JP2007223965A - Epidermal keratinocyte endotherin-1 production inhibitor - Google Patents
Epidermal keratinocyte endotherin-1 production inhibitor Download PDFInfo
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本発明は、シミ、ソバカスなどの疾患の治療または予防に利用できる表皮角化細胞エンドセリン-1産生抑制剤及び美白化粧料に関する。 The present invention relates to an epidermal keratinocyte endothelin-1 production inhibitor and a whitening cosmetic that can be used for the treatment or prevention of diseases such as spots and buckwheat.
エンドセリン-1は血管内皮細胞の産生する強力な血管収縮ペプチドで21残基のアミノ酸からなる。エンドセリン-1は血管内皮細胞に存在するエンドセリン変換酵素の働きでビッグエンドセリンから生成する。エンドセリン-1は標的細胞のエンドセリン受容体を介してホスホリパーゼＣの活性化、カルシウムチャンネルの開口など多様な細胞内シグナル伝達系を活性化する。その結果、ホルモン・神経伝達物質分泌調節、細胞増殖・分化作用などさまざまな作用が奏される。一般的に、エンドセリン-1の濃度上昇は高血圧症や心疾患などの原因となる。 Endothelin-1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and consists of 21 amino acids. Endothelin-1 is produced from big endothelin by the action of endothelin converting enzyme present in vascular endothelial cells. Endothelin-1 activates various intracellular signal transduction systems such as activation of phospholipase C and opening of calcium channels via the endothelin receptor of target cells. As a result, various effects such as regulation of hormone / neurotransmitter secretion and cell proliferation / differentiation are exhibited. Generally, an increase in the concentration of endothelin-1 causes hypertension, heart disease, and the like.
エンドセリン-1はまた、血管以外の種々の組織細胞で産生され、様々な生理的役割を担っている。例えば、紫外線が肌にあたると、表皮角化細胞（ケラチノサイトとも言う）でエンドセリン-1が産生され（非特許文献１参照）、産生されたエンドセリン-1は、表皮メラニン細胞（メラノサイトとも言う）を刺激して、シミ、ソバカスの原因となるメラニンを盛んに合成させる。したがって、表皮角化細胞のエンドセリン-1の合成を抑制することができればシミ、ソバカスを治療または予防することができる。
かかる市場の要求に応えるべく、エンドセリン-1産生抑制剤が研究されてきた。従来エンドセリン-1産生抑制剤として検討されたものは化学合成品が中心であるが、安全性を考慮すれば天然物で同様の効果を有するものが望まれる。
Endothelin-1 is also produced in various tissue cells other than blood vessels and plays various physiological roles. For example, when ultraviolet rays hit the skin, endothelin-1 is produced by epidermal keratinocytes (also called keratinocytes) (see Non-Patent Document 1), and the produced endothelin-1 stimulates epidermal melanocytes (also called melanocytes). Then, melanin causing freckles and freckles is actively synthesized. Therefore, if the synthesis of endothelin-1 in epidermal keratinocytes can be suppressed, it is possible to treat or prevent spots and buckwheat.
Endothelin-1 production inhibitors have been studied to meet such market demands. Conventionally, what has been studied as an endothelin-1 production inhibitor is mainly a chemically synthesized product, but considering safety, a natural product having the same effect is desired.
これまでに天然物として、赤ワイン抽出物に含まれるポリフェノール（ピセアタンノール、デルフィニジンなど）（非特許文献２参照）、大豆イソフラボン（ゲニステイン）（非特許文献３参照）、ルテオリンおよびクダモノトケイソウ抽出物（特許文献１）が血管内皮細胞エンドセリン-1の産生抑制剤として検討されてはいるものの、紫外線照射時（あるいは紫外線非照射時）の表皮角化細胞のエンドセリン-1産生を抑制することは報告されていない。
現在、市場にある美白化粧品の多くはチロシナーゼ活性を抑制する働きを持つが、カミツレの花から抽出して得られるカミツレエキスにはエンドセリン-1の働きを抑える作用（エンドセリン-1拮抗薬）があり、結果としてメラニンの産生を抑制することから新しいタイプの美白化粧品として販売されている。また、エンドセリン拮抗薬が請求項に記載された公開特許公報として特許文献２−５が例示される。
As a natural product, polyphenols (piceatannol, delphinidin, etc.) contained in red wine extract (see Non-Patent Document 2), soy isoflavone (Genistein) (see Non-Patent Document 3), luteolin and Kudamono Toiiso extract ( Although Patent Document 1) has been studied as an inhibitor of vascular endothelial cell endothelin-1 production, it has been reported to suppress endothelin-1 production of epidermal keratinocytes during ultraviolet irradiation (or non-ultraviolet irradiation). Not.
