JP6567382B2 - Material screening method and kit - Google Patents
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- JP6567382B2 JP6567382B2 JP2015193535A JP2015193535A JP6567382B2 JP 6567382 B2 JP6567382 B2 JP 6567382B2 JP 2015193535 A JP2015193535 A JP 2015193535A JP 2015193535 A JP2015193535 A JP 2015193535A JP 6567382 B2 JP6567382 B2 JP 6567382B2
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Description
本発明は、物理刺激によって生じるくすみの改善効果を有する素材のスクリーニング方法及びキットに関する。具体的には、一酸化窒素（ＮＯ）を指標とした、物理刺激によって生じるくすみの改善効果を有する素材のスクリーニング方法及びキットに関する。 The present invention relates to a screening method and kit for a material having an effect of improving dullness caused by physical stimulation. Specifically, the present invention relates to a screening method and a kit for a material having an effect of improving dullness caused by physical stimulation using nitric oxide (NO) as an index.
メラニンは、紫外線による細胞核のＤＮＡの損傷を防ぐ等、生体内で重要な役割を果たしており、一定量のメラニンは生体にとって必要と言える。
一方で、シミ、ソバカス、くすみ、色むらの少ない肌は、女性の永遠の憧れであり、市場においては、それらに対する効果や即効性に優れた皮膚外用剤が常に求められてきた。
ところが、昨今、高い効果とは裏腹に、その過剰な効果がかえって病的な症状を引き起こすことが社会的な問題となってきた。
Melanin plays an important role in the living body, such as preventing damage to DNA in the cell nucleus due to ultraviolet rays, and it can be said that a certain amount of melanin is necessary for the living body.
On the other hand, skin with little spots, freckles, dullness, and uneven color is an eternal admiration of women, and in the market, there has always been a demand for a skin external preparation excellent in effect and immediate effect on them.
However, recently, contrary to the high effect, it has become a social problem that the excessive effect on the contrary causes pathological symptoms.
その為、現在では、色素沈着等皮膚に異常が起きている部分は改善し、異常が起きていない部分には影響が少ない素材の提供が求められるようになってきた。 Therefore, at present, it has been demanded to provide a material that is improved in a part where skin abnormality such as pigmentation has occurred and has little influence on a part where abnormality does not occur.
肌のくすみとは、女性の肌の悩みの一つであり、シミやソバカス等の色素沈着とは異なり、必ずしもメラニン色素の沈着によるもののみを指すわけではない。くすみとは、一般に肌に透明感がなく明るさや艶が感じられない肌状態のことを指す。くすみの発生部位で見られる現象としては、角層の水分量低下やきめの乱れ、角層の重層化などの表面状態の悪化や、明度の低下などが知られており、その原因には、一時的な体調不良や精神的ストレス、紫外線の影響、加齢による機能低下、肌の摩擦や圧迫などの物理刺激に起因する肌荒れや血行不良、メラニン量の増加などが複合的に関与していると考えられている。 Skin dullness is one of the problems of women's skin, and unlike pigmentation such as spots and freckles, it does not necessarily refer only to melanin pigmentation. Dullness generally refers to a skin condition in which the skin is not transparent and does not feel brightness or gloss. As the phenomenon seen in the dull spots, known are the deterioration of the surface condition such as the decrease in the amount of moisture in the stratum corneum, the disturbance of the texture, the stratification of the stratum corneum, and the decrease in brightness. There are multiple factors such as temporary poor physical condition, mental stress, effects of ultraviolet rays, functional deterioration due to aging, rough skin due to physical stimulation such as skin friction and pressure, poor circulation, and increased melanin. It is believed that.
我々の皮膚は、顔等を洗う、メークをする、衣服や服飾品を身につける、目を擦る等、日常生活において、様々な物理刺激にさらされている。これらの物理刺激は、若年者・老齢者、或いは、紫外線照射の有無、ストレスの有無に関わらず日々発生し、反復されている。このような日常的に繰り返される物理刺激によって生じるくすみは、仮に紫外線を全く浴びない乳幼児であっても、摩擦・圧迫によって発生する点で、紫外線照射や加齢等の他の原因によるくすみとは区別される。 Our skin is exposed to various physical stimuli in daily life, such as washing the face, making makeup, wearing clothes and clothing, and rubbing eyes. These physical stimuli are generated and repeated every day, regardless of whether they are young or old, or with or without UV irradiation or stress. The dullness caused by such daily repeated physical stimulation is caused by friction and pressure even in infants who are not exposed to ultraviolet rays at all. What is dullness caused by other causes such as ultraviolet irradiation and aging? Differentiated.
肌の摩擦・圧迫などの物理刺激よって発生したくすみ部位では、メラニン色素の沈着が生じていることが報告されている（非特許文献１）が、そのメラニン色素沈着過程については良く分かっていなかった。 Melanin pigmentation has been reported to occur in dull areas caused by physical stimuli such as skin friction and pressure (Non-Patent Document 1), but the melanin pigmentation process was not well understood. .
このような状況であるにも関わらず、従来のくすみの対策は、その発生原因を区別することなく、メーキャップによるカバーやメラニン生成抑制・角質除去といった対症療法が行われていた。メーキャップによるカバーでは、もちろんくすみがある、という問題の解決には至らない。また、物理刺激により生じたくすみに対して、メラニン生成抑制を目的とする美白剤を使用した場合には、くすみ部位だけでなく、その周囲のメラニン生成も抑制されるためか、肌全体としては皮膚色が明るくなるものの、くすみ部位とその周囲の皮膚色の差は解消されず、くすみを改善するという当初の目的が達成されないということが経験的に知られていた。美白剤をくすみ部位に限局して使用する方法も考えられるが、くすみは、シミやソバカスなど、他の色素沈着とは異なって境界が不明瞭であり、色素沈着の程度の勾配に合わせて美白剤を適用するという方法は現実的には難しい。
このように物理刺激によって生じるくすみの原因に着目した解決は行なわれておらず、物理刺激によるくすみに効果がある素材のスクリーニング方法も確立されていなかった。
Despite this situation, conventional measures for dullness have been symptomatic treatments such as covering with makeup, suppression of melanin production, and removal of keratin without distinguishing the cause of the occurrence. Of course, the makeup cover does not solve the problem of dullness. In addition, when using a whitening agent for the purpose of suppressing melanin production against dullness caused by physical stimulation, not only the dull part but also the surrounding melanin production is suppressed. It has been empirically known that although the skin color becomes brighter, the difference between the dull site and the surrounding skin color is not eliminated, and the original purpose of improving dullness is not achieved. A method of using a whitening agent confined to the dull area is also conceivable, but unlike other pigmentation such as spots and freckles, dullness is unclear, and whitening is performed according to the gradient of the degree of pigmentation. The method of applying the agent is practically difficult.
