JP2007127444A - Method of evaluating moistureproofness - Google Patents
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本発明は、人の肌の保湿を目的とした化粧品や日用品の保湿性能評価方法に関する。 The present invention relates to a method for evaluating the moisturizing performance of cosmetics and daily necessities for the purpose of moisturizing human skin.
皮膚の最外層に位置する角層は物理的に生体を保護、異物の進入を防御、あるいは生体内への水の浸入や生体からの水の蒸散を防いでいる。数週間かけて皮膚内部から表層に到達して形成される角層には、生体の様々な状態が反映される。特に皮膚病変部では皮膚内部の変化が角層の形成や機能に作用して角層水分量が変化することがある。湿疹性病変部では角層水分量は増加し、アトピー性乾皮症や乾癬ではターンオーバーの変調を反映して角層水分量が著しく低下する。また日焼けなどの炎症後にも角層水分量は低下して、時間とともにカサカサとした感触となる。逆に保湿剤の塗布によって短時間で角層水分量は増加し、塗布物質が有効であればその減衰は単に水を塗布したケースより緩やかになる。そういった物質を長期的に連用し、皮膚の状態が改善されれば、無塗布状態（素肌）の角層水分量も増加する。このような角層水分量は測定時の皮膚コンディション、受けたダメージの程度、あるいはスキンケア効果の重要な指標となっている。また、湿疹部や外傷、強い炎症時など滲出液がでるようなケースを除き、一般的にコンディショニングの良い肌では角層水分量が多く、バリア機能も優れていて異物や刺激物質によるダメージからの防御機能も高いことが多い。逆にバリア機能の低い皮膚では、皮膚水分の蒸散が激しく、角層水分量は低いことが多い。すなわち角層水分量は、角層機能の重要なバロメーターともなっている。この角層水分量を正しく、容易に、且つ、人に対して安全に測定する方法が当該製品の性能評価に望まれている。 The stratum corneum located at the outermost layer of the skin physically protects the living body, prevents the entry of foreign substances, or prevents water from entering the living body and transpiration of water from the living body. Various states of the living body are reflected in the stratum corneum formed by reaching the surface layer from within the skin over several weeks. Especially in skin lesions, changes in the skin may affect the formation and function of the stratum corneum and the stratum corneum moisture content may change. In the eczema lesion, the stratum corneum water content increases, and in atopic psoriasis and psoriasis, the stratum corneum water content decreases remarkably reflecting the modulation of turnover. In addition, the amount of stratum corneum moisture decreases after inflammation such as sunburn, and it feels crisp over time. On the contrary, the moisture content of the stratum corneum increases in a short time due to the application of the moisturizing agent, and if the applied substance is effective, its attenuation becomes more gradual than the case where water is simply applied. If such substances are used for a long period of time and the skin condition is improved, the amount of stratum corneum moisture in the uncoated state (bare skin) also increases. Such stratum corneum moisture content is an important indicator of the skin condition at the time of measurement, the degree of damage received, or the skin care effect. In addition, excluding cases where exudates occur due to eczema, trauma, strong inflammation, etc., skin with good conditioning generally has a high moisture content in the stratum corneum and an excellent barrier function to prevent damage from foreign substances and irritants. The defense function is often high. On the contrary, in skin with a low barrier function, transpiration of skin moisture is intense and the stratum corneum moisture content is often low. That is, the stratum corneum water content is also an important barometer of the stratum corneum function. A method for measuring the stratum corneum moisture content correctly, easily and safely for humans is desired for the performance evaluation of the product.
角層水分量の測定には、皮膚を測定に適する状態に整えることが必要である。そのためEU（EEMCO）における角層水和評価ガイドラインがある。ガイドラインには長期水和評価と短期水和評価があり、いずれも発汗しにくい環境下で一定時間馴化、被験者が１２〜１５名などの条件下で行うとされている。 For the measurement of the stratum corneum moisture content, it is necessary to prepare the skin in a state suitable for the measurement. Therefore, there are guidelines for evaluating stratum corneum hydration in the EU (EEMCO). The guidelines include long-term hydration evaluation and short-term hydration evaluation, both of which are acclimatized for a certain period of time in an environment where sweating is difficult, and are performed under conditions such as 12 to 15 subjects.
非侵襲的に角層水分量の測定方法する様々な方法が試みられている。しかし、実用的には市販されている汎用機器による「キャパシタンス測定法」と「コンダクタンス測定法」のいずれかの方法が選択されることが多い。いずれも皮膚に交流電流を流し、角層の電気的特性に着目し水分量に応じた値が得られる方法である。 Various methods for noninvasively measuring the stratum corneum moisture content have been attempted. However, in practice, either the “capacitance measurement method” or the “conductance measurement method” using a commercially available general-purpose device is often selected. In either case, an alternating current is passed through the skin, and the value corresponding to the amount of water is obtained by focusing on the electrical characteristics of the stratum corneum.
以上のように保湿性評価には角層水分量を測定することが代表的な方法とされているが、再現性を得るために被験者数を有る程度の大人数が必要である点、皮膚のコンディショニングが重要であり、被験者の体調、評価する季節などの外乱因子が結果に影響を与える点、試験をスムーズに進めるための験者の準備など経済面などの問題がある。 As described above, measuring moisture content of the stratum corneum is a typical method for evaluating moisturizing properties. However, in order to obtain reproducibility, a large number of subjects with a certain number of subjects is required. Conditioning is important, and there are problems such as the physical condition of the subject, the disturbance factors such as the season to be evaluated affect the results, and economic problems such as preparation of the examiner to smoothly proceed with the test.
