JP3746986B2 - Whitening agent and skin external preparation for whitening - Google Patents

Description

【００１２】
ミフェプリストンの作用について、（１）細胞内チロシナーゼ活性の測定、（２）細胞数の測定（ＭＴＴ活性）を行ない評価した。測定方法を以下に示す。
（１）細胞内チロシナーゼ活性の測定方法
正常ヒト表皮メラニン細胞（クラボウ）を２４ウェルプレートに２万個／ウェルとなるように分植し、４８時間後に培地をＭＣＤＢ１５３＋５％ＦＢＳ＋４ｎＭ ＰＭＡに交換し、さらに４８時間後に各種試験検体を含む同様の培地を作用した。
【００１３】
試験検体は、ミフェプリストン２０μＭ、フォルスコリン１０μＭ、ミフェプリストン２０μＭ＋フォルスコリン１０μＭを用いる検体と、ミフェプリストン２０μＭ、α−ＭＳＨ（メラノサイト刺激ホルモン）５００ｎＭ、ミフェプリストン２０μＭ＋α−ＭＳＨ５００ｎＭを用いる検体とをそれぞれ準備した。
フォルスコリンはメラニン合成を活発にする細胞内セカンドメッセンジャーとして知られるｃＡＭＰを、アデニル酸シクラーゼを刺激することでその量を増やす物質として研究に頻用されているため、また、α−ＭＳＨはケラチノサイトあるいはメラノサイト自身から放出され、パラクラインあるいはオートクラインにメラニン合成を活発にすることが知られているため、フォルスコリンまたはα−ＭＳＨによる細胞内チロシナーゼ活性の増大をミフェプリストンがどの程度抑制できるかを調査するために用いた。
【００１４】
４８時間後に培地を除去し、１％ＴｒｉｔｏｎＸ−１００を含む１００ｍＭリン酸緩衝液（ｐＨ６．８）０．１ｍｌを添加後、軽く超音波処理し、０．１％Ｄｏｐａを含む５０ｍＭリン酸緩衝液（ｐＨ６．８）０．２ｍｌを添加し、３７℃、３時間反応し、４５０ｎｍの吸光度を測定した。市販のマッシュルーム由来チロシナーゼをスタンダードとして作成した検量線からウェルあたりのチロシナーゼ量を算出し、コントロールとの相対値として表わした。結果を表１に示す。
【００１５】
（２）細胞数の測定（ＭＴＴ活性）方法
上記細胞内チロシナーゼ活性の測定方法と同様にして各種試験検体を作用して４８時間後に培地を除去し、０．０５％ＭＴＴ：臭化３−（４，５−ジメチル−２−チアゾリル）−２，５−ジフェニル−２Ｈ−テトラゾリウムを含むＭＣＤＢ１５３＋５％ＦＢＳ＋４ｎＭ ＰＭＡ０．３ｍｌに交換し、さらに４時間３７℃で培養した。０．０４ＮＨＣＩを含むイソプロピルアルコール０．３ｍｌを添加し、不溶物を溶解し、６５５ｎｍをリファレンスとして５４０ｎｍの吸光度を測定した。あらかじめ作成した細胞数と吸光度の検量線から細胞数を算出し、コントロールとの相対値として表わした。結果を表１に示す。
【００１６】
【表１】

【００１７】
また、代表的な美白剤であるアルブチンを比較例に用いて上記と同様な方法で細胞内チロシナーゼ活性およびＭＴＴ活性の測定を行なった。結果を表２に示す。
【００１８】
【表２】