Thus, there has been no solution focusing on the cause of dullness caused by physical stimulation, and no screening method for a material effective for dullness due to physical stimulation has been established.
ところで、ＮＯは、窒素酸化物（ＮＯｘ）の一種であり、生体内で恒常的に存在することが知られている。また、紫外線照射により生体内でＮＯが発生することも知られている。 By the way, NO is a kind of nitrogen oxide (NOx) and is known to exist constantly in the living body. It is also known that NO is generated in the living body by ultraviolet irradiation.
生体で恒常的に存在するＮＯは、Ｌ−アルギニン、ＮＡＤＰＨ、テトラヒドロビオプテリン、カルモジュリン等を基質としてＮＯ合成酵素により作られ、極めて重要な生理活性分子として機能している。例えば、血管内皮由来弛緩因子（ＥＤＲＦ）の正体はＮＯであり、その他、神経系における情報伝達物質や免疫系での異物攻撃の役割を担っていることが知られている。それに対し、紫外線照射により発生したＮＯはメラノサイトを刺激し、メラニン生成を促進することが知られている（非特許文献２）。 NO constantly present in the living body is produced by NO synthase using L-arginine, NADPH, tetrahydrobiopterin, calmodulin, etc. as a substrate, and functions as an extremely important physiologically active molecule. For example, the true identity of vascular endothelium-derived relaxing factor (EDRF) is NO, and it is also known that it plays a role of a foreign body attack in the information system in the nervous system and in the immune system. In contrast, it is known that NO generated by ultraviolet irradiation stimulates melanocytes and promotes melanin production (Non-patent Document 2).
一方、物理刺激によってケラチノサイトが活性化されＮＯが生成されることが知られているが（非特許文献３）、物理刺激によって発生したＮＯが生体内でどのような役割を担っているかは知られていなかった。 On the other hand, it is known that keratinocytes are activated and NO is generated by physical stimulation (Non-Patent Document 3), but it is known what role NO generated by physical stimulation plays in vivo. It wasn't.
このような状況下、本発明者らは、物理刺激によるくすみを改善するには、最終結果物であるメラニン（合成反応経路を含む）そのものに影響を及ぼす素材ではなく、かつ、物理刺激に起因するくすみの発生経路を解明し、物理刺激によって生じたくすみを積極的に改善する素材を用いることが有用であるとの結論に至った。 Under such circumstances, in order to improve dullness due to physical stimulation, the present inventors are not a material that affects the final melanin (including the synthetic reaction pathway) itself, and that is caused by physical stimulation. It was concluded that it is useful to clarify the path of dullness and to use materials that positively improve dullness caused by physical stimulation.
本発明は、物理刺激によって生じるくすみを改善する効果を有する素材を見出すスクリーニング方法及びキットの提供を課題としている。 An object of the present invention is to provide a screening method and a kit for finding a material having an effect of improving dullness caused by physical stimulation.
本発明者らは上述の課題解決のために鋭意研究した結果、物理刺激により発生したＮＯがメラニン生成を促進していることを発見し、ＮＯを指標に用いることで上記課題を解決した。 As a result of intensive studies for solving the above-mentioned problems, the present inventors have found that NO generated by physical stimulation promotes melanin production, and solved the above problems by using NO as an index.
本発明によれば、物理刺激に起因する肌のくすみを改善する素材を選択することが可能になり、これにより選択された素材は、物理刺激によるくすみ部位の皮膚色と、その周囲の皮膚色の相対的な差を少なくすることができ、物理刺激に起因する肌のくすみを根本的に解決することができる。 According to the present invention, it is possible to select a material that improves the dullness of the skin caused by physical stimulation, and the selected material includes the skin color of the dull part due to physical stimulation and the surrounding skin color. Relative dullness can be reduced, and skin dullness caused by physical stimulation can be fundamentally solved.
本発明は、くすみの中でも物理刺激によって生じるくすみを改善する素材のスクリーニング方法及びそれを使用したキットに関する発明であって、物理刺激によって生じたＮＯを指標とするものである。 The present invention relates to a screening method for a material that improves dullness caused by physical stimulation among dullness and a kit using the same, and uses NO generated by physical stimulation as an index.
本発明において「くすみ」とは、近隣の皮膚色と比較して相対的に明度が下がっている状態である部位をさす。 In the present invention, “dullness” refers to a portion in which the brightness is relatively lowered as compared with the skin color in the vicinity.
本発明において「物理刺激」とは、繰り返し行なわれるか否かは問わず、その強弱に関係なく、摩擦、圧迫等の物理的要因に発生する刺激をいい、紫外線や薬剤等の化学的刺激やストレス等の精神的刺激を除く意味である。 In the present invention, “physical stimulation” refers to stimulation that occurs due to physical factors such as friction and compression, regardless of its strength, regardless of whether or not it is repeatedly performed. It means to exclude mental stimuli such as stress.
本発明において「物理刺激によって生じるくすみを改善する」とは、物理刺激によって生じるくすみを解消するという意味であり、防御、抑制、改善を含む概念である。 In the present invention, “to improve dullness caused by physical stimulation” means to eliminate dullness caused by physical stimulation, and is a concept including defense, suppression, and improvement.
本発明において「ＮＯを指標にする」とは、被験素材適用前後のＮＯ量を指標にするという意味であり、消去率、生成促進率のいずれでも良い。消去率を指標とする場合はＮＯ消去率が高いものを選択すればよく、生成促進率を指標とする場合は、ＮＯ生成促進率が低いものを選択すれば良いだけである。言い換えれば、最終的に被験素材の適用によってＮＯの存在量が下がったものを選択するという趣旨である。 In the present invention, “uses NO as an index” means that the amount of NO before and after application of the test material is used as an index, and may be either an erasure rate or a generation promotion rate. When the erasing rate is used as an index, a high NO erasing rate may be selected, and when the generation promotion rate is used as an index, only a low NO generation accelerating rate may be selected. In other words, the intention is to select the one whose NO abundance has finally decreased due to the application of the test material.
