrudは、キモトリプシン様セリンプロテアーゼがＳＣに存在していて剥離に関与していることを報告している（非特許文献１）。この酵素は、ヒトの足裏のSCから角層キモトリプシン酵素として精製され、角層キモトリプシン酵素をコードしているcDNAは、ヒトケラチノサイトcDNAライブラリーから単離された（非特許文献２；非特許文献３）。本発明者は、キモトリプシン酵素だけでなく、トリプシン様セリンプロテアーゼも足裏以外でのＳＣの剥離プロセスに関与していることを示した。最近の研究により、15種類のセリンプロテアーゼ（それをカリクレイン１～15（KLK1～15）と呼ぶ）をコードしているヒト組織カリクレイン遺伝子ファミリーが染色体19q13.4にクラスターとして位置していることが明らかにされた（非特許文献４；非特許文献５）。これらセリンプロテアーゼの多くはヒト組織で広く発現しており、その中に皮膚が含まれる。剥離への関与が以前に明らかにされたこれらプロテアーゼは、カリクレインファミリーのメンバーとして新たな命名されている。すなわち、角層キモトリプシン酵素はカリクレイン７（KLK7、hK7）と命名され（非特許文献６）、トリプシン様酵素はカリクレイン5（KLK5、hK5）と命名された（非特許文献７）。ＫＬＫ５とＫＬＫ７が剥離において極めて重要な機能を有する。 Lundstrom and Egelrud have reported that chymotrypsin-like serine protease exists in SC and is involved in detachment (Non-patent Document 1). This enzyme was purified from human sole SC as a stratum corneum chymotrypsin enzyme, and a cDNA encoding the stratum corneum chymotrypsin enzyme was isolated from a human keratinocyte cDNA library (Non-patent document 2; Non-patent document). 3). The present inventor has shown that not only chymotrypsin enzyme but also trypsin-like serine protease is involved in the SC exfoliation process other than the sole. Recent studies reveal that the human tissue kallikrein gene family encoding 15 serine proteases (called kallikreins 1-15 (KLK1-15)) is located as a cluster on chromosome 19q13.4 (Non-patent document 4; Non-patent document 5). Many of these serine proteases are widely expressed in human tissues, including skin. These proteases, previously revealed to be involved in detachment, are newly named as members of the kallikrein family. That is, the stratum corneum chymotrypsin enzyme was named kallikrein 7 (KLK7, hK7) (Non-Patent Document 6), and the trypsin-like enzyme was named kallikrein 5 (KLK5, hK5) (Non-Patent Document 7). KLK5 and KLK7 have a very important function in peeling.
　その一方で、異なる３種類のトリプシノーゲンがヒト膵液に存在する。これらトリプシノーゲンは、その等電位点に基づき、カチオン性トリプシノーゲン、アニオン性トリプシノーゲン、メソトリプシノーゲンと呼ばれている。最近の研究により、これらトリプシノーゲンは、それぞれ異なる遺伝子ＰＲＳＳ１、ＰＲＳＳ２、ＰＲＳＳ３によりコードされる（非特許文献８）。ＰＲＳＳ１遺伝子産物であるトリプシノーゲン１と、ＰＲＳＳ２遺伝子産物であるトリプシノーゲン２は、主要な消化性膵臓プロテアーゼである。ＰＲＳＳ３はメソトリプシノーゲン遺伝子であり、少なくとも２つのスプライシング変異体が生成される（トリプシノーゲン３と４）。これらのトリプシノーゲンはすべて、様々な種に由来する膵臓トリプシノーゲンに存在する非常に高度に保存された、エンテロペプチダーゼによって活性化される推定の切断部位配列ＤＤＤＤＫ－１を有する（LightとJanska、1989年）。トリプシノーゲンは膵臓だけでなく他の組織でも発現しており、その中にはさまざまな組織の上皮細胞とヒトの脳が含まれる。興味深いことに、メソトリプシンは他の２つのトリプシンとは基質特異性と阻害剤感受性が異なっている。メソトリプシンはタンパク質基質を余り切断しない。その最も特徴的な性質は、天然のトリプシンインヒビターであるα１－トリプシンインヒビター、膵臓分泌トリプシンインヒビター（Kazalタイプ）、ダイズトリプシンインヒビター（Kunitzタイプ）などに対する耐性である。これらの知見は、トリプシンの、腸での消化に加えて、さまざまな生理学的反応における関与の可能性を示唆している。 