本発明は、上記のような課題を解決しようとするものであって、人の保湿性評価試験結果と対応が取れるin vitro試験にて保湿性効能を示す化粧品、日用品、医薬品、衣料品などの繊維加工製品に関する簡便で再現性の高い保湿性評価方法を提供することを課題とする。 The present invention is intended to solve the above-mentioned problems, such as cosmetics, daily necessities, pharmaceuticals, clothing, etc. that show moisturizing effect in an in vitro test that can correspond to human moisturizing evaluation test results. It is an object of the present invention to provide a simple and highly reproducible method for evaluating moisturizing properties of processed fiber products.
本発明者らは鋭意検討した結果、以下に示すような手段により、上記課題を解決できることを見出し、本発明に到達した。すなわち、本発明は、人の肌の保湿を目的とした化粧品や日用品の保湿性能評価方法に関する。
１．3次元的組織細胞培養物に被験物質を接触させて、該被験物質の人の肌に対する保湿効果を予測する保湿性評価方法。
２．3次元的組織細胞培養物が、バリア能を有する構造を持つ3次元的組織細胞培養物であることを特徴とする１の保湿性評価方法。
３．3次元的組織細胞培養物が、ヒト由来組織、動物由来組織及び3次元にインビトロ再構築された組織細胞培養物からなる群より選ばれた１種以上であることを特徴とする１又は２の保湿性評価方法。
４．バリア能を低下させた3次元的組織細胞培養物を用いることを特徴とする１〜３のいずれかの保湿性評価方法。
５．界面活性剤及び／または有機溶剤に接触させて3次元的組織細胞培養物のバリア能を低下させることを特徴とする４の保湿性評価方法。
６．3次元的組織細胞培養物を低湿度環境で乾燥することを特徴とする１〜５のいずれかの保湿性評価方法。
７．相対湿度が０％RH以上９０％RH以下の環境下で、被験物質と接触させることを特徴とする１〜６のいずれかの保湿性評価方法。
８．被験物質が液体、固体、気体、ゲル状物もしくはこれらの混合物あることを特徴とする１〜７のいずれかの保湿性評価方法。
９．被験物質が液体、固体、気体、ゲル状物もしくはこれらの混合物を繊維製品へ付与したものであることを特徴とする１〜７のいずれかの保湿性評価方法。
１０．3次元的組織細胞培養物を、界面活性剤及び／または有機溶剤に接触させる第一工程と、相対湿度が０％RH以上９０％RH以下の環境下で被験物質と接触させる第二工程と、該3次元的組織細胞培養物の生細胞数を確認する第三工程とを備えた保湿性評価方法。
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention. That is, the present invention relates to a method for evaluating the moisturizing performance of cosmetics and daily necessities for the purpose of moisturizing human skin.
1. A moisturizing evaluation method for predicting the moisturizing effect of a test substance on human skin by bringing the test substance into contact with a three-dimensional tissue cell culture.
2. The moisturizing evaluation method according to 1, wherein the three-dimensional tissue cell culture is a three-dimensional tissue cell culture having a structure having a barrier ability.
3. The three-dimensional tissue cell culture is one or more selected from the group consisting of human-derived tissue, animal-derived tissue, and tissue cell culture reconstructed in three dimensions in vitro 1 or 2. Moisturizing evaluation method of 2.
4). The method for evaluating moisturizing properties according to any one of 1 to 3, wherein a three-dimensional tissue cell culture with reduced barrier ability is used.
5. 4. The method for evaluating moisture retention according to 4, wherein the barrier ability of a three-dimensional tissue cell culture is lowered by contact with a surfactant and / or an organic solvent.
6. The method for evaluating moisture retention according to any one of 1 to 5, wherein the three-dimensional tissue cell culture is dried in a low humidity environment.
7). The method for evaluating moisturizing properties according to any one of 1 to 6, wherein the method is brought into contact with a test substance in an environment having a relative humidity of 0% RH to 90% RH.
8). The method of evaluating moisture retention according to any one of 1 to 7, wherein the test substance is a liquid, a solid, a gas, a gel or a mixture thereof.
9. The method of evaluating moisture retention according to any one of 1 to 7, wherein the test substance is a liquid, solid, gas, gel or a mixture thereof applied to the fiber product.
10. A first step of contacting a three-dimensional tissue cell culture with a surfactant and / or an organic solvent, and a second step of contacting the test substance in an environment having a relative humidity of 0% RH to 90% RH. And a third step of confirming the number of viable cells in the three-dimensional tissue cell culture.
本発明による保湿性評価方法により、3次元的組織培養物を用いることから、人での保湿性評価に対して再現性が高く、評価時間も短いため経済性に優れた保湿性評価方法を提供することができる。 Since the three-dimensional tissue culture is used by the moisturizing evaluation method according to the present invention, the reproducibility is high for human moisturizing evaluation, and the evaluation time is short. can do.
