JP2013178114A - Inspection method of irritation given to skin by cosmetic raw material or cosmetic - Google Patents
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Abstract
Description
本発明は、化粧品原料又は化粧品がヒト皮膚に与える微小刺激の検査方法に関する。 The present invention relates to a method for testing minute irritation that cosmetic raw materials or cosmetics give to human skin.
化粧品は日常的に、また長期間継続して皮膚に塗布するため、原料も含めてその安全性には細心の注意が必要とされる。化粧品や、化粧品原料の安全性を担保するための試験として、厚生労働省は、昭和６２年に「新規原料の安全性確保のためのガイドライン」を通知し、確認すべき安全性試験を定めている（非特許文献１：厚生省薬務局審査第２課事務連絡）。これによれば、実施すべき試験として、１．急性毒性試験、２．皮膚一次刺激性、３．連続皮膚刺激性、４．感作性、５．光毒性、６．光感作性、７．目刺激性、８．変異原性９．ヒトパッチ以上の９項目の試験である。 Since cosmetics are applied to the skin on a daily basis and continuously for a long period of time, careful attention is required for their safety, including the ingredients. As a test to ensure the safety of cosmetics and cosmetic raw materials, the Ministry of Health, Labor and Welfare notified the "Guidelines for ensuring the safety of new raw materials" in 1987 and established safety tests to be confirmed. (Non-patent literature 1: Ministry of Health and Welfare Pharmaceutical Affairs Bureau Examination Section 2 administrative communication). According to this, as tests to be carried out: 1. Acute toxicity test 1. Primary skin irritation 3. continuous skin irritation; 4. Sensitization, Phototoxicity, 6. 6. Photosensitization, Eye irritation, 8. Mutagenicity9. This is a 9-item test over human patches.
しかし、このような多種多様な、安全性試験を実施しても、化粧品による炎症や肌荒れなどが発生することが良く知られている（非特許文献２：化粧品事典２４０〜２５１ページ、日本化粧品技術者協会編集、丸善株式会社、平成１５年１２月１５日発行）。このような、従来の安全性試験で予測不能な皮膚のトラブルの原因は、たとえ安全な成分であるとして化粧品に配合されている成分であっても長期間の連用によって、皮膚に微小なストレスを与え続け、それらのストレスが蓄積されて皮膚トラブルにつながることが知られている。このような微小なストレスを与えるか否かは上記の一連の安全性試験では検出できない。
例えば、化粧品に通常防腐剤として配合される成分であるパラベン（パラヒドロキシ安息香酸エステル）はこのような微小ストレスの原因物質として良く研究されている。パラベンは上記試験ではいずれも安全と評価されている。しかし、パラベン配合化粧料ではしばしばパラベンアレルギーと呼ばれる症状が発生する（非特許文献３：日皮協情報、Ｎｏ．４８ ２００２年５月）。このため、パラベンの使用を避けてフェノキシエタノールを使用する化粧品が多く見られる。フェノキシエタノールは、グリコールエーテルの一種であり、微かな芳香を有する化合物であり、上記のパラベンよりも皮膚刺激性が少ない防腐剤として知られている。しかし、このフェノキシエタノールを含む化粧品を塗布した皮膚に強い痛みと接触性蕁麻疹が発生する例が知られている（非特許文献３：Mitani T,香粧会誌,12(4),238-247,1998)。また、正常な皮膚を持つ１１歳から６４歳までの男女にフェノキシエタノールを含む化粧料を塗布したところ統計的有意にスティンギング（Stinging）と呼ばれる痛みを伴う微小刺激を皮膚に与えることを確認している（非特許文献３参照）。
However, it is well known that even when such a wide variety of safety tests are performed, inflammation and rough skin due to cosmetics occur (Non-patent Document 2: pages 240-251 of cosmetics encyclopedia, Japanese cosmetics technology) Edited by the Association of Persons, Maruzen Corporation, issued December 15, 2003) The cause of skin problems unpredictable in conventional safety tests is that even if it is a component that is formulated in cosmetics as a safe component, it causes minute stress to the skin by prolonged use. It is known that these stresses accumulate and lead to skin problems. Whether or not such a minute stress is applied cannot be detected by the above series of safety tests.
