JP2015204780A - Screening method of whitening component - Google Patents
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Abstract
Description
本発明は、線維芽細胞を用いることを特徴とする美白成分のスクリーニング方法に関するものである。 The present invention relates to a screening method for whitening components, characterized by using fibroblasts.
一般に、シミ、ソバカス、日焼け等にみられる皮膚の色素沈着は、ホルモンの異常や紫外線の刺激により、皮膚内に存在するメラニン色素生成細胞（メラノサイト）がメラニン色素を過剰に生成し、これが皮膚内に沈着することが原因と考えられている。このような色素沈着を防ぐ方法の一つに、メラニンの過剰な生成を抑制する方法が知られている。そして従来から、メラニンの過剰な生成を抑制する目的で、アルブチン、ハイドロキノン、コウジ酸、トラネキサム酸及びこれらの誘導体等が用いられてきた。 In general, skin pigmentation in spots, freckles, sunburn, etc. is caused by excessive melanin pigment produced by melanin pigment cells (melanocytes) present in the skin due to hormonal abnormalities or stimulation of ultraviolet rays. It is thought to be caused by deposition. As one method for preventing such pigmentation, a method for suppressing excessive production of melanin is known. Conventionally, arbutin, hydroquinone, kojic acid, tranexamic acid, and derivatives thereof have been used for the purpose of suppressing excessive production of melanin.
ｎｅｕｒｅｇｕｌｉｎ１（ＮＲＧ１）は、中枢神経系において、神経細胞やグリア細胞の増殖、分化を調節している神経栄養因子であるが、近年、真皮の線維芽細胞において、その発現がみられること、又、メラノサイトに作用してメラニン生成を引き起こすことが分かってきた（非特許文献１及び２）。 neurogulin 1 (NRG1) is a neurotrophic factor that regulates proliferation and differentiation of nerve cells and glial cells in the central nervous system, but its expression is recently seen in dermal fibroblasts, It has been found that it acts on melanocytes to cause melanin production (Non-patent Documents 1 and 2).
美白成分のスクリーニング方法としては、Ｂ１６マウスメラノーマを用いたメラニン生成抑制試験によるスクリーニング方法（特許文献１）、メラノサイト内でのメラノソーム構造タンパク質ｇｐ１００を指標とするスクリーニング方法（特許文献２）、アドレノメジュリン結合受容体を介するシグナル伝達系への不活性化作用を指標とするスクリーニング方法（特許文献３）等が既に知られている。しかしながら、これらは全て表皮に存在するケラチノサイトやメラノサイトが指標であった。 As a screening method for whitening components, a screening method based on a melanin production inhibition test using B16 mouse melanoma (Patent Document 1), a screening method using melanosome structural protein gp100 in melanocytes as an index (Patent Document 2), adrenomedullin A screening method (Patent Document 3) and the like using an inactivation effect on a signal transduction system via a binding receptor as an index is already known. However, all of these indicators were keratinocytes and melanocytes present in the epidermis.
最近の研究から、皮膚色が濃いほど真皮中のＮＲＧ１の発現が高いことが明らかとなり、表皮以外の因子がメラニン生成に関与していることが分かってきた（非特許文献１及び２）。ところが、今までに、真皮に存在する線維芽細胞から産生される因子を指標とする美白成分のスクリーニング方法は開発されていなかった。 Recent studies have revealed that the darker the skin color, the higher the expression of NRG1 in the dermis and that factors other than the epidermis are involved in melanin production (Non-patent Documents 1 and 2). However, until now, no screening method for a whitening component has been developed that uses a factor produced from fibroblasts present in the dermis as an index.
そこで、本発明は、優れた美白成分の発見に繋がる新規の技術を確立することを課題とする。特に、ケラチノサイトやメラノサイトを用いる既知の作用機序に基づいた美白作用を利用するだけでは、優れた美白成分を発見することは困難であると考えられる。即ち、本発明は、新規の作用機序に基づいた美白作用を評価し得る方法を確立することを課題とする。 Then, this invention makes it a subject to establish the novel technique which leads to discovery of the outstanding whitening component. In particular, it is considered difficult to find an excellent whitening component only by using a whitening action based on a known mechanism of action using keratinocytes and melanocytes. That is, an object of the present invention is to establish a method capable of evaluating a whitening action based on a novel action mechanism.