以下に、本発明を詳細に説明する。本発明の保湿性評価方法は、3次元的組織細胞培養物の表面に被験物を添加し、被験物の細胞に対する反応からその被験物の人の肌に対する保湿効果を予測する方法に関するものである。3次元的組織細胞培養物中の細胞は、ヒトと同様に水分を含み細胞が活性している。この水分が乾燥して失われると細胞は活性が低下し、最終的に死滅する。また、皮膚細胞の乾燥は皮膚最表層の角層により守られている。皮膚細胞の乾燥は角層のバリア能低下によるものともいえる。すなわち、細胞の活性度合いを用いて、角層のバリア能つまり肌保湿性を予測することが出来るものと考える。 The present invention is described in detail below. The moisturizing evaluation method of the present invention relates to a method for predicting the moisturizing effect of a test subject on human skin from the reaction of the test subject to cells by adding the test subject to the surface of a three-dimensional tissue cell culture. . Cells in a three-dimensional tissue cell culture contain water and are active like humans. When this moisture is lost by drying, the cells become less active and eventually die. Moreover, the drying of the skin cells is protected by the stratum corneum, the outermost layer of the skin. It can be said that the drying of skin cells is due to a decrease in the barrier ability of the stratum corneum. That is, it is considered that the barrier ability of the stratum corneum, that is, the skin moisturizing property can be predicted using the degree of cell activity.
本発明で用いる3次元的組織細胞培養物とは、ヒト由来組織、動物由来組織および3次元にインビトロ再構築された組織細胞培養物であり、バリア能を有するものであれば特に問わない。 The three-dimensional tissue cell culture used in the present invention is a human-derived tissue, an animal-derived tissue, or a tissue cell culture reconstructed in vitro in three dimensions, and is not particularly limited as long as it has a barrier ability.
３次元とは、皮膚構造を再構築させたものであり、表皮層と真皮層からなる多細胞層の構造物、あるいは、表皮層のみの多細胞層の構造物である。表皮層は、表皮細胞層が積み重なった構造を有し、所定の分化した表皮細胞群で構成されたものである。3次元的組織細胞培養物は培養人工皮膚であってもよい。 The three-dimensional structure is a reconstructed skin structure, which is a multi-cellular layer structure composed of an epidermis layer and a dermis layer, or a multi-cell layer structure consisting of only the epidermis layer. The epidermal layer has a structure in which epidermal cell layers are stacked, and is composed of a predetermined group of differentiated epidermal cells. The three-dimensional tissue cell culture may be cultured artificial skin.
本発明の培養人工皮膚とは、天然の皮膚組織に類似したものであって、生体より単離された皮膚の細胞から、細胞培養技術を用いて皮膚構造を再構築させたものであり、表皮層と真皮層からなる多細胞層の構造物、あるいは、表皮層のみの多細胞層の構造物である。表皮層は、表皮細胞層が積み重なった構造を有し、所定の分化した表皮細胞群で構成され、各細胞は生存している。 The cultured artificial skin of the present invention is similar to natural skin tissue, and is obtained by reconstructing the skin structure from skin cells isolated from a living body using a cell culture technique. It is a multi-cellular layer structure consisting of a layer and a dermis layer, or a multi-cell layer structure consisting of only the epidermis layer. The epidermis layer has a structure in which epidermis cell layers are stacked, is composed of a predetermined group of differentiated epidermis cells, and each cell is alive.
培養人工皮膚の構造は、具体的には、コラーゲン等の細胞外マトリックス、またはそれに皮膚線維芽細胞を含有する基材上に基底細胞層、有棘細胞層、顆粒細胞層、角質層よりなる表皮層が重層した構造を持ち、例えば、ベル（Bell) により報告されたものは、真皮から採取された線維芽細胞をコラーゲンゲル中で培養し、この上に表皮から採取した表皮細胞を培養するものである(Bell ら、Science, Vol.211, p.1052, 1981年) （図１参照）。また、コラーゲンスポンジの上に、表皮細胞を培養させ、製造する方法がある(Boyceら、Surgery, Vol.103, p.421, 1988 年) 。培養人工皮膚の市販品としては、ＬＳＥ（Living Skin Equivalent、東洋紡績株式会社製) や EpiDerm (MatTek Corporation製) などがある。 Specifically, the structure of the cultured artificial skin is an epidermis composed of an basal cell layer, spinous cell layer, granule cell layer, and stratum corneum on an extracellular matrix such as collagen or a substrate containing dermal fibroblasts. Layers have a layered structure, for example, reported by Bell, in which fibroblasts collected from the dermis are cultured in a collagen gel, and then epidermal cells collected from the epidermis are cultured thereon (Bell et al., Science, Vol. 211, p. 1052, 1981) (see FIG. 1). In addition, there is a method of culturing and producing epidermal cells on a collagen sponge (Boyce et al., Surgery, Vol. 103, p.421, 1988). Commercially available cultured artificial skin includes LSE (Living Skin Equivalent, manufactured by Toyobo Co., Ltd.) and EpiDerm (manufactured by MatTek Corporation).
