JP2016003198A - Kallikrein 7 production promoter - Google Patents
Kallikrein 7 production promoter Download PDFInfo
- Publication number
- JP2016003198A JP2016003198A JP2014123582A JP2014123582A JP2016003198A JP 2016003198 A JP2016003198 A JP 2016003198A JP 2014123582 A JP2014123582 A JP 2014123582A JP 2014123582 A JP2014123582 A JP 2014123582A JP 2016003198 A JP2016003198 A JP 2016003198A
- Authority
- JP
- Japan
- Prior art keywords
- klk7
- production
- manufactured
- acid
- thioredoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
本発明は、皮膚老化に伴って減少するカリクレイン７（Ｋａｌｌｉｋｒｅｉｎ ７）（以下、ＫＬＫ７という。）の産生を促進する剤に関する。 The present invention relates to an agent that promotes the production of Kallikrein 7 (hereinafter referred to as KLK7) that decreases with skin aging.
皮膚から採取した角層におけるＫＬＫ７の発現量は、目尻の弾力性の低下に伴って有意に減少すること、及び目尻のしわ体積率の増加に伴って有意に減少することが知られている（特許文献１）。
また、ヒト表皮角化細胞におけるＫＬＫ７発現量は、カルシウム、ビタミンＤ３、レチノイン酸といった細胞分化に影響を与える分子によって変動することが知られている（非特許文献１）。また、ＫＬＫ７は、銅イオンや亜鉛イオン等の遷移金属イオンがＫＬＫ７アミノ酸側鎖のヒスチジン残基にμＭオーダーで共有結合することで阻害されることが知られている（非特許文献２）。
ＫＬＫ７産生促進剤としては、既に塩化カルシウム、レチノイン酸が知られているが、さらに皮膚外用剤への応用に適した物質の探索が望まれている。
It is known that the expression level of KLK7 in the stratum corneum collected from the skin is significantly decreased as the elasticity of the corner of the eye decreases, and is significantly decreased as the volume ratio of the wrinkle of the corner of the eye is increased ( Patent Document 1).
In addition, it is known that the expression level of KLK7 in human epidermal keratinocytes varies depending on molecules that affect cell differentiation such as calcium, vitamin D 3 and retinoic acid (Non-patent Document 1). In addition, KLK7 is known to be inhibited when a transition metal ion such as a copper ion or a zinc ion is covalently bonded to the histidine residue of the KLK7 amino acid side chain on the order of μM (Non-patent Document 2).
Calcium chloride and retinoic acid are already known as KLK7 production promoters, but further search for substances suitable for application to external preparations for skin is desired.
皮膚老化に伴って減少するＫＬＫ７の産生を促進する剤を提供する。 The present invention provides an agent that promotes the production of KLK7 that decreases with skin aging.
本発明は以下の構成である。
（１）チオレドキシン、シリビン、６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物、パントテン酸またはその塩、セダノリド、環状フォスファチジン酸から選ばれる１種以上からなるカリクレイン７産生促進剤。
（２）（１）に記載のカリクレイン７産生促進剤を含有する組成物。
（３）医薬品、化粧品、食品、飲料のいずれかであることを特徴とする（２）に記載の組成物。
The present invention has the following configuration.
(1) A kallikrein 7 production promoter comprising at least one selected from thioredoxin, silybin, 6-methylsulfinylhexyl isothiocyanate-containing wasabi extract, pantothenic acid or a salt thereof, sedanolide, and cyclic phosphatidic acid.
(2) A composition containing the kallikrein 7 production promoter according to (1).
(3) The composition according to (2), which is any one of pharmaceuticals, cosmetics, foods, and beverages.
本発明により、ＫＬＫ７の産生を促進する剤を提供することができる。 According to the present invention, an agent that promotes the production of KLK7 can be provided.
以下、本発明について詳細に説明する。
ＫＬＫ７は分子質量２７，５２５Ｄａの分泌タンパク質である。ＫＬＫ７は、角質層の細胞間どうしの結合部分を切断することにより皮膚表面からの細胞の落屑を促し、組織中では皮膚で多く発現される一方、脳、乳腺、脊髄、腎臓等でも発現が見られる。
皮膚の弾力性低下や、シワの増加は、皮膚内のＫＬＫ７量が低下しているか否かを指標として評価することができる。ＫＬＫ７産生促進剤により、皮膚内のＫＬＫ７量を増加させることで皮膚の抗老化作用が期待できる。
Hereinafter, the present invention will be described in detail.
KLK7 is a secreted protein with a molecular mass of 27,525 Da. KLK7 promotes cell desquamation from the skin surface by cleaving the cell-cell junctions in the stratum corneum, and is highly expressed in the skin in tissues, but is also expressed in the brain, breast, spinal cord, kidney, etc. It is done.
A decrease in the elasticity of the skin and an increase in wrinkles can be evaluated using as an index whether or not the amount of KLK7 in the skin is decreased. An anti-aging effect on the skin can be expected by increasing the amount of KLK7 in the skin with the KLK7 production promoter.
本発明のＫＬＫ７産生促進剤は、チオレドキシン、シリビン、６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物、パントテン酸またはその塩、セダノリド、環状フォスファチジン酸から選ばれる一種以上からなる。 The KLK7 production promoter of the present invention comprises at least one selected from thioredoxin, silybin, 6-methylsulfinylhexyl isothiocyanate-containing wasabi extract, pantothenic acid or a salt thereof, sedanolide, and cyclic phosphatidic acid.
