JP3657373B2 - Antipigmentation treatment - Google Patents

Description

【０００９】
本発明の色素沈着症予防治療剤を調製する場合、フェニルヒドロキノンの配合量は、特に制限されないが、全重量当たり、好ましくは0.1〜5重量%、より好ましくは0.25〜2重量%であることができる。この配合量は、皮膚の吸収率や残留率を高くする適切な基剤と共に用いた場合はさらに低濃度で有効である。なお、その配合量が5重量%を越える場合、長期間の使用で白斑を生じる場合がある。
【００１０】
本発明の色素沈着症予防治療剤は、公知の方法で各種軟膏剤（油脂性、乳剤性、水溶性）、ローション剤等の剤形に調製することができる。また、これらの剤形の調製において使用することができる構成成分の種類、配合量等は、当業者に知られる慣用例に従って適宜決定されることができる。
【００１１】
これらの構成成分の種類、配合量等は、限定されるべきではなく、その目的の剤形を調製し得ることが知られている任意の種類、配合量等であることができる。尚、かかる色素沈着症予防治療剤の調製においては、慣用のメラニン生成抑制剤、紫外線吸収剤、紫外線散乱剤、抗炎症剤、ビタミン剤、ステロイド剤等を合わせて配合してもよい。
【００１２】
本発明の有効成分であるフェニルヒドロキノンは、光や温度上昇により、またフェニルヒドロキノンを含んだ製剤がアルカリ性になると酸化を受けやすくなり、やや安定性に欠ける。そのため、冷暗所保存するか、酸化防止剤（例えばアスコルビン酸、ジブチルヒドロキシトルエン、クエン酸、没食子酸プロピル、トコフェロール等）や製剤の基剤を酸性にする薬剤（例えば、亜硫酸水素ナトリウム、クエン酸、アスコルビン酸等）を加えると安定性を増すことができる。
【００１３】
本発明の色素沈着症予防治療剤の有効成分であるフェニルヒドロキノンの皮膚白色化やメラニン生成抑制効果は、後記各試験例によりヒドロキノンより約２倍強く、一方、文献上に報告されているオルトフェニルフェノールにはそれらの効果は認められないことが判明した。
【００１４】
本発明の色素沈着症予防治療剤の投与は、上記の各種剤形に代表される皮膚外用剤として経皮的に行うことが例示される。また、投与量としては、患者の症状、年齢、剤形により異なるが、軟膏剤の場合、例えば１％フェニルヒドロキノン含有のものを適量、１日１〜２回塗布すればよい。
【００１５】
本発明の色素沈着症予防治療剤の有効成分であるフェニルヒドロキノンは、高濃度を長期間にわたって連続使用しない限り、副作用として考えられる白斑の残存を生ずることはない。
【００１６】
【実施例】
以下の実施例、試験例によって本発明をさらに説明するが、本発明の範囲は、これらの実施例により何ら限定されるものではない。
〔実施例１〕（軟膏１）