本発明者らは、上記課題の解決に向け鋭意検討を行った結果、神経栄養因子ＮＲＧ１を指標にし、表皮に存在するケラチノサイトやメラノサイトではなく、真皮に存在する線維芽細胞に着目した新規の美白成分のスクリーニング方法を完成するに至った。ＮＲＧ１は、真皮由来のメラニン生成促進因子であると考えられ、メラノサイトにおけるメラニンの過剰な生成を抑制するには、この真皮由来メラニン生成促進因子の制御も重要である。更に、かかる方法により評価し、選択した成分を含有した組成物が、色素沈着の予防又は改善効果を発揮することを確認した。 As a result of intensive studies aimed at solving the above problems, the present inventors have made a novel whitening using the neurotrophic factor NRG1 as an index and focusing on fibroblasts present in the dermis rather than keratinocytes and melanocytes present in the epidermis. It came to complete the screening method of an ingredient. NRG1 is considered to be a dermal-derived melanin production-promoting factor, and control of this dermis-derived melanin production-promoting factor is also important for suppressing excessive production of melanin in melanocytes. Furthermore, it evaluated by this method and it confirmed that the composition containing the selected component exhibited the prevention or improvement effect of pigmentation.
即ち、本発明は以下の（１）〜（５）から成る。 That is, the present invention comprises the following (1) to (5).
（１）線維芽細胞を用いることを特徴とする美白成分のスクリーニング方法。
（２）線維芽細胞における神経栄養因子の産生量を指標とする（１）記載の方法。
（３）神経栄養因子がｎｅｕｒｅｇｌｉｎ１（ＮＲＧ１）であることを特徴とする（２）記載の方法。
（４）神経栄養因子の産生抑制効果を指標とする（２）又は（３）記載の方法。
（５）予め酸化ストレスを負荷した線維芽細胞を用いることを特徴とする（１）〜（４）のいずれか一項記載の方法。
(1) A screening method for whitening components, comprising using fibroblasts.
(2) The method according to (1), wherein the production amount of neurotrophic factor in fibroblasts is used as an index.
(3) The method according to (2), wherein the neurotrophic factor is neuroglin1 (NRG1).
(4) The method according to (2) or (3), wherein the production inhibitory effect of neurotrophic factor is used as an index.
(5) The method according to any one of (1) to (4), wherein fibroblasts previously loaded with oxidative stress are used.
本発明によれば、線維芽細胞における神経栄養因子ＮＲＧ１を指標とすることにより、化粧料や皮膚外用剤等に用いる美白成分の評価又はスクリーニングが可能となる。 According to the present invention, by using the neurotrophic factor NRG1 in fibroblasts as an index, it is possible to evaluate or screen for whitening components used in cosmetics, skin external preparations and the like.
本発明における美白成分のスクリーニング方法とは、美白効果のある成分を選抜する方法である。本発明の方法によりスクリーニングされた美白成分は、化粧料又は皮膚外用剤等に配合及び調製することができる。 The whitening component screening method in the present invention is a method for selecting a component having a whitening effect. The whitening component screened by the method of the present invention can be blended and prepared in cosmetics or skin external preparations.
本発明における神経栄養因子とは、神経細胞へ栄養を送り届け、神経の機能維持や成長等の要因となっている因子のことである。代表的なものとして、ｎｅｕｒｅｇｕｌｉｎ、ｎｅｕｒｏｔｒｏｐｈｉｎ、ｂｒａｉｎ−ｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ、ｎｅｒｖｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ、ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ等が挙げられる。好ましくは、ｎｅｕｒｅｇｕｌｉｎである。 The neurotrophic factor in the present invention refers to a factor that delivers nutrients to nerve cells and is a factor for maintaining the function and growth of nerves. Typical examples include neurogulin, neurotrophin, brain-derived neurotrophic factor, nave growth factor, and linear neurotrophic factor. Preferably, it is neurogulin.
本発明におけるｎｅｕｒｅｇｌｉｎ（ＮＲＧ）とは神経細胞の軸索成長因子であり、神経やグリアの増殖・転移・運命決定に重要な役割を果たしている。今までのところ、ＮＲＧのサブタイプとしてＮＲＧ１、２、３及び４が知られている。最近の研究から、ＮＲＧ１は神経系だけではなく皮膚の線維芽細胞にも発現しており、皮膚色が濃いほどその発現が高く、ＮＲＧ１タンパク質をメラノサイトに作用させるとメラニン生成に影響を及ぼすことが報告されている。 In the present invention, neuroglin (NRG) is an axon growth factor for nerve cells, and plays an important role in determining proliferation, metastasis and fate of nerves and glia. So far, NRG 1, 2, 3, and 4 are known as subtypes of NRG. From recent studies, NRG1 is expressed not only in the nervous system but also in the fibroblasts of the skin. The darker the skin color, the higher the expression, and the effect of NRG1 protein on melanocytes can affect melanin production. It has been reported.