On the other hand, three different types of trypsinogen are present in human pancreatic juice. These trypsinogens are called cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen based on their equipotential points. According to recent studies, these trypsinogens are encoded by different genes PRSS1, PRSS2, and PRSS3 (Non-patent Document 8). Trypsinogen 1 which is a PRSS1 gene product and trypsinogen 2 which is a PRSS2 gene product are major digestive pancreatic proteases. PRSS3 is a mesotrypsinogen gene and at least two splicing variants are generated (trypsinogen 3 and 4). All of these trypsinogens have the very highly conserved enteropeptidase-activated putative cleavage site sequence DDDDK-1 present in pancreatic trypsinogen from various species (Light and Janska, 1989) . Trypsinogen is expressed not only in the pancreas but also in other tissues, including epithelial cells of various tissues and the human brain. Interestingly, mesotrypsin differs from the other two trypsins in substrate specificity and inhibitor sensitivity. Mesotrypsin does not cleave protein substrates very much. Its most characteristic property is resistance to natural trypsin inhibitor α1-trypsin inhibitor, pancreatic secretory trypsin inhibitor (Kazal type), soybean trypsin inhibitor (Kunitz type) and the like. These findings suggest a possible involvement of trypsin in various physiological responses in addition to intestinal digestion.
　本発明者は、ヒトケラチノサイトｃＤＮＡライブラリーからトリプシノーゲン遺伝子のｃＤＮＡクローニングを行い、その結果メソトリプシノーゲン遺伝子ＰＲＳＳ３の２つのスプライシングアイソフォームを単離し、その一方はトリプシノーゲン４（脳トリプシノーゲン）と同一であり、そして他方はＮ末端領域をコードするエキソンにおいてのみ相違して、それをトリプシノーゲン５の命名した（非特許文献９）。 The inventor performed cDNA cloning of the trypsinogen gene from a human keratinocyte cDNA library, resulting in the isolation of two splicing isoforms of the mesotrypsinogen gene PRSS3, one of which is identical to trypsinogen 4 (brain trypsinogen), and The other was different only in the exon encoding the N-terminal region and was named trypsinogen 5 (Non-Patent Document 9).
　両アイソフォーム共に、活性化配列ＤＤＤＤＫ－１を有す。ＤＤＤＤＫ－１は、切断部位での主に負に帯電した残基の存在を理由に、トリプシン自身ではほとんど切断されない。対照的に、エンテロペプチダーゼはＤＤＤＤＫ－１配列に対して特異性が高い（非特許文献１０）。例えば、牛トリプシノーゲンに対するエンテロペプチダーゼの触媒効率は、牛トリプシンに対するそれの34,000倍である。したがって、エンテロペプチダーゼがトリプシノーゲンをトリプシンに生理学的に変換する唯一の酵素である。エンテロペプチダーゼが表皮の顆粒層にもっぱら存在することも見出された。これらのトリプシノーゲンとその活性化酵素エンテロペプチダーゼの発現及び存在箇所は、メソトリプシンがケラチノサイトの末端分化に関与する見解と一致する。 Both isoforms have the activation sequence DDDDK-1. DDDDK-1 is hardly cleaved by trypsin itself because of the presence of mainly negatively charged residues at the cleavage site. In contrast, enteropeptidase is highly specific for the DDDDK-1 sequence (Non-Patent Document 10). For example, the catalytic efficiency of enteropeptidase for bovine trypsinogen is 34,000 times that for bovine trypsin. Thus, enteropeptidase is the only enzyme that physiologically converts trypsinogen to trypsin. It has also been found that enteropeptidase is present exclusively in the granular layer of the epidermis. The expression and location of these trypsinogens and their activating enzyme enteropeptidase is consistent with the view that mesotrypsin is involved in terminal differentiation of keratinocytes.