本発明のチオレドキシンとして、ヒト組換え体チオレドキシン、チオレドキシンを高含有する清酒抽出物を用いることができる。
ヒト組換え体チオレドキシンとはヒトオリゴペプチド−４と称されるE.coliを使った組換え体のペプチドで、市販品としては、オリエンタル酵母工業株式会社製「組換え型ヒト・チオレドキシン」やアリスタヘルスアンドニュートリションサイエンス株式会社製「ｒｈｕＴＲＸ」を用いることができる。
チオレドキシンを高含有する清酒抽出物としては、市販品のアリスタヘルスアンドニュートリションサイエンス株式会社製「清酒ＴＲＸ」を用いることができる。
As a thioredoxin of the present invention, a human recombinant thioredoxin, a sake extract containing a high amount of thioredoxin can be used.
Human recombinant thioredoxin is a recombinant peptide using E. coli called human oligopeptide-4, and commercially available products include “recombinant human thioredoxin” manufactured by Oriental Yeast Co., Ltd. and Arista. “RhuTRX” manufactured by Health and Nutrition Science Co., Ltd. can be used.
As the sake extract containing a high amount of thioredoxin, a commercially available product “Sake TRX” manufactured by Arista Health and Nutrition Sciences Co., Ltd. can be used.
本発明に用いるシリビンは、抗酸化能、肝臓保護、抗老化作用などが知られているマリアアザミ（Silybum marianum）シリマリン混合物に含まれるフラボノリグナンである。本発明に用いるシリビンとして、シリビンを含有するマリアアザミシリマリン混合物を用いることができる。シリビンとして、市販品のシリビン（Sigma-Aldrich社製）を用いることができる。 The silybin used in the present invention is a flavonolignan contained in a Silybum marianum silymarin mixture, which is known for its antioxidant capacity, liver protection, anti-aging action and the like. As silybin used in the present invention, a mariamiami silymarin mixture containing silybin can be used. As silybin, commercially available silybin (manufactured by Sigma-Aldrich) can be used.
本発明には、６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物を用いられる。６−メチルスルフィニルヘキシルイソチオシアネートはワサビに含まれる成分で、ＮＡＤＰＨオキシダーゼに作用して活性酸素生成を阻害して生体内抗酸化物質（グルタチオン、チオレドキシン）を誘導することが知られている食品成分である。６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物として、市販品を使用することが可能であり、例えば金印株式会社製「金印ワサビスルフィニルＫＰＣ−１」が挙げられる。 In the present invention, 6-methylsulfinylhexyl isothiocyanate-containing horseradish extract is used. 6-Methylsulfinylhexyl isothiocyanate is a component contained in horseradish, a food ingredient known to induce in vivo antioxidants (glutathione, thioredoxin) by acting on NADPH oxidase to inhibit active oxygen production. is there. A commercially available product can be used as the 6-methylsulfinylhexyl isothiocyanate-containing horseradish extract, and examples thereof include “Kinsei Wasabi Rufinil KPC-1” manufactured by Kinshi Co., Ltd.
本発明に用いるパントテン酸は、肉類、小麦胚芽、腎臓、肝臓、ナッツ類、ビール酵母などに含まれている水溶性のビタミンである。パントテン酸はカルシウム塩等の金属塩として用いることができ、パントテン酸カルシウムは化学的な合成法で作られている食品添加物である。パントテン酸またはその塩が不足すると、栄養障害、低血糖症、血液や皮膚の障害などが生じることがある。パントテン酸またはその塩として、市販品を用いることができ、例えば、D-Pantothenic acid hemi calcium salt（パントテン酸カルシウム、Sigma-Aldrich社製）が挙げられる。 Pantothenic acid used in the present invention is a water-soluble vitamin contained in meat, wheat germ, kidney, liver, nuts, brewer's yeast and the like. Pantothenic acid can be used as a metal salt such as a calcium salt, and calcium pantothenate is a food additive made by a chemical synthesis method. Insufficient pantothenic acid or its salt may cause nutritional disorders, hypoglycemia, blood and skin disorders, and the like. A commercially available product can be used as pantothenic acid or a salt thereof, and examples thereof include D-Pantothenic acid hemi calcium salt (calcium pantothenate, manufactured by Sigma-Aldrich).
本発明に用いるセダノリドは、糖尿病の治療及び予防剤として知られている化合物であり、Apium graveolens（セロリ）やLigusticum officinale（センキョウ）に含まれる成分である。セダノリドとして、市販品を用いることができ、例えば、(＋)-Sedanolide（セダノリド、Enzo Life Science社製）が挙げられる。 The sedanolide used in the present invention is a compound known as a therapeutic and prophylactic agent for diabetes, and is a component contained in Apium graveolens (celery) and Ligusticum officinale (senkyo). A commercially available product can be used as cedanolide, and examples thereof include (+)-Sedanolide (Cedanolide, manufactured by Enzo Life Science).
本発明に用いる環状フォスファチジン酸は、大豆を出発物質として製造される環状フォスファチジン酸で、ヒアルロン酸産生増強効果を有することが知られている。環状ファオスファチジン酸として市販品を用いることができ、例えば、ＳＡＮＳＨＯ株式会社製「ｃＰＡ」、日油株式会社製「水添大豆環状リン脂質ＣＰ７」が挙げられる。 The cyclic phosphatidic acid used in the present invention is a cyclic phosphatidic acid produced using soybean as a starting material, and is known to have a hyaluronic acid production enhancing effect. Commercially available products can be used as the cyclic phasophatic acid, and examples thereof include “cPA” manufactured by SANSHO Co., Ltd. and “hydrogenated soybean cyclic phospholipid CP7” manufactured by NOF Corporation.