For example, paraben (parahydroxybenzoic acid ester), which is a component that is usually blended in cosmetics as a preservative, has been well studied as a causative substance of such micro stress. Parabens are all considered safe in the above tests. However, a paraben-containing cosmetic often causes a symptom called paraben allergy (Non-patent Document 3: JSAI Information, No. 48, May 2002). For this reason, there are many cosmetics that avoid the use of parabens and use phenoxyethanol. Phenoxyethanol is a kind of glycol ether, a compound having a slight fragrance, and is known as a preservative with less skin irritation than the above-mentioned parabens. However, there are known examples in which strong pain and contact urticaria occur on the skin to which the cosmetic containing phenoxyethanol is applied (Non-patent Document 3: Mitani T, Cosmetic Society Journal, 12 (4), 238-247, 1998). In addition, when cosmetics containing phenoxyethanol were applied to men and women between the ages of 11 and 64 with normal skin, it was confirmed that they gave a statistically significant painful stimulus called stinging to the skin. (See Non-Patent Document 3).
このような健康な皮膚を持つ人より外界刺激に対する抵抗性が低く、容易に皮膚トラブルを生ずる皮膚を皮膚科学分野では敏感肌と呼ぶ。一般にもこの呼称が普及している（非特許文献４：伊藤正敏,Fragrance Jounal,30(10),11-16,2002)。
従来は、このような刺激に対しては皮膚のバリアー機能を増強することが有効とされ、そのような物質の探索が積極的に行われてきた（特許文献１：特開２０１１−１６２４６９号公報、特許文献２：特開２０１１−１６７１８号公報等参照）
Such a skin having a lower resistance to external stimuli than a person having healthy skin and easily causing skin troubles is called sensitive skin in the field of dermatology. This name is also widely used (Non-Patent Document 4: Masatoshi Ito, Fragrance Jounal, 30 (10), 11-16, 2002).
Conventionally, it has been effective to enhance the barrier function of the skin against such a stimulus, and such a substance has been actively searched for (Patent Document 1: JP 2011-162469 A). Patent Document 2: Japanese Patent Application Laid-Open No. 2011-16718)
本発明は、化粧品原料または化粧品が肌に与える微小な刺激を検出する検査方法を提供することを課題とする。 An object of the present invention is to provide an inspection method for detecting a minute stimulus given to a skin by a cosmetic raw material or cosmetics.
本発明は、従来の皮膚バリアー機能の強化という発想とはまったく異なるもので、皮膚の微小刺激の原因となる物質を化粧品原料または化粧品から除くための技術を提供するものである。
すなわち、本発明は以下の構成である。
（１）皮膚由来の表皮角化細胞を培養し、この培養細胞に被検物質を添加し、ヒートショックプロテイン（ＨＳＰ）２７の発現量を被検物質無添加の表皮角化細胞中のＨＳＰ２７の発現量と比較することにより、化粧品原料又は化粧品の肌に与える刺激を検出する方法。
（２）表皮角化細胞の培養細胞に被検物質を添加する添加濃度が、当該被検物質が示す表皮角化細胞５０％死濃度（ＥＣ５０）の１０分の１以下の濃度である（１）記載の方法。
（３）ＨＳＰ２７の発現量の検出がウエスタンブロッティング法によるものである（１）又は（２）記載の方法。
（４）被検物質添加後３日目に検査を実施する（１）〜（３）のいずれかに記載の方法。
The present invention is completely different from the conventional idea of enhancing the skin barrier function, and provides a technique for removing substances that cause minute skin irritation from cosmetic raw materials or cosmetics.
That is, the present invention has the following configuration.
(1) Skin-derived epidermal keratinocytes are cultured, a test substance is added to the cultured cells, and the expression level of heat shock protein (HSP) 27 is determined based on the expression of HSP27 in the epidermis keratinocytes to which no test substance is added. A method of detecting a stimulus given to a cosmetic raw material or cosmetic skin by comparing with an expression level.