【００１９】
表１により、ミフェプリストンはフォルスコリンやα−ＭＳＨによる細胞数の上昇にも僅かに影響を与えるものの、細胞内チロシナーゼ活性の上昇に与える影響がはるかに大きいことが分かる。
また、表２に示すアルブチンとの比較により、ミフェプリストンの細胞内チロシナーゼ活性を下げる作用は、すでに美白剤として使われているアルブチンを１０倍濃度の２００ｍＭで作用するよりも強い。したがって、ミフェプリストンは優れたメラニン生成抑制効果を有する。
【００２０】
本発明の美白用皮膚外用剤は、メラノサイト内の酵素チロシナーゼ量を減少させる物質を必須成分として含んでいればよい。特にミフェプリストンを含んでいることが好ましい。
配合量は、有効成分を０．００５〜２重量％含有することが好ましい。０．００５重量％未満では美白効果がなく、２重量％をこえると美白効果の向上が望めなくなる。
【００２１】
なお、酵素チロシナーゼ量を減少させる物質の他に、他の化粧品原料、例えばコウジ酸、アスコルビン酸、ハイドロキノン、チオール系化合物等の美白剤、スクワラン、ホホバ油等の液状油、ミツロウ、セチルアルコール等の固体油、各種の活性剤、グリセリン、1,3-ブチレングリコール等の保湿剤や各種薬剤等とを添加してさまざまな剤形の美白用皮膚外用剤に調整できる。例えばローション、クリーム、乳液、パック等の目的に応じた利用形態で使用できる。
【００２２】
【実施例】
実施例１
ローションの例について説明する。配合成分と配合量（重量％）を以下に示す。
オリーブ油 ０．５
ミフェプリストン ０．５
ポリオキシエチレン（ 20 E.0 ）ソルビタンモノステアレート ２．０
ポリオキシエチレン（ 60 E.0 ）硬化ヒマシ油 ２．０
エタノール １０．０
ヒアルロン酸ナトリウム水溶液（ 1.0重量％） ５．０
精製水 ８０．０
【００２３】
精製水におよびヒアルロン酸ナトリウム水溶液加え 70 ℃に加熱調整する。オリーブ油にポリオキシエチレン（ 20 E.0 ）ソルビタンモノステアレートおよびポリオキシエチレン（ 60 E.0 ）硬化ヒマシ油を加え 70 ℃に加熱調整する。この油相を先に調整した水相に加え予備乳化し、さらにミフェプリストンおよびエタノールを加えてホモミキサーにて乳化粒子を均一にした後、脱気、濾過、冷却して実施例１のローションを得た。
【００２４】
実施例２クリームの例について説明する。配合成分と配合量（重量％）を以下に示す。
Ａ成分：
スクワラン ２０．０
オリーブ油 ２．０
ミンク油 １．０
ホホバ油 ５．０
ミツロウ ５．０
セトステアリルアルコール ２．０
グリセリンモノステアレート １．０
ソルビタンモノステアレート ２．０
Ｂ成分：
精製水 ４７．９
ポリオキシエチレン（ 20 E.0 ）ソルビタンモノステアレート ２．Ｏ
ポリオキシエチレン（ 60 E.0 ）硬化ヒマシ油 １．０
グリセリン ５．０
ミフェプリストン ０．５
ヒアルロン酸ナトリウム水溶液（ 1.0重量％） ５．０
パラオキシ安息香酸メチル ０．１
【００２５】
Ａ成分とＢ成分とをそれぞれ計量し、それぞれ 70 ℃まで加温し、Ｂ成分にＡ成分を攪拌しつつ徐々に加えたのち、ゆっくり攪拌しつつ 30 ℃まで冷却して実施例２のクリームを得た。
【００２６】
実施例１および実施例２で得られた化粧品は、使用テストの結果、ミフェプリストンを除き、水に代えた比較例に比較して、美白効果が見られた。
【００２７】
【発明の効果】
本発明は、メラノサイト内の酵素チロシナーゼ量を減少させる物質を含むので、メラニン産生を抑制できる。
また、該物質が上述したステロイド化合物であるので、特にミフェプリストンであるので、メラニン産生をより抑制できる。
メラニン産生を抑制できるので、優れた美白効果を有する美白用皮膚外用剤が得られる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a whitening agent and a whitening skin external preparation, and more particularly to a whitening agent that acts on and reduces the amount of the enzyme tyrosinase in human melanocytes and a whitening skin external preparation using the whitening agent.
[0002]
[Prior art]
Skin pigmentation, freckles, and other pigmentation occur due to excessive production of melanin pigment in epidermal melanocytes, triggered by hormonal abnormalities or stimulation of ultraviolet rays.
Conventionally, substances that suppress the formation of melanin pigments to prevent spots and freckles have been studied, and substances that inhibit the activity of the enzyme tyrosinase that catalyzes the first step of the biochemical reaction of the melanin pigment formation process, such as hydroquinone, Known as whitening agents are kojic acid, or ascorbic acid or cysteine which suppresses the darkening process of melanin pigments due to oxidation. Arbutin has an activity of inhibiting the activity of the enzyme tyrosinase or an inhibitory effect on melanin biosynthesis, and is known as a typical whitening agent. These whitening agents are blended with skin external preparations such as ointments, creams, lotions and the like to obtain skin external preparations for whitening, and are applied to the topic.
[0003]
[Problems to be solved by the invention]
However, many of these have problems in terms of stability, safety, odor and the like, and the expected effects are weak and still not satisfactory. The present invention has been made to address such problems, and provides a whitening agent that exhibits excellent whitening effects while being excellent in stability and safety, and a whitening skin external preparation using the whitening agent. With the goal.
[0004]
[Means for Solving the Problems]
The whitening agent according to the present invention includes mifepristone.
[0005]
The skin whitening preparation for whitening according to the present invention contains a substance that decreases the amount of enzyme tyrosinase in melanocytes, particularly mifepristone.
[0006]
Production of the enzyme tyrosinase that promotes melanin pigment synthesis in epidermal melanocytes is considered as follows.