【００１７】
〔実施例２〕（軟膏２）

【００１８】
〔実施例３〕（軟膏３）

【００１９】
〔実施例４〕（ローション剤）

【００２０】
次に掲げる各試験例に用いた実験動物、被検物質、塗布方法および皮膚の白色化の評価方法を以下に示す。
【００２１】
〔参考例〕
１．実験動物
黒色モルモット JY-4（1984年に国立予防衛生研究所より東京都立衛生研究所に導入し、以後兄妹交配により維持されている）10〜15週齢の雌雄を各試験例当たり４匹用いた。本動物は、表皮にドーパ（L-β-3,4-dihydroxyphenylalanine：DOPA）陽性の活性化メラノサイトをもち、皮膚色は黒灰色である。
【００２２】
２．被検物質
フェニルヒドロキノンおよび比較対照物質としてオルトフェニルフェノール、ヒドロキノン、アルブチンおよびコウジ酸を用いた。被検物質の各濃度はW/V％ [100ml中の含量 (g)を示す] で調整された。
【００２３】
３．塗布方法
被毛をバリカンとシェーバーで剪毛した背部皮膚に黒色マーカーで 4x4cmあるいは 4x3cmの四角に区切った部位（前者は動物１匹当たり６箇所、後者は動物１匹当たり８箇所）を設けた。その区画にマイクロピペットで、100μlの被検物質溶液およびその溶媒を１日１回、週６日、５週間連続して塗布した。塗布部位の被毛は週１回剪毛した。
また、白色化した皮膚の回復試験については、13週間連続して塗布を行った群も設けた。
【００２４】
４．皮膚白色化の評価
皮膚白色化を調べるために以下の評価法を用い白色度を判定した。
４段階評価法：
− ：白色化を認めない
± ：僅かな白色化を認める
＋ ：十分な白色化を認める
＋＋：顕著な白色化を認める
さらに、試験例３〜５の実験については肉眼的白色化の評価を確認するために分光測色計（CM-2022、ミノルタカメラ (株) 製）を用い判定した。
【００２５】
分光測色計による皮膚色の評価：表色系には1976年に国際照明委員会（略称：CIE）で規格化されたL*a*b*を用いて測定した。L*は明度、a*,b*は色相と彩度を示す色度を表し、L*値は適当な範囲では皮膚のメラニン含量に大まかに比例する数値であることが期待できる。L*値の１区画５箇所の機器による自動平均化の計測を行い、各群のモルモット４匹当たりの平均値±標準偏差を算出し、Dunnettの多重比較検定法で溶媒対照群との間の有意差検定を行った。
【００２６】
〔試験例１〕（フェニルヒドロキノンとオルトフェニルフェノールの皮膚白色化作用の検討）
本発明の色素沈着症予防治療剤の有効成分であるフェニルヒドロキノンと今まで文献で報告されているオルトフェニルフェノールの皮膚白色化作用に対する効果を評価する実験を行った。
【００２７】

この結果を表１に示す（図１参照）。
【００２８】
【表１】

【００２９】
表１および図１に示すように、オルトフェニルフェノールには皮膚の白色化作用はみられず、フェニルヒドロキノンで白色化作用がみられた。
【００３０】
〔試験例２〕（フェニルヒドロキノンで白色化した皮膚の回復試験）
フェニルヒドロキノンについて連続塗布終了後、フェニルヒドロキノンにより白色化した区画の皮膚が、一定の期間を置いて、元の皮膚色に回復するか否かを調べた。
塗布期間は５週で終了した動物の群と、塗布をさらに１３週まで続けた動物の群を作製し、各々、その後さらに１３週間、無処置で飼育し、色調を調べた。この結果を表２に示す。
【００３１】
【表２】

【００３２】
表２に示すように５％フェニルヒドロキノン溶液（溶媒：エタノール）を１３週間塗布した群の１匹にわずかな白斑の残存が認められ、その他の群では白色化は消失し元の皮膚色に戻った。
【００３３】
〔試験例３〕（フェニルヒドロキノンとヒドロキノンの皮膚白色化作用の比較検討１）
本発明の色素沈着症予防治療剤の有効成分であるフェニルヒドロキノンとメラニン生成抑制作用を有することが知られているヒドロキノンの両者につき、皮膚白色化作用を比較する実験を行った。
【００３４】

この結果の肉眼的判定を表３、分光測色計による評価を表４に示す（図２参照）。
【００３５】
【表３】

【００３６】
【表４】

【００３７】
表３、４および図２に示すように、フェニルヒドロキノンは、ヒドロキノンよりも強い（およそ２倍）皮膚白色化作用を有している。
【００３８】
〔試験例４〕（フェニルヒドロキノンとヒドロキノンの皮膚白色化作用の比較検討２）
試験例３の実験よりも濃度をあげてフェニルヒドロキノンとヒドロキノンを比較した。

この結果の肉眼的判定を表５、分光測色計による評価を表６に示す（図３参照）。
【００３９】
【表５】

【００４０】
【表６】

【００４１】
表５、６および図３に示すように、フェニルヒドロキノンは、ヒドロキノンよりも強い（およそ２倍）皮膚白色化作用を有している。
【００４２】
〔試験例５〕（フェニルヒドロキノンとコウジ酸およびアルブチンの皮膚白色化作用の比較検討）
本発明の色素沈着症予防治療剤の有効成分であるフェニルヒドロキノンとメラニン生成抑制作用を有することが知られているコウジ酸（特開昭63-270619号参照）およびヒドロキノン誘導体のアルブチン（特開昭60-16906号参照）を使用して、皮膚白色化作用を比較する実験を行った。
【００４３】