本発明における神経栄養因子の産生抑制効果とは、真皮に存在する線維芽細胞にて産生される神経栄養因子ＮＲＧ１を指標とし、何らかの成分によりその産生を抑制する効果のことである。この目的のために、ＮＲＧ１を測定する必要がある。この測定に用いられる方法は特に限定されず、何れかの方法を用いることができる。代表的なものとして、リアルタイムＲＴ−ＰＣＲ法によりＮＲＧ１ ｍＲＮＡ発現量の測定を行う方法、ウェスタンブロッティング法によりＮＲＧ１タンパク質量の測定を行う方法、ＥＬＩＳＡ法によりＮＲＧ１タンパク質量の測定を行う方法等が挙げられる。好ましくは、リアルタイムＲＴ−ＰＣＲ法によりＮＲＧ１ ｍＲＮＡ発現量の測定を行う方法である。 The production inhibitory effect of the neurotrophic factor in the present invention is an effect of inhibiting the production by some component using the neurotrophic factor NRG1 produced in fibroblasts existing in the dermis as an index. For this purpose, NRG1 needs to be measured. The method used for this measurement is not particularly limited, and any method can be used. Representative examples include a method for measuring NRG1 mRNA expression level by real-time RT-PCR, a method for measuring NRG1 protein level by Western blotting, a method for measuring NRG1 protein level by ELISA, and the like. . Preferably, the NRG1 mRNA expression level is measured by a real-time RT-PCR method.
本発明における酸化ストレスとは、酸化反応により引き起こされる生体にとって有害な作用のことである。細胞を用いた試験においては、紫外線、活性酸素、過酸化脂質等で曝露することにより酸化ストレスを負荷する手法が用いられている。好ましくは、過酸化水素（Ｈ２Ｏ２）を用いた手法である。 The oxidative stress in the present invention is an action harmful to a living body caused by an oxidative reaction. In a test using cells, a technique of applying oxidative stress by exposure to ultraviolet rays, active oxygen, lipid peroxide, or the like is used. A method using hydrogen peroxide (H 2 O 2 ) is preferable.
次に、本発明を詳細に説明するため、具体的な実施例を挙げて説明する。これらの実施例は効果を具体的に説明するもので、発明の範囲を限定するものではない。実施例中の配合量は重量％である。 Next, in order to describe the present invention in detail, specific examples will be given and described. These examples specifically illustrate the effect and do not limit the scope of the invention. The compounding quantity in an Example is weight%.
スクリーニング対象薬剤として、月下美人抽出物を使用した。月下美人とは、サボテン科クジャクサボテンの原種（Ｅｐｉｐｈｙｌｌｕｍ ｏｘｙｐｅｔａｌｕｍ Ｈａｗ．）及びその近縁種であり、その園芸品種の全草や花の乾燥物等を利用することができる。 Tsukishita Bijin Extract was used as a screening target drug. The beauty of the moon is a genus of cactiaceae peafowl cactus (Epiphyllum oxypetalum Haw.) And related species thereof, and the whole cultivar and dried flowers can be used.
月下美人の製造例として、下記の抽出を行うことができる。 The following extraction can be performed as an example of the production of a moonlight beauty.
製造例１ 月下美人抽出物
月下美人の花の乾燥物１００ｇに精製水３Ｌを加え、９５〜１００℃で２時間抽出した。濾過した後、濾液を減圧濃縮し、更に凍結乾燥して月下美人抽出物４８ｇを得た。
Production Example 1 Tsukishita Bijin Extract 3 g of purified water was added to 100 g of the dried ginger flower, and extracted at 95-100 ° C for 2 hours. After filtration, the filtrate was concentrated under reduced pressure and further freeze-dried to obtain 48 g of a moon moon beauty extract.
スクリーニング対象薬剤として用いる月下美人抽出物は、処方例として下記の製剤化を行うことができる。 As a prescription example, the beauty of the moon moon extract used as a screening target drug can be formulated as follows.
処方例１ クリーム
処方 配合量（％）
１．月下美人抽出物（製造例１） ０．１
２．スクワラン ５．５
３．オリーブ油 ３．０
４．ステアリン酸 ２．０
５．ミツロウ ２．０
６．ミリスチン酸オクチルドデシル ３．５
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ベヘニルアルコール １．５
９．モノステアリン酸グリセリン ２．５
１０．香料 ０．１
１１．パラオキシ安息香酸メチル ０．２５
１２．１，３‐ブチレングリコール ８．５
１３．精製水にて全量を１００とする
［製造方法］成分２〜９を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１１〜１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分１０を加え、更に３０℃まで冷却後、製品とする。
Formulation Example 1 Cream Formulation Amount (%)
1. Moonlight Beauty Extract (Production Example 1) 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.25
12.1,3-Butylene glycol 8.5
13. [Manufacturing method] Components 2 to 9 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 11 to 13 are heated and dissolved and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.