　末端分化の際、多くの現象がタンパク質分解作用に関連している。最外層の顆粒細胞において、核と全てのオルガネラは消失し、ケラチンの分子量は減少し、そしてプロフィラグリンはフラグリンへとプロセッシングされる。最近の研究では、セリンプロテアーゼインヒビターであるリンホ－エピセリアル－カザール－タイプ５インヒビター（ＬＥＫＴＩ）が角層剥離に重要であることが示されている。ＬＥＫＴＩ遺伝子の突然変異体ＳＰＩＮＫ５がNetherton症候群として知られる重篤な先天性魚鱗癬様紅皮症を有する患者の欠陥遺伝子として同定されている。主要角層剥離プロテアーゼ（ＫＬＫ５，ＫＬＫ７）はＬＥＫＴＩにより強力に阻害される（非特許文献１１）。ＳＰＩＮＫ５欠損マウスは表皮プロテアーゼの過剰亢進を示し、またデスモグレン１の過剰分解を通じてNetherton症候群に似た症状を示す。複数の表皮カリクレインがデスモグレン１の切断を通じて角層剥離に関与していることもあり、それらはＬＥＫＴＩにより調節される。 Many phenomena are related to proteolytic action during terminal differentiation. In the outermost granule cells, the nucleus and all organelles disappear, the molecular weight of keratin decreases, and profilagrin is processed into fragrin. Recent studies have shown that the serine protease inhibitor lympho-epithelial-casal-type 5 inhibitor (LEKTI) is important for stratum corneum detachment. A mutant SPINK5 of the LEKTI gene has been identified as a defective gene in a patient with severe congenital ichthyosis-like erythroderma known as Netherton syndrome. The main stratum corneum peeling protease (KLK5, KLK7) is strongly inhibited by LEKTI (Non-patent Document 11). SPINK5-deficient mice show hyperproliferation of epidermal protease and show symptoms similar to Netherton syndrome through excessive degradation of desmoglen-1. Multiple epidermal kallikreins may be involved in stratum corneum detachment through desmoglen 1 cleavage, and they are regulated by LEKTI.
　角層剥離は、細胞接着の働きを示すデスモゾームにＫＬＫが作用してそれを分解すること生じ、ＬＥＫＴＩがＫＬＫの作用を抑制すると角層剥離も抑えられる。これまで、メソトリプシンはＬＥＫＴＩを分解することでのみ角層剥離を促進するものと考えられていた。 The stratum corneum detachment occurs when KLK acts on a desmosome that exhibits cell adhesion and decomposes it, and when LEKTI suppresses the action of KLK, stratum corneum detachment is also suppressed. Until now, mesotrypsin was thought to promote stratum corneum peeling only by degrading LEKTI.
　本発明の課題は、新たな角層剥離促進剤の提供にある。角層剥離促進剤は文字通り角層剥離を促進することで、肌荒れ改善効果などが発揮される。 An object of the present invention is to provide a new stratum corneum peeling accelerator. The stratum corneum peeling accelerator literally promotes the peeling of the stratum corneum, thereby exhibiting an effect of improving rough skin.
　本発明者は、メソトリプシンが、ＬＥＫＴＩを分解するだけでなく、驚くべきことに角層剥離に寄与するＫＬＫ７を直接活性化することを見出した。よって、メソトリプシンの発現を亢進する候補薬剤をスクリーニングした結果、イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインがメソトリプシン発現亢進効果を有することを見出し、本発明を完成するに至った：
（１）イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれるメソトリプシン発現亢進剤。
（２）（１）のメソトリプシン発現亢進剤を含む角層剥離促進剤。
（３）角層剥離促進を所望する対象者にイチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれる成分を肌に適用することにより角層剥離促進方法。
（４）イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれる成分を肌に適用することで肌荒れの予防及び／又は改善のための美容方法。
（５）イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれる成分の角層剥離促進のための使用。 The inventors have found that mesotrypsin not only degrades LEKTI, but surprisingly directly activates KLK7, which contributes to stratum corneum detachment. Therefore, as a result of screening candidate drugs that enhance the expression of mesotrypsin, it was found that Ginkgo biloba extract, Yukinosita extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine have an effect of enhancing mesotrypsin expression, thereby completing the present invention. Was:
(1) A mesotrypsin expression enhancer selected from the group consisting of ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine.