Currently, many whitening cosmetics on the market have the effect of suppressing tyrosinase activity, but chamomile extract obtained by extracting from chamomile flowers has the action of suppressing the action of endothelin-1 (endothelin-1 antagonist) As a result, it is marketed as a new type of whitening cosmetic because it suppresses the production of melanin. Moreover, patent document 2-5 is illustrated as an open patent publication with which the endothelin antagonist was described in the claim.
また、紫外線により生じる皮膚傷害を防止する化粧料として、表皮角化細胞から産生されるadult T cell leukemia derived factor（表皮メラニン細胞に働いてメラニン産生を促進させる）を抑制する効果を有する物質のなかにフラボン類及び/又はその配糖体が記載されている（特許文献６）。
このように美白効果のある物質は種々知られているが、紫外線照射時（あるいは紫外線非照射時）の表皮角化細胞のエンドセリン-1産生を抑制する天然物は報告されておらず、かかる原理に基づくエンドセリン-1産生抑制剤が必要とされている。
In addition, as a cosmetic that prevents skin damage caused by ultraviolet rays, it is a substance that has the effect of suppressing adult T cell leukemia derived factor (which works on epidermal melanocytes and promotes melanin production) produced from epidermal keratinocytes. Describes flavones and / or glycosides thereof (Patent Document 6).
Various substances with such whitening effects are known, but no natural product that suppresses endothelin-1 production of epidermal keratinocytes when irradiated with ultraviolet light (or when not irradiated with ultraviolet light) has not been reported. There is a need for endothelin-1 production inhibitors based on the above.
この出願に関係する先行技術文献情報としては次のものがある。
本発明の課題は、天然物由来の、安全でかつ有効性の高い表皮角化細胞エンドセリン-1産生抑制剤を提供することである。 An object of the present invention is to provide a safe and highly effective epidermis keratinocyte endothelin-1 production inhibitor derived from a natural product.
本発明者らは、天然物由来の物質中から、表皮角化細胞のエンドセリン-1産生を効果的に抑制する物質を鋭意探索した結果、スクテラレイン（Scutellarein）、ユーパフォリン（Eupafolin）及びシネンセチン（Sinensetin）が、表皮角化細胞エンドセリン-1産生を効果的に抑制するとともに、これらが安全で、美白化粧料の有効成分としても有効であることを見いだし、本発明を完成させた。
すなわち本発明以下のとおりである。
（１）スクテラレイン（Scutellarein）、ユーパフォリン（Eupafolin）又はシネンセチン（Sinensetin）のいずれか一種以上を有効成分として含有することを特徴とする表皮角化細胞エンドセリン-1産生抑制剤。
（２）スクテラレイン（Scutellarein）、ユーパフォリン（Eupafolin）又はシネンセチン（Sinensetin）のいずれか一種以上を有効成分として含有することを特徴とする、美白化粧料。
As a result of diligent search for substances that effectively suppress endothelin-1 production of epidermal keratinocytes among substances derived from natural products, the present inventors have found that Scutellarein, Eupaforin, and Sinensetin. However, the present inventors have found that, while effectively inhibiting the production of epithelial keratinocyte endothelin-1, they are safe and effective as an active ingredient of whitening cosmetics, and the present invention has been completed.
That is, the present invention is as follows.
(1) An epidermis keratinocyte endothelin-1 production inhibitor containing any one or more of Scutellarein, Eupaforin, and Sinensetin as an active ingredient.
(2) Whitening cosmetics characterized by containing any one or more of Scutellarein, Eupaforin and Sinensetin as an active ingredient.
本発明の使用化合物であるスクテラレイン（Scutellarein）、ユーパフォリン（Eupafolin）及びシネンセチン（Sinensetin）は、いずれも植物由来のものであって、安全性に優れ、高い表皮角化細胞エンドセリン-1産生抑制作用を有する。したがって、本発明によれば、より有用な表皮角化細胞エンドセリン-1産生抑制在及び美白化粧料が提供できる。 The compounds used in the present invention, scutellarein, eupafolin, and sinensetin, are all derived from plants and have excellent safety and high inhibitory effect on endothelin-1 production of epidermal keratinocytes. Have. Therefore, according to the present invention, it is possible to provide a more useful epidermal keratinocyte endothelin-1 production inhibitor and whitening cosmetic.
以下、上記発明について詳細に説明するが、本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜実施し得る。
本発明は、スクテラレイン（scutellarein）、ユーパフォリン（eupafolin）、及びシネンセチン（sinensetin）の少なくとも一種以上を有効成分として含有する表皮角化細胞エンドセリン-1産生抑制剤、及び美白化粧料に関する。
本発明で使用するスクテラレイン（scutellarein）、ユーパフォリン（eupafolin）、及びシネンセチン（sinensetin）はいずれも、特に、紫外線で刺激した表皮角化細胞に作用してエンドセリン-1産生を抑制する効果を有する点で有用ではあるが、紫外線非照射下においても、表皮角化細胞におけるエンドセリン-1産生を抑制する。また、この表皮角化細胞におけるエンドセリン−１産生抑制作用に基づき、皮膚の美白作用をする。
Hereinafter, although the said invention is demonstrated in detail, the range of this invention is not restrained by these description and can implement suitably in the range which does not impair the meaning of this invention except the following illustration.