本発明では、まず、培養人工皮膚を例えば、シャーレ又は培養プレートに入れ、さらに栄養培地を培養皮膚底面に接触するように加え、皮膚表面は、空気に曝された状態で培養できるようにする（図２参照）。栄養培地としては、必須細胞栄養素を含む基礎培地、あるいはその基礎培地に種々の添加物を含む培地であり、その一例としては、ハムF-12(HamF-12) 培地とダルベッコ修正イーグル培地(DMEM)の混合培地に、牛胎児血清などの添加物を加えた培地、例えば、ＬＳＥ用アッセイ培地（東洋紡績株式会社製）などを用いる。培養人工皮膚は、基本的には、実験全体にわたり、約３７℃付近で、約５〜１０％二酸化炭素下で維持される。 In the present invention, first, cultured artificial skin is placed in, for example, a petri dish or a culture plate, and a nutrient medium is added so as to come into contact with the bottom of the cultured skin so that the skin surface can be cultured while being exposed to air ( (See FIG. 2). The nutrient medium is a basal medium containing essential cell nutrients, or a medium containing various additives in the basal medium, and examples thereof include Ham F-12 (HamF-12) medium and Dulbecco's modified Eagle medium (DMEM). ), A medium obtained by adding an additive such as fetal calf serum, for example, an LSE assay medium (manufactured by Toyobo Co., Ltd.) or the like. Cultured artificial skin is basically maintained under about 5-10% carbon dioxide at about 37 ° C. throughout the experiment.
バリア能とは、3次元的組織細胞培養物における人の皮膚角層部分と同じ機能の部位を指す。通常、その最表面がこれに該当する。バリア能は健康な肌を維持するために必要な機能である。バリア能が低下した肌はドライスキンと呼ばれている。ドライスキンとは、角層の水分保持機能の低下、水分蒸散量の増加などにより、角層中の水分量が低下した潤いのない、かさついた肌の状態をいう。このような皮膚はアトピー性皮膚炎、乾皮症（皮脂減少症）、胆汁うっ滞性肝障害、尿毒症などで観察され、難治性のかゆみを呈することが多い。角層の水分保持には皮脂、角質細胞間脂質、遊離アミノ酸（天然保湿因子またはnatural moisturizing factor：NMF）の３大保湿因子が関与している。3次元的組織細胞培養物のバリア能として必要な点は、左記３大保湿因子の内、少なくとも１つが含まれていること、且つ、この保湿因子により、バリア能の下層に位置する真皮を構成する部位、表皮を構成する部位からの外気への水分蒸散が低く抑えられているものが好ましい。 Barrier ability refers to a site having the same function as a human skin stratum corneum in a three-dimensional tissue cell culture. Usually, the outermost surface corresponds to this. Barrier ability is a function necessary for maintaining healthy skin. Skin with reduced barrier ability is called dry skin. Dry skin refers to the state of skin with no moisture, with no moisture, due to a decrease in moisture content in the stratum corneum due to a decrease in the moisture retention function of the stratum corneum, an increase in the amount of moisture transpiration. Such skin is observed in atopic dermatitis, xeroderma (sebum seborrhoea), cholestatic liver disorder, uremia, etc., and often exhibits intractable itching. Moisture retention in the stratum corneum involves three major moisturizing factors, sebum, keratin intercellular lipids, and free amino acids (natural moisturizing factor: NMF). What is necessary for the barrier ability of three-dimensional tissue cell culture is that at least one of the three major moisturizing factors shown in the left column is included, and the dermis located under the barrier ability is constituted by this moisturizing factor. It is preferable that the moisture transpiration to the outside air from the part to be formed and the part constituting the epidermis is kept low.
被験物の細胞に対する反応は、バリア能に対する影響、細胞活性化、細胞増殖性など生体組織や細胞に対して刺激が加われた時に生じる保湿性に関わる種々の反応を意味する。例としては、これに限定されるものではないが、被験物質の各曝露時間後の皮膚に対する刺激度としては、細胞生存率や刺激により細胞が産生する因子量などを指標と用いて用いてもよい。例えば、被験物質の細胞に対する反応の測定方法としては、生細胞数、テトラゾリウム塩、乳酸脱水素酵素（ＬＤＨ）、プロスタグランジンＥ2（ＰＧＥ2）、インターロイキン１（ＩＬ−１）、インターロイキン８（ＩＬ−８）等の測定法によって行っても良い。 The reaction of a test substance to cells means various reactions related to moisture retention that occur when a stimulus is applied to a living tissue or cell, such as influence on barrier ability, cell activation, and cell proliferation. As an example, but not limited to this, the degree of irritation to the skin after each exposure time of the test substance may be used by using as an index the cell viability or the amount of factors produced by the cells by stimulation. Good. For example, the method for measuring the reaction of a test substance to cells includes the number of living cells, tetrazolium salt, lactate dehydrogenase (LDH), prostaglandin E2 (PGE2), interleukin 1 (IL-1), interleukin 8 ( It may be performed by a measurement method such as IL-8).