この結果の肉眼的判定を表７、分光測色計による評価を表８に示す（図４参照）。
【００４４】
【表７】

【００４５】
【表８】

【００４６】
表７、８および図４に示すように、フェニルヒドロキノンは、アルブチンおよびコウジ酸よりも（４倍以上）強い皮膚白色化作用を有している。
【００４７】
〔試験例６〕（皮膚垂直面の表皮メラノサイトの顕微鏡による観察）
試験例５の実験の皮膚を採取し、凍結切片作製用包埋剤で包埋し、液体窒素で凍結した。その組織について、クリオスタット（cryostat）を用いて凍結切片標本を作製し、10%中性緩衝ホルマリン液で５分固定し、水洗後、0.1%ドーパ溶液（溶媒：pH 6.8のリン酸緩衝液）で37℃において6 時間反応させた。その後、水洗し、ケルンエヒトロート（Kernechtrot）による核染色を行い、脱水後、封入剤で封入し、光学顕微鏡で観察した。その結果、表皮のドーパ陽性メラノサイトはアルブチンおよびコウジ酸の1%および4%の両濃度群およびフェニルヒドロキノンの1%群において溶媒対照群と比較してあまり変化なく観察されたが、フェニルヒドロキノンの4%群では顕著に減少した。アルブチン, コウジ酸, フェニルヒドロキノンの4%群についての光学顕微鏡観察の結果をそれぞれ図５，６，７に示す。
【００４８】
また、試験例１における5%フェニルヒドロキノン塗布群、溶媒対照群、および無処置群の電子顕微鏡的観察では、各群の皮膚組織細片を２％グルタールアルデヒド・１％オスミウム酸二重固定法で固定してエタノール上昇系列で脱水し、エポキシ樹脂包埋し、樹脂重合後、超薄切し、その切片を酢酸ウラン・クエン酸鉛二重電子染色して透過型電子顕微鏡で観察し撮影した。その結果、５％フェニルヒドロキノン塗布群の皮膚では表皮メラノサイトの胞体内のメラノゾーム数の明らかな減少および残存メラノゾームの破壊像が観察された。無処置群および溶媒対照群にはそのような像は観察されなかった。５％フェニルヒドロキノン群および無処置群の皮膚メラノサイトの電子顕微鏡像をそれぞれ図８，９に示す。
【００４９】
文献より、アルブチンやコウジ酸のメラニン生成抑制作用はメラニン産生に必用なチロシナーゼ酵素の活性阻害が考えられている（秋保暁ら，アルブチンのメラニン生成抑制作用，B16メラノーマ培養細胞による生化学的研究，日本皮膚科学会雑誌，101，609-613，1991 および比嘉良喬，コウジ酸のmelanin生成抑制作用について，Fragrance Journal, 63, 40-44, 1983）。しかしヒドロキノンの作用ついては、Jimbowら（J. Invest. Dermatol., 62, 436-449, 1974 ）が電子顕微鏡による観察から、比較的高い選択性をもってメラノサイトの傷害によりメラニン生成を抑制することを報告している。今回のフェニルヒドロキノン塗布により、肉眼的に観察された皮膚白色化、組織学的に観察した表皮のドーパ陽性メラノサイトの著しい減少および電子顕微鏡的に観察したメラノゾームの破壊像などから、フェニルヒドロキノンの皮膚白色化は表皮メラノサイトの顕著なメラニン生成抑制によることは確実である。
【００５０】
【発明の効果】
本発明によれば、紫外線、ホルモンバランス、中毒等の各種の原因で生じるシミ・ソバカスなどの色素沈着症の予防・治療、ならびに皮膚の美白効果に優れ、かつ安全性にも優れた新規の色素沈着症予防治療剤が提供される。
【図面の簡単な説明】
【図１】フェニルヒドロキノンの1%、5% 溶液、オルトフェニルフェノールの1%、5%溶液および溶媒（エタノール）を5週間塗布した黒色モルモットJY-4の背部皮膚の外観（生物の形態）を示す写真である。
【図２】フェニルヒドロキノンの0.25%、0.5%、1%溶液、ヒドロキノンの0.25%、0.5%、1%溶液および溶媒（エタノール）を5週間塗布した黒色モルモットJY-4の背部皮膚の外観（生物の形態）を示す写真である。
【図３】フェニルヒドロキノンの1%、2%、4%溶液、ヒドロキノンの1%、2%、4%溶液および溶媒（エタノール）を5週間塗布した黒色モルモットJY-4の背部皮膚の外観（生物の形態）を示す写真である。
【図４】フェニルヒドロキノンの1%、4%溶液、アルブチンの1%、4%溶液、コウジ酸の1%、4%溶液および溶媒（ジメチルスルホキシド：エタノールが１：４の混合液）を5週間塗布した黒色モルモットJY-4の背部皮膚の外観（生物の形態）を示す写真である。
【図５】 4%アルブチンを5週間塗布した部分の黒色モルモットJY-4の背部皮膚について、凍結組織標本を作製し、ドーパ反応およびケルンエヒトロートによる核染色を行った組織像を示す顕微鏡写真である。
【図６】 4%コウジ酸を5週間塗布した部分の黒色モルモットJY-4の背部皮膚について、凍結組織標本を作製し、ドーパ反応およびケルンエヒトロートによる核染色を行った組織像を示す顕微鏡写真である。
【図７】 4%フェニルヒドロキノンを5週間塗布した部分の黒色モルモットJY-4の背部皮膚について、凍結組織標本を作製し、ドーパ反応およびケルンエヒトロートによる核染色を行った組織像を示す顕微鏡写真である。
【図８】 5%フェニルヒドロキノンを5週間塗布した部分の黒色モルモットJY-4の背部皮膚について、グルタールアルデヒド・オスミウム酸二重固定を行いエポキシ樹脂包埋して電子顕微鏡試料を作製し、表皮メラノサイトを撮影した電子顕微鏡写真である。
【図９】無処置の部分の黒色モルモットJY-4の背部皮膚について、グルタールアルデヒド・オスミウム酸二重固定を行いエポキシ樹脂包埋して電子顕微鏡試料を作製し、表皮メラノサイトを撮影した電子顕微鏡写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention is a pigmentation prevention / treatment agent containing phenylhydroquinone as an active ingredient. The agent for preventing and treating pigmentation of the present invention is effective for the prevention and treatment of various pigmentation diseases typified by spots and freckles and whitening of the skin, by virtue of the melanin production inhibitory action and depigmentation action.
[0002]
[Prior art]
There are various causes of pigmentation of human skin represented by stains and freckles, ranging from pathological to aging and ultraviolet rays. The development of pigmentation treatment agents and whitening cosmetics that control these is one of the important issues in the field of pharmaceuticals and cosmetics. For the purpose of controlling pigmentation, ascorbic acid derivatives or complex compounds thereof (for example, JP-A 63-198674), kojic acid (JP-A 63-2770619), hydroquinone and derivatives thereof (Arbutin: JP-A 60-60) 16906), linoleic acid (JP-A 64-85907), plant components (JP-A 1-149706), animal components (JP-A 1-143887), and the like have been used. In the US, hydroquinone has been approved by the FDA as an over-the-counter (OTC) drug for safety and efficacy (Federal Register, 47, 39108, 1982) and is sold in general pharmacies. However, in Japan, it is only used in some clinics in terms of primary skin irritation and sensitization, but its effectiveness has been evaluated by dermatologists.
[0003]
In terms of the chemical structure of hydroquinone, phenylhydroquinone, also known as 2,5-dihydroxybiphenyl, has a phenyl group bonded to the 2-position. Ortho-phenylphenol, also known as 2-hydroxybiphenyl, which has a 5-position hydroxyl group out of the two hydroxyl groups of phenylhydroquinone, is currently used in Japan as a citrus fungicide. It is designated as an additive and is also used as a preservative and disinfectant for cosmetics. Into et al. (Bull. Pharm. Res. Inst., 76, 5-13, 1968) and Kahn (Arch. Dermatol., 102, 177-187, 1970). However, it has not been known so far that phenylhydroquinone has a skin whitening action or a melanin suppressing action.
[0004]
[Problems to be solved by the invention]
The problems of the present invention are that the conventional pigmentation treatment agents and whitening cosmetics listed above have insufficient effects, and interest in prevention and treatment of pigmentation represented by stains and freckles has increased. In view of the actual situation, it is to provide a drug having a more effective whitening effect.
[0005]
[Means for Solving the Problems]
As a result of continuing the search for a substance having a whitening effect that can solve the above-mentioned problems, the present inventors have found that phenylhydroquinone is a compound having a remarkable whitening effect, thereby completing the present invention. It came.
That is, the present invention is a pigmentation prevention / treatment agent containing phenylhydroquinone as an active ingredient.
[0006]
The action of the anti-pigmentation therapeutic agent of the present invention is to prevent the occurrence of pigmentation such as melasma, spots and freckles due to ultraviolet rays by ultraviolet rays, and to depigment the pigmentation that has occurred. It means treating.
Hereinafter, the present invention will be described in detail.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The active ingredient of the pigmentation prevention and treatment agent of the present invention is phenylhydroquinone (also known as phenylhydroquinol), also known as phenylhydroquinol, 2,5-dihydroxybiphenyl, 2,5- Dihydroxydiphenyl (2,5-dihydroxydiphenyl), 1,1 '-(biphenyl) -2,5-diol [1,1'-(biphenyl) -2,5-diol], 1,1 '-(diphenyl)- 2,5-diol [1,1 '-(diphenyl) -2,5-diol] and the like. Its chemical formula is C ₁₂ H ₁₀ O ₂ and its structural formula is shown below.
[0008]
[Chemical 1]

[0009]
When preparing the pigmentation prevention / treatment agent of the present invention, the amount of phenylhydroquinone is not particularly limited, but is preferably 0.1 to 5% by weight, more preferably 0.25 to 2% by weight, based on the total weight. it can. This amount is effective at a lower concentration when used together with an appropriate base that increases the absorption rate and residual rate of the skin. If the blending amount exceeds 5% by weight, vitiligo may occur when used for a long time.
[0010]
The agent for preventing and treating pigmentation of the present invention can be prepared in various ointments (oil-based, emulsion-based, water-soluble), lotion and the like by known methods. Moreover, the kind of component which can be used in preparation of these dosage forms, a compounding quantity, etc. can be suitably determined according to the common example known to those skilled in the art.
[0011]
The types, blending amounts, and the like of these components should not be limited, and can be any types, blending amounts, and the like that are known to be able to prepare the target dosage form. In the preparation of such a pigmentation prevention and treatment agent, a conventional melanin production inhibitor, ultraviolet absorber, ultraviolet scattering agent, anti-inflammatory agent, vitamin agent, steroid agent and the like may be combined.
[0012]
Phenylhydroquinone, which is an active ingredient of the present invention, is susceptible to oxidation when the preparation containing phenylhydroquinone becomes alkaline due to light or temperature rise, and is slightly less stable. Therefore, store in a cool dark place, or use an antioxidant (eg, ascorbic acid, dibutylhydroxytoluene, citric acid, propyl gallate, tocopherol, etc.) or an agent that makes the formulation base acidic (eg, sodium bisulfite, citric acid, ascorbine) The addition of acid etc. can increase the stability.
[0013]
The skin whitening and melanin production inhibitory effects of phenylhydroquinone, which is an active ingredient of the pigmentation prevention and treatment agent of the present invention, are about twice as strong as hydroquinone in each test example described below, while orthophenyl reported in the literature. Phenol was found to have no such effect.
[0014]
Administration of the pigmentation prevention / treatment agent of the present invention is exemplified by being performed transdermally as a skin external preparation typified by the above various dosage forms. The dosage varies depending on the patient's symptoms, age and dosage form, but in the case of an ointment, for example, an appropriate amount containing 1% phenylhydroquinone may be applied once or twice a day.
[0015]
Phenylhydroquinone, which is an active ingredient of the agent for preventing and treating pigmentation of the present invention, does not cause vitiligo remaining as a side effect unless a high concentration is continuously used over a long period of time.
[0016]
【Example】
The following examples and test examples further illustrate the present invention, but the scope of the present invention is not limited by these examples.
[Example 1] (Ointment 1)

[0017]
[Example 2] (Ointment 2)

[0018]
[Example 3] (Ointment 3)

[0019]
[Example 4] (Lotion)

[0020]
The experimental animals, test substances, application methods, and skin whitening evaluation methods used in the following test examples are shown below.
[0021]
[Reference example]
1. Experimental animal black guinea pig JY-4 (introduced by the National Institute of Preventive Health in 1984 to the Tokyo Metropolitan Institute of Public Health and subsequently maintained by brother-sister mating) 10-15 weeks old males and females for each test case It was. This animal has activated melanocytes positive for dopa (L-β-3,4-dihydroxyphenylalanine: DOPA) in the epidermis, and the skin color is black-gray.
[0022]
2. The test substance phenylhydroquinone and orthophenylphenol, hydroquinone, arbutin and kojic acid were used as comparative control substances. Each concentration of the test substance was adjusted with W / V% [indicating a content (g) in 100 ml].
[0023]
3. Application method The back skin was shaved with a hair clipper and a shaver, and was divided into 4x4cm or 4x3cm squares with the black marker (the former was 6 sites per animal and the latter was 8 sites per animal). 100 μl of the test substance solution and its solvent were applied to the compartment once a day for 6 days a week for 5 weeks with a micropipette. The coat at the application site was shaved once a week.
Moreover, about the recovery test of the whitened skin, the group which applied 13 weeks continuously was also provided.
[0024]
4). Evaluation of skin whitening In order to examine skin whitening, the following evaluation method was used to determine whiteness.
Four-level evaluation method:
−: No whitening is recognized ±: Slight whitening is recognized +: Sufficient whitening is recognized ++: Remarkable whitening is observed Further, in the experiments of Test Examples 3 to 5, the evaluation of macroscopic whitening was confirmed. Therefore, a spectrocolorimeter (CM-2022, manufactured by Minolta Camera Co., Ltd.) was used for determination.
[0025]
Evaluation of skin color by spectrocolorimeter: The color system was measured using L * a * b * standardized by the International Commission on Illumination (abbreviation: CIE) in 1976. L * represents lightness, and a * and b * represent chromaticity indicating hue and saturation, and the L * value can be expected to be a value roughly proportional to the melanin content of the skin in an appropriate range. The L * value was measured by automatic averaging with 5 devices per compartment, and the mean value ± standard deviation of each group of 4 guinea pigs was calculated. A significant difference test was performed.
[0026]
[Test Example 1] (Examination of skin whitening effect of phenylhydroquinone and orthophenylphenol)
Experiments were conducted to evaluate the effect of phenylhydroquinone, which is an active ingredient of the pigmentation prevention and treatment agent of the present invention, and orthophenylphenol, which has been reported in the literature, on the skin whitening action.
[0027]

The results are shown in Table 1 (see FIG. 1).
[0028]
[Table 1]

[0029]
As shown in Table 1 and FIG. 1, orthophenylphenol had no skin whitening effect, and phenylhydroquinone had a whitening effect.
[0030]
[Test Example 2] (Recovery test of skin whitened with phenylhydroquinone)
After the continuous application of phenylhydroquinone, it was examined whether or not the skin of the section whitened with phenylhydroquinone was restored to the original skin color after a certain period of time.
A group of animals whose application period ended at 5 weeks and a group of animals whose application was continued for up to 13 weeks were prepared, and each of them was reared without treatment for further 13 weeks, and the color tone was examined. The results are shown in Table 2.
[0031]
[Table 2]

[0032]
As shown in Table 2, slight white spots remained in one of the groups applied with a 5% phenylhydroquinone solution (solvent: ethanol) for 13 weeks, and whitening disappeared and the original skin color was restored in the other groups. It was.
[0033]
[Test Example 3] (Comparative study 1 of skin whitening action of phenylhydroquinone and hydroquinone 1)
Experiments were conducted to compare the skin whitening effect of both phenylhydroquinone, which is an active ingredient of the agent for preventing and treating pigmentation of the present invention, and hydroquinone known to have a melanin production inhibitory effect.
[0034]

Table 3 shows the macroscopic determination of this result, and Table 4 shows the evaluation by the spectrocolorimeter (see FIG. 2).
[0035]
[Table 3]

[0036]
[Table 4]

[0037]
As shown in Tables 3 and 4 and FIG. 2, phenylhydroquinone has a skin whitening effect stronger (approximately twice) than hydroquinone.
[0038]
[Test Example 4] (Comparative study 2 of skin whitening action of phenylhydroquinone and hydroquinone 2)
Phenylhydroquinone and hydroquinone were compared with each other at a higher concentration than the experiment of Test Example 3.

Table 5 shows the macroscopic determination of this result, and Table 6 shows the evaluation by the spectrocolorimeter (see FIG. 3).
[0039]
[Table 5]

[0040]
[Table 6]

[0041]
As shown in Tables 5 and 6 and FIG. 3, phenylhydroquinone has a skin whitening effect stronger (approximately twice) than hydroquinone.
[0042]
[Test Example 5] (Comparison study of skin whitening effect of phenylhydroquinone, kojic acid and arbutin)
Phenylhydroquinone, which is an active ingredient of the anti-pigmentation therapeutic agent of the present invention, kojic acid known to have an inhibitory action on melanin production (see JP-A 63-270619) and arbutin, a hydroquinone derivative (JP-A No. 60-16906) was used to compare the skin whitening effect.
[0043]

Table 7 shows the macroscopic determination of this result, and Table 8 shows the evaluation by the spectrocolorimeter (see FIG. 4).
[0044]
[Table 7]

[0045]
[Table 8]

[0046]
As shown in Tables 7 and 8 and FIG. 4, phenylhydroquinone has a skin whitening effect stronger (4 times or more) than arbutin and kojic acid.
[0047]
[Test Example 6] (observation of epidermal melanocytes on the vertical surface of the skin with a microscope)
The skin of the experiment of Test Example 5 was collected, embedded with an embedding agent for preparing frozen sections, and frozen with liquid nitrogen. For the tissue, a frozen section sample is prepared using cryostat, fixed with 10% neutral buffered formalin solution for 5 minutes, washed with water, 0.1% dopa solution (solvent: phosphate buffer solution at pH 6.8). For 6 hours at 37 ° C. Thereafter, it was washed with water, subjected to nuclear staining with Kernechtrot, dehydrated, sealed with a mounting medium, and observed with an optical microscope. As a result, epidermal dopa-positive melanocytes were observed in both 1% and 4% concentration groups of arbutin and kojic acid and 1% group of phenylhydroquinone with little change compared to the solvent control group, but 4% of phenylhydroquinone. There was a marked decrease in the% group. The results of optical microscope observation for the 4% group of arbutin, kojic acid, and phenylhydroquinone are shown in FIGS.
[0048]
In addition, in the electron microscopic observation of the 5% phenylhydroquinone application group, the solvent control group, and the non-treatment group in Test Example 1, 2% glutaraldehyde / 1% osmate acid double fixation method was used for each group of skin tissue strips. Fixed with, dehydrated with ethanol ascending series, embedded in epoxy resin, polymerized, ultra-thin cut, sliced uranium acetate / lead citrate double electron stained and observed with transmission electron microscope and photographed . As a result, in the skin of the 5% phenylhydroquinone application group, a clear decrease in the number of melanosomes in the endoplasmic reticulum melanocytes and destruction of the remaining melanosomes were observed. Such an image was not observed in the untreated group and the solvent control group. Electron microscopic images of skin melanocytes in the 5% phenylhydroquinone group and the untreated group are shown in FIGS.
[0049]
From the literature, melanin production inhibitory action of arbutin and kojic acid is considered to inhibit the activity of tyrosinase enzyme necessary for melanin production (Akibo, et al., Melanin production inhibitory action of arbutin, biochemical study using cultured B16 melanoma cells, Journal of the Japanese Dermatological Association, 101, 609-613, 1991 and Ryo Higa, Kojic acid on the inhibitory action on melanin production, Fragrance Journal, 63, 40-44, 1983). However, with regard to the action of hydroquinone, Jimbow et al. (J. Invest. Dermatol., 62, 436-449, 1974) reported that melanin production was inhibited by melanocyte injury with relatively high selectivity, as observed by electron microscopy. ing. With this application of phenylhydroquinone, the whitening of the skin of phenylhydroquinone was observed based on the whitening of the skin observed macroscopically, the marked decrease in dopa-positive melanocytes of the epidermis and the destruction of melanosomes observed by electron microscopy. It is certain that the conversion is due to the marked inhibition of melanin production by epidermal melanocytes.
[0050]
【The invention's effect】
According to the present invention, a novel pigment excellent in the prevention and treatment of pigmentation diseases such as spots and freckles caused by various causes such as ultraviolet rays, hormone balance, and poisoning, as well as the skin whitening effect and excellent in safety. An agent for preventing and treating deposition disease is provided.
[Brief description of the drawings]
FIG. 1 shows the appearance (biological form) of the back skin of black guinea pig JY-4 to which 1%, 5% solution of phenylhydroquinone, 1%, 5% solution of orthophenylphenol and solvent (ethanol) were applied for 5 weeks. It is a photograph shown.
[Fig. 2] Appearance of the back skin of black guinea pig JY-4 to which 0.25%, 0.5%, 1% solution of phenylhydroquinone, 0.25%, 0.5%, 1% solution of hydroquinone and solvent (ethanol) were applied for 5 weeks (biological) It is a photograph which shows a form.
[Fig. 3] Appearance of back skin of black guinea pig JY-4 to which 1%, 2%, 4% solution of phenylhydroquinone, 1%, 2%, 4% solution of hydroquinone and solvent (ethanol) were applied for 5 weeks. It is a photograph which shows a form.
[Fig. 4] 1%, 4% solution of phenylhydroquinone, 1%, 4% solution of arbutin, 1%, 4% solution of kojic acid and solvent (dimethyl sulfoxide: ethanol 1: 4 mixture) for 5 weeks It is a photograph which shows the external appearance (biological form) of the back skin of the apply | coated black guinea pig JY-4.
FIG. 5: Photomicrograph showing a tissue image of a frozen tissue specimen prepared from the back skin of black guinea pig JY-4 where 4% arbutin was applied for 5 weeks, and subjected to nuclear staining with Dopa reaction and Cologne Echlot It is.
FIG. 6 is a microscope showing a frozen tissue specimen of a black guinea pig JY-4 dorsal skin where 4% kojic acid was applied for 5 weeks, and a tissue image obtained by nuclear staining with a Dopa reaction and a Cologne Echrotroth. It is a photograph.
FIG. 7 is a microscope showing a frozen tissue specimen of a black guinea pig JY-4 dorsal skin where 4% phenylhydroquinone was applied for 5 weeks, and a tissue image obtained by nuclear staining with a Dopa reaction and a Cologne Echlot funnel. It is a photograph.
[Fig.8] The back skin of black guinea pig JY-4 where 5% phenylhydroquinone was applied for 5 weeks was double-fixed with glutaraldehyde and osmate and embedded in an epoxy resin to prepare an electron microscope sample. It is the electron micrograph which image | photographed the melanocyte.
FIG. 9 shows an electron microscope in which the skin of black guinea pig JY-4 in an untreated part was double-fixed with glutaraldehyde and osmate and embedded in an epoxy resin to prepare an electron microscope sample, and an epidermis melanocyte was photographed. It is a photograph.

Claims (2)

A pigmentation preventive or therapeutic agent comprising phenylhydroquinone as an active ingredient. The agent for preventing or treating pigmentation according to claim 1, which is in the form of an external preparation for skin.

Images