The present invention relates to an epidermal keratinocyte endothelin-1 production inhibitor containing at least one or more of scutellarein, eupafolin, and sinensetin as an active ingredient, and a whitening cosmetic.
All of scutellarein, eupafolin, and sinensetin used in the present invention particularly have an effect of suppressing endothelin-1 production by acting on epidermal keratinocytes stimulated with ultraviolet rays. Although useful, it suppresses endothelin-1 production in epidermal keratinocytes even when not irradiated with ultraviolet rays. Moreover, based on the endothelin-1 production inhibitory effect in this epidermal keratinocyte, it has the skin whitening effect | action.
スクテラレイン（Scutellarein；4',5,6,7-テトラヒドロキシフラボン）はフラボノイドの一種であり、Scutellaria barbata（半枝蓮）などのシソ科植物に含まれている天然物である。
また、ユーパフォリン（eupafolin；6-メトキシルテオリン）もフラボノイドの一種でありキク科のEupatorium cannabinumなどに含まれている天然物であり、シネンセチン（Sinensetin；3',4',5,6,7-ペンタメトキシフラボン）もフラボノイドの一種でありシソ科のクミスクチン（Orthosiphon aristatus）や柑橘類の果皮などに含まれている天然物である。
Scutellarein (4 ', 5,6,7-tetrahydroxyflavone) is a kind of flavonoid and is a natural product contained in Lamiaceae plants such as Scutellaria barbata.
In addition, eupafolin (eupafolin; 6-methoxylteolin) is a kind of flavonoid and is a natural product contained in Eupatorium cannabinum of the family Asteraceae. Sinensetin (Sinensetin; Methoxyflavone) is a kind of flavonoid, and is a natural product contained in the persimmon skin, such as Orthosiphon aristatus and citrus peel.
これらスクテラレイン（scutellarein）、ユーパフォリン（eupafolin）、またはシネンセチン（sinensetin）がエンドセリン-1の産生を抑制する効果を有することは従来知られておらず、また、ポリフェノール類が表皮角化細胞のエンドセリン-1産生を抑制することも知られていない。
本発明におけるスクテラレイン（scutellarein）、ユーパフォリン（eupafolin）、シネンセチン（sinensetin）は市販品を用いることもできるが、種々の植物から単離して用いてもよい。
It has not been known that these scutellarein, eupafolin, or sinensetin has the effect of suppressing the production of endothelin-1, and polyphenols are also known as endothelin-1 of epidermal keratinocytes. It is also not known to suppress production.
Commercially available products can be used for scutellarein, eupafolin and sinensetin in the present invention, but they may be isolated from various plants.
本発明の表皮角化細胞エンドセリン-1産生抑制剤は、それ自体を直接適用してもよく、また、溶液状、可溶化状、乳化状、粉末状、ペースト状、ムース状、ジェル状にして、化粧水、乳液、クリーム、パック、軟膏等として、例えば皮膚外用剤としてあるいは美白化粧料の形態で、皮膚に適用することができる。
また、本発明の表皮角化細胞のエンドセリン-1産生抑制剤には必要に応じて本発明の効果を損なわない範囲で、通常、化粧料や医薬部外品、外用医薬品等の製剤に使用される成分、すなわち、精製水、アルコール類、水溶性高分子、油剤、界面活性剤、ゲル化剤、保湿剤、ビタミン類、抗菌剤、香料、塩類、ＰＨ調整剤等を加えることができる。
本発明の表皮角化細胞エンドセリン-1産生抑制剤の含有量は0.0001％〜1％が好ましく、より好ましくは0.0005％〜0.1％である。この場合、スクテラレイン（scutellarein、ユーパフォリン（eupafolin）またはシネンセチン（sinensetin）をそれぞれ単独で、あるいはこれらの２種、3種を適宜組み合わせて用いることができる。
The epidermal keratinocyte endothelin-1 production inhibitor of the present invention may be directly applied as it is, or in the form of a solution, a solubilized form, an emulsified form, a powder form, a paste form, a mousse form, or a gel form. It can be applied to the skin as a skin lotion, emulsion, cream, pack, ointment, etc., for example, as a skin external preparation or in the form of a whitening cosmetic.
Moreover, the endothelin-1 production inhibitor of epidermal keratinocytes of the present invention is usually used in preparations such as cosmetics, quasi-drugs, and external medicines as long as the effects of the present invention are not impaired as necessary. In other words, purified water, alcohols, water-soluble polymers, oil agents, surfactants, gelling agents, moisturizers, vitamins, antibacterial agents, fragrances, salts, pH adjusting agents and the like can be added.