本発明におけるスクリーニング方法では、強制的にＮＯを発生させた環境下で被験素材を適用させても良いし、既に一定のＮＯが存在する環境下において被験素材を適用させても良い。必ずしも細胞を用いて試験を行う必要はない。試験管レベルでＮＯの存在量の変化を確認しても良い。いずれの場合であっても、被験素材の適用によりＮＯ量の減少効果が高いものを選択することで、物理刺激によるくすみを改善する素材をスクリーニングすることが出来る。
より厳密に物理刺激によって発生したＮＯを消去する素材を選択するには、人為的に発生させたＮＯ量を測定し、人為的に増加させた分だけのＮＯを減少させる素材を選択することが望ましい。
もっとも、厳密に人為的に増加させた分だけを減らす素材だけを選択する必要はなく、選択した素材の皮膚内への浸透程度を考慮することができる。
In the screening method of the present invention, the test material may be applied in an environment where NO is forcibly generated, or the test material may be applied in an environment where a certain amount of NO already exists. It is not always necessary to conduct tests using cells. Changes in the amount of NO present may be confirmed at the test tube level. In any case, a material that improves dullness due to physical stimulation can be screened by selecting a material that has a high NO reduction effect by applying the test material.
In order to select a material that eliminates NO generated by physical stimulation more strictly, it is necessary to measure the amount of artificially generated NO and select a material that reduces NO by the amount artificially increased. desirable.
However, it is not necessary to select only a material that reduces the amount that is strictly increased artificially, and the degree of penetration of the selected material into the skin can be considered.
ＮＯを発生させる方法は、公知の方法を用いれば良い。例えばＮＯ発生剤を用いれば良く、ＮＯＣ−５（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３−（３−ａｍｉｎｏｐｒｏｐｙｌ）−３−ｉｓｏｐｒｏｐｙｌ−１−ｔｒｉａｚｅｎｅ）（同仁化学）、ＮＯＣ−７（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３−（Ｎ−ｍｅｔｈｙｌ−３−ａｍｉｎｏｐｒｏｐｙｌ）−３−ｍｅｔｈｙｌ−１−ｔｒｉａｚｅｎｅ）（同仁化学）、ＮＯＣ−１２（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３−（Ｎ−ｅｔｈｙｌ−２−ａｍｉｎｏｅｔｈｙｌ）−３−ｅｔｈｙｌ−１−ｔｒｉａｚｅｎｅ）（同仁化学）、ＮＯＣ−１８（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３，３−ｂｉｓ（２−ａｍｉｎｏｅｔｈｙｌ）−１−ｔｒｉａｚｅｎｅ）（同仁化学）等を使用することができる。 A known method may be used as a method of generating NO. For example, a NO generator may be used, and NOC-5 (1-Hydroxy-2-oxo-3- (3-aminopropyl) -3-isopropyl-1-triazene) (Dojindo), NOC-7 (1-Hydroxy- 2-oxo-3- (N-methyl-3-aminopropyl) -3-methyl-1-triazene) (Dojindo), NOC-12 (1-Hydroxy-2-oxo-3- (N-ethyl-2-) aminoethyl) -3-ethyl-1-triazene) (Dojindo), NOC-18 (1-Hydroxy-2-oxo-3,3-bis (2-aminoethyl) -1-triazene) (Dojindo), etc. can do.
ＮＯの存在量を確認する方法は、公知の方法を用いれば良い。例えばＮＯの酸化物であるＮＯ２ −を測定することでＮＯ量を測定するＧｒｉｅｓｓ法を用いて、ＮＯ消去効果の評価をすることが出来る。Ｇｒｉｅｓｓ法による測定は、市販のＮＯ２／ＮＯ３ Ａｓｓａｙ Ｋｉｔ−ＣＩＩ（Ｃｏｌｏｒｉｍｅｔｒｉｃ）（同仁化学）やＮｉｔｒｉｃ Ｏｘｉｄｅ Ｆｌｕｏｒｏｍｅｔｒｉｃ Ａｓｓａｙ Ｋｉｔ（ＢｉｏＶｉｓｉｏｎ）等を使用することができる。 A known method may be used as a method for confirming the abundance of NO. For example, the NO erasing effect can be evaluated using the Griess method of measuring the amount of NO by measuring NO 2 − which is an oxide of NO. For the measurement by the Griess method, commercially available NO 2 / NO 3 Assay Kit-CII (Colorimetric) (Dojin Chemical), Nitric Oxide Fluorometric Assay Kit (BioVision) and the like can be used.
上記のようにして選択された被験素材の中から、更にＮＯ以外に起因するメラニンの産生能を指標として加えることで、より好ましい素材を選択することが出来る。
物理刺激によるくすみを改善するには、ＮＯを指標とすれば十分であるが、皮膚外用剤に使用される各種素材は、単独の効果だけでなく複数の効果を有することが多い。その為、ＮＯ消去効果を有する素材の中には、例えばチロシナーゼ活性阻害能を合わせて有する素材が存在する。このような素材は、理論的には、物理刺激に起因するくすみを改善するだけでなく、物理刺激とは関係なく生成されるメラニンの生成を阻害することも可能である。そうすると、本来必要であるメラニンまでも生成阻害することになり、過剰な効果を引き起こす可能性がある。そこで、ＮＯを指標とする以外に、ＮＯ以外に起因するメラニンの産生能を指標として加えることで、過剰な効果を引き起こす可能性ある素材を排除することが可能になり、より好ましいと言える。
もっとも、前述したＮＯ消去効果と同様、選択した素材の皮膚への浸透度を考慮することができる。
From the test materials selected as described above, a more preferable material can be selected by adding, as an index, the ability to produce melanin other than NO.
In order to improve dullness due to physical stimulation, it is sufficient to use NO as an index. However, various materials used for external preparations for skin often have multiple effects as well as single effects. For this reason, among materials having an NO elimination effect, for example, there are materials having both tyrosinase activity inhibition ability. Theoretically, such a material not only improves dullness caused by physical stimulation, but also can inhibit the production of melanin that is generated independently of physical stimulation. If it does so, it will inhibit generation | occurrence | production even the melanin which is originally required, and may cause an excessive effect. Therefore, in addition to using NO as an index, adding melanin producing ability derived from other than NO as an index makes it possible to eliminate materials that may cause excessive effects, which is more preferable.
However, like the NO elimination effect described above, the degree of penetration of the selected material into the skin can be considered.