本発明のＫＬＫ７産生促進剤を含有する組成物として医薬品、化粧品、食品、飲料などを例示できる。その投与経路も、経口投与、経皮投与のいずれもが可能である。なかでも経皮投与が特に好ましく、化粧品や皮膚外用医薬などの経皮組成物の形態を採用することが好ましい。経口投与などにより、有効成分を皮膚に到達させることもできる。 Examples of the composition containing the KLK7 production promoter of the present invention include pharmaceuticals, cosmetics, foods, and beverages. The administration route can be either oral administration or transdermal administration. Of these, transdermal administration is particularly preferable, and it is preferable to adopt the form of a transdermal composition such as cosmetics and external medicine for skin. The active ingredient can also reach the skin by oral administration or the like.
本発明のＫＬＫ７産生促進剤である、チオレドキシン、シリビン、６−メチルスルフィニルヘキシルイソチオシアネート、パントテン酸またはその塩、セダノリド、環状フォスファチジン酸を含む組成物の製剤化にあたっては、通常の食品、医薬品、化粧料などの製剤化で使用される任意成分を含有することが出来る。この様な任意成分としては、経口投与組成物であれば、例えば、乳糖や白糖などの賦形剤、デンプン、セルロース、アラビアゴム、ヒドロキシプロピルセルロースなどの結合剤、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロースカルシウムなどの崩壊剤、大豆レシチン、ショ糖脂肪酸エステルなどの界面活性剤、マルチトールやソルビトールなどの甘味剤、クエン酸などの酸味剤、リン酸塩などの緩衝剤、シェラックやツェインなどの皮膜形成剤、タルク、ロウ類などの滑沢剤、軽質無水ケイ酸、乾燥水酸化アルミニウムゲルなどの流動促進剤、生理食塩水、ブドウ糖水溶液などの希釈剤、矯味矯臭剤、着色剤、殺菌剤、防腐剤、香料などが好適に例示出来る。経皮投与組成物であれば、スクワラン、ワセリン、マイクロクリスタリンワックスなどの炭化水素類、ホホバ油、カルナウバワックス、オレイン酸オクチルドデシルなどのエステル類、オリーブ油、牛脂、椰子油などのトリグリセライド類、ステアリン酸、オレイン酸、レチノイン酸などの脂肪酸、エタノール、イソプロパノール等の低級アルコール、オレイルアルコール、ステアリルアルコール、オクチルドデカノール等の高級アルコール、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、アルキルベタイン塩等の両性界面活性剤類、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ソルビタン脂肪酸エステル、脂肪酸モノグリセライド、これらのポリオキシエチレン付加物、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン脂肪酸エステル、ポリオキシエチレンヒマシ油、ポリオキシエチレン硬化ヒマシ油等の非イオン界面活性剤類、ジプロピレングリコール、ポリエチレングリコール、グリセリン、１，３−ブタンジオール、１，２−ペンタンジオール等の多価アルコール類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等を含有することができる。 In formulating a composition containing thioredoxin, silybin, 6-methylsulfinylhexyl isothiocyanate, pantothenic acid or a salt thereof, sedanolide, and cyclic phosphatidic acid, which are KLK7 production promoters of the present invention, ordinary foods and pharmaceuticals , Optional ingredients used in the formulation of cosmetics and the like can be contained. As such an optional component, if it is a composition for oral administration, for example, excipients such as lactose and sucrose, binders such as starch, cellulose, gum arabic, and hydroxypropyl cellulose, sodium carboxymethylcellulose, carboxymethylcellulose calcium, etc. Disintegrants, soy lecithin, surfactants such as sucrose fatty acid esters, sweeteners such as maltitol and sorbitol, sour agents such as citric acid, buffering agents such as phosphate, film forming agents such as shellac and zein, Lubricants such as talc and waxes, light anhydrous silicic acid, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline and aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, A fragrance | flavor etc. can illustrate suitably. For transdermal administration compositions, hydrocarbons such as squalane, petrolatum, and microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow, coconut oil, stearin Acids, fatty acids such as oleic acid and retinoic acid, lower alcohols such as ethanol and isopropanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate , Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, poly Nonionic surfactants such as xylethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, dipropylene glycol, polyethylene glycol, glycerin, 1,3-butanediol, 1, Polyhydric alcohols such as 2-pentanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders and the like can be contained.
以下に実施例を示し、本発明を詳細に説明する。
「ＫＬＫ７産生促進効果の測定方法」
細胞は、NHEK-Neo（ヒト表皮角化細胞、新生児皮膚由来、Life Technologies Japan社製）を用いた。培養液は、EpiLife（登録商標）Medium with 60μM Calcium（Life Technologies Japan社製）にＨｕｍｅｄｉａ−ＫＧ２増殖添加剤セット（倉敷紡績株式会社製）を加えた培地を用いた。
The following examples illustrate the invention in detail.