The content of the epidermal keratinocyte endothelin-1 production inhibitor of the present invention is preferably 0.0001% to 1%, more preferably 0.0005% to 0.1%. In this case, scutellarein, eupafolin, or sinensetin can be used alone, or two or three of these can be used in appropriate combination.
本発明の表皮角化細胞エンドセリン-1産生抑制剤は、上記したように、美白化粧料として用いることができ、このためには、典型的には皮膚に直接塗布して用いられるが、こればかりでなく、美白効果のある医薬としても用いることもできる。また、さまざまな食品中に添加して美白効果のある機能性食品、特定保健用食品などとしても用いることができる。かかる食品として、清涼飲料、乳酸飲料、スープ、ジャム、菓子類などが挙げられる。
本発明の表皮角化細胞エンドセリン-1産生抑制剤の投与量は、特に限定されるものではなく、例えば、皮膚に直接塗布して用いる場合には表皮角化細胞エンドセリン-1産生抑制剤を0.0001％〜1％含有する化粧品を1日あたり１〜4回塗布する。
As described above, the epidermal keratinocyte endothelin-1 production inhibitor of the present invention can be used as a whitening cosmetic, and for this purpose, it is typically applied directly to the skin, but only this is used. It can also be used as a medicine having a whitening effect. In addition, it can be added to various foods and used as a functional food having a whitening effect, a food for specified health use, and the like. Examples of such foods include soft drinks, lactic acid drinks, soups, jams, and confectionery.
The dosage of the epidermal keratinocyte endothelin-1 production inhibitor of the present invention is not particularly limited. For example, when used by directly applying to the skin, the epidermal keratinocyte endothelin-1 production inhibitor is 0.0001. Cosmetics containing 1% to 1% are applied 1 to 4 times per day.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。
〔試験例１〕（scutellareinが紫外線照射で刺激したヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
正常ヒト新生児包皮表皮角化細胞（クラボウ株式会社から購入）を増殖用無血清液体培地HuMedia-KG2（クラボウ株式会社から購入、増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、１mlのPBS(-)溶液で2回洗浄し、1ml のPBS(-)溶液を添加した。フナコシ社より購入した紫外線照射機（EL Lamp UVM-16、波長302 nm）を用いて、このプレートに紫外線を5 mJ/cm2照射した。紫外線の照射量はフナコシ社のUVX RadiometerによりUV-Bの領域である310 nmの強度を測定した。
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
[Test Example 1] (Effect of scutellarein suppressing endothelin-1 production in human epidermal keratinocytes stimulated by ultraviolet irradiation)
Normal human newborn foreskin epidermis keratinocytes (purchased from Kurabo Corporation) Serum-free liquid medium for proliferation HuMedia-KG2 (purchased from Kurabo Corporation, insulin, hEGF, hydrocortisone, bovine pituitary extract as growth additives, Using gentamicin and amphotericin B), start culture at 37 ° C in the presence of 5% CO 2 using a 24-well plate (2 cm 2 per well) (the number of cells at the start of culture is 2.8 cells per well) x10 4 ) The culture medium was changed after 1 day, and the culture was further continued for 2 days. Next, the medium in each well of the plate was discarded, washed twice with 1 ml of PBS (−) solution, and 1 ml of PBS (−) solution was added. The plate was irradiated with 5 mJ / cm 2 of ultraviolet rays using an ultraviolet irradiator (EL Lamp UVM-16, wavelength 302 nm) purchased from Funakoshi. The UV irradiation intensity was measured at 310 nm, which is the UV-B region, using a Funakoshi UVX Radiometer.
直ちにPBS(-)溶液を除去し、380μlの増殖用無血清液体培地HuMedia-KB2（クラボウ株式会社から購入、増殖添加剤不含）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はscutellarein を10% DMSO (dimethyl sulfoxide)溶液に溶解し100、200、400μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。この24穴プレートを5％CO2存在下、37℃で24時間静置した。その後、上清300μlを回収して、遠心により混入した細胞を除去した。上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表１に示す。結果はn=3の平均値（平均値±標準偏差、紫外線照射した対照区とscutellarein添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。また、紫外線照射なしの対照区はn=6で行った。scutellareinにエンドセリン-1産生抑制効果が認められた。 Immediately remove the PBS (-) solution and add 380 μl of serum-free liquid medium for growth HuMedia-KB2 (purchased from Kurabo Industries Inc., without growth additives) to each well with 400 μl of a solution containing 20 μl of the sample solution. . The above 20 μl sample solution was prepared by dissolving scutellarein in 10% DMSO (dimethyl sulfoxide) solution to a concentration of 100, 200, 400 μM, and using a 10% DMSO solution as a control (the DMSO concentration in the reaction solution was 0.5%). The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 . Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation. The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Bioscience). Table 1 shows the concentration of endothelin-1 in the supernatant. The result is the average value of n = 3 (mean value ± standard deviation, * P <0.05, ** p <0.01, *** when a significant difference is observed between the UV-irradiated control group and the scutellarein addition group p <0.001). Further, the control group without ultraviolet irradiation was carried out at n = 6. Scutellarein was found to inhibit endothelin-1 production.