被験物質の細胞に対する反応の測定方法にテトラゾリウム塩を利用する場合には、乳酸脱水素酵素（ＬＤＨ）などの細胞内脱水素酵素の作用により遊離した水素を電子キャリアを介して受容し還元されてホルマザンとなるものが好ましい。例えば、2-(4-indophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride（ＩＮＴ）、2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide inner salt（ＸＴＴ）、3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyl tetrazolium bromide（ＭＴＴ）などを用いることができる。テトラゾリウム塩は、3次元的組織細胞培養物の生細胞のミトコンドリアの脱水素酵素によって還元されてホルマザンを生成する。例えば、テトラゾリウム塩としてＩＮＴを用いた場合は赤紫色のホルマザンが生成するので、５００ｎｍ付近における吸光度を測定すればよく、MTTを用いた場合は青紫色のホルマザンが生成するので、５６２ｎｍ付近における吸光度を測定すればよく、それぞれ生成するホルマザンに適当な波長の吸光度を測定すればよい。ここで、脱水素酵素の活性と3次元的組織細胞培養物の生細胞数との間にも相関関係があるため、結局、ホルマザンを定量することにより3次元的組織細胞培養物の生細胞数がわかる。 When a tetrazolium salt is used as a method for measuring the reaction of a test substance to cells, hydrogen released by the action of intracellular dehydrogenase such as lactate dehydrogenase (LDH) is received through an electron carrier and reduced. What becomes formazan is preferable. For example, 2- (4-indophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (INT), 2,3-bis (2-Methoxy-4-nitro-5-sulfophenyl) -2H -tetrazolium-5-carboxyanilide inner salt (XTT), 3- (4,5-dimethylthyazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), etc. can be used. Tetrazolium salts are reduced by mitochondrial dehydrogenase in living cells of three-dimensional tissue cell cultures to produce formazan. For example, when INT is used as the tetrazolium salt, reddish purple formazan is produced, so it is only necessary to measure the absorbance near 500 nm. When MTT is used, blue purple formazan is produced. What is necessary is just to measure the light absorbency of a wavelength suitable for the formazan which each produces | generates. Here, since there is also a correlation between the activity of dehydrogenase and the number of living cells in the three-dimensional tissue cell culture, the number of living cells in the three-dimensional tissue cell culture was eventually determined by quantifying formazan. I understand.
プロスタグランジンＥ2（ＰＧＥ2）、インターロイキン１（ＩＬ−１）、インターロイキン８（ＩＬ−８）等の測定はエンザイムイムノアッセイ法などをもちいて測定しても良い。 Prostaglandin E2 (PGE2), interleukin 1 (IL-1), interleukin 8 (IL-8), etc. may be measured using an enzyme immunoassay method or the like.
3次元的組織細胞培養物へ被験物質を接触させる手段としては、繊維製品、紙製品やガラス繊維などにしみこませて接触させたり、フイルムやガラス製品などに塗布したりして接触させてもよい。ヒト皮膚パッチテスト等に用いられるフィンチャンバー、コットンパッド、濾紙、スポンジ、ガーゼ、不織布等にしみ込ませて接触させても良い。また、直接、被験物を皮膚表面に接触させても良い。被験物は、液体、気体、固体、ゲル状物、これらには、界面活性剤(アニオン系、カチオン系、または非イオン系)とこれらの界面活性剤を含む製品、例えば、シャンプー、洗剤、コンディショナー、食器洗浄液、スキンクレンザー、クリーニング剤およびスキンケア品目などがある。上記記載から明らかな様に、医薬品、化学品およびこれらの原料などの皮膚と接触する他の成分または製品を適用しても良い。 As a means of bringing the test substance into contact with the three-dimensional tissue cell culture, it may be infiltrated into a fiber product, paper product, glass fiber, etc., or applied to a film or glass product, etc. . A fin chamber, a cotton pad, a filter paper, a sponge, a gauze, a non-woven fabric, etc., used for a human skin patch test or the like may be soaked and contacted. Moreover, you may make a test substance contact the skin surface directly. The test object is a liquid, gas, solid, gel, which includes a surfactant (anionic, cationic, or nonionic) and a product containing these surfactants, such as shampoos, detergents, conditioners , Dishwashing liquids, skin cleansers, cleaning agents and skin care items. As is apparent from the above description, other ingredients or products that come into contact with the skin, such as pharmaceuticals, chemicals and their raw materials, may be applied.
本発明の特徴の一つに、低湿度の環境下に被験物質暴露後の組織をおいてインキュベートすることにある。通常の3次元的組織細胞培養物のインキュベートには３７℃で飽和湿度の環境下で行われる。しかしながら、本発明の試験方法は、皮膚の乾燥による細胞の活性低下からの被験物質暴露による回復効果を見ることが目的の一つである。従って、3次元的組織細胞培養物を乾燥下におくのがよい。そのため、インキュベートの条件は温度が３７℃付近で、相対湿度が０％ＲＨから９０％RH、好ましくは０％ＲＨから８０％RH、さらに好ましくは１０％ＲＨから５０％ＲＨ、特に好ましくは１０％ＲＨ〜３０％ＲＨの乾燥した環境下で実施する事が望ましい。 One of the features of the present invention resides in incubating the tissue after exposure to the test substance in a low humidity environment. Incubation of normal three-dimensional tissue cell culture is performed at 37 ° C. in a saturated humidity environment. However, one of the purposes of the test method of the present invention is to observe the recovery effect by exposure to a test substance from the decrease in cell activity due to dry skin. Therefore, it is better to keep the three-dimensional tissue cell culture dry. Therefore, the incubation conditions are such that the temperature is around 37 ° C. and the relative humidity is 0% RH to 90% RH, preferably 0% RH to 80% RH, more preferably 10% RH to 50% RH, and particularly preferably 10%. It is desirable to carry out in a dry environment of RH to 30% RH.