具体的な手法として、予めＮＯ量を減少させる素材を選択した後、ＮＯ以外に起因するメラニンの産生を抑制しない素材を選択しても良いし、ＮＯ以外に起因するメラニンの産生を抑制しない素材を選択した後に、ＮＯ量を減少させる素材を選択しても良い。これらを同時に行なってもよい。いずれの場合においても、被験素材の適用によって、ＮＯの存在量を減少させるが、ＮＯ以外に起因するメラニンの産生を抑制する能力がない素材を選択すればよい。 As a specific method, after selecting a material that reduces NO amount in advance, a material that does not suppress melanin production caused by other than NO may be selected, or a material that does not inhibit melanin production caused by other than NO After selecting, a material that reduces the amount of NO may be selected. These may be performed simultaneously. In any case, the application of the test material reduces the abundance of NO, but a material that does not have the ability to suppress the production of melanin caused by something other than NO may be selected.
ＮＯ以外を発生原因とするメラニン生成能を指標にするとは、紫外線照射やチロシナーゼ等の各種酵素活性、情報伝達物質等、ＮＯ以外を原因とするメラニン生成抑制能を指標にするという意味であり、これらを原因とするメラニン生成を抑制する能力を積極的に有していないものを選択するという趣旨である。全く有していないものが望ましいが、多少効果が確認出来ても差し支えない。本指標は、最終的に選択した素材が、過剰な効果を有していないことを期待するものであるから、その限度において多少の効果が見られるものでも使用できる。 Using melanin producing ability as a cause other than NO as an index means that various enzyme activities such as ultraviolet irradiation and tyrosinase, information transmission substances, etc., and melanin production inhibiting ability caused by something other than NO as an index, The purpose is to select those that do not actively have the ability to suppress melanin production caused by these. Although it is desirable that it does not have at all, there is no problem even if the effect can be confirmed to some extent. Since this index expects that the finally selected material does not have an excessive effect, it can be used even if it shows some effect within that limit.
ＮＯ以外を発生原因とするメラニン生成抑制能の有無を確認するには、公知の方法を用いれば良い。例えばＢ１６メラノーマ細胞を用いたメラニン生成抑制評価法、チロシナーゼ活性抑制評価法、くすみが発生していない部位の角層細胞中のメラニン顆粒染色法等が挙げられる。これらの方法において、メラニン生成抑制能がない素材を選択すれば良い。必ずしも細胞を用いて試験を行う必要はない。試験管レベルで、確認を行なってもよい。いずれの場合であっても、被験素材の適用した際に、ＮＯ以外を原因とするメラニン生成抑制能がないもの選択すれば良い。 A known method may be used to confirm the presence or absence of the ability to suppress melanin production caused by other than NO. Examples thereof include a method for evaluating inhibition of melanin production using B16 melanoma cells, a method for evaluating inhibition of tyrosinase activity, and a method for staining melanin granules in horny layer cells where no dullness has occurred. In these methods, a material having no ability to suppress melanin production may be selected. It is not always necessary to conduct tests using cells. Confirmation may be performed at the test tube level. In any case, when the test material is applied, a material having no ability to suppress melanin production caused by other than NO may be selected.
ここで、「ＮＯ以外を原因とするメラニン生成抑制能がない」とは、一般に効果が期待できないと判定できるレベルを指し、例えば、精製水等の陰性対象と比較した場合に、統計的な有意差がみられない等によって判別できる。 Here, “there is no ability to suppress melanin production caused by other than NO” refers to a level at which it can be generally determined that the effect cannot be expected, and is statistically significant when compared with a negative target such as purified water, for example. It can be determined by no difference.
被験素材は、特に制限はない。動物由来エキス、菌類の培養物、又はこれらの酵素等処理物、化合物又はその誘導体等であっても被検物質として用いることが出来、液状の他、粉末状、ジェル状等であっても差し支えない。また、そのままでは培地に溶解しない場合は、界面活性剤等の可溶化剤を適宜使用することにより溶解させることで被験素材として用いることができる。抽出の方法は、特に限定されない。添加濃度については、細胞を用いる場合は、エキス等を添加から２４時間後に明らかに細胞が死滅していなければ、どの濃度でも問題ない。 The test material is not particularly limited. It can be used as a test substance even if it is an animal-derived extract, a culture of fungi, a processed product such as these enzymes, a compound or a derivative thereof, and it may be in the form of powder, gel, etc. in addition to liquid. Absent. Moreover, when it does not melt | dissolve in a culture medium as it is, it can use as solubilizing materials by making it melt | dissolve by using suitably solubilizing agents, such as surfactant. The extraction method is not particularly limited. With regard to the addition concentration, when cells are used, any concentration is acceptable as long as the cells are not clearly killed 24 hours after the addition of the extract or the like.
本発明のスクリーニング方法を用いたキットは、当該方法を使用していれば特に限定はされない。一つのキットで本発明のスクリーニング方法を具備するものでも良いし、二つ以上キットに分かれていても差し支えない。例えば、三次元培養ヒト皮膚モデルとこれに適した培地、使用する重りをセットにしたもの等が考えられる。 The kit using the screening method of the present invention is not particularly limited as long as the method is used. One kit may comprise the screening method of the present invention, or two or more kits may be separated. For example, a three-dimensional cultured human skin model, a medium suitable for this, and a set of weights to be used can be considered.
以下、本発明を実施例について具体的に説明するが、本発明はこれらの実施例により限定されるものではない。また、特記しない限り配合量は質量％で示す。 EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples. Unless otherwise specified, the blending amount is expressed in mass%.
＜三次元培養ヒト皮膚モデルを用いた実験＞
三次元培養ヒト皮膚モデルを用いて、荷重負荷がメラニン生成に与える影響を確認した。
三次元培養ヒト皮膚モデルＭＥＬ−３００Ａ（倉敷紡績）を３７℃、５％ＣＯ２下で培養し、２〜３日に１回培地ＥＰＩ−１００ＬＬＭＭ（倉敷紡績）の交換を行った。
ヒト皮膚における物理刺激の再現として、三次元培養ヒト皮膚モデルに荷重をかけ、荷重をかけない群と比較することでその影響を確認した。荷重をかける群では、毎回の培地交換後に重り（約５．０ｇ）を各皮膚モデルカップの上に３０分間静置し、その後重りを取り除き、培養を継続した。
サンプルを添加する場合は、毎回の培地交換後の培地中に、サンプル（被験素材又は公知の一酸化窒素消去剤）を各終濃度になるように添加した後、重り（約５．０ｇ）を各皮膚モデルカップの上に３０分間静置し、その後重りを取り除き、培養を継続した。
培地交換から次の培地交換前までを１回の培養とし、上記培養を7回繰り返した。
メラニン生成量は、培養開始３週間後に下記の実験手法を用いて測定した。
尚、公知の一酸化窒素消去剤は、Ｃａｒｂｏｘｙ−ＰＴＩＯ（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３−（３−ａｍｉｎｏｐｒｏｐｙｌ）−３−ｉｓｏｐｒｏｐｙｌ−１−ｔｒｉａｚｅｎｅ）（同仁化学）をＰＢＳ（−）で溶解させて、終濃度が５０μＭとなるように培地中に添加した。
<Experiment using three-dimensional cultured human skin model>
Using a three-dimensional cultured human skin model, the effect of loading on melanin production was confirmed.