(1) Stimulate melanocytes from the outside such as ultraviolet rays, or cause hormonal abnormalities.
(2) A blueprint for synthesis of the enzyme tyrosinase (tyrosinase protein) is transcribed from DNA in the cell nucleus to tyrosinase mRNA and transmitted to the cytoplasm.
(3) Produces the enzyme tyrosinase.
[0007]
The enzyme tyrosinase plays a central role in melanin synthesis in melanocytes. Conventionally, a component that acts directly on this enzyme to inhibit the enzyme activity, such as kojic acid, has been searched and used as a whitening agent.
The present inventor does not inhibit the enzyme activity by directly acting on the enzyme tyrosinase in the melanocyte, but the amount of melanin pigment synthesis can be suppressed by decreasing the amount. I found out that I can do it.
Considering the mechanism of enzyme tyrosinase production described above, the decrease in the amount of enzyme tyrosinase in melanocytes suppresses transcription of tyrosinase mRNA, or even if the enzyme tyrosinase is made, it is not stable and is readily degraded. It is thought that. Since the enzyme tyrosinase is a protein, degradation always occurs normally, but it is thought that the degradation speed is increased.
[0008]
Other whitening agents have also been searched for components that whiten cells. However, since the cells that have been used are not normal human melanocytes but mainly mouse melanomas (the melanocytes are cancerous), they become white due to toxic effects on cells, and white due to fluctuations in pH. It was difficult to search for substances that have a real whitening effect on normal melanocytes of people who do not turn white.
The whitening agent containing the substance for reducing the amount of enzyme tyrosinase in human melanocytes according to the present invention reduces the amount of enzyme tyrosinase itself, and thus has an effect on human normal melanocytes.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The substance that reduces the amount of the enzyme tyrosinase in human melanocytes may be a substance that acts on human melanocytes to suppress the production of the enzyme tyrosinase, or a substance that rapidly degrades the enzyme tyrosinase. For example, the substance that suppresses the production of the enzyme tyrosinase includes a substance that can suppress the transcription of tyrosinase mRNA.
In addition, it binds to the progesterone / glucocorticoid receptor and inhibits the effect of the agonist, but itself does not exert the effect even when bound to the receptor. The antagonist, also called antagonist or blocker, is an enzyme tyrosinase level. As a substance that reduces
[0010]
Examples of substances having an effect of acting on human melanocytes to reduce intracellular tyrosinase activity include steroid compounds having progesterone / glucocorticoid receptor antagonist activity. Its structure is represented by the above formula (I). In R ₁ , the derivative of a cycloalkyl group or a phenyl group containing 3 to 6 carbon atoms includes hydroxy, halogen, trifluoromethyl group, alkyl having 6 or less carbon atoms, alkoxy, sulfoxide or sulfone. And alkylthio groups that are oxidized or non-oxidized.
In R ₄ , an alkenyl or alkynyl group derivative represents an amino, alkylamino, dialkylamino, halogen, alkylthio, alkoxy, trialkylsilyl, or a group substituted by a cyano group.
As for A and B, when R ₄ represents a group containing an amino functional group, it includes an addition salt with an acid that is acceptable on a pharmaceutical or whitening skin external preparation.
[0011]
An example of a specific substance that reduces the amount of the enzyme tyrosinase is mifepristone.
Mifepristone is 17β-hydroxy-11β- (4-dimethylaminophenyl) -17α- (1-propynyl) estradi-4,9-dien-3-one, also referred to as RU486, and has the formula (II ).
[Chemical 3]