次に、表１の濃度範囲のscutellarein添加24時間における細胞増殖に対する影響をロッシュ社のMTT (3-(4,5-ジメチルチアゾール-2-イル)-2,5-ジフェニルテトラゾリウムブロミド)アッセイキットにより測定したところ、この濃度範囲のscutellareinは細胞増殖に対して全く影響のないことが確認できた。 Next, the effect on cell proliferation in scutellarein addition in the concentration range shown in Table 1 for 24 hours was measured using Roche's MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay kit. As a result of measurement, it was confirmed that scutellarein in this concentration range had no influence on cell proliferation.
〔試験例２〕（scutellareinがヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
正常ヒト新生児包皮表皮角化細胞（クラボウ株式会社から購入）を増殖用無血清液体培地HuMedia-KG2（クラボウ株式会社から購入、増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、1 mlの増殖用無血清液体培地HuMedia-KB2（クラボウ株式会社から購入、増殖添加剤不含）で２回洗浄した後、380μlの増殖用無血清液体培地（上記と同一のHuMedia-KB2）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はscutellarein を10% DMSO溶液に溶解し50、100、200、400μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。
[Test Example 2] (Effect of scutellarein suppressing endothelin-1 production in human epidermal keratinocytes)
Normal human newborn foreskin epidermis keratinocytes (purchased from Kurabo Corporation) Serum-free liquid medium for proliferation HuMedia-KG2 (purchased from Kurabo Corporation, insulin, hEGF, hydrocortisone, bovine pituitary extract as growth additives, Using gentamicin and amphotericin B), start culture at 37 ° C in the presence of 5% CO 2 using a 24-well plate (2 cm 2 per well) (the number of cells at the start of culture is 2.8 cells per well) x10 4 ) The culture medium was changed after 1 day, and the culture was further continued for 2 days. Next, discard the medium in each well of the plate, wash twice with 1 ml of growth-free serum-free liquid medium HuMedia-KB2 (purchased from Kurabo Corporation, without growth additives), and then add 380 μl of serum-free liquid for growth. 400 μl of a solution obtained by adding 20 μl of the sample solution to the medium (the same HuMedia-KB2 as above) was added to each well. The 20 μl sample solution was prepared by dissolving scutellarein in a 10% DMSO solution to a concentration of 50, 100, 200, or 400 μM. A 10% DMSO solution was used as a control (the DMSO concentration in the reaction solution was 0.5%). become).
この24穴プレートを5％CO2存在下、37℃で24時間静置した。その後、上清300μlを回収して、遠心により混入した細胞を除去した。上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表２に示す。結果はn=４の平均値（平均値±標準偏差、対照区とscutellarein添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。紫外線で刺激しない状態でもエンドセリン-1産生抑制効果がscutellareinに認められた。 The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 . Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation. The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Bioscience). Table 2 shows the concentration of endothelin-1 in the supernatant. The result is the average value of n = 4 (mean value ± standard deviation, * P <0.05, ** p <0.01, *** p <0.001 when a significant difference is found between the control group and the scutellarein addition group ). Scutellarein was found to inhibit endothelin-1 production even when not stimulated by ultraviolet light.
〔試験例３〕（eupafolinが紫外線照射で刺激したヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
試験例１と同様に、正常ヒト新生児包皮表皮角化細胞を増殖用無血清液体培地HuMedia-KG2（増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、１mlのPBS(-)溶液で2回洗浄し、1ml のPBS(-)溶液を添加した。紫外線照射機（EL Lamp UVM-16、波長302 nm）を用いて、このプレートに紫外線を5 mJ/cm2照射した。紫外線の照射量はUVX RadiometerによりUV-Bの領域である310 nmの強度を測定した。直ちにPBS(-)溶液を除去し、380μlの増殖用無血清液体培地HuMedia-KB2（増殖添加剤不含）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はeupafolin を10% DMSO溶液に溶解し100、200、400μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。
[Test Example 3] (Eupafolin suppresses endothelin-1 production in human epidermal keratinocytes stimulated by UV irradiation)
As in Test Example 1, normal human newborn foreskin epidermis keratinocytes containing serum-free liquid medium HuMedia-KG2 for growth (Insulin, hEGF, hydrocortisone, bovine pituitary extract, gentamicin, amphotericin B as growth additives) ) Using a 24-well plate (2 cm 2 per well), start culturing at 37 ° C. in the presence of 5% CO 2 (number of cells at the start of culture is 2.8 × 10 4 per well), 1 After a day, the medium was changed and further cultured for 2 days. Next, the medium in each well of the plate was discarded, washed twice with 1 ml of PBS (−) solution, and 1 ml of PBS (−) solution was added. The plate was irradiated with 5 mJ / cm 2 of ultraviolet rays using an ultraviolet irradiator (EL Lamp UVM-16, wavelength 302 nm). The intensity of irradiation at 310 nm, which is the UV-B region, was measured by UVX Radiometer. The PBS (−) solution was immediately removed, and 400 μl of a solution obtained by adding 20 μl of the sample solution to 380 μl of serum-free liquid medium for growth HuMedia-KB2 (without growth additives) was added to each well. The above 20 μl sample solution was prepared by dissolving eupafolin in 10% DMSO solution to a concentration of 100, 200, 400 μM, and using 10% DMSO solution as a control (the DMSO concentration in the reaction solution is 0.5%) ).