(2) The addition concentration at which the test substance is added to the cultured cells of epidermal keratinocytes is a concentration of 1/10 or less of the 50% epidermal keratinocyte death concentration (EC50) indicated by the test substance (1 ) The method described.
(3) The method according to (1) or (2), wherein the detection of the expression level of HSP27 is by Western blotting.
(4) The method according to any one of (1) to (3), wherein the test is performed on the third day after the addition of the test substance.
本発明の評価方法によれば、従来の検査方法では検出できなかった化粧品原料又は化粧品の肌に及ぼす微小な刺激を検出することが可能となる。またこのような刺激の有無を検査することによって、化粧品が肌に持続的に与える刺激のない、あるいは刺激を軽減した化粧品を設計することができる。さらにまた、敏感肌の患者に適した化粧品を提供することが可能となる。 According to the evaluation method of the present invention, it is possible to detect minute stimuli on cosmetic skin or cosmetic skin that could not be detected by a conventional inspection method. Further, by examining the presence or absence of such a stimulus, it is possible to design a cosmetic product that has no stimulus that the cosmetic product continuously gives to the skin or that reduces the stimulus. Furthermore, it is possible to provide cosmetics suitable for patients with sensitive skin.
以下、本発明について詳細に説明する。
ＨＳＰ２７とは一連のヒートショック蛋白質ファミリーの一つとして知られている分子量２７ＫＤの蛋白質で分子シャペロンとしての機能を有している。そして細胞の保護機能を担っているといわれている。
Hereinafter, the present invention will be described in detail.
HSP27 is a protein with a molecular weight of 27 KD, which is known as one of a series of heat shock protein families, and has a function as a molecular chaperone. And it is said to be responsible for cell protection.
角層は、皮膚の一番上にある組織であって、角化細胞からなり、体の外からの異物や刺激から皮膚を守る働きを有している。
本発明の検査方法は、この角化細胞を培養し、角化細胞中に発現するＨＳＰ２７の増加を検出することで、化粧品原料や化粧品などの皮膚に対する微小刺激の有無を判定する方法である。この試験に用いる表皮角化細胞としては、自己増殖性を有する正常な表皮角化細胞であれば使用可能であり、マウス、ブタ、ラットなどの異種の動物の皮膚角化細胞でも使用可能である。しかしヒト皮膚に対する刺激の有無を判断するものであり、ヒトの細胞を用いるのが好ましい。動物の表皮角化細胞を用いる場合には、動物より表皮角化細胞を採取し、常法に従って処理して用いることもできる。あるいは、既に市販されているものを購入して利用することもできる。
The stratum corneum is a tissue on the top of the skin, and is composed of keratinocytes, and has a function of protecting the skin from foreign substances and irritation from outside the body.
The examination method of the present invention is a method for cultivating keratinocytes and detecting the increase in HSP27 expressed in the keratinocytes to determine the presence or absence of microstimulation on the skin of cosmetic raw materials or cosmetics. As the epidermal keratinocytes used in this test, normal epidermal keratinocytes having self-proliferative properties can be used, and skin keratinocytes of different animals such as mice, pigs, and rats can also be used. . However, it is used to determine the presence or absence of irritation to human skin, and it is preferable to use human cells. When using animal epidermis keratinocytes, epidermis keratinocytes can be collected from the animal and treated according to a conventional method. Or what is already marketed can also be purchased and utilized.