[0012]
The action of mifepristone was evaluated by (1) measuring intracellular tyrosinase activity and (2) measuring the number of cells (MTT activity). The measuring method is shown below.
(1) Method for measuring intracellular tyrosinase activity Normal human epidermal melanocytes (Kurabo) are planted in a 24-well plate at 20,000 cells / well, and after 48 hours, the medium is replaced with MCDB153 + 5% FBS + 4 nM PMA. 48 hours later, the same medium containing various test specimens was acted.
[0013]
The test sample is a sample using mifepristone 20 μM, forskolin 10 μM, mifepristone 20 μM + forskolin 10 μM, a sample using mifepristone 20 μM, α-MSH (melanocyte stimulating hormone) 500 nM, mifepristone 20 μM + α-MSH 500 nM, Prepared each.
Forskolin is frequently used in research as a substance that increases the amount of cAMP known as an intracellular second messenger that activates melanin synthesis by stimulating adenylate cyclase, and α-MSH is also used for keratinocytes or melanocytes. Investigate how much mifepristone can suppress the increase of intracellular tyrosinase activity by forskolin or α-MSH because it is released from itself and is known to activate melanin synthesis in paracrine or autocrine Used to do.
[0014]
After 48 hours, the medium was removed and 0.1 ml of 100 mM phosphate buffer (pH 6.8) containing 1% Triton X-100 was added, followed by light sonication and 50 mM phosphate buffer containing 0.1% Dopa. (PH 6.8) 0.2 ml was added, reacted at 37 ° C. for 3 hours, and absorbance at 450 nm was measured. The amount of tyrosinase per well was calculated from a calibration curve prepared using commercially available mushroom-derived tyrosinase as a standard, and was expressed as a relative value to the control. The results are shown in Table 1.
[0015]
(2) Method for measuring the number of cells (MTT activity) In the same manner as the method for measuring intracellular tyrosinase activity, the medium was removed 48 hours after acting various test specimens, and 0.05% MTT: 3- (3- The medium was replaced with 0.3 ml of MCDB153 + 5% FBS + 4 nM PMA containing 4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium, and further cultured at 37 ° C. for 4 hours. 0.3 ml of isopropyl alcohol containing 0.04 NHCI was added to dissolve insoluble matters, and the absorbance at 540 nm was measured using 655 nm as a reference. The number of cells was calculated from a previously prepared calibration curve of the number of cells and absorbance and expressed as a relative value to the control. The results are shown in Table 1.
[0016]
[Table 1]

[0017]
In addition, intracellular tyrosinase activity and MTT activity were measured in the same manner as described above using arbutin, which is a typical whitening agent, as a comparative example. The results are shown in Table 2.
[0018]
[Table 2]

[0019]
Table 1 shows that mifepristone slightly affects the increase in the number of cells due to forskolin and α-MSH, but has a much larger effect on the increase in intracellular tyrosinase activity.
Moreover, the comparison with the arbutin shown in Table 2 shows that the action of lowering the intracellular tyrosinase activity of mifepristone is stronger than that of arbutin already used as a whitening agent at a concentration of 200 mM. Therefore, mifepristone has an excellent melanin production inhibitory effect.
[0020]
The whitening skin external preparation of the present invention may contain a substance that reduces the amount of enzyme tyrosinase in melanocytes as an essential component. In particular, it preferably contains mifepristone.
The amount is preferably 0.005 to 2% by weight of the active ingredient. If it is less than 0.005% by weight, there is no whitening effect, and if it exceeds 2% by weight, no improvement in whitening effect can be expected.
[0021]
In addition to substances that reduce the amount of enzyme tyrosinase, other cosmetic ingredients such as whitening agents such as kojic acid, ascorbic acid, hydroquinone, thiol compounds, liquid oils such as squalane and jojoba oil, beeswax, cetyl alcohol, etc. Addition of solid oil, various active agents, humectants such as glycerin and 1,3-butylene glycol, and various agents can be used to prepare skin external preparations for whitening in various dosage forms. For example, it can be used in a usage form according to the purpose, such as lotion, cream, milky lotion, and pack.
[0022]
【Example】
Example 1
An example of a lotion will be described. The blending components and blending amount (% by weight) are shown below.
Olive oil 0.5
Mifepristone 0.5
Polyoxyethylene (20 E.0) sorbitan monostearate 2.0
Polyoxyethylene (60 E.0) hydrogenated castor oil 2.0
Ethanol 10.0
Sodium hyaluronate aqueous solution (1.0 wt%) 5.0
Purified water 80.0
[0023]
Add to purified water and sodium hyaluronate solution and heat to 70 ° C. Add olive oil to polyoxyethylene (20 E.0) sorbitan monostearate and polyoxyethylene (60 E.0) hydrogenated castor oil and heat to 70 ° C. The oil phase is pre-emulsified in addition to the previously prepared aqueous phase, and further mifepristone and ethanol are added to make the emulsified particles uniform with a homomixer, followed by deaeration, filtration and cooling to obtain the lotion of Example 1. Got.
[0024]
Example 2 An example of the cream will be described. The blending components and blending amount (% by weight) are shown below.
A component:
Squalane 20.0
Olive oil 2.0
Mink oil 1.0
Jojoba oil 5.0
Beeswax 5.0
Cetostearyl alcohol 2.0
Glycerol monostearate 1.0
Sorbitan monostearate 2.0
B component:
Purified water 47.9
1. Polyoxyethylene (20 E.0) sorbitan monostearate O
Polyoxyethylene (60 E.0) hydrogenated castor oil 1.0
Glycerin 5.0
Mifepristone 0.5
Sodium hyaluronate aqueous solution (1.0 wt%) 5.0
Methyl paraoxybenzoate 0.1
[0025]
A component and B component are weighed, each is heated to 70 ° C, A component is gradually added to B component with stirring, and then cooled to 30 ° C with slow stirring to give the cream of Example 2 Obtained.
[0026]
As a result of the use test, the cosmetics obtained in Example 1 and Example 2 showed a whitening effect as compared with the comparative example in which water was replaced except for mifepristone.
[0027]
【The invention's effect】
Since this invention contains the substance which reduces the amount of enzyme tyrosinase in a melanocyte, it can suppress melanin production.
In addition, since the substance is the steroid compound described above, and particularly mifepristone, melanin production can be further suppressed.
Since melanin production can be suppressed, a whitening skin external preparation having an excellent whitening effect can be obtained.

Claims (2)

  A whitening agent characterized by containing mifepristone.   A whitening skin external preparation comprising the whitening agent according to claim 1.