この24穴プレートを5％CO2存在下、37℃で24時間静置した。その後、上清300μlを回収して、遠心により混入した細胞を除去した。上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表３に示す。結果はn=3の平均値（平均値±標準偏差、紫外線照射した対照区とeupafolin添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。また、紫外線照射なしの対照区はn=6で行った。eupafolinにエンドセリン-1産生抑制効果が認められた。
次に、表３の濃度範囲のeupafolin添加24時間における細胞増殖に対する影響をMTTアッセイキットにより測定したところ、この濃度範囲のeupafolinは細胞増殖に対して全く影響のないことが確認できた。
The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 . Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation. The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Bioscience). Table 3 shows the concentration of endothelin-1 in the supernatant. The result is the average value of n = 3 (mean value ± standard deviation, * P <0.05, ** p <0.01, *** when significant difference was found between the UV-irradiated control group and eupafolin addition group p <0.001). Further, the control group without ultraviolet irradiation was carried out at n = 6. eupafolin was found to inhibit endothelin-1 production.
Next, when the effect on cell proliferation at 24 hours after addition of eupafolin in the concentration range shown in Table 3 was measured with an MTT assay kit, it was confirmed that eupafolin in this concentration range had no effect on cell proliferation.
〔試験例４〕（eupafolinがヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
試験例２と同様に、正常ヒト新生児包皮表皮角化細胞を増殖用無血清液体培地HuMedia-KG2（増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、1 mlの増殖用無血清液体培地HuMedia-KB2（クラボウ株式会社から購入、増殖添加剤不含）で２回洗浄した後、380μlの増殖用無血清液体培地（上記と同一のHuMedia-KB2）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はeupafolin を10% DMSO溶液に溶解し50、100、200、400μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。この24穴プレートを5％CO2存在下、37℃で24時間静置した。その後、上清300μlを回収して、遠心により混入した細胞を除去した。［０００１］上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表４に示す。結果はn=4の平均値（平均値±標準偏差、対照区とeupafolin添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。紫外線で刺激しない状態でもエンドセリン-1産生抑制効果がeupafolinに認められた。
[Test Example 4] (Eupafolin suppresses endothelin-1 production in human epidermal keratinocytes)
As in Test Example 2, normal human newborn foreskin epidermis keratinocytes containing serum-free liquid medium HuMedia-KG2 for growth (Insulin, hEGF, hydrocortisone, bovine pituitary extract, gentamicin, amphotericin B as growth additives) ) Using a 24-well plate (2 cm 2 per well), start culturing at 37 ° C. in the presence of 5% CO 2 (number of cells at the start of culture is 2.8 × 10 4 per well), 1 After a day, the medium was changed and further cultured for 2 days. Next, discard the medium in each well of the plate, wash twice with 1 ml of growth-free serum-free liquid medium HuMedia-KB2 (purchased from Kurabo Corporation, without growth additives), and then add 380 μl of serum-free liquid for growth. 400 μl of a solution obtained by adding 20 μl of the sample solution to the medium (the same HuMedia-KB2 as above) was added to each well. The above 20 μl sample solution was prepared by dissolving eupafolin in 10% DMSO solution to a concentration of 50, 100, 200, 400 μM, and using 10% DMSO solution as a control (DMSO concentration in the reaction solution was 0.5%) become). The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 . Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation. [0001] The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Biosciences). Table 4 shows the concentration of endothelin-1 in the supernatant. The result is the mean value of n = 4 (mean ± standard deviation, * P <0.05, ** p <0.01, *** p <0.001 if a significant difference was found between the control group and eupafolin addition group ). Even if it was not stimulated by ultraviolet rays, the effect of inhibiting endothelin-1 production was observed in eupafolin.
〔試験例５〕（sinensetinが紫外線照射で刺激したヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
試験例１と同様に、正常ヒト新生児包皮表皮角化細胞を増殖用無血清液体培地HuMedia-KG2（増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、１mlのPBS(-)溶液で2回洗浄し、1ml のPBS(-)溶液を添加した。紫外線照射機（EL Lamp UVM-16、波長302 nm）を用いて、このプレートに紫外線を5 mJ/cm2照射した。紫外線の照射量はUVX RadiometerによりUV-Bの領域である310 nmの強度を測定した。直ちにPBS(-)溶液を除去し、380μlの増殖用無血清液体培地HuMedia-KB2（増殖添加剤不含）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はsinensetin を10% DMSO溶液に溶解し100、200、400μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。この24穴プレートを5％CO2存在下、37℃で24時間静置した。その後、上清300μlを回収して、遠心により混入した細胞を除去した。
[Test Example 5] (Sinensetin suppresses endothelin-1 production in human epidermal keratinocytes stimulated by ultraviolet irradiation)
As in Test Example 1, normal human newborn foreskin epidermis keratinocytes containing serum-free liquid medium HuMedia-KG2 for growth (Insulin, hEGF, hydrocortisone, bovine pituitary extract, gentamicin, amphotericin B as growth additives) ) Using a 24-well plate (2 cm 2 per well), start culturing at 37 ° C. in the presence of 5% CO 2 (number of cells at the start of culture is 2.8 × 10 4 per well), 1 After a day, the medium was changed and further cultured for 2 days. Next, the medium in each well of the plate was discarded, washed twice with 1 ml of PBS (−) solution, and 1 ml of PBS (−) solution was added. The plate was irradiated with 5 mJ / cm 2 of ultraviolet rays using an ultraviolet irradiator (EL Lamp UVM-16, wavelength 302 nm). The intensity of irradiation at 310 nm, which is the UV-B region, was measured by UVX Radiometer. The PBS (−) solution was immediately removed, and 400 μl of a solution obtained by adding 20 μl of the sample solution to 380 μl of serum-free liquid medium for growth HuMedia-KB2 (without growth additives) was added to each well. The 20 μl sample solution was prepared by dissolving sinensetin in a 10% DMSO solution to a concentration of 100, 200, or 400 μM. A 10% DMSO solution was used as a control (the DMSO concentration in the reaction solution was 0.5%). ). The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 . Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation.
上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表５に示す。結果はn=3の平均値（平均値±標準偏差、紫外線照射した対照区とsinensetin添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。また、紫外線照射なしの対照区はn=6で行った。sinensetinのエンドセリン-1産生抑制効果については20μMの濃度で有意差が認められた。
次に、表５の濃度範囲のsinensetin添加24時間における細胞増殖に対する影響をMTTアッセイキットにより測定したところ、この濃度範囲のsinensetinは細胞増殖に対して全く影響のないことが確認できた。
The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Bioscience). Table 5 shows the concentration of endothelin-1 in the supernatant. The result is the average value of n = 3 (mean value ± standard deviation, * P <0.05, ** p <0.01, *** when a significant difference is observed between the UV-irradiated control group and the sinensetin-added group p <0.001). Further, the control group without ultraviolet irradiation was carried out at n = 6. There was a significant difference in the inhibitory effect of sinensetin on endothelin-1 production at a concentration of 20 μM.
Next, when the influence on the cell proliferation at 24 hours after addition of sinensetin in the concentration range of Table 5 was measured by the MTT assay kit, it was confirmed that sinensetin in this concentration range had no influence on the cell proliferation.
〔試験例６〕（sinensetinがヒト表皮角化細胞のエンドセリン-1産生を抑制する効果）
試験例２と同様に、正常ヒト新生児包皮表皮角化細胞を増殖用無血清液体培地HuMedia-KG2（増殖添加剤としてインスリン、hEGF、ハイドロコーチゾン、ウシ脳下垂体抽出液、ゲンタマイシン、アンフォテリシンBを含む）にて、24穴（１穴2cm2）のプレートを用いて、5％ CO2存在下、37℃で培養を開始し（培養開始時の細胞数は１穴あたり2.8x104個）、１日後に培地の交換を行い、さらに２日間培養を行った。次にプレートの各穴の培地を捨て、1 mlの増殖用無血清液体培地HuMedia-KB2（クラボウ株式会社から購入、増殖添加剤不含）で２回洗浄した後、380μlの増殖用無血清液体培地（上記と同一のHuMedia-KB2）に20μl の試料溶液を加えた溶液400μlを各穴に添加した。上記の20μlの試料溶液はsinensetin を10% DMSO溶液に溶解し100、200、400、800μMの濃度としたものであり、対照として10% DMSO溶液を用いた（反応液中のDMSO濃度は0.5％になる）。この24穴プレートを5％CO2存在下、37℃で24時間静置した。
[Test Example 6] (Sinensetin suppresses endothelin-1 production in human epidermal keratinocytes)
As in Test Example 2, normal human newborn foreskin epidermis keratinocytes containing serum-free liquid medium HuMedia-KG2 for growth (Insulin, hEGF, hydrocortisone, bovine pituitary extract, gentamicin, amphotericin B as growth additives) ) Using a 24-well plate (2 cm 2 per well), start culturing at 37 ° C. in the presence of 5% CO 2 (number of cells at the start of culture is 2.8 × 10 4 per well), 1 After a day, the medium was changed and further cultured for 2 days. Next, discard the medium in each well of the plate, wash twice with 1 ml of growth-free serum-free liquid medium HuMedia-KB2 (purchased from Kurabo Corporation, without growth additives), and then add 380 μl of serum-free liquid for growth. 400 μl of a solution obtained by adding 20 μl of the sample solution to the medium (the same HuMedia-KB2 as above) was added to each well. The 20 μl sample solution was prepared by dissolving sinensetin in a 10% DMSO solution to a concentration of 100, 200, 400, or 800 μM. A 10% DMSO solution was used as a control (the DMSO concentration in the reaction solution was 0.5%). become). The 24-well plate was allowed to stand at 37 ° C. for 24 hours in the presence of 5% CO 2 .
その後、上清300μlを回収して、遠心により混入した細胞を除去した。上清中のエンドセリン-1量は、エンドセリン-1 ELISAキット（アマシャムバイオサイエンス社より購入）を用いた酵素免疫測定法により測定した。上清中のエンドセリン-1濃度を表６に示す。結果はn=4の平均値（平均値±標準偏差、対照区とsinensetin添加区との間に有意差が認められた場合は*P<0.05、**p<0.01、***p<0.001）で示した。紫外線で刺激しない状態でもエンドセリン-1産生抑制効果がsinensetinに認められた。 Thereafter, 300 μl of the supernatant was recovered, and the contaminated cells were removed by centrifugation. The amount of endothelin-1 in the supernatant was measured by an enzyme immunoassay using an endothelin-1 ELISA kit (purchased from Amersham Bioscience). Table 6 shows the concentration of endothelin-1 in the supernatant. The result is the average value of n = 4 (mean ± standard deviation, * P <0.05, ** p <0.01, *** p <0.001 if a significant difference was found between the control group and sinensetin addition group ). Even when not stimulated by ultraviolet rays, endothelin-1 production was suppressed by sinensetin.
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A whitening cosmetic characterized by containing any one or more of Scutellarein, Eupaforin, and Sinensetin as an active ingredient.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011092180A (en) * | 2009-02-26 | 2011-05-12 | Toyobo Co Ltd | Medium suitable for ultraviolet radiation experiment using 3-dimensional cultured skin model |
| JP2015013849A (en) * | 2013-06-06 | 2015-01-22 | 株式会社東洋新薬 | Hair-growing composition, hair dermal papilla cell growth promotion composition, fgf-7 production promotion composition, vegf production promotion composition, blood flow promotion composition for skin surface, and cosmetic |
| JP2017521411A (en) * | 2014-06-30 | 2017-08-03 | 株式会社アモーレパシフィックＡｍｏｒｅｐａｃｉｆｉｃ Ｃｏｒｐｏｒａｔｉｏｎ | Whitening composition containing Scutellaria alpina extract |
| JP2021123534A (en) * | 2020-01-31 | 2021-08-30 | 株式会社ナリス化粧品 | Keratinocyte PAR-2 expression inhibitor and melanocyte dendrite formation inhibitor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2011092180A (en) * | 2009-02-26 | 2011-05-12 | Toyobo Co Ltd | Medium suitable for ultraviolet radiation experiment using 3-dimensional cultured skin model |
| JP2015013849A (en) * | 2013-06-06 | 2015-01-22 | 株式会社東洋新薬 | Hair-growing composition, hair dermal papilla cell growth promotion composition, fgf-7 production promotion composition, vegf production promotion composition, blood flow promotion composition for skin surface, and cosmetic |
| JP2017521411A (en) * | 2014-06-30 | 2017-08-03 | 株式会社アモーレパシフィックＡｍｏｒｅｐａｃｉｆｉｃ Ｃｏｒｐｏｒａｔｉｏｎ | Whitening composition containing Scutellaria alpina extract |
| JP2021123534A (en) * | 2020-01-31 | 2021-08-30 | 株式会社ナリス化粧品 | Keratinocyte PAR-2 expression inhibitor and melanocyte dendrite formation inhibitor |
| JP7399730B2 (en) | 2020-01-31 | 2023-12-18 | 株式会社ナリス化粧品 | Keratinocyte PAR-2 expression inhibitor and melanocyte dendrite formation inhibitor |
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