"Measurement method of KLK7 production promotion effect"
NHEK-Neo (human epidermal keratinocytes, derived from newborn skin, manufactured by Life Technologies Japan) was used as the cells. As a culture solution, a medium in which an EpiLife (registered trademark) Medium with 60 μM Calcium (manufactured by Life Technologies Japan) was added with a Media-KG2 proliferation additive set (manufactured by Kurashiki Boseki Co., Ltd.) was used.
NHEK-Neoを50,000cells/wellの細胞密度で平板12well plateに播種し、一晩３７℃５％ＣＯ２インキュベーターにて細胞定着後、培養液を交換時に４日間成分を暴露して３７℃５％ＣＯ２インキュベーターにて培養した。培養終了後、培養液を回収し、PBS(−)で細胞を洗浄後、Cell lysis Bufferで細胞を溶解し、Cell Lysateとしてサンプル保存した。
Cell Lysate サンプルはPierce（登録商標）BCA Protein Assay Kit（Pierce社製）にて全タンパク量を定量した。
また、ＫＬＫ７タンパク発現量は、ELISA法で測定した。
NHEK-Neo was seeded on a 12-well plate at a cell density of 50,000 cells / well, and after overnight cell fixation in a 37 ° C 5% CO 2 incubator, the components were exposed for 4 days when the culture solution was changed to 37 ° C 5% The cells were cultured in a CO 2 incubator. After completion of the culture, the culture solution was collected, the cells were washed with PBS (−), lysed with Cell lysis buffer, and the sample was stored as Cell Lysate.
Cell Lysate samples were quantified with Pierce (registered trademark) BCA Protein Assay Kit (Pierce).
Further, the expression level of KLK7 protein was measured by ELISA.
市販のELISAプレート（Coster（登録商標） 96well EIA/RIA plate、Fisher Scientific社製）にCapture抗体として、Anti-hKLK7 goat polyclonal antibody（R&D systems社製）1.11μg/ml（希釈培養液はPBS（−）、和光純薬工業株式会社製）を１００μＬ／ｗｅｌｌ滴下し、シーリングして２０℃で一晩インキュベートした。
ELISAプレートは、Phosphate Buffered Saline (PBS) with Tween（登録商標）20 (PBS-T) （タカラバイオ株式会社製）３００μＬ／ｗｅｌｌで３回洗浄した。ブロッキング溶液はReagent Diluent Concentrate（×10蒸留水希釈、R&D systems社製）２００μＬ／ｗｅｌｌを滴下し、シーリングして３７℃１ｈインキュベートした。ブロッキング終了後、ELISAプレートをPBS-T ３００μＬ／ｗｅｌｌで３回洗浄した。Cell Lysateサンプルを50倍Pierce RIPA Buffer(サーモフィッシャーサイエンティフィック株式会社製)で希釈した溶液を１００μＬ／ｗｅｌｌ添加した。併せて、別途のｗｅｌｌに抗原として精製ＫＬＫ７タンパク試料（KLK7,Recombinant,CF,Human、R&D systems社製）を250,000pg/mlから公比１／２で希釈して７点でＫＬＫ７タンパク発現量の検量線を作成した。この抗原−抗体反応は３７℃１．５ｈインキュベートで行った。抗原−抗体反応終了後、ELISAプレートをPBS-T ３００μＬ／ｗｅｌｌで３回洗浄した。Ｄｅｔｅｃｔｉｏｎ抗体として、anti-Kallilrein7,goat-poly,Biotin (R&D systems社製)０．２μｇ／ｍｌ（希釈培養液はPBS-T、タカラバイオ株式会社製）を１００μＬ／ｗｅｌｌ滴下し、３７℃１ｈインキュベートした。反応終了後、ELISAプレートをPBS-T ３００μＬ／ｗｅｌｌで３回洗浄した。引き続き、Streptavidin-HRP反応としてStreptavidin-HRP（R&D Systems社製）の５００倍希釈溶液（希釈培養液はPBS-T、タカラバイオ株式会社製）を１００μＬ／ｗｅｌｌ滴下し、３７℃３０ｍｉｎインキュベートした。反応終了後、ELISAプレートをPBS-T ３００μＬ／ｗｅｌｌで３回洗浄した。発色反応として、室温にてTMB One Solution（Promega社製）を１００μＬ／ｗｅｌｌ滴下し、１０分間反応させ、反応終了液として０．５Ｎ硫酸を１００μＬ／ｗｅｌｌを加えて、全量２００μＬ／ｗｅｌｌで吸光度４５０ｎｍをプレートリーダー（装置名：SPECTRAMAX190、モレキュラーデバイス社製）にて読み取って、検量線に対するＫＬＫ７タンパク発現量を算出した。
予め測定した全タンパク量との比でＫＬＫ７タンパク発現量（pg/μg protein）を求めた。
Anti-hKLK7 goat polyclonal antibody (manufactured by R & D systems) 1.11 μg / ml as a capture antibody on a commercially available ELISA plate (Coster (registered trademark) 96 well EIA / RIA plate, manufactured by Fisher Scientific) ), Wako Pure Chemical Industries, Ltd.) was added dropwise at 100 μL / well, sealed, and incubated at 20 ° C. overnight.