(2) A stratum corneum peeling promoter comprising the mesotrypsin expression enhancer of (1).
(3) A method for promoting exfoliation of stratum corneum by applying to the skin a component selected from the group consisting of Ginkgo biloba extract, Yukinoshita extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine to a subject who desires exfoliation promotion.
(4) A cosmetic method for preventing and / or improving rough skin by applying to the skin a component selected from the group consisting of ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine.
(5) Use of a component selected from the group consisting of ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine for promoting stratum corneum peeling.
　本発明によれば、イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインはメソトリプシンを活性化し、それにより角層剥離促進効果が発揮され、その結果角層の剥離を通じて肌荒れ改善などが図られる。 According to the present invention, ginkgo biloba extract, saxifrage extract, izayoi rose extract, dipotassium glycyrrhizinate and ectoine activate mesotrypsin, thereby exerting a stratum corneum peeling promoting effect, and as a result, rough skin is improved through peeling of the stratum corneum. Figured.
メソトリプシンがＫＬＫ７の前駆体（proKLK7）を活性化する能力を有することを示す。FIG. 5 shows that mesotrypsin has the ability to activate the precursor of KLK7 (proKLK7). 各種生薬及び薬剤のメソトリプシン発現促進効果を示す。図中、各生薬エキス名の末尾の数値は濃度（μg/ml）を表わす。The mesotrypsin expression promotion effect of various crude drugs and drugs is shown. In the figure, the numerical value at the end of each herbal extract name represents the concentration (μg / ml).
角層剥離促進剤
　１つの観点において、本発明はイチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれるメソトリプシン発現亢進剤を含む角層剥離促進剤を提供する。 Corneal layer exfoliation promoter In one aspect, the present invention provides a stratum corneum exfoliation promoter comprising a mesotrypsin expression enhancer selected from the group consisting of ginkgo biloba extract, saxifrage extract, Isaiyo rose extract, dipotassium glycyrrhizinate and ectoine.
　イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキスについては皮膚外用剤に使用されているものはあるが、いずれのエキスも角層剥離促進効果を有することは知られていない。尚、イザヨイバラエキスはセラミド合成促進効果（特開２００６－１１１５６０）、コラゲナーゼ阻害効果（特開２００６－２４１１４８）などを有することは公知である。グリチルリチン酸ジカリウム及びエクトインも同様に、皮膚外用剤に使用されているものはあるが、いずれのエキスも角層剥離促進効果を有することは知られていない。 Although there are some ginkgo biloba extract, yukinoshita extract, and izayoi rose extract that are used for external preparations for skin, none of them is known to have a stratum corneum peeling promoting effect. It is known that Izayoi rose extract has a ceramide synthesis promoting effect (Japanese Patent Laid-Open No. 2006-111560), a collagenase inhibitory effect (Japanese Patent Laid-Open No. 2006-241148), and the like. Similarly, dipotassium glycyrrhizinate and ectoin are also used for external preparations for skin, but none of the extracts is known to have a stratum corneum peeling promoting effect.