A three-dimensional cultured human skin model MEL-300A (Kurashiki Spin) was cultured at 37 ° C. under 5% CO 2 , and the medium EPI-100LLMM (Kurashiki Spin) was changed once every 2-3 days.
As a reproduction of physical stimulation in human skin, the effect was confirmed by applying a load to a three-dimensional cultured human skin model and comparing it with a group not applied with a load. In the group to which a load was applied, the weight (about 5.0 g) was allowed to stand on each skin model cup for 30 minutes after each medium change, and then the weight was removed and the culture was continued.
When adding a sample, add a sample (test material or a known nitric oxide scavenger) to each final concentration in the medium after each medium exchange, and then add a weight (about 5.0 g). It was allowed to stand for 30 minutes on each skin model cup, after which the weight was removed and the culture was continued.
The culture was performed from one medium exchange to the next before the next medium exchange, and the above culture was repeated seven times.
The amount of melanin produced was measured using the following experimental method 3 weeks after the start of culture.
As a known nitric oxide scavenger, Carboxy-PTIO (1-Hydroxy-2-oxo-3- (3-aminopropyl) -3-isopropyl-1-triazene) (Dojin Chemical) is dissolved in PBS (-). And added to the medium so that the final concentration was 50 μM.
＜メラニン生成量測定＞
三次元培養ヒト皮膚モデルをＰＢＳ（−）で洗浄後、カップから細胞を剥がしてエッペンチューブに入れ、２Ｎ ＮａＯＨａｑ８００μＬを添加した。その後、１００℃下で３０分間煮沸し、３５００ｒｐｍで５分間遠心分離した。この上清を９６ウェルプレートに３５０μＬ移し、４０５ｎｍ、６５５ｎｍの吸光度（Ａ４０５、Ａ６５５）を測定した。さらに、この上清４０μＬを別の９６ウェルプレートに移し、ＢＣＡ法に従い、５４０ｎｍ、６５５ｎｍの吸光度（Ｂ５４０、Ｂ６５５）を測定しタンパク量を測定した。得られた吸光度から以下の式によりメラニン生成促進率を算出した。
<Measurement of melanin production>
After washing the three-dimensional cultured human skin model with PBS (−), the cells were peeled off from the cup, placed in an Eppendorf tube, and 2N NaOHaq 800 μL was added. Then, it boiled for 30 minutes under 100 degreeC, and centrifuged for 5 minutes at 3500 rpm. 350 μL of this supernatant was transferred to a 96-well plate, and the absorbance (A405, A655) at 405 nm and 655 nm was measured. Furthermore, 40 μL of this supernatant was transferred to another 96-well plate, and the absorbance (B540, B655) at 540 nm and 655 nm was measured according to the BCA method to measure the amount of protein. From the obtained absorbance, the melanin production promotion rate was calculated by the following formula.
図１は、ヒト皮膚において繰り返し行われる物理刺激を、三次元培養ヒト皮膚モデルを用いて再現した結果である。
三次元培養ヒト皮膚モデルに重りにより荷重をかけたときのメラニン生成量は、荷重をかけていないときと比べて、有意に増加するということが確認された。このことから、本方法により、繰り返し行われる物理刺激によって生じる、色素沈着を含むくすみ現状を再現することができた（図１）。
FIG. 1 shows the result of reproducing physical stimulation repeatedly performed on human skin using a three-dimensional cultured human skin model.
It was confirmed that the amount of melanin produced when a weight was applied to the three-dimensional cultured human skin model was significantly increased compared to when no weight was applied. From this, the present condition was able to reproduce the dull state including pigmentation caused by repeated physical stimulation (FIG. 1).
〔００３３〕の実験の１回目の荷重負荷直後の培地を採取し（荷重をかけない群では、１回目の荷重をかけた直後と同時期に培地を採取）、下記の実験手法を用いて培地中のＮＯ量を測定した（図２）。 The medium immediately after the first loading of the experiment of [0033] was collected (in the group where no load was applied, the medium was collected immediately after the first loading), and the medium was obtained using the following experimental method. The amount of NO was measured (FIG. 2).
＜ＮＯ量測定試験（Ｎｉｔｒｉｃ Ｏｘｉｄｅ Ｆｌｕｏｒｏｍｅｔｒｉｃ Ａｓｓａｙ）＞
Ｎｉｔｒｉｃ Ｏｘｉｄｅ Ｆｌｕｏｒｏｍｅｔｒｉｃ Ａｓｓａｙ（ＢｉｏＶｉｓｉｏｎ）を用いて、蛍光プレートリーダーで３６０ｎｍの励起波長、４６５ｎｍの蛍光波長での蛍光強度を測定し培地中に含まれるＮＯ量を測定した。皮膚モデルに荷重負荷をかけていない場合のＮＯ量を１００％とした場合における相対比をＮＯ量（％）として算出した。
<NOx Measurement Test (Nitric Oxide Fluorometric Assay)>
Using a Nitric Oxide Fluorometric Assay (BioVision), the fluorescence intensity at an excitation wavelength of 360 nm and a fluorescence wavelength of 465 nm was measured with a fluorescence plate reader, and the amount of NO contained in the medium was measured. The relative ratio when the NO amount when no load was applied to the skin model was taken as 100% was calculated as the NO amount (%).
図２は、細胞から培地に放出されるＮＯ量を、荷重をかけなかった場合を１００％とした場合の相対値を比較したものである。
三次元培養ヒト皮膚モデルに荷重をかけたときの培地から検出されるＮＯ量は、荷重負荷によって増加した。また、荷重負荷によって増加したＮＯ量は、公知のＮＯ消去剤であるＣａｒｂｏｘｙ−ＰＴＩＯの添加により消去されることが確認された。
FIG. 2 is a comparison of relative values when the amount of NO released from the cells into the medium is 100% when no load is applied.
The amount of NO detected from the culture medium when a load was applied to the three-dimensional cultured human skin model increased with the load. In addition, it was confirmed that the NO amount increased by load application was erased by the addition of Carboxy-PTIO, which is a known NO scavenger.