The ELISA plate was washed three times with 300 μL / well of Phosphate Buffered Saline (PBS) with Tween (registered trademark) 20 (PBS-T) (Takara Bio Inc.). As a blocking solution, 200 μL / well of Reagent Diluent Concentrate (× 10 diluted with distilled water, manufactured by R & D systems) was added dropwise, sealed, and incubated at 37 ° C. for 1 h. After the blocking, the ELISA plate was washed 3 times with PBS-T 300 μL / well. A solution obtained by diluting the Cell Lysate sample with 50 times Pierce RIPA Buffer (manufactured by Thermo Fisher Scientific Co., Ltd.) was added at 100 μL / well. In addition, a purified KLK7 protein sample (KLK7, Recombinant, CF, Human, manufactured by R & D systems) as an antigen in a separate well was diluted from 250,000 pg / ml to a common ratio of 1/2, and the expression level of KLK7 protein was determined at 7 points. A calibration curve was created. This antigen-antibody reaction was performed by incubation at 37 ° C. for 1.5 hours. After completion of the antigen-antibody reaction, the ELISA plate was washed 3 times with PBS-T 300 μL / well. Anti-Kallilrein 7, goat-poly, Biotin (manufactured by R & D systems) 0.2 μg / ml (PBS-T, manufactured by Takara Bio Inc.) as a detection antibody was added dropwise at 100 μL / well and incubated at 37 ° C. for 1 hour. did. After completion of the reaction, the ELISA plate was washed three times with 300 μL / well of PBS-T. Subsequently, as a Streptavidin-HRP reaction, a 500-fold diluted solution of Streptavidin-HRP (manufactured by R & D Systems) (PBS-T, manufactured by Takara Bio Inc.) was added dropwise at 100 μL / well and incubated at 37 ° C. for 30 min. After completion of the reaction, the ELISA plate was washed three times with 300 μL / well of PBS-T. As a color development reaction, 100 μL / well of TMB One Solution (Promega) was added dropwise at room temperature, reacted for 10 minutes, 100 N / well of 0.5 N sulfuric acid was added as a reaction end solution, and the absorbance was 450 nm with a total amount of 200 μL / well. Was read with a plate reader (device name: SPECTRAMAX190, manufactured by Molecular Devices), and the expression level of KLK7 protein relative to the calibration curve was calculated.
The expression level of KLK7 protein (pg / μg protein) was determined by the ratio to the total protein amount measured in advance.
「実施例１」
チオレドキシンのＫＬＫ７産生促進効果
チオレドキシンとして、ヒト組換え体チオレドキシン（rhuTRX、アリスタヘルスアンドニュートリションサイエンス株式会社製）を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図１に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。rhuTRXは１〜１０μｇ／ｍｌの範囲で、濃度依存的に細胞内ＫＬＫ７を増加させた。
また、既にＫＬＫ７産生促進が知られている塩化カルシウムは１ｍＭから０．１ｍＭにおいて、レチノイド（All trans レチノイン酸、和光純薬工業株式会社製）は１００ｎＭから１０ｎＭ添加でそれぞれＫＬＫ７発現量が増加していることを確認し、試験系が妥当であることを確認した。図１中のＣａＣｌ２は塩化カルシウムを表し、ＲＡはAll trans レチノイン酸を表す。なお、All trans レチノイン酸は脂溶性に富む化合物で、そのままでは培養液に溶解できないので、まずAll trans レチノイン酸をＤＭＳＯ（ジメチルスルホキシド）に溶解し、そのＤＭＳＯ溶液を培養液に希釈したため、レチノイドの試験系は、ＤＭＳＯを０．０２５％含有している。
ヒト組み換え体チオレドキシンは、塩化カルシウム、レチノイドと比較して、ＫＬＫ７の産生を促進した。
"Example 1"
KLK7 production promoting effect of thioredoxin KLK7 production promoting effect was measured using human recombinant thioredoxin (rhuTRX, manufactured by Arista Health and Nutrition Science Co., Ltd.) as thioredoxin. The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD for each group. rhuTRX increased intracellular KLK7 concentration-dependently in the range of 1-10 μg / ml.
In addition, calcium chloride, which is already known to promote production of KLK7, increases in expression level of KLK7 from 1 mM to 0.1 mM, and retinoid (All trans retinoic acid, manufactured by Wako Pure Chemical Industries, Ltd.) increases from 100 nM to 10 nM. It was confirmed that the test system was valid. In FIG. 1, CaCl 2 represents calcium chloride, and RA represents All trans retinoic acid. All trans retinoic acid is a compound rich in fat solubility and cannot be dissolved in the culture medium as it is. Therefore, all trans retinoic acid was first dissolved in DMSO (dimethyl sulfoxide), and the DMSO solution was diluted in the culture medium. The test system contains 0.025% DMSO.
Human recombinant thioredoxin promoted the production of KLK7 as compared with calcium chloride and retinoid.
チオレドキシンを高含有する清酒抽出物（アリスタヘルスアンドニュートリションサイエンス株式会社製 清酒ＴＲＸ）を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図２に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。清酒ＴＲＸは５μｇ／ｍｌから１５μｇ／ｍｌにおいて、濃度依存的に細胞内ＫＬＫ７の産生を促進した。 Using a sake extract containing a high amount of thioredoxin (Sake Sake TRX manufactured by Arista Health and Nutrition Science Co., Ltd.), the KLK7 production promoting effect was measured. The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD in each group. Sake TRX promoted intracellular KLK7 production in a concentration-dependent manner at 5 μg / ml to 15 μg / ml.