　上記抽出物は常法により得ることができ、例えばその起源となる植物の一部又は全部を抽出溶媒とともに常温で又は加熱して浸漬または加熱還流した後、濾過し、濃縮して得ることができる。溶媒抽出の前に、抽出部位を乾燥させてもよい。抽出溶媒としては、通常抽出に用いられる溶媒であれば任意に用いることができ、例えば、有機溶媒、例えばメタノール、エタノール、プロピレングリコール、１，３－ブチレングリコール、グリセリン等のアルコール類、含水アルコール類、クロロホルム、ジクロルエタン、四塩化炭素、アセトン、酢酸エチル、ヘキサン等、あるいは水性溶媒、例えば水、生理食塩水、リン酸緩衝液、ホウ酸緩衝液等を、それぞれ単独で、あるいは組み合わせて用いることができる。好ましくは、溶媒として、水、メタノール、エタノール、１，３－ブチレングリコールから選ばれる１種または２種以上が好適に使用される。 The above extract can be obtained by a conventional method. For example, a part or all of a plant that is the origin of the extract can be obtained by soaking or heating and refluxing together with an extraction solvent at room temperature or after heating, filtering and concentrating. . Prior to solvent extraction, the extraction site may be dried. As the extraction solvent, any solvent can be used as long as it is usually used for extraction. For example, organic solvents such as alcohols such as methanol, ethanol, propylene glycol, 1,3-butylene glycol and glycerin, hydrous alcohols , Chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, or an aqueous solvent such as water, physiological saline, phosphate buffer, borate buffer, etc., either alone or in combination. it can. Preferably, one or more kinds selected from water, methanol, ethanol, and 1,3-butylene glycol are preferably used as the solvent.
　上記溶媒で抽出して得られた抽出物をそのまま、あるいは例えば凍結乾燥などにより濃縮したエキスを使用でき、また必要であれば吸着法、例えばイオン交換樹脂を用いて不純物を除去したものや、ポーラスポリマー（例えばアンバーライトＸＡＤ－２）のカラムにて吸着させた後、所望の溶媒で溶出し、さらに濃縮したものも使用することができる。 The extract obtained by extraction with the above solvent can be used as it is or, for example, an extract concentrated by lyophilization or the like, and if necessary, an adsorbent method, for example, an ion exchange resin removed impurities, A polymer (eg, Amberlite XAD-2) adsorbed on a column, eluted with a desired solvent, and further concentrated can be used.
　イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキスの抽出物やグリチルリチン酸ジカリウム、エクトインなどの薬剤は、濃度依存的にメソトリプシン発現亢進作用を示す。従って、このような観点からは、本発明の角層剥離促進剤中のイチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及び／又はエクトインの配合量は、剤全量中、乾燥物として０．０００１～２０．０質量％、好ましくは０．０００１～１０．０質量％である。 Drugs such as Ginkgo biloba leaf extract, Yukinoshita extract, Isaiyoi rose extract, dipotassium glycyrrhizinate and ectoine show mesotrypsin expression enhancing action in a concentration-dependent manner. Therefore, from such a viewpoint, the amount of ginkgo biloba extract, saxifrage extract, Izayoi rose extract, dipotassium glycyrrhizinate and / or ectoine in the stratum corneum peeling promoter of the present invention is 0. The content is 0001 to 20.0 mass%, preferably 0.0001 to 10.0 mass%.
　本発明に係る角層剥離促進剤は、常法に従って製造することができ、また同角層剥離促進剤を構成する成分として、上記エキス１種又は２種以上単独でも調製可能であるが、通常医薬部外品を含む化粧品や医薬品等の皮膚外用剤等に用いられる成分、例えば油分、界面活性剤、粉末、色材、水、アルコール類、増粘剤、キレート剤、シリコーン類、酸化防止剤、紫外線吸収剤、保湿剤、香料、各種薬効成分、防腐剤、ｐＨ調整剤、中和剤等必要に応じて適宜配合される。 The stratum corneum peeling accelerator according to the present invention can be produced according to a conventional method, and as the component constituting the stratum corneum peeling accelerator, it can be prepared by one or more of the above-mentioned extracts. Ingredients used in skin preparations such as cosmetics and pharmaceuticals including quasi drugs, such as oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants UV absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers, and the like are appropriately blended as necessary.
