JP2019055932A - Composition for dj-1 protein production promotion - Google Patents
Composition for dj-1 protein production promotion Download PDFInfo
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- JP2019055932A JP2019055932A JP2017217996A JP2017217996A JP2019055932A JP 2019055932 A JP2019055932 A JP 2019055932A JP 2017217996 A JP2017217996 A JP 2017217996A JP 2017217996 A JP2017217996 A JP 2017217996A JP 2019055932 A JP2019055932 A JP 2019055932A
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- protein
- skin
- curcumin
- resveratrol
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Abstract
Description
本発明は、ＤＪ−１タンパク質の産生を促進する組成物、及び皮膚細胞に対する外部刺激抵抗性を増強する組成物に関する。 The present invention relates to a composition that promotes the production of DJ-1 protein and a composition that enhances resistance to external stimuli against skin cells.
ＤＪ−１タンパク質は、公知の生理活性タンパク質で、神経細胞や皮膚細胞を含む広範なヒト細胞に存在しており、１８９のアミノ酸からなる。ＤＪ−１タンパク質はガン遺伝子産物であり、家族性パーキンソン病と関連があることが知られている（非特許文献１）。
さらに、ＤＪ−１タンパク質は酸化ストレスによる神経細胞死を抑制する効果を有することが知られている。すなわち、酸化ストレスを与える６−ヒドロキシドーパミンを注入されたパーキンソン病モデルラットに、６−ヒドロキシドーパミンの注入と同時又は注入後にＤＪ−１タンパク質を注入すると、ドーパミン神経細胞死が抑制され、行動異常が改善されることが知られている（特許文献１）。
特許文献１には、酸化ストレスによる神経細胞死を抑制する効果をＤＪ−１が有することが記載されている。そしてＤＪ−１に結合し、活性化する化合物を提案している。
The DJ-1 protein is a known physiologically active protein and is present in a wide range of human cells including nerve cells and skin cells, and consists of 189 amino acids. DJ-1 protein is an oncogene product and is known to be associated with familial Parkinson's disease (Non-patent Document 1).
Furthermore, it is known that DJ-1 protein has an effect of suppressing neuronal cell death due to oxidative stress. That is, when DJ-1 protein is injected into Parkinson's disease model rats injected with 6-hydroxydopamine giving oxidative stress at the same time as or after 6-hydroxydopamine injection, dopamine neuronal cell death is suppressed and behavioral abnormalities are observed. It is known to be improved (Patent Document 1).
Patent Document 1 describes that DJ-1 has an effect of suppressing neuronal cell death caused by oxidative stress. And the compound which couple | bonds with DJ-1 and activates is proposed.
またＤＪ−１タンパク質は、皮膚の抵抗性に関与していることが知られている。本発明者らは、皮膚角層中のＤＪ−１タンパク質量を測定することによって紫外線に曝露したときの発赤の発生に対する抵抗性の指標とすることができること（特許文献２）、紫外線曝露歴の評価指標とすることができること（特許文献３）、あるいは発毛状態の評価指標とできること（特許文献４）などを明らかにしている。 DJ-1 protein is known to be involved in skin resistance. The present inventors can measure the amount of DJ-1 protein in the stratum corneum as an index of resistance to the occurrence of redness when exposed to ultraviolet rays (Patent Document 2). It is clarified that it can be used as an evaluation index (Patent Document 3), or an evaluation index of a hair growth state (Patent Document 4).
本発明は、皮膚の外部刺激に対する抵抗性に関与するＤＪ−１タンパク質の産生を促進する物質を探索する過程で見出されたものである。また、本発明者らは紫外線が皮膚細胞に及ぼす影響を検討する過程でＤＪ−１タンパク質が増加した細胞は紫外線抵抗性が増強していることを見いだした。
すなわち本発明は、皮膚細胞のＤＪ−１タンパク質産生を促進する新たな組成物を提供すること、及び紫外線抵抗性を皮膚細胞に付与するための組成物を提供することを課題とする。
The present invention was discovered in the process of searching for a substance that promotes the production of DJ-1 protein involved in resistance to external stimulation of the skin. In addition, the present inventors have found that cells with increased DJ-1 protein have enhanced UV resistance in the process of examining the effect of UV rays on skin cells.
That is, an object of the present invention is to provide a new composition for promoting the production of DJ-1 protein in skin cells and to provide a composition for imparting ultraviolet resistance to skin cells.
本発明は以下の構成である。
（１）クルクミン類及び／又はレスベラトロール類を有効成分として含有するＤＪ−１タンパク質産生促進用組成物。
（２）（１）に記載の組成物を含むＤＪ−１タンパク質産生促進のための皮膚外用剤。
（３）（１）に記載の組成物を含む皮膚細胞の外部刺激抵抗性を増強するための組成物。
（４）外部刺激が紫外線によるものである（３）に記載の組成物。
（５）紫外線曝露によって誘発される赤み（発赤）発生に対する抵抗性向上剤。
The present invention has the following configuration.
(1) A composition for promoting DJ-1 protein production comprising curcumin and / or resveratrol as an active ingredient.
(2) A skin external preparation for promoting DJ-1 protein