更に、本発明の特徴として、バリア能を低下させた3次元的組織細胞培養物に対して被験物を貼付することも含まれる。バリア能の低下とは、角層の保湿成分を溶出し、表皮細胞から外気への水分蒸散量を増加することである。3次元的組織細胞培養物は本来健康な皮膚をモデルとしている。健常者に対して保湿性の評価を行っても、本来健康な肌を取り戻すことが目的である保湿剤の効果は期待出来ないと考えられる。そこで、バリア能の保湿性成分を適度に溶出させ、表皮細胞からの水分蒸散量を増加促進させ、3次元的組織細胞培養物を短時間に乾燥させる方法が好ましい。本発明のバリア能破壊には界面活性剤や有機溶剤を用いることが有効であることを見出した。 Furthermore, as a feature of the present invention, attaching a test object to a three-dimensional tissue cell culture with reduced barrier ability is also included. The decrease in the barrier ability is to elute the moisturizing component of the stratum corneum and increase the amount of water transpiration from the epidermal cells to the outside air. Three-dimensional tissue cell cultures are modeled after healthy skin. Even if the moisturizing property is evaluated for healthy persons, it is considered that the effect of the moisturizing agent, which is originally intended to recover healthy skin, cannot be expected. Therefore, a method of appropriately eluting the moisturizing component having the barrier ability, promoting the increase of water transpiration from the epidermal cells, and drying the three-dimensional tissue cell culture in a short time is preferable. It has been found that the use of a surfactant or an organic solvent is effective for breaking the barrier ability of the present invention.
界面活性剤には特に限定はなく、アニオン性（アルキルカルボン酸塩、アルキルスルホン酸塩、アルキル硫酸エステル塩、アルキルリン酸エステル塩）、非イオン性（エーテル型非イオン界面活性剤、エーテルエステル型非イオン界面活性剤、エステル型非イオン界面活性剤、ブロックポリマー型非イオン界面活性剤、含窒素型非イオン界面活性剤）及び両性（カルボン酸型両性界面活性剤（アミノ型、ベタイン型）、硫酸エステル型両性界面活性剤、スルホン酸型両性界面活性剤、リン酸エステル型両性界面活性剤）その他の界面活性剤（天然界面活性剤、タンパク質加水分解物の誘導体、高分子界面活性剤、チタン・ケイ素を含む界面活性剤、フッ化炭素系界面活性剤）の活性剤を用いることができる。好ましくは、ＳＤＳ（Sodium dodecylsulfate）水溶液、Ｔｒｉｔｏｎ Ｘ−１００水溶液などを用いることができる。 There is no particular limitation on the surfactant, anionic (alkyl carboxylate, alkyl sulfonate, alkyl sulfate ester salt, alkyl phosphate ester salt), nonionic (ether type nonionic surfactant, ether ester type) Nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing type nonionic surfactant) and amphoteric (carboxylic acid type amphoteric surfactant (amino type, betaine type), Sulfate ester amphoteric surfactant, sulfonic acid amphoteric surfactant, phosphate ester amphoteric surfactant) and other surfactants (natural surfactants, protein hydrolyzate derivatives, polymer surfactants, titanium) (Surfactants containing silicon, fluorocarbon surfactants) can be used. Preferably, SDS (Sodium dodecylsulfate) aqueous solution, Triton X-100 aqueous solution, etc. can be used.
有機溶剤としては、エタノール、メタノール、トルエン、アセトンなどを用いても良いがこれらに限定されるものではない。好ましくは、アセトンがよく、さらに好ましくはエタノールがよい。また、界面活性剤と有機溶剤を混合して用いても良い。 As the organic solvent, ethanol, methanol, toluene, acetone or the like may be used, but is not limited thereto. Acetone is preferable, and ethanol is more preferable. Further, a surfactant and an organic solvent may be mixed and used.
これらの界面活性剤や有機溶剤を3次元的組織細胞培養物に被験物を貼付する前に添加し、一定時間後除去し洗浄することで、バリア能が低下した3次元的組織細胞培養物を得ることが出来る。 These surfactants and organic solvents are added before applying the test substance to the 3D tissue cell culture, removed after a certain period of time, and washed to obtain a 3D tissue cell culture with reduced barrier ability. Can be obtained.
たとえば、ＳＤＳおよびＴｒｉｔｏｎＸ−１００を用いる場合の水溶液濃度は、０．０１％〜５％が好ましく、さらに好ましくは０．０３％〜４％がよく、特に好ましくは０．０５％から３％にすることが望ましい。５％を超えるとバリア能および表皮細胞全体を死滅させてしまい、０．０１％を下回るとバリア能低下に至らない可能性があり好ましくないが、本発明の効果を損なわない範囲で使用してもよい。これら界面活性剤を3次元的組織細胞培養物の接触させておく時間は、１０秒〜１０分が好ましく、さらに好ましくは３０秒〜８分がよく、特に好ましくは１分から５分が望ましい。有機溶剤に関しても3次元的組織細胞培養物に接触させる時間は１０秒〜１０分が好ましく、さらに好ましくは３０秒〜８分がよく、特に好ましくは１分から５分が望ましい。試験点数が多い場合、バリア能破壊時間を短く設定することが作業時間の面から困難であるため、接触時間は３分〜５分が適当であるが、これに限定されるものではない。 For example, when using SDS and Triton X-100, the aqueous solution concentration is preferably 0.01% to 5%, more preferably 0.03% to 4%, and particularly preferably 0.05% to 3%. It is desirable. If it exceeds 5%, the barrier ability and the entire epidermal cell are killed. If it is less than 0.01%, the barrier ability may not be lowered, which is not preferable, but it is used within the range not impairing the effect of the present invention. Also good. The time for which these surfactants are kept in contact with the three-dimensional tissue cell culture is preferably 10 seconds to 10 minutes, more preferably 30 seconds to 8 minutes, and particularly preferably 1 minute to 5 minutes. The time for contacting the organic solvent with the three-dimensional tissue cell culture is preferably 10 seconds to 10 minutes, more preferably 30 seconds to 8 minutes, and particularly preferably 1 minute to 5 minutes. When the number of test points is large, it is difficult to set the barrier ability breaking time short from the viewpoint of working time. Therefore, the contact time is suitably 3 minutes to 5 minutes, but is not limited thereto.