ヒト皮膚角化細胞としては、初代培養や継代培養されたものであってもよいが、ケラチノサイトとしての特性を失わないように注意しなければならない。好ましくは継代が１０代以内、特に好ましくは４〜５代以内の培養細胞を用いると良い。多くのメーカーより凍結角化細胞が市販されておりこれを利用することができる。凍結細胞は、解凍後培養液で培養しコンフルエントになるまで増殖させ、これをトリプシン処理して培養容器から剥離、単細胞化したのちハーベストしてプラスチックの９６穴ウエルあるいは６ウエルの細胞培養プレートなどに播種し、１〜２日培養し、このウエルを用いて試験を行う。培養液は表皮角化細胞の培養に適した複数の組成のものが市販されており、いずれのものであっても使用できる。代表的な培養液として、「Humedia−KG2」（倉敷紡績株式会社製）、「Epilife」（Life Technology社製）を例示することができる。 The human skin keratinocytes may be primary cultured or subcultured, but care must be taken not to lose the characteristics of keratinocytes. It is preferable to use cultured cells whose passage is within 10 generations, particularly preferably within 4 to 5 generations. Frozen keratinocytes are commercially available from many manufacturers and can be used. Frozen cells are thawed and cultured in a culture solution until they become confluent. They are treated with trypsin, detached from the culture vessel, converted into single cells, and harvested into a plastic 96-well or 6-well cell culture plate. Seed and cultured for 1-2 days and test using this well. A culture solution having a plurality of compositions suitable for culturing epidermal keratinocytes is commercially available, and any one can be used. As typical culture solutions, “Humedia-KG2” (manufactured by Kurashiki Boseki Co., Ltd.) and “Epilife” (manufactured by Life Technology) can be exemplified.
上述した細胞を播種した９６ウエルプレートを二酸化炭素インキュベーター中で２４時間培養し、その後培養溶液で希釈した被検物質を０．２〜２０質量％の濃度範囲で、ウエルに添加し、約８〜２４時間培養し、細胞毒性を確認する。被検物質の希釈濃度系列からそれぞれの被検物質のＥＣ５０（細胞が５０％死亡する濃度）を求める。
次いで、細胞を播種した６ウエルの細胞培養プレートを二酸化炭素インキュベーター中で２４時間培養し、その後培養溶液でこのＥＣ５０の１０分の１の濃度になるように希釈した被検物質を６ウエルの細胞培養プレートに加え、３〜５日間培養を行う。培養終了後、細胞を回収しイムノアッセイ法又はウエスタンブロット法、あるいはＰＣＲ法などによって、ＨＳＰ２７の蛋白質又は遺伝子のコピー数を測定してＨＳＰ２７の発現量を測定する。同様にして被検物質を添加しなかったウエル殻も細胞を回収して、ＨＳＰ２７を測定する。被検物質を添加したウエルのＨＳＰ２７の発現量が、被検物質を添加しなかったウエルの発現量より高値を示した場合を皮膚の微小刺激が陽性であると判定する。
The 96-well plate seeded with the above cells was cultured in a carbon dioxide incubator for 24 hours, and then a test substance diluted with a culture solution was added to the well at a concentration range of 0.2 to 20% by mass, and about 8 to Incubate for 24 hours to confirm cytotoxicity. The EC50 (concentration at which cells die 50%) of each test substance is determined from the series of dilution concentrations of the test substance.
Subsequently, the 6-well cell culture plate seeded with the cells is cultured in a carbon dioxide incubator for 24 hours, and then the test substance diluted with the culture solution to a concentration of 1/10 of this EC50 is added to the 6-well cells. Incubate for 3-5 days in addition to the culture plate. After completion of the culture, the cells are collected, and the expression level of HSP27 is measured by measuring the copy number of the protein or gene of HSP27 by an immunoassay method, Western blot method, PCR method or the like. Similarly, the cells of the well shell to which the test substance is not added are also collected and HSP27 is measured. When the expression level of HSP27 in the well to which the test substance is added is higher than the expression level in the well to which the test substance is not added, it is determined that the skin microstimulation is positive.
以下に試験例、実施例を示し、本発明をより詳細に説明する。
試験例 ：メチルパラベンの示す微小刺激の限界確認試験
メチルパラベンを対象としてヒト表皮角化細胞に与える刺激のレベルを測定した。
（１）細胞の調整
凍結ヒト皮膚角化細胞（Lonza社製）を解凍し、培養液としてEpifile（Life Technologies社製）用いてＴ−２５フラスコに播種した。これを３７℃の二酸化炭素インキュベーターでほぼコンフルエント状態になるまで培養した。
その後細胞をトリプシン処理して単細胞化し、９６ウエル細胞培養プレートに１ウエルあたり１０４個になるように播種し２４時間培養した。この９６ウエルプレートを以下の細胞毒性確認試験に用いた。また、６ウエル細胞培養プレートに１ウエル当り１０５個になるように播種し、２４時間培養した。この６ウエルプレートを以下の微小刺激試験に用いた。
Hereinafter, the present invention will be described in more detail with reference to test examples and examples.