　本発明の角層剥離促進剤の剤型は特に限定されるものではなく、例えば、溶液系、可溶化系、乳化系、粉末分散系、水－油二層系、水－油－粉末三層系、軟膏、ゲル、エアゾール等の任意の剤型をとることができる。また、使用形態も特に限定されるものではなく、例えば、化粧水、乳液、クリーム、エッセンス、ゼリー、ジェル、軟膏、パック、マスク、ファンデーション等の任意の形態をとることができる。 The dosage form of the stratum corneum peeling accelerator of the present invention is not particularly limited, and for example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system. Arbitrary dosage forms such as systems, ointments, gels, aerosols and the like can be taken. Also, the form of use is not particularly limited, and can take any form such as lotion, milky lotion, cream, essence, jelly, gel, ointment, pack, mask, foundation, and the like.
　本発明の角層剥離促進剤は肌に適用することで、肌荒れの予防及び／又は改善を図るための美容方法に利用できる。かかる美容方法における本発明の角層剥離促進剤の用法、用量も特に限定されるものではなく、剤型や処置する肌のしわの状態により適宜決定されるが、典型的には、１日当たり数回、例えば１回～５回、適量、例えば１平方ｃｍ2当たり0.1ｍｌから１ｍｌ、肌に直接すり込むか、又その適量をガーゼなどに染み込ませてから肌に貼付することができる。 The stratum corneum peeling promoter of the present invention can be applied to the skin and used in a cosmetic method for preventing and / or improving rough skin. The usage and dosage of the stratum corneum peeling promoter of the present invention in such a cosmetic method is not particularly limited, and is appropriately determined depending on the dosage form and the state of wrinkles on the skin to be treated. Appropriate amount, eg 0.1 to 1 ml per square cm 2 , can be rubbed directly into the skin, or the appropriate amount can be soaked in gauze or the like and applied to the skin.
　次に実施例によって本発明をさらに詳細に説明する。なお、本発明はこれにより限定されるものではない。 Next, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited by this.
（１）メソトリプシンによるＫＬＫ７の活性化は、簡単には以下の手順で調べた。
Figure JPOXMLDOC01-appb-T000001
 合成基質（chromogenic substrate）
1）S-2444：pyroGlu-Gly-Arg-p-nitroanilide
2）S2586:Arg-Pro-Tyr-p-nitroanilide
用いた酵素の濃度
エンテロペプチダーゼ:2 μg/ml
メソトリプシノーゲン：1 μg/ml
アッセイ法
　まず、上記１のメソトリプシノーゲンの活性化法により、エンテロペプチダーゼでメソトリプシノーゲンの活性化を行う。メソトリプシンの活性の確認には、合成基質Ｓ－２４４４などを用いるが、proKLK7の活性化においては、インキュベーション後、活性化されたメソトリプシンを含む溶液、5 μlをproKLK7とPBSを含む溶液に直接加えて室温で10分放置した後、基質溶液（S-2586）を加え37℃で30分インキュベーションし、遊離したp-ニトロアニリド（pNA）を405 nmで測定した。その結果、メソトリプシンが濃度依存式にＫＬＫ７を活性化することがわかった（図１）。 (1) Activation of KLK7 by mesotrypsin was simply examined by the following procedure.
Figure JPOXMLDOC01-appb-T000001
 Synthetic substrate (chromogenic substrate)
1) S-2444: pyroGlu-Gly-Arg-p-nitroanilide
2) S2586: Arg-Pro-Tyr-p-nitroanilide
Concentration of enzyme used Enteropeptidase: 2 μg / ml
Mesotrypsinogen: 1 μg / ml
Assay Method First, mesotrypsinogen is activated with enteropeptidase according to the method for activating mesotrypsinogen described in 1 above. To confirm the activity of mesotrypsin, synthetic substrate S-2444 or the like is used. In the activation of proKLK7, after incubation, 5 μl of the solution containing activated mesotrypsin is directly added to the solution containing proKLK7 and PBS. In addition, after leaving at room temperature for 10 minutes, a substrate solution (S-2586) was added and incubated at 37 ° C. for 30 minutes, and the released p-nitroanilide (pNA) was measured at 405 nm. As a result, it was found that mesotrypsin activates KLK7 in a concentration-dependent manner (FIG. 1).