以下に本発明の実施例を具体的に説明するが、本発明はこれらの実施例にのみ限定されるものではない。 Examples of the present invention will be specifically described below, but the present invention is not limited to these examples.
本発明の3次元的組織細胞培養物や培養人工皮膚は、前記の方法をもちいて調整しても構わないが、市販品を使用しても構わない。例としては、テストスキン（東洋紡績株式会社製）、EP1-100（倉敷紡績株式会社製)、ビトロライフスキン（グンゼ株式会社製）を使用しても構わない。 The three-dimensional tissue cell culture and cultured artificial skin of the present invention may be prepared using the above-described method, but commercially available products may be used. As an example, a test skin (manufactured by Toyobo Co., Ltd.), EP1-100 (manufactured by Kurashiki Textile Co., Ltd.), or Vitro Life Skin (manufactured by Gunze Co., Ltd.) may be used.
（実施例１）
テストスキン（東洋紡績株式会社製）ＬＳＥ−００３キット付属の取り扱い説明の要領に沿って組織を取り出す。薬剤暴露部位を確保するリングをＬＳＥ組織表面に接着させ、ドデシル硫酸ナトリウム（ＳＤＳ）０．１%水溶液をリング内に添加し５分間室温で静置する。その後、ＳＤＳをアスピレーターで除去し、アッセイ培地３ｍｌをピペットで吹きかけ洗浄。この操作にて、角層の保湿成分が溶出し、乾燥肌が作成された。
Example 1
Take out the tissue according to the instructions of the test skin (manufactured by Toyobo Co., Ltd.) LSE-003 kit. A ring that secures the site of drug exposure is adhered to the LSE tissue surface, and a 0.1% aqueous solution of sodium dodecyl sulfate (SDS) is added to the ring and allowed to stand at room temperature for 5 minutes. Thereafter, SDS is removed with an aspirator, and 3 ml of assay medium is sprayed with a pipette and washed. By this operation, the moisturizing component of the stratum corneum was eluted and a dry skin was created.
次に、被験物として、純水、保湿性化粧液（株式会社ファンケル製 フェナティ化粧液しっとり）を各８０μｌマイクロピペットにてＬＳＥ（Living Skin Equivalent)組織表面に添加。室温で６０分静置した後、試料をアスピレーターにて吸引除去。続いて温度３７℃、相対湿度１５%ＲＨから２０％RHに調整したCO2インキュベーターにＬＳＥ組織をアッセイ培地が入っていないアッセイトレイに乗せて状態で入れ、２４時間インキュベートした。その後、ＬＳＥ組織をＣＯ２インキュベーターより取り出し、ＬＳＥ−００３キット付属の取り扱い説明の要領に沿って、テトラゾリウム塩（ＭＴＴ）試薬０．３３３ｇ／ｍｌが含まれるアッセイ培地の混合液１．２ｍｌをアッセイトレイに入れ、温度３７℃、相対湿度１５%ＲＨから２０％RHに調整したCO2インキュベーター内で３時間インキュベートした。ＭＴＴ処理後、ＬＳＥ組織中央部を８ｍｍφのバイオプシーパンチを用いてポリカーボネートの膜ごとくり抜き、切片を小試験管に移し、０．０４Ｎ塩酸―イソプロパノールを７００μｌ加え、暗所にて２時間抽出。抽出終了後、攪拌し、十分混和後、抽出された青紫色のホルマザンの５６２ｎｍの吸光度を測定。ブランクとしては、抽出に用いる酸性イソプロパノール液を用いた。 Next, pure water and moisturizing cosmetic liquid (Fenkel Co., Ltd. fenati cosmetic liquid moist) were added to the surface of the LSE (Living Skin Equivalent) tissue with each 80 μl micropipette. After standing at room temperature for 60 minutes, the sample was removed by suction with an aspirator. Subsequently, the LSE tissue was placed in a CO 2 incubator adjusted to a temperature of 37 ° C. and a relative humidity of 15% RH to 20% RH in an assay tray without assay medium, and incubated for 24 hours. After that, remove the LSE tissue from the CO2 incubator, and add 1.2 ml of the assay medium mixture containing 0.333 g / ml of tetrazolium salt (MTT) reagent to the assay tray according to the instructions in the instruction manual attached to the LSE-003 kit. And incubated for 3 hours in a CO 2 incubator adjusted to a temperature of 37 ° C. and a relative humidity of 15% RH to 20% RH. After the MTT treatment, the LSE tissue central part was cut out together with the polycarbonate film using a biopsy punch of 8 mmφ, the section was transferred to a small test tube, 700 μl of 0.04N hydrochloric acid-isopropanol was added, and extracted in the dark for 2 hours. After completion of extraction, the mixture was stirred and thoroughly mixed, and the absorbance of the extracted blue-violet formazan was measured at 562 nm. As a blank, an acidic isopropanol solution used for extraction was used.