図３は、〔００３３〕において三次元培養ヒト皮膚モデルに荷重をかけた群に加えて、一般のＮＯ消去剤をサンプルとして添加した場合のメラニン生成促進率を確認した結果である。荷重負荷によって生成が促進されたメラニン量が、ＮＯ消去剤の添加によって減少していることが確認された。 FIG. 3 shows the results of confirming the melanin production promotion rate when a general NO scavenger is added as a sample in addition to the group in which a load is applied to the three-dimensional cultured human skin model in [0033]. It was confirmed that the amount of melanin whose production was promoted by load application was reduced by the addition of NO scavenger.
以上の結果から、三次元培養ヒト皮膚モデルに対する荷重負荷は、メラニン生成を促し、ここにＮＯが関与することを確認した。このことにより、まぶた等のくすみ発生にはアイメークなどの繰り返し行われる物理刺激によって生じるＮＯが関与している可能性が示唆された。 From the above results, it was confirmed that the load applied to the three-dimensional cultured human skin model promotes melanin production, and NO is involved here. This suggests that NO generated by repeated physical stimulation such as eye makeup may be involved in the generation of dullness in eyelids and the like.
ＮＯ発生剤としてＮＯＣ−５（１−Ｈｙｄｒｏｘｙ−２−ｏｘｏ−３−（３−ａｍｉｎｏｐｒｏｐｙｌ）−３−ｉｓｏｐｒｏｐｙｌ−１−ｔｒｉａｚｅｎｅ）（同仁化学）を使用し、下記の実験手法を用いてＮＯ消去効果を有する素材をスクリーニングした。 NOC-5 (1-Hydroxy-2-oxo-3- (3-aminopropyl) -3-isopropyl-1-triazene) (Dojin Chemical) was used as a NO generator and the following experimental method was used to eliminate NO. The material with
＜被験素材の調製＞
被験物質の植物バベンソウ（Ｖｅｒｂｅｎａ ｏｆｆｉｃｉｎａｌｉｓ）の葉及び茎、ウラジロガシ（Ｑｕｅｒｃｕｓ ｓａｌｉｃｉｎａ）の葉、柿（Ｄｉｏｓｐｙｒｏｓ ｋａｋｉ Ｔｈｕｎｂｅｒｇ）の葉、クロモジ（Ｌｉｎｄｅｒａ ｕｍｂｅｌｌａｔａ）の葉及び茎、ナズナ（Ｃａｐｓｅｌｌａ ｂｕｒｓａ-ｐａｓｔｏｒｉｓ）の葉及び茎、ヤナギマツタケ（Ａｇｒｏｃｙｂｅ ｃｙｌｉｎｄｒａｃｅａ）の子実体、ゴボウ（Ａｒｃｔｉｕｍ ｌａｐｐａ Ｌ.）の根の抽出物は、乾燥重量１ｇに１０倍量の５０％エタノールを１０ｇ加えて６０℃、３時間加熱抽出した。抽出後、ろ過をし、植物原体を取り除いた後、固形蒸発残分が１％になるように蒸留水で希釈した。以上の操作によって、固形蒸発残分が１％になるように調製した植物抽出物を得た。ワスレグサ花発酵液は、ワスレグサ（Ｈｅｍｅｒｏｃａｌｌｉｓ ｆｕｌｖａ及びＨｅｍｅｒｏｃａｌｌｉｓ ｆｌａｖａ及びＨｅｍｅｒｏｃａｌｌｉｓ ｍｉｎｏｒ）の蕾粉末の水懸濁液を酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）で発酵させることにより、その抽出液を得た。
<Preparation of test material>
Leaves and stems of the test substance plant, Verbena officinalis, leaves of Quercus salicina, leaves of Diospyros kaki Thunberg, leaves and stems of Lindera umbellata, ora sap In addition, the stem, the fruit body of Agrocybe cylindracea, and the root extract of burdock (Arcium lappa L.) were extracted by heating at 60 ° C. for 3 hours by adding 10 g of 50% ethanol to 10 g of dry weight. . After extraction, the mixture was filtered to remove the plant material, and diluted with distilled water so that the solid evaporation residue was 1%. By the above operation, a plant extract prepared so that the solid evaporation residue was 1% was obtained. The forget-me-not fermented liquor was obtained by fermenting an aqueous suspension of forget-me-nots (Hemerocallis fulva and Hemerocallis flava and Hemerocallis minor) in yeast (Saccharomyces cerevisiae).
＜ＮＯ消去効果評価試験（Ｇｒｉｅｓｓ法）＞
９６ウェルプレートにＰＢＳ（−）を１０μＬ、緩衝液（ｐＨ＝７．６）を４０μＬ、〔００４５〕で調製した被験素材を１０μＬ添加した。その後、５０μＭに調製したＮＯＣ−５（同仁化学）溶液４０μＬを添加して、室温で３０分間反応させた。反応終了後、Ｇｒｉｅｓｓ法に従い、マイクロプレートリーダーで５４０ｎｍの吸光度を測定した。
サンプル群として被験素材添加群の吸光度をＳ５４０、コントロール群として被験素材無添加群の吸光度をＣ５４０、ブランク群としてＰＢＳ（−）及び緩衝液のみの群の吸光度をＢ５４０として以下の式でＮＯ消去率（％）を算出した。
<NO elimination effect evaluation test (Griess method)>
10 μL of PBS (−), 40 μL of a buffer solution (pH = 7.6), and 10 μL of a test material prepared in [0045] were added to a 96-well plate. Thereafter, 40 μL of a NOC-5 (Dojin Chemical) solution prepared to 50 μM was added and reacted at room temperature for 30 minutes. After completion of the reaction, the absorbance at 540 nm was measured with a microplate reader according to the Griess method.
The absorbance of the test material added group as the sample group is S540, the absorbance of the test material non-added group is C540 as the control group, the absorbance of the PBS (−) and buffer only group is B540 as the blank group, and the NO elimination rate by the following formula (%) Was calculated.
効果が高かったものとして、エタノール水溶液で抽出して得られたクロモジ（葉及び茎）、ウラジロガシ（葉）、バベンソウ（葉及び茎）、柿（葉）の抽出物や酵母で発酵させて得られたワスレグサ花発酵液が挙げられ、これらについては、ＮＯ消去効果ありと判定した。中でもワスレグサ花発酵液は、ＮＯ消去率が３６．７％と特に高い結果を得た。
一方、ナズナ（葉及び茎）、ヤナギマツタケ（子実体）、ゴボウ（根）の抽出物は、ＮＯ消去率が２〜５％と低く、これらはＮＯ消去効果なしと判定した。結果を〔表１〕に示す。
It was obtained by fermenting with an extract of yeast or leaves (stems), radish (leaves), babensou (leaves and stems), and persimmons (leaves) extracted from an aqueous ethanol solution and yeast. The forget-me-not flower fermented liquid was mentioned, and about these, it determined with NO elimination effect. Above all, the forget-me-not flower fermented liquid had a particularly high NO elimination rate of 36.7%.