「実施例２」
シリビンのＫＬＫ７産生促進効果
シリビン（Sigma-Aldrich社製）のＫＬＫ７産生促進効果を測定した。測定結果を図３に示す。ＫＬＫ７タンパク発現量は各群n=2の平均値で表記した。シリビンは０．０５ｍｇ／ｍｌ添加で細胞内ＫＬＫ７の産生を促進した。その他にビタミンＣ（和光純薬工業株式会社製）、ビタミンＥ（関東化学株式会社製）、ＣｏＱ１０（Sigma社製）、アスタキサンチン（Sigma社製）、レスベラトロール（和光純薬工業株式会社製）、αリポ酸（Sigma社製）の効果を調べたが、ＫＬＫ７産生促進効果は認められなかった。この試験系で、コントロール以外のサンプルは、ＤＭＳＯを０．０２５％含有する。
"Example 2"
KLK7 production promoting effect of silybin The KLK7 production promoting effect of silybin (Sigma-Aldrich) was measured. The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as the average value of each group n = 2. Silybin promoted the production of intracellular KLK7 by addition of 0.05 mg / ml. In addition, vitamin C (manufactured by Wako Pure Chemical Industries, Ltd.), vitamin E (manufactured by Kanto Chemical Co., Ltd.), CoQ10 (manufactured by Sigma), astaxanthin (manufactured by Sigma), resveratrol (manufactured by Wako Pure Chemical Industries, Ltd.) The effect of α lipoic acid (manufactured by Sigma) was examined, but no KLK7 production promoting effect was observed. In this test system, the sample other than the control contains 0.025% DMSO.
「実施例３」
６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物のＫＬＫ７産生促進効果
６−メチルスルフィニルヘキシルイソチオシアネート含有ワサビ抽出物として、金印株式会社製、金印ワサビスルフィニルＫＰＣ−１を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図４に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。金印ワサビスルフィニルＫＰＣ−１は５μｇ／ｍｌから５０μｇ／ｍｌにおいて、細胞内ＫＬＫ７の産生を促進した。図４中の「ＫＰＣ−１」は金印株式会社製 金印ワサビスルフィニルＫＰＣ−１を表す。
"Example 3"
KLK7 production promoting effect of wasabi extract containing 6-methylsulfinylhexyl isothiocyanate KLK7 production promoting effect using 6-methylsulfinylhexyl isothiocyanate-containing wasabi extract made by Kinshi Co., Ltd. Was measured. The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD for each group. Gold stamp Wasabisulfinyl KPC-1 promoted intracellular KLK7 production at 5 to 50 μg / ml. “KPC-1” in FIG. 4 represents Gold Seal Wasabisulfinyl KPC-1 manufactured by Kinshi Co., Ltd.
「実施例４」
パントテン酸カルシウムのＫＬＫ７産生促進効果
パントテン酸カルシウム（Sigma-Aldrich社製 D-Pantothenic acid hemi calcium salt）を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図５に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。パントテン酸カルシウムは１０μｇ／ｍｌから１００μｇ／ｍｌにおいて濃度依存的に細胞内ＫＬＫ７タンパク発現量を増加させた。
Example 4
KLK7 production promoting effect of calcium pantothenate The KLK7 production promoting effect was measured using calcium pantothenate (D-Pantothenic acid hemi calcium salt manufactured by Sigma-Aldrich). The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD for each group. Calcium pantothenate increased intracellular KLK7 protein expression in a concentration-dependent manner from 10 μg / ml to 100 μg / ml.
「実施例５」
セダノリドのＫＬＫ７産生促進効果
セダノリド（Enzo Life Science社製 (＋)−Sedanolide）を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図６に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。セダノリドは０．５μｇ／ｍｌから５μｇ／ｍｌにおいて濃度依存的に細胞内ＫＬＫ７タンパク発現量を増加させた。
"Example 5"
Effect of sedanolide on promoting KLK7 production Using sedanolide ((+)-Sedanolide manufactured by Enzo Life Science), the effect of promoting KLK7 production was measured. The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD for each group. Sedanolide increased the expression level of intracellular KLK7 protein in a concentration-dependent manner from 0.5 μg / ml to 5 μg / ml.