比較例１ 従来のクリーム１
処方例１において、月下美人抽出物（製造例１）をシラカバ抽出物（一丸ファルコス）に置き換えたものを従来のクリーム１とした。
Comparative Example 1 Conventional cream 1
In Formulation Example 1, a conventional cream 1 was obtained by replacing the moon beauty extract (Production Example 1) with a birch extract (Ichimaru Falcos).
比較例２ 従来のクリーム２
処方例１において、月下美人抽出物（製造例１）を精製水に置き換えたものを従来のクリーム２とした。
Comparative Example 2 Conventional Cream 2
In Formulation Example 1, a conventional cream 2 was prepared by replacing the beautiful moon extract (Production Example 1) with purified water.
次に、本発明を詳細に説明するため、具体的な実験例を挙げて説明する。本発明はこれら実験例に何ら制約されるものではない。 Next, in order to describe the present invention in detail, a specific experimental example will be described. The present invention is not limited to these experimental examples.
実験例１ 線維芽細胞におけるＮＲＧ１タンパク質の検出
ヒト皮膚線維芽細胞（ＮＢ１ＲＧＢ）を１０％ホルマリンにて固定後、ＮＲＧ１タンパク質の免疫染色を行った。使用した一次抗体はＮＲＧ１（Ｒ＆Ｄ ＳＹＳＴＥＭＳ）、二次抗体はＡｌｅｘａ Ｆｌｕｏｒ５９４ Ｄｏｎｋｅｙ Ａｎｔｉ−Ｇｏａｔ ＩｇＧ（Ｈ＋Ｌ）（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）である。染色後、蛍光顕微鏡にて観察した。
Experimental Example 1 Detection of NRG1 protein in fibroblasts Human skin fibroblasts (NB1RGB) were fixed with 10% formalin and then immunostained for NRG1 protein. The primary antibody used was NRG1 (R & D SYSTEMS), and the secondary antibody was Alexa Fluor 594 Donkey Anti-Goat IgG (H + L) (Molecular Probes). After staining, it was observed with a fluorescence microscope.
実験結果を図１に示した。その結果、線維芽細胞において、ＮＲＧ１タンパク質が検出された。従って、線維芽細胞において、確かにＮＲＧ１が発現していることが示された。 The experimental results are shown in FIG. As a result, NRG1 protein was detected in fibroblasts. Therefore, it was shown that NRG1 is certainly expressed in fibroblasts.
実験例２ ＮＲＧ１タンパク質のメラニン生成促進効果
メラニン生成においては、チロシナーゼ（ＴＹＲ）、チロシナーゼ関連タンパク質１（ＴＲＰ１）及びチロシナーゼ関連タンパク質２（ＴＲＰ２）が主要な酵素として関与し、これら酵素の発現制御には小眼球症関連転写因子（ＭＩＴＦ）が重要な役割を担っている。コンフルエントな状態のヒト正常メラノサイト（ＮＨＥＭ）に、最終濃度が１００及び２００ｎｇ／ｍＬになるように調製したＮＲＧ１（ＰＲＯＳＰＥＣ）を添加して２４時間培養後、ＲＮＡｉｓｏ ｐｌｕｓ（ＴＡＫＡＲＡ）を用いて総ＲＮＡの抽出を行った。総ＲＮＡを基に、リアルタイムＲＴ−ＰＣＲ法により、ＴＹＲ、ＴＲＰ１、ＴＲＰ２及びＭＩＴＦ ｍＲＮＡ発現量の測定を行った。リアルタイムＲＴ−ＰＣＲ法にはＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用い、ＴＹＲ、ＴＲＰ１、ＴＲＰ２及びＭＩＴＦ用のプライマーは以下に示すものを用いた。内部標準として、β−アクチンを用いた。リアルタイムＲＴ−ＰＣＲの操作は定められた方法に従い、ＴＹＲ、ＴＲＰ１、ＴＲＰ２及びＭＩＴＦ ｍＲＮＡの発現量を内部標準であるβ−アクチン ｍＲＮＡの発現量に対する割合として求めた。ＴＹＲ、ＴＲＰ１、ＴＲＰ２及びＭＩＴＦの発現率は、未添加のｍＲＮＡ発現量に対するＮＲＧ１添加群のｍＲＮＡ発現量の比率として算出した。
Experimental Example 2 Effect of NRG1 Protein on Promoting Melanin Production In melanin production, tyrosinase (TYR), tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2) are involved as major enzymes, and the expression control of these enzymes Microphthalmia-related transcription factor (MITF) plays an important role. NRG1 (PROSPEC) prepared to a final concentration of 100 and 200 ng / mL is added to human normal melanocytes (NHEM) in a confluent state, cultured for 24 hours, and then total RNA is extracted using RNAiso plus (TAKARA). Extraction was performed. Based on the total RNA, TYR, TRP1, TRP2, and MITF mRNA expression levels were measured by real-time RT-PCR. SYBR Select Master Mix (Life Technologies) was used for the real-time RT-PCR method, and the following primers were used for TYR, TRP1, TRP2, and MITF. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression levels of TYR, TRP1, TRP2, and MITF mRNA were determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of TYR, TRP1, TRP2, and MITF was calculated as the ratio of the mRNA expression level of the NRG1 added group to the unadded mRNA expression level.