On the other hand, extracts of Nazuna (leaves and stems), willow matsutake (fruit bodies), and burdock (root) have a low NO elimination rate of 2 to 5%, and these were judged to have no NO elimination effect. The results are shown in [Table 1].
マウスメラノーマ細胞（Ｂ１６ｍｅｌａｎｏｍａ細胞）５．０×１０４ｃｅｌｌｓ／ｍＬを０．２ｍＬ１０％ＦＢＳ含有ダルベッコ改変イーグル培地（ＧＩＢＣＯ）に分散し、１２ウェルプレートに１ｍＬ播種し、３７℃、５％ＣＯ２下で培養した。２４時間後、被検物質（乾燥残分）を最終濃度５０ｐｐｍ、１００ｐｐｍとなるように添加した。細胞播種から９６時間培養後、培地を捨てＰＢＳ（−）で洗浄し、２Ｎ ＮａＯＨを５００μＬ添加した。１００℃下で３０分間煮沸し、３５００ｒｐｍで１分間遠心分離した。この上清を９６ウェルプレートに３５０μＬ移し、４０５ｎｍ、６５５ｎｍ（Ａ４０５、Ａ６５５）の吸光度を測定した。さらに、この上清４０μＬを９６ウェルプレートに移し、ＢＣＡ法に従い、５４０ｎｍ、６５５ｎｍ（Ｂ５４０、Ｂ６５５）の吸光度を測定しタンパク量を測定した。得られた吸光度から〔式１〕〔式２〕によりメラニン生成抑制率を算出した。 Mouse melanoma cells (B16 melanoma cells) 5.0 × 10 4 cells / mL were dispersed in Dulbecco's modified Eagle medium (GIBCO) containing 0.2 mL 10% FBS, and 1 mL was seeded in a 12-well plate, at 37 ° C. under 5% CO 2. In culture. After 24 hours, the test substance (dry residue) was added to a final concentration of 50 ppm and 100 ppm. After culturing for 96 hours after cell seeding, the medium was discarded and washed with PBS (−), and 500 μL of 2N NaOH was added. It was boiled at 100 ° C. for 30 minutes and centrifuged at 3500 rpm for 1 minute. 350 μL of this supernatant was transferred to a 96-well plate, and the absorbance at 405 nm and 655 nm (A405, A655) was measured. Furthermore, 40 μL of this supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm and 655 nm (B540, B655) according to the BCA method to measure the amount of protein. The inhibition rate of melanin production was calculated from the obtained absorbance according to [Formula 1] and [Formula 2].
〔表１〕より、ＮＯ消去効果の高かった素材〔クロモジ（葉及び茎）抽出物、ウラジロガシ（葉）抽出物、バベンソウ（葉及び茎）抽出物、柿（葉）抽出物、ワスレグサ花発酵液〕をＢ１６ｍｅｌａｎｏｍａに添加し、メラニン生成抑制効果を確認した。その結果、クロモジ抽出物、柿（葉）抽出物は、１００ｐｐｍにおいてコントロール（陰性対象）に比べ、タンパク当りのメラニン量は８５〜８８％のメラニン生成抑制効果が確認された。これらについては、メラニン生成抑制効果ありと判定した。
一方、バベンソウ（葉及び茎）抽出物、ウラジロガシ（葉）抽出物、ワスレグサ花発酵液は、低濃度においても、高濃度においてもコントロール（陰性対象）と同程度の値を示したので、これらについては、ＮＯ以外に起因するメラニン生成を抑制する効果はなしと判定した。結果を〔表２〕に示す。
From [Table 1], materials with higher NO elimination effects [chromodium (leaves and stems) extract, radish (leaves) extract, babensou (leaves and stems) extract, persimmon (leaves) extract, forget-me-not flower fermentation liquid ] Was added to B16 melanoma, and the melanin production inhibitory effect was confirmed. As a result, chrominum extract and persimmon (leaf) extract were confirmed to have a melanin production inhibitory effect of 85 to 88% of the amount of melanin per protein compared to the control (negative target) at 100 ppm. These were determined to have a melanin production inhibitory effect.
On the other hand, the extract of babensou (leaves and stems), the extract of radish (leaves), and forget-me-not flowers showed the same value as the control (negative target) at both low and high concentrations. Determined that there was no effect of suppressing melanin production caused by other than NO. The results are shown in [Table 2].
ＮＯ消去効果が高く、ＮＯ以外に起因するメラニンの生成を抑制する効果がない成分として選択されたワスレグサ花発酵液に関し、〔００３３〕の方法を用いて、荷重をかけた細胞での効果を確認した。その結果、荷重負荷によって促進されたメラニン量がワスレグサ花発酵液濃度依存的に抑制することが観察された。これにより、ワスレグサ花発酵液は、物理刺激によって発生したＮＯを消去することでメラニン生成抑制効果を発揮し、過度な効果ではないことが推測された。結果を〔表３〕に示す。 Regarding forget-me-not flower fermented liquor selected as a component that has a high NO-erasing effect and has no effect of suppressing the production of melanin caused by other than NO, the effect on loaded cells was confirmed using the method of [0033] did. As a result, it was observed that the amount of melanin promoted by the load was suppressed depending on the concentration of forget-me-not flowers. Thereby, it was estimated that the forget-me-not flower fermented liquid exhibits a melanin production inhibitory effect by eliminating NO generated by physical stimulation and is not an excessive effect. The results are shown in [Table 3].
＜人での効果試験＞
物理刺激としてアイメークをする習慣があり、まぶたのくすみが気になる２０〜５０代の女性８名に前述のワスレグサ花発酵液を配合した化粧水を８週間使用していただき、使用前後の皮膚色を比較した。試験は、ワスレグサ花発酵液を２％配合した化粧水と、ワスレグサ花発酵液を精製水に置き換えた化粧水を左右半顔ずつそれぞれ朝晩１日２回塗布していただき、４週間、８週間後に分光測色計（ＹＯＫＯＧＡＷＡ）を用いて皮膚色を測定した。また、試験は二重盲検法にて実施した。
使用した化粧水の処方は以下の通り。
<Effectiveness test in humans>
8 females in their 20s and 50s who have a habit of eye makeup as a physical stimulus and are concerned about dullness of the eyelids should use skin lotion containing the above-mentioned forget-me-not flower fermentation liquid for 8 weeks. Compared. The test was performed by applying a lotion containing 2% forget-me-not flower fermentation liquid and a lotion obtained by replacing the forget-me-not flower fermentation liquid with purified water twice a day for each left and right half face each morning and evening. Skin color was measured using a spectrocolorimeter (YOKOGAWA). The test was conducted in a double blind manner.