ＳＤＳで表面処理をせず、且つ、被験物を添加しなかった組織の吸光度、ＳＤＳで表面処理行い、被験物を添加しなかった組織の吸光度、ＳＤＳで表面処理後、被験物を添加した組織の吸光度の測定値を図３に示す。ＳＤＳと被験物を添加しなかったものの吸光度に対して、ＳＤＳを添加し被験物を添加しなかったものの吸光度は、やや低下しており、細胞の活性が低下していることが明らかである。一方、ＳＤＳで表面処理後、被験物である保湿性化粧水を添加したものの吸光度は、被験物が純水のものに対して高く、細胞活性が相対的に高いことが分かる。 Absorbance of tissue without surface treatment with SDS and no test substance added, Absorbance of tissue with surface treatment with SDS and no test substance added, tissue after surface treatment with SDS and addition of test substance The measured value of the absorbance is shown in FIG. It is clear that the absorbance of the sample to which SDS was added and the sample was not added is somewhat lower than that of the sample to which SDS and the test sample were not added, and the cell activity was reduced. On the other hand, after the surface treatment with SDS, the absorbance of the test sample to which the moisturizing lotion was added was higher than that of the pure test sample and the cell activity was relatively high.
上記方法で得られた結果と人の肌の保湿性の対応を次の方法で確認した。被験物に純水、保湿性化粧液（株式会社ファンケル製 フェナティ（登録商標）化粧液しっとり）をＳＤＳ０．１％水溶液で表面処理した肌に塗布し、EU（EEMCO）における角層水和評価ガイドラインに沿って８時間暴露試験を行った。被験者は２０代、３０代の女性計８名を用いた。角層水分量はキャパシタンス測定法であるSKICON（登録商標）―２００を使用して測定した。その結果を図４に示す。保湿性化粧液を塗布した角層水分量は、純水のものに対して高く、保湿性化粧液の塗布による保湿効果が認められた。 The correspondence between the results obtained by the above method and the moisture retention of human skin was confirmed by the following method. Applying pure water and moisturizing cosmetic liquid (Funaty (registered trademark) cosmetic liquid moist) manufactured by FANCL Co., Ltd. to the surface of the skin with a 0.1% aqueous solution of SDS, and the EU (EEMCO) guidelines for evaluating horny layer hydration And an exposure test was conducted for 8 hours. The test subjects used a total of 8 women in their 20s and 30s. The stratum corneum water content was measured using a capacitance measuring method SKICON (registered trademark) -200. The result is shown in FIG. The moisture content of the stratum corneum to which the moisturizing cosmetic liquid was applied was higher than that of pure water, and the moisturizing effect by application of the moisturizing cosmetic liquid was recognized.
肌角層水分測定による保湿性化粧液の保湿性効果と、3次元的組織細胞培養物を用いた被験物暴露後の組織の抽出液吸光度には密接な関係があり、本発明の3次元的組織細胞培養物を用いた方法は、ヒトの肌の保湿性評価を定量的、簡易的、且つ経済的に実施できる有効な保湿性評価方法である。 There is a close relationship between the moisturizing effect of the moisturizing cosmetic liquid by skin stratum corneum moisture measurement and the extract absorbance of the tissue after exposure to the test substance using a three-dimensional tissue cell culture. The method using the tissue cell culture is an effective method for evaluating the moisturizing property of human skin, which can quantitatively, easily and economically evaluate the moisturizing property of human skin.
本発明による保湿性評価方法により、3次元的組織培養物を用いることから、人での保湿性評価に対して再現性が高く、評価時間も短いため経済性に優れた保湿性評価方法を提供することができ、産業界に大きく寄与することが期待される。 Since the three-dimensional tissue culture is used by the moisturizing evaluation method according to the present invention, the reproducibility is high for human moisturizing evaluation, and the evaluation time is short. It is expected to contribute greatly to the industry.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2010172200A (en) * | 2009-01-27 | 2010-08-12 | Shiseido Co Ltd | Rough skin model using dry skin, and method for evaluating medicine for enhancing horny cell layer transparency with the same |
| JP2011024489A (en) * | 2009-07-27 | 2011-02-10 | Morinaga & Co Ltd | Method for evaluating moistening effect |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06180311A (en) * | 1991-01-28 | 1994-06-28 | Procter & Gamble Co:The | Method of evaluating skin irritability |
| JP2005505312A (en) * | 2001-03-02 | 2005-02-24 | ストラタテック コーポレーション | Improved skin substitute and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06180311A (en) * | 1991-01-28 | 1994-06-28 | Procter & Gamble Co:The | Method of evaluating skin irritability |
| JP2005505312A (en) * | 2001-03-02 | 2005-02-24 | ストラタテック コーポレーション | Improved skin substitute and use thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010172200A (en) * | 2009-01-27 | 2010-08-12 | Shiseido Co Ltd | Rough skin model using dry skin, and method for evaluating medicine for enhancing horny cell layer transparency with the same |
| JP2011024489A (en) * | 2009-07-27 | 2011-02-10 | Morinaga & Co Ltd | Method for evaluating moistening effect |
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