ＴＹＲ用のプライマーセット
ＴＧＣＧＧＴＧＧＧＡＡＣＡＡＧＡＡＡＴＣ（配列番号１）
ＧＡＡＧＡＡＴＧＡＴＧＣＴＧＧＧＣＴＧＡＧＴ（配列番号２）
ＴＲＰ１用のプライマーセット
ＣＧＡＡＡＣＡＣＡＧＴＧＧＡＡＧＧＴＴＡＣＡＧＴ（配列番号３）
ＣＴＣＣＴＣＡＧＣＣＡＴＴＣＡＴＣＡＡＡＧＡＣＴ（配列番号４）
ＴＲＰ２用のプライマーセット
ＧＧＡＡＴＧＣＴＴＴＧＧＡＡＧＧＧＴＴＴＧ（配列番号５）
ＡＡＡＧＣＧＴＴＴＧＴＣＣＣＧＴＴＣＡＧ（配列番号６）
ＭＩＴＦ用のプライマーセット
ＧＡＧＧＣＡＧＴＧＧＴＴＴＧＧＧＣＴＴ（配列番号７）
ＡＡＴＴＣＴＧＣＡＣＣＣＧＧＧＡＡＴＣ（配列番号８）
β―アクチン用のプライマーセット
ＣＡＣＴＣＴＴＣＣＡＧＣＣＴＴＣＣＴＴＣＣ（配列番号９）
ＧＴＧＴＴＧＧＣＧＴＡＣＡＧＧＴＣＴＴＴＧ（配列番号１０）
Primer set TGCGGTGGGAACAAGAAATC for TYR (SEQ ID NO: 1)
GAAGAATGATGCTGGGCTGAGT (SEQ ID NO: 2)
Primer set CGAAACACAGTGGAAGGTTACAGT for TRP1 (SEQ ID NO: 3)
CTCCTCAGCCCTTCATCAAAAGACT (SEQ ID NO: 4)
Primer set GGAATGCTTTGGAAGGGTTTG for TRP2 (SEQ ID NO: 5)
AAAGCGTTTGTCCCGTTCAG (SEQ ID NO: 6)
Primer set GAGGCAGTGGTTTGGGCTT for MITF (SEQ ID NO: 7)
AATTCTGCACCCGGGAATC (SEQ ID NO: 8)
Primer set CACTCTCCCAGCCTCTCTCCC for β-actin (SEQ ID NO: 9)
GTGTTGCGCGTACAGGTCTTTG (SEQ ID NO: 10)
実験結果を表１〜４に示した。その結果、ＮＲＧ１タンパク質は、メラノサイトにおけるＴＹＲ、ＴＲＰ１、ＴＲＰ２及びＭＩＴＦの発現を促進した。従って、ＮＲＧ１は、メラノサイトに作用してメラニン生成促進効果を有することが示された。 The experimental results are shown in Tables 1-4. As a result, NRG1 protein promoted the expression of TYR, TRP1, TRP2, and MITF in melanocytes. Therefore, it was shown that NRG1 acts on melanocytes and has a melanin production promoting effect.
実験例３ 酸化ストレスによるＮＲＧ１生成促進効果
コンフルエントな状態のヒト皮膚線維芽細胞（ＮＢ１ＲＧＢ）に、最終濃度が２５０及び５００μＭになるように調製した過酸化水素（Ｈ２Ｏ２）を添加して２４時間培養後、ＲＮＡｉｓｏ ｐｌｕｓ（ＴＡＫＡＲＡ）を用いて総ＲＮＡの抽出を行った。総ＲＮＡを基に、リアルタイムＲＴ−ＰＣＲ法により、ＮＲＧ１ ｍＲＮＡ発現量の測定を行った。リアルタイムＲＴ−ＰＣＲ法にはＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用い、ＮＲＧ１用のプライマーは以下に示すものを用いた。内部標準として、β−アクチンを用いた。リアルタイムＲＴ−ＰＣＲの操作は定められた方法に従い、ＮＲＧ１ ｍＲＮＡの発現量を内部標準であるβ−アクチン ｍＲＮＡの発現量に対する割合として求めた。ＮＲＧ１の発現率は、未添加のｍＲＮＡの発現量に対するＨ２Ｏ２添加群のｍＲＮＡの発現量の比率として算出した。
Experimental Example 3 Effect of Promoting NRG1 Production by Oxidative Stress Hydrogen peroxide (H 2 O 2 ) prepared to final concentrations of 250 and 500 μM was added to human skin fibroblasts (NB1RGB) in a confluent state by adding 24 After time culture, total RNA was extracted using RNAiso plus (TAKARA). Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the following primers were used for NRG1. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the H 2 O 2 added group to the expression level of unadded mRNA.