（２）メソトリプシン発現促進効果を示す生薬のスクリーニング
　正常な包皮に由来するヒトケラチノサイト（Cascade Biologics社、ポートランド、メリーランド州）を、上皮増殖因子（0.1ng/ml）、インスリン（10μg/ml）、ヒドロコルチゾン（0.5μg/ml）、ウシ下垂体抽出物（0.4％）、ゲンタマイシン（50μg/ml）、アンホテリシンB（50ng/ml）を補足したMCDB 153培地からなるケラチノサイト増殖培地の中で、各種生薬エキス（丸善製薬より入手）及び薬剤（4-20 μg/ml）の存在下で24時間室温で培養した。コントロールとしては、0.1% 1，3－ブチレングリコールを用いた。 (2) Screening for crude drugs showing mesotrypsin expression promoting effect Human keratinocytes derived from normal foreskin (Cascade Biologics, Portland, Maryland), epidermal growth factor (0.1 ng / ml), insulin (10 μg / ml) ), Hydrocortisone (0.5 μg / ml), bovine pituitary extract (0.4%), gentamicin (50 μg / ml), amphotericin B (50 ng / ml) supplemented with MCDB 153 medium, various keratinocyte growth media The cells were cultured at room temperature for 24 hours in the presence of a crude drug extract (obtained from Maruzen Pharmaceutical) and a drug (4-20 μg / ml). As a control, 0.1% 1,3-butylene glycol was used.
ＲＴ－ＰＣＲ
　上記のとおりに培養したヒトケラチノサイトから単離した全RNA（500ng）を、ランダム・ヘキサマーとSuperscript II RNアーゼH-逆転写酵素（Gibco-BRL社、ゲサースバーグ、メリーランド州）を用いて逆転写した後、Taq DNAポリメラーゼ（Takara社、京都、日本国）と下記のプライマーを用いてPCR増幅した。94℃で30秒間、60℃で1分間、72℃で1分間という増幅サイクルを40回実行した。 RT-PCR
Total RNA (500 ng) isolated from human keratinocytes cultured as described above was reverse transcribed using random hexamers and Superscript II RNase H-reverse transcriptase (Gibco-BRL, Getersberg, MD) Thereafter, PCR amplification was performed using Taq DNA polymerase (Takara, Kyoto, Japan) and the following primers. 40 amplification cycles of 94 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute were performed.
　図２の結果より、コントロールと比較して、イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインが、メソトリプシンの発現を有意に亢進させることが分かる。従って、これらの抽出がいずれもメソトリプシンの発現を促進し、それ故ＫＬＫ７の活性を亢進させ、その結果角層剥離促進効果を示すことが示唆された。
フォワードプライマー: gacaatgacatcatgctgatcaaactctc　（配列番号１）
リバースプライマー： cctcaaggaagcccacacagaac　（配列番号２） From the results shown in FIG. 2, it can be seen that ginkgo biloba extract, saxifrage extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine significantly enhance the expression of mesotrypsin as compared with the control. Therefore, it was suggested that all of these extractions promote the expression of mesotrypsin and hence enhance the activity of KLK7, and as a result, show the effect of promoting stratum corneum peeling.
Forward primer: gacaatgacatcatgctgatcaaactctc (SEQ ID NO: 1)
Reverse primer: cctcaaggaagcccacacagaac (SEQ ID NO: 2)

Claims (2)

  1. 　イチョウ葉エキス、ユキノシタエキス、イザヨイバラエキス、グリチルリチン酸ジカリウム及びエクトインから成る群から選ばれる１又は複数種のメソトリプシン発現亢進剤。 1 or more types of mesotrypsin expression enhancer selected from the group consisting of Ginkgo biloba leaf extract, Yukinoshita extract, Izayoi rose extract, dipotassium glycyrrhizinate and ectoine.
  2. 　請求項１記載のメソトリプシン発現亢進剤を含む角層剥離促進剤。 A stratum corneum peeling promoter comprising the mesotrypsin expression enhancer according to claim 1.
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