The formula of the lotion used is as follows.
＜化粧水＞（質量％）
（１）１，３−ブチレングリコール １０．００
（２）グリセリン ２．００
（３）モノラウリン酸ポリオキシエチレンソルビタン（２０Ｅ．Ｏ．） ０．１５
（４）エタノール ５．００
（５）ワスレグサ花発酵液（又は精製水） ２．００
（６）防腐剤 適 量
（７）精製水 残 部
合計 １００
＜製法＞（１）〜（７）を常法により混合・攪拌し、化粧水を得た。
<Lotion> (mass%)
(1) 1,3-butylene glycol 10.00
(2) Glycerin 2.00
(3) Polyoxyethylene sorbitan monolaurate (20E.O.) 0.15
(4) Ethanol 5.00
(5) Forget-me-not flower fermentation liquid (or purified water) 2.00
(6) Preservative appropriate amount (7) Purified water remaining total 100
<Production Method> (1) to (7) were mixed and stirred by a conventional method to obtain a lotion.
測定部位は、アイメークなどで日頃物理刺激を受ける頻度が高いまぶたと頻度が低い頬で行った。冒頭にも論述したが、くすみとは、周囲の皮膚色と比較して暗く見える現象である。そこで、周囲との皮膚色の差を評価するために、まぶた、頬それぞれの皮膚色と、これらと隣接する部位として、目の横の皮膚色を引いた値をくすみ度合いと定義した。また、このくすみ度合いは〔式５〕に示す式で定義し算出した。 The measurement site was the eyelid which is frequently subjected to physical stimulation such as eye makeup and the cheek which is less frequent. As discussed at the beginning, dullness is a phenomenon that appears darker than the surrounding skin color. Therefore, in order to evaluate the difference in skin color from the surroundings, the skin color of each of the eyelids and cheeks, and the value obtained by subtracting the skin color of the side of the eye as a part adjacent to these were defined as the degree of dullness. The dullness was defined and calculated by the equation shown in [Equation 5].
被験者のまぶたおよび頬の試験開始前のＬ＊値について見てみると、まぶたのくすみ度合いは頬のくすみ度合いに比べて値が低く、皮膚色が暗いことがわかった（図４）。 Looking at the L * values before the test on the subject's eyelids and cheeks, it was found that the degree of dullness of the eyelids was lower than the degree of dullness of the cheeks and the skin color was dark (FIG. 4).
Ｌ＊値について、日常的に物理刺激を受けＮＯが生じていると考えられるまぶたにおいて、ワスレグサ花発酵液を配合した化粧水を塗布したところ、配合していない化粧水を塗布した場合と比べて、有意なＬ＊値の増加が見られ、まぶたにおいてくすみ度合いが改善されたことがわかった（図５、図６、図７）。 About L * value, when applying lotion containing blended forget-me-not flower on the eyelids that are thought to be subject to physical stimulation on a daily basis, compared to the case of applying lotion not blended It was found that a significant increase in L * value was observed and the dullness was improved in the eyelids (FIGS. 5, 6, and 7).
日常的に物理刺激を受けＮＯが生じていると考えられるまぶたにおいて、ＮＯ消去効果の高いワスレグサ花発酵液を配合した化粧水を塗布したところ、まぶたのくすみの度合いが小さくなり、物理刺激によるくすみ改善効果が確認できた。実際のくすみの変化を写真に示す（図８）。 Applying lotion containing blended forget-me-not flower that has a high NO-eliminating effect on the eyelids that are thought to produce NO upon daily physical stimulation, the dullness of the eyelids is reduced and dullness due to physical stimulation The improvement effect was confirmed. The actual change in dullness is shown in the photograph (Fig. 8).
本発明の方法によれば、物理刺激によるくすみ形成抑制効果のある素材をスクリーニングすることができる。従って、物理刺激によるくすみ形成の予防及び／又は治療用の医薬や化粧品の開発に有用である。
According to the method of the present invention, a material having an effect of suppressing dull formation due to physical stimulation can be screened. Therefore, it is useful for the development of medicaments and cosmetics for preventing and / or treating dull formation due to physical stimulation.
Claims (3)
（２） 人為的に発生させた一酸化窒素以外に起因したメラニン生成を抑制する効果を有 しない素材を選別するステップ
を含む 物理刺激によって生じるくすみを改善する効果を有する素材のスクリーニング方法。 (1) Step of selecting a material having an effect of eliminating nitric oxide
(2) A step of selecting a material that has no effect of suppressing melanin production caused by other than artificially generated nitric oxide.
A screening method for a material having an effect of improving dullness caused by physical stimulation.
（１） 被験素材を添加するステップ
（２） ケラチノサイトとメラニン生成能を有する細胞とを併存させた状態で、 ケラチノサイトに物理刺激を与える群（A群）と、 ケラチノサイトに物理刺激を与えない群（B群）を設けるステップ を含み、 A群ではメラニン生成抑制効果を示し、かつ、B群ではメラニン生成抑制効果を有しない被験素材を選択する物理刺激によって生じるくすみを改善する効果を有する素材のスクリーニング方法。 In a screening method using cells capable of producing melanin
(1) Step of adding test material
(2) In the state where keratinocytes and cells having melanin-producing ability coexist, the step of providing a group (Group A) that gives physical stimulation to keratinocytes and a group (Group B) that does not give physical stimulation to keratinocytes, A screening method for a material having an effect of improving dullness caused by physical stimulation in which A group exhibits a melanin production inhibitory effect and B group selects a test material that does not have a melanin production inhibitory effect.
ケラチノサイトに物理刺激を与えて培養する手段と
ケラチノサイトに物理刺激を与えないで培養する手段とを有する
物理刺激によって生じるくすみを改善する効果を有する素材のスクリーニングキット。 A kit for use in the screening method according to claim 2,
Means for cultivating keratinocytes with physical stimulation;
Means for culturing keratinocytes without physical stimulation
A screening kit for materials that have the effect of improving dullness caused by physical stimulation .
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