「実施例６」
環状フォスファチジン酸のＫＬＫ７産生促進効果
環状フォスファチジン酸（ＳＡＮＳＨＯ株式会社製 ｃＰＡ）を用いて、ＫＬＫ７産生促進効果を測定した。測定結果を図７に示す。ＫＬＫ７タンパク発現量は各群n=3, mean±S.D.で表記した。環状フォフファチジン酸は５μｇ／ｍｌから１５μｇ／ｍｌにおいて濃度依存的に細胞内ＫＬＫ７タンパク発現量を増加させた。
"Example 6"
KLK7 production promoting effect of cyclic phosphatidic acid KLK7 production promoting effect was measured using cyclic phosphatidic acid (cPA manufactured by SANSHO Co., Ltd.). The measurement results are shown in FIG. The expression level of KLK7 protein was expressed as n = 3, mean ± SD for each group. Cyclic phosphatidic acid increased the expression level of intracellular KLK7 protein in a concentration-dependent manner at 5 μg / ml to 15 μg / ml.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014123582A JP6609401B2 (en) | 2014-06-16 | 2014-06-16 | Kallikrein 7 production promoter |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014123582A JP6609401B2 (en) | 2014-06-16 | 2014-06-16 | Kallikrein 7 production promoter |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2016003198A true JP2016003198A (en) | 2016-01-12 |
| JP6609401B2 JP6609401B2 (en) | 2019-11-20 |
Family
ID=55222748
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014123582A Active JP6609401B2 (en) | 2014-06-16 | 2014-06-16 | Kallikrein 7 production promoter |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6609401B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016216436A (en) * | 2015-05-18 | 2016-12-22 | 株式会社ファンケル | Skin viscoelasticity improver |
| JP2017125117A (en) * | 2016-01-13 | 2017-07-20 | 金印株式会社 | Antioxidant |
| JP2018131405A (en) * | 2017-02-15 | 2018-08-23 | 株式会社ファンケル | Kallikrein 7 production-promoting composition |
| CN112294686A (en) * | 2019-07-29 | 2021-02-02 | 友华生技医药股份有限公司 | Cosmetic composition for preventing and/or improving skin aging and application thereof |
| WO2023233405A1 (en) * | 2022-05-31 | 2023-12-07 | Ahava - Dead Sea Laboratories Ltd. | Skin care compositions |
Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992021323A1 (en) * | 1991-05-31 | 1992-12-10 | The Procter & Gamble Company | Use of lysophosphate acids for the treatment of skin wrinkles |
| JPH0912471A (en) * | 1995-06-29 | 1997-01-14 | Noevir Co Ltd | Skin preparation for external use |
| JP2002234845A (en) * | 2001-02-09 | 2002-08-23 | Naris Cosmetics Co Ltd | Skin care preparation |
| JP2003292432A (en) * | 2002-04-04 | 2003-10-15 | Noevir Co Ltd | Skin care preparation |
| JP2004091397A (en) * | 2002-08-30 | 2004-03-25 | Fancl Corp | Skin aging-preventive composition for preventing and ameliorating epidermis flattening |
| JP2004131431A (en) * | 2002-10-11 | 2004-04-30 | Fancl Corp | Composition for preventing or ameliorating ultraviolet damage |
| JP2006008562A (en) * | 2004-06-24 | 2006-01-12 | Kinjirushi Kk | Elastase activity inhibitor and cosmetic |
| JP2006176440A (en) * | 2004-12-22 | 2006-07-06 | Kinjirushi Kk | Bleaching or aging-preventing agent composition for skin and cosmetic |
| JP2006241005A (en) * | 2005-03-01 | 2006-09-14 | Kinjirushi Kk | Skin aging-preventing agent and cosmetic |
| JP2007099650A (en) * | 2005-10-03 | 2007-04-19 | Fancl Corp | Composition for removing abnormal protein |
| JP2007230893A (en) * | 2006-02-28 | 2007-09-13 | Univ Of Tokyo | Method for producing sedanolide |
| JP2008222643A (en) * | 2007-03-13 | 2008-09-25 | Nof Corp | Liposome for cosmetic use |
| JP2010065009A (en) * | 2008-09-12 | 2010-03-25 | Maruzen Pharmaceut Co Ltd | Skin brightener and anti-aging agent, and skin cosmetic |
| JP2010065025A (en) * | 2008-08-13 | 2010-03-25 | Seiren Co Ltd | Composition for protecting horny layer desquamation enzyme |
| JP2011001269A (en) * | 2009-06-16 | 2011-01-06 | Chikako Nishigori | External preparation for preventing or treating ultraviolet-induced dermatitis or primary irritant dermatitis |
| WO2012115247A1 (en) * | 2011-02-25 | 2012-08-30 | 株式会社資生堂 | Stratum corneum peeling accelerator |
| JP5362219B2 (en) * | 2005-08-23 | 2013-12-11 | 株式会社ファンケル | Skin aging marker and its utilization technology |
-
2014
- 2014-06-16 JP JP2014123582A patent/JP6609401B2/en active Active
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992021323A1 (en) * | 1991-05-31 | 1992-12-10 | The Procter & Gamble Company | Use of lysophosphate acids for the treatment of skin wrinkles |
| JPH0912471A (en) * | 1995-06-29 | 1997-01-14 | Noevir Co Ltd | Skin preparation for external use |
| JP2002234845A (en) * | 2001-02-09 | 2002-08-23 | Naris Cosmetics Co Ltd | Skin care preparation |
| JP2003292432A (en) * | 2002-04-04 | 2003-10-15 | Noevir Co Ltd | Skin care preparation |
| JP2004091397A (en) * | 2002-08-30 | 2004-03-25 | Fancl Corp | Skin aging-preventive composition for preventing and ameliorating epidermis flattening |
| JP2004131431A (en) * | 2002-10-11 | 2004-04-30 | Fancl Corp | Composition for preventing or ameliorating ultraviolet damage |
| JP2006008562A (en) * | 2004-06-24 | 2006-01-12 | Kinjirushi Kk | Elastase activity inhibitor and cosmetic |
| JP2006176440A (en) * | 2004-12-22 | 2006-07-06 | Kinjirushi Kk | Bleaching or aging-preventing agent composition for skin and cosmetic |
| JP2006241005A (en) * | 2005-03-01 | 2006-09-14 | Kinjirushi Kk | Skin aging-preventing agent and cosmetic |
| JP5362219B2 (en) * | 2005-08-23 | 2013-12-11 | 株式会社ファンケル | Skin aging marker and its utilization technology |
| JP2007099650A (en) * | 2005-10-03 | 2007-04-19 | Fancl Corp | Composition for removing abnormal protein |