ＮＲＧ１用のプライマーセット
ＧＣＣＣＡＴＣＡＣＴＣＣＡＣＴＡＣＴＧＴＣＡ（配列番号１１）
ＧＣＴＴＴＣＡＧＴＧＴＧＴＣＣＧＴＴＧＣＴ（配列番号１２）
Primer set GCCCATCACTCCACTACTGTCA for NRG1 (SEQ ID NO: 11)
GCTTTCAGTGTGTCCGTTGCT (SEQ ID NO: 12)
実験結果を表５に示した。その結果、Ｈ２Ｏ２添加による酸化ストレスは、ＮＲＧ１の発現を促進した。従って、線維芽細胞に酸化ストレスを負荷すると、ＮＲＧ１の産生が亢進されることが示された。 The experimental results are shown in Table 5. As a result, the oxidative stress due to the addition of H 2 O 2 promoted the expression of NRG1. Therefore, it was shown that when oxidative stress is applied to fibroblasts, the production of NRG1 is enhanced.
実験例４ ＮＲＧ１の産生量を指標とする美白成分のスクリーニング方法
コンフルエントな状態のヒト皮膚線維芽細胞（ＮＢ１ＲＧＢ）に、最終濃度が５０、１００及び２００μｇ／ｍＬになるように調製した月下美人抽出物（製造例１）及びメラニン生成抑制効果を有することが知られているシラカバ抽出物（比較例３、一丸ファルコス）を添加して２４時間培養後、ＲＮＡｉｓｏ ｐｌｕｓ（ＴＡＫＡＲＡ）を用いて総ＲＮＡの抽出を行った。総ＲＮＡを基に、リアルタイムＲＴ−ＰＣＲ法により、ＮＲＧ１ ｍＲＮＡ発現量の測定を行った。リアルタイムＲＴ−ＰＣＲ法にはＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用い、ＮＲＧ１用のプライマーは実験例３と同様のものを用いた。内部標準として、β−アクチンを用いた。リアルタイムＲＴ−ＰＣＲの操作は定められた方法に従い、ＮＲＧ１ ｍＲＮＡの発現量を内部標準であるβ−アクチン ｍＲＮＡの発現量に対する割合として求めた。ＮＲＧ１の発現率は、未添加のｍＲＮＡの発現量に対する試料添加群のｍＲＮＡの発現量の比率として算出した。
Experimental Example 4 Screening method for whitening ingredients using NRG1 production as an index Extraction of beauty of the moon under beautiful human skin fibroblasts (NB1RGB) prepared to final concentrations of 50, 100 and 200 μg / mL Product (Production Example 1) and birch extract (Comparative Example 3, Ichimaru Falcos), which is known to have a melanin production inhibitory effect, were cultured for 24 hours, and then total RNA was extracted using RNAiso plus (TAKARA). Extraction was performed. Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the primer for NRG1 was the same as in Experimental Example 3. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the sample addition group to the expression level of unadded mRNA.
実験結果を表６に示した。その結果、シラカバ抽出物はＮＲＧ１の発現に影響を及ぼさなかったが、月下美人抽出物はＮＲＧ１の発現を顕著に抑制した。従って、ＮＲＧ１産生に対し、月下美人抽出物は優れた抑制効果を示した。 The experimental results are shown in Table 6. As a result, the birch extract had no effect on the expression of NRG1, but the Tsukishita beauties extract significantly suppressed the expression of NRG1. Therefore, Tsukishita Beauty extract showed an excellent inhibitory effect on NRG1 production.
実験例５ 酸化ストレスを負荷することを特徴とする美白成分のスクリーニング方法
コンフルエントな状態のヒト皮膚線維芽細胞（ＮＢ１ＲＧＢ）に、最終濃度が５００μＭになるように調製した過酸化水素（Ｈ２Ｏ２）と同時に、最終濃度が１００及び２００μｇ／ｍＬになるように調製した月下美人抽出物（製造例１）を添加した。２４時間培養後、ＲＮＡｉｓｏ ｐｌｕｓ（ＴＡＫＡＲＡ）を用いて総ＲＮＡの抽出を行った。総ＲＮＡを基に、リアルタイムＲＴ−ＰＣＲ法により、ＮＲＧ１ ｍＲＮＡ発現量の測定を行った。リアルタイムＲＴ−ＰＣＲ法にはＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用い、ＮＲＧ１用のプライマーは実験例３と同様のものを用いた。内部標準として、β−アクチンを用いた。リアルタイムＲＴ−ＰＣＲの操作は定められた方法に従い、ＮＲＧ１ ｍＲＮＡの発現量を内部標準であるβ−アクチン ｍＲＮＡの発現量に対する割合として求めた。ＮＲＧ１の発現率は、未添加のｍＲＮＡの発現量に対する試料添加群のｍＲＮＡの発現量の比率として算出した。
Experimental Example 5 Screening Method for Whitening Component Characterized by Loading Oxidative Stress Hydrogen peroxide (H 2 O 2) prepared to a final concentration of 500 μM in confluent human skin fibroblasts (NB1RGB) At the same time, Tsukishita Bijin extract (Production Example 1) prepared so as to have final concentrations of 100 and 200 μg / mL was added. After incubation for 24 hours, total RNA was extracted using RNAiso plus (TAKARA). Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the primer for NRG1 was the same as in Experimental Example 3. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the sample addition group to the expression level of unadded mRNA.
実験結果を表７に示した。その結果、月下美人抽出物は酸化ストレスによるＮＲＧ１の発現亢進を抑制した。従って、酸化ストレスを負荷した時に増加するＮＲＧ１に対し、月下美人抽出物は優れた抑制効果を示した。 The experimental results are shown in Table 7. As a result, the beautiful moon extract suppressed the increase in NRG1 expression due to oxidative stress. Therefore, Tsukishita Beauty Extract showed an excellent inhibitory effect against NRG1 that increases when oxidative stress is applied.
実験例６ Ｂ１６マウスメラノーマを用いたメラニン生成抑制試験
マウスメラノーマ由来細胞株であるＢ１６細胞を６ｃｍディッシュに３×１０４個播種し、１０％ＦＢＳを含むＭＥＭ ｗｉｔｈ ＮＥＡＡにて５％ＣＯ２、３７℃条件下で５日間培養した。その時、最終濃度が５０、１００及び２００μｇ／ｍＬになるように調製した月下美人抽出物（製造例１）及びメラニン生成抑制効果を有することが知られているシラカバ抽出物（比較例３、一丸ファルコス）を添加した。次に、細胞をＰＢＳ（−）にて２回洗浄した後、ＰＢＳ（−）１ｍＬを加え、ラバーポリスマンにて集めた。遠心操作をして得られたペレットにＰＢＳ（−）０．５ｍＬを加え、超音波破砕操作をしてペレットを溶解させた。タンパク量はＬｏｗｒｙ法｛Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，１９３，２６５−２７５（１９５１）｝により測定した。又、１ｍｇタンパクあたりのメラニン量を測定する場合、タンパク定量用に取った残りの細胞破砕溶液に４Ｎ水酸化ナトリウム０．５ｍＬを加え、６０℃で２時間反応させた後Ｏ．Ｄ．４７５を測定し、検量線からメラニン定量を行った。データは１ｍｇタンパクあたりのメラニン量を算出し、試料未添加のメラニン生成量をコントロールとし、コントロールに対する試料添加時のメラニン生成量の値からメラニン生成抑制率を算出した。
Experimental Example 6 Inhibition test of melanin production using B16 mouse melanoma 3 × 10 4 B16 cells, which are a mouse melanoma-derived cell line, were seeded in a 6 cm dish, and 5% CO 2 , 37 in MEM with NEAA containing 10% FBS. The cells were cultured for 5 days under the condition of 0C. At that time, the beautiful moon extract (Production Example 1) prepared to have final concentrations of 50, 100 and 200 μg / mL and the birch extract known to have a melanin production inhibitory effect (Comparative Example 3, Ichimaru) Falcos) was added. Next, after the cells were washed twice with PBS (−), 1 mL of PBS (−) was added and collected with a rubber policeman. PBS (-) 0.5mL was added to the pellet obtained by centrifugation, and the pellet was dissolved by ultrasonic crushing operation. The amount of protein was determined by the Lowry method {J. Biol. Chem. , 193, 265-275 (1951)}. When measuring the amount of melanin per mg of protein, 0.5 mL of 4N sodium hydroxide was added to the remaining cell disruption solution taken for protein determination and reacted at 60 ° C. for 2 hours. D. 475 was measured, and melanin was determined from the calibration curve. The data calculated the amount of melanin per 1 mg protein, the amount of melanin not added to the sample was taken as a control, and the inhibition rate of melanin generation was calculated from the value of the amount of melanin generated when the sample was added to the control.
これらの試験結果を表８に示す。その結果、月下美人抽出物はメラノーマにおけるメラニン生成に影響を及ぼさなかったが、シラカバ抽出物はメラニン生成を抑制した。従って、メラノーマにおけるメラニン生成に対し、月下美人抽出物は抑制効果を示さなかった。 These test results are shown in Table 8. As a result, the beautiful moon extract did not affect the melanin production in melanoma, but the birch extract suppressed the melanin production. Therefore, Tsukishita Beauty extract did not show an inhibitory effect on melanin production in melanoma.
実験例７ 使用試験
処方例１のクリーム、比較例１の従来のクリーム１及び比較例２の従来のクリーム２を用いて、各々女性３０人（２０〜４５歳）を対象に３カ月間の使用試験を行った。使用後、シミ及びソバカスの改善についてのアンケート調査を行って、美白効果を判定した。アンケートの評価基準は、有効なものを「優」、やや有効なものを「良」、わずかに有効なものを「可」、無効なものを「不可」として評価した。
Experimental Example 7 Use Test Using the cream of Formulation Example 1, the conventional cream 1 of Comparative Example 1 and the conventional cream 2 of Comparative Example 2, each for 30 women (20-45 years) for 3 months A test was conducted. After use, a questionnaire survey on the improvement of spots and freckles was conducted to determine the whitening effect. The evaluation criteria of the questionnaire were evaluated as “excellent” for valid, “good” for slightly effective, “good” for slightly effective, and “impossible” for invalid.
これらの結果を表９に示した。処方例１の月下美人抽出物を含有する本発明のクリームは優れた美白効果を示した。又、比較例１のシラカバ抽出物を含有する従来のクリーム１は美白効果を示したが、処方例１の月下美人抽出物を含有する本発明のクリームよりも効果は低かった。尚、試験期間中皮膚トラブルは一人もなく、安全性においても問題なかった。 These results are shown in Table 9. The cream of the present invention containing the extract of beautiful moon moon of Formulation Example 1 showed an excellent whitening effect. Moreover, although the conventional cream 1 containing the birch extract of the comparative example 1 showed the whitening effect, the effect was lower than the cream of the present invention containing the moon beautiful woman extract of the prescription example 1. During the test period, there was no skin problem and there was no problem with safety.
前記図１及び表１〜４より、ＮＲＧ１は線維芽細胞から産生されてメラニン生成に関与していることが確認された。そして、前記表５より、酸化剤曝露にてＮＲＧ１の発現が亢進されることが分かった。又、前記表９より、月下美人抽出物は最終的に美白効果を有する薬剤であることが認められた。しかしながら、前記表８より、そのメカニズムはメラノサイトのみによるものではないことが分かった。一方、前記表６及び７より、月下美人抽出物の美白効果は、真皮線維芽細胞におけるＮＲＧ１の発現量と相関があることが認められた。 From FIG. 1 and Tables 1 to 4, it was confirmed that NRG1 was produced from fibroblasts and involved in melanin production. From Table 5, it was found that the expression of NRG1 was enhanced by exposure to the oxidizing agent. Further, from Table 9, it was confirmed that the moonlight beauty extract is a drug having a whitening effect. However, from Table 8, it was found that the mechanism is not solely due to melanocytes. On the other hand, from Tables 6 and 7 above, it was confirmed that the whitening effect of the moon moon bean extract was correlated with the expression level of NRG1 in dermal fibroblasts.
以上より、線維芽細胞における神経栄養因子ＮＲＧ１発現量を指標とすることで美白成分のスクリーニングが可能となった。又、同様に、神経栄養因子ＮＲＧ１の産生抑制効果を指標とすることで美白成分のスクリーニングが可能となった。 From the above, it became possible to screen for whitening components by using the expression level of neurotrophic factor NRG1 in fibroblasts as an index. Similarly, the whitening component can be screened by using the production inhibitory effect of the neurotrophic factor NRG1 as an index.
本発明の線維芽細胞を用いることを特徴とする美白成分のスクリーニング方法は、これまでのケラチノサイトやメラノサイトを用いる美白成分のスクリーニング方法では評価することができなかった化粧料や皮膚外用剤等に用いる美白成分の開発に用いられるものである。 The screening method for whitening ingredients characterized by using the fibroblasts of the present invention is used for cosmetics and skin external preparations that could not be evaluated by conventional screening methods for whitening ingredients using keratinocytes and melanocytes. It is used for the development of whitening ingredients.
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| JP6992220B2 (en) | 2017-06-23 | 2022-02-15 | ポーラ化成工業株式会社 | Screening method for pigmentation inhibitors |
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