Test example: Limiting confirmation test of microstimulation exhibited by methylparaben The level of stimulation given to human epidermal keratinocytes was measured using methylparaben as a target.
(1) Preparation of cells Frozen human skin keratinocytes (Lonza) were thawed and seeded in a T-25 flask using Epifile (Life Technologies) as a culture solution. This was cultured in a carbon dioxide incubator at 37 ° C. until almost confluent.
Thereafter, the cells were treated with trypsin to form single cells, seeded in a 96-well cell culture plate at 10 4 cells per well, and cultured for 24 hours. This 96-well plate was used for the following cytotoxicity confirmation test. Moreover, it seed | inoculated so that it might become 10 < 5 > per well to a 6 well cell culture plate, and cultured for 24 hours. This 6-well plate was used for the following microstimulation tests.
（２）細胞毒性確認試験
メチルパラベン（和光純薬株式会社、以下ＭＰという）を上記の培養に用いた培養液で０．２％から８段階の希釈し、最大希釈濃度０．０００１５６２５％の濃度になるようにウエルに加え約２４時間培養した。
２４時間経過後培養液を交換しさらに、MTT(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide; Thiazolyl blue)溶液を加え約３時間培養して生細胞を染色した。そしてリン酸緩衝液（ＰＢＳ）で洗浄後、イソプロパノールを１００μｌ/well添加して室温で１０分間振とうした。このイソプロパノール溶液の着色度を５７０ｎｍの吸光度（ＯＤ）を測定し、得られたＯＤをＭＰを添加しなかったウエルのＯＤ度で除した値を細胞生存率とした。段階希釈系列から求めたＭＰのＥＣ５０値は０．１質量％であった。
(2) Cytotoxicity confirmation test Methylparaben (Wako Pure Chemical Industries, Ltd., hereinafter referred to as MP) is diluted from 0.2% to 8 stages with the culture solution used in the above culture to a maximum dilution concentration of 0.00015625%. In addition to the wells, the cells were cultured for about 24 hours.
After 24 hours, the culture solution was changed, and MTT (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; Thiazolyl blue) solution was added and cultured for about 3 hours to stain living cells. . Then, after washing with a phosphate buffer (PBS), 100 μl / well of isopropanol was added and shaken at room temperature for 10 minutes. The absorbance (OD) at 570 nm was measured for the coloring degree of this isopropanol solution, and the value obtained by dividing the obtained OD by the OD degree of the well to which MP was not added was defined as the cell viability. The EC50 value of MP determined from the serial dilution series was 0.1% by mass.
（３）微小刺激試験
細胞を播種し、２４時間培養した６ウエルのプレートの培養液を、０．０１質量％、０．００３質量％になるようＭＰが含まれるように上記の培養液で希釈したものと交換し、表皮角化細胞を低濃度のＭＰで刺激した。対照としてＭＰを添加しないウエルを置いた。３日後に培養液を除去し、ＰＢＳで洗浄後、界面活性剤（Igepal CA-630 SIGMA社製）を含む細胞溶解液を１００μl/well加え、細胞をスクレーパーでこすって、細胞破砕液を回収した。
(3) Microstimulation test The culture solution of a 6-well plate seeded with cells and cultured for 24 hours was diluted with the above culture solution so as to contain MP so as to be 0.01% by mass and 0.003% by mass. The epidermal keratinocytes were stimulated with a low concentration of MP. As a control, wells without MP were placed. After 3 days, the culture solution was removed, washed with PBS, 100 μl / well of a cell lysate containing a surfactant (Igepal CA-630 SIGMA) was added, and the cells were scraped with a scraper to recover a cell disruption solution. .
細胞回収液をウエスタンブロッティングプロトコール（ＣＳＴジャパン社）に従って、ＳＤＳゲル電気泳動を行った。このＳＤＳゲルをHybond-P PVDFメンブレン（ＧＥヘルスケア バイオサイエンス）へ転写した。メンブレンを５％スキムミルク溶液でブロッキングし、１次抗体としてＨＳＰ２７：mouse monoclonal antibody (Stressgen社製)を４℃一晩で反応させ、翌日ＰＢＳで洗浄後、２次抗体Goat-Anti-IgG mouse（Invitrogen社製）で１時間反応させ、ECL plus western Blotting Detection system（ＧＥヘルスケア バイオサイエンス社製）を用いて検出した。
検出結果は３ウエルの平均値を求めさらに、ＭＰ無添加のウエルの測定値を１００とする相対値で表した。結果を図１に示す。
この結果からＭＰは０．０１質量％濃度（ＥＣ５０値の１０％）ではＨＳＰ２７の発現を誘導するが０．００３質量％濃度ではＨＳＰ２７の発現を誘導しないことがわかった。すなわち、ＨＳＰ２７の発現を指標とする微小刺激試験では、細胞毒性を示すＥＣ５０の１０分の１以上の濃度で引き起こされる影響を検出できることがわかった。従って、この試験方法を使用すれば低刺激性の物質を選別することが可能となる。
The cell collection solution was subjected to SDS gel electrophoresis according to Western blotting protocol (CST Japan). This SDS gel was transferred to Hybond-P PVDF membrane (GE Healthcare Bioscience). The membrane was blocked with a 5% skim milk solution, and HSP27: mouse monoclonal antibody (manufactured by Stressgen) was reacted as a primary antibody at 4 ° C. overnight, washed with PBS the next day, and then secondary antibody Goat-Anti-IgG mouse (Invitrogen For 1 hour and detected using an ECL plus western Blotting Detection system (GE Healthcare Bioscience).
The detection result was expressed as a relative value obtained by calculating an average value of 3 wells and setting the measured value of wells without MP to 100. The results are shown in FIG.
From this result, it was found that MP induces the expression of HSP27 at a concentration of 0.01% by mass (10% of the EC50 value), but does not induce the expression of HSP27 at a concentration of 0.003% by mass. That is, it was found that the microstimulation test using the expression of HSP27 as an index can detect an effect caused by a concentration of 1/10 or more of EC50 indicating cytotoxicity. Therefore, if this test method is used, it becomes possible to select a substance having low irritation.
化粧品原料の刺激性の検出例
上述したとおり、メチルパラベンは安全な防腐剤として広く普及しているが、所謂敏感肌の女性はメチルパラベンなどのパラベン類が原因となる肌荒れや痛みをしばしば感じている。このためメチルパラベンに代えてフェノキシエタノールが使用される。
このフェノキシエタノール（以下ＰＥという）と同様に化粧品に配合される成分で、皮膚刺激や肌荒れの原因とならないことが良く知られている成分として、１，３−ブチレングリコール(以下ＢＧという)が保湿剤や抗菌剤として使用されている。また美白化粧料にしばしば配合されるアスコルビン酸誘導体である安全性の高いアスコルビン酸２グルコシド(以下ＡＡ２Ｇという)がある。これらの３物質について、本発明の検査方法で行った。
As the above-described example of detection of irritation of cosmetics raw materials, methyl paraben is widely used as a safe preservative, women of so-called sensitive skin have often felt the rough skin and pain that parabens such as methyl paraben causes. For this reason, phenoxyethanol is used instead of methylparaben.
1,3-butylene glycol (hereinafter referred to as BG) is a moisturizer as a component that is blended in cosmetics in the same manner as phenoxyethanol (hereinafter referred to as PE) and is well known not to cause skin irritation and rough skin. It is used as an antibacterial agent. There is also a highly safe ascorbic acid 2-glucoside (hereinafter referred to as AA2G) which is an ascorbic acid derivative often blended in whitening cosmetics. About these three substances, it carried out with the inspection method of the present invention.
（１）評価濃度の設定
下記表１の段階希釈系列で希釈し、ヒト表皮角化細胞に対する細胞毒性を検査した。
(1) Setting of evaluation concentration It diluted with the serial dilution series of following Table 1, and examined the cytotoxicity with respect to a human epidermal keratinocyte.
試験例と同様にＥＣ５０値を測定し、さらに下記の表２とおりＥＣ５０値の１０分の１並びに３分の１の濃度を試験濃度に設定した。 The EC50 value was measured in the same manner as in the test example, and the concentrations of 1/10 and 1/3 of the EC50 value were set as test concentrations as shown in Table 2 below.
（２）試験結果
試験例と同様にして各化粧品原料のヒト角化細胞のＨＳＰ２７発現量を誘導する濃度を測定した。測定結果を図２〜図４に示す。
ＰＥはＥＣ５０値の１０分の１濃度でＨＳＰ２７の発現を誘導し、ＢＧ及びＡＡ２Ｇは誘導しなかった。この結果は従来経験的に言われている化粧品原料としての安全性の評価結果によく一致している。従って、本方法によって化粧品原料又は化粧品の微小刺激を細胞毒性の数値にもとづく具体的な作用濃度として数値化することが可能となった。
(2) Test result The concentration which induces the expression level of HSP27 in human keratinocytes of each cosmetic raw material was measured in the same manner as in the test examples. The measurement results are shown in FIGS.
PE induced HSP27 expression at a concentration of 1/10 the EC50 value, but not BG and AA2G. This result is in good agreement with the result of safety evaluation as a cosmetic raw material, which has been said from experience. Therefore, this method makes it possible to quantify the cosmetic raw material or cosmetic microstimulation as a specific working concentration based on the cytotoxicity value.
Claims (4)
The method according to claim 1, wherein the test is performed on the third day after the addition of the test substance.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003207506A (en) * | 2002-01-15 | 2003-07-25 | Toyobo Co Ltd | In vitro testing method using living tissue |
| JP2003523201A (en) * | 2000-02-18 | 2003-08-05 | ドイチェス クレブスフォルシュンクスツェントルム スチフトゥング デス エッフェントリヒェン レヒツ | Genetically engineered skin cell types and toxicity assays |
| WO2011083110A2 (en) * | 2010-01-08 | 2011-07-14 | Chanel Parfums Beaute | Use of at least one extract of flowers of camellia japonica alba plena for moisturizing the skin |
| JP2011520768A (en) * | 2007-06-11 | 2011-07-21 | シャネル パフュームズ ビューテ | Cosmetic use of an active agent for stimulating the expression of FN3K and / or FN3KRP for improving the barrier function of the skin |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003523201A (en) * | 2000-02-18 | 2003-08-05 | ドイチェス クレブスフォルシュンクスツェントルム スチフトゥング デス エッフェントリヒェン レヒツ | Genetically engineered skin cell types and toxicity assays |
| JP2003207506A (en) * | 2002-01-15 | 2003-07-25 | Toyobo Co Ltd | In vitro testing method using living tissue |
| JP2011520768A (en) * | 2007-06-11 | 2011-07-21 | シャネル パフュームズ ビューテ | Cosmetic use of an active agent for stimulating the expression of FN3K and / or FN3KRP for improving the barrier function of the skin |
| WO2011083110A2 (en) * | 2010-01-08 | 2011-07-14 | Chanel Parfums Beaute | Use of at least one extract of flowers of camellia japonica alba plena for moisturizing the skin |
| JP2013516441A (en) * | 2010-01-08 | 2013-05-13 | シャネル パフュームズ ビューテ | Use of at least one extract of Camellia Japan Alba Plena flower to moisturize the skin |
Non-Patent Citations (1)
| Title |
|---|
| JPN6016007885; ＪＥＡＮＭＡＩＲＥ Ｃ、外５名: 'Ｗｈａｔ ｉｓ ｔｈｅ Ｍｏｓｔ Ｓｕｉｔａｂｌｅ Ｓｔｒａｔｅｇｙ ｆｏｒ Ｓｔｒｅｓｓ Ｐｒｏｔｅｉｎｓ ｉｎ Ｃｏｓｍｅｔｉｃｓ？' ＩＦＳＣＣ Ｍａｇ Ｖｏｌ．６ Ｎｏ．３, 200307, Ｐａｇｅ．２２１-２２６ * |
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