| JP2007230893A (en) * | 2006-02-28 | 2007-09-13 | Univ Of Tokyo | Method for producing sedanolide |
| JP2008222643A (en) * | 2007-03-13 | 2008-09-25 | Nof Corp | Liposome for cosmetic use |
| JP2010065025A (en) * | 2008-08-13 | 2010-03-25 | Seiren Co Ltd | Composition for protecting horny layer desquamation enzyme |
| JP2010065009A (en) * | 2008-09-12 | 2010-03-25 | Maruzen Pharmaceut Co Ltd | Skin brightener and anti-aging agent, and skin cosmetic |
| JP2011001269A (en) * | 2009-06-16 | 2011-01-06 | Chikako Nishigori | External preparation for preventing or treating ultraviolet-induced dermatitis or primary irritant dermatitis |
| WO2012115247A1 (en) * | 2011-02-25 | 2012-08-30 | 株式会社資生堂 | Stratum corneum peeling accelerator |
Non-Patent Citations (3)
| Title |
|---|
| "京大発ベンチャー、清酒由来成分の事業化本格化、黄桜から化粧品", 化学工業日報, JPN6019002142, 1 April 2013 (2013-04-01), pages 5, ISSN: 0003964075 * |
| "化粧品原料 アリスタヘルス＆ニュートリション", 化学工業日報, JPN6019002143, 15 March 2013 (2013-03-15), pages 8, ISSN: 0003964076 * |
| "日本酒成分入り化粧品、黄桜、４０〜６０代女性向け。", 日本経済新聞 地方経済面 京都・滋賀, JPN6019002145, 4 April 2013 (2013-04-04), pages 45, ISSN: 0003964077 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016216436A (en) * | 2015-05-18 | 2016-12-22 | 株式会社ファンケル | Skin viscoelasticity improver |
| JP2017125117A (en) * | 2016-01-13 | 2017-07-20 | 金印株式会社 | Antioxidant |
| JP2018131405A (en) * | 2017-02-15 | 2018-08-23 | 株式会社ファンケル | Kallikrein 7 production-promoting composition |
| CN112294686A (en) * | 2019-07-29 | 2021-02-02 | 友华生技医药股份有限公司 | Cosmetic composition for preventing and/or improving skin aging and application thereof |
| EP3771462A1 (en) * | 2019-07-29 | 2021-02-03 | Orient Europharma Co Ltd | Cosmetic compositions to prevent and/or ameliorate skin aging and methods of applications |
| CN112294686B (en) * | 2019-07-29 | 2024-01-16 | 友华生技医药股份有限公司 | Cosmetic composition for preventing and/or improving skin aging and application thereof |
| WO2023233405A1 (en) * | 2022-05-31 | 2023-12-07 | Ahava - Dead Sea Laboratories Ltd. | Skin care compositions |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6609401B2 (en) | 2019-11-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6609401B2 (en) | Kallikrein 7 production promoter | |
| Piccolomini et al. | High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells | |
| JP2002193738A (en) | Use of at least one extract from at least one azalea plant in composition for treating symptom of skin aging | |
| TW201302242A (en) | Collagen production promoter, hyaluronic acid production promoter, fibroblast proliferation promoter and anti-wrinkle agent | |
| JP2018043999A (en) | Collagen hydrolysate and use thereof | |
| EP3372220B1 (en) | Use of gingerone or derivatives thereof for reducing or delaying the signs of skin ageing | |
| JP2008056569A (en) | External preparation for skin | |
| JP2013241398A (en) | β-GLUCOCEREBROSIDASE ACTIVITY ENHANCER, CERAMIDE PRODUCTION ENHANCER, AND SKIN BARRIER FUNCTION IMPROVING AGENT | |
| WO2019211567A1 (en) | Composition comprising alpha-lipoic acid or an alpha-lipoic acid salt, a vitamin c derivative and hyaluronic acid and its use | |
| JP2017043594A (en) | Hair-growing composition | |
| JP6032918B2 (en) | Topical skin preparation | |
| JPWO2016121962A1 (en) | Melanin production inhibitor | |
| JP6580342B2 (en) | Tropoelastin expression promoter and elastin production promoter | |
| JP6370094B2 (en) | Composition for suppressing yellowing of skin | |
| KR20170078270A (en) | Composition for improving skin conditions comprising Ethyl ferulate | |
| JP5918168B2 (en) | β-Glucocerebrosidase activity enhancer | |
| EP3130332A1 (en) | Anti-aging external skin preparation | |
| KR101916531B1 (en) | albinism preventing and melanotrichia promoting composition comprising extracts of Tenebrio molitor | |
| WO2014208093A1 (en) | Moisturizing-related gene expression promoting agent related to improving skin moisturizing function | |
| KR101515158B1 (en) | Cosmetic composition containing disulfanyl hypopigmenting peptide | |
| JP2013257347A (en) | Screening method and antiaging composition | |
| JP2013166731A (en) | Endothelin activity inhibitor and whitening agent | |
| KR20140118345A (en) | Composition for renewing skin containing vacuum-distillated chamomile extract as effective component | |
| JP2010159252A (en) | Melanosome traffick inhibitor, and method for the same | |
| US11318079B2 (en) | Polyhydroxylated (1-phenyl-2-phenyl) ethylene derivatives as an anti-ageing and photoprotective agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20170131 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20171124 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20171219 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20180214 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180413 Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20180413 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20180925 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20181114 Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181114 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20181121 |
|
| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20190125 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190704 Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20190704 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20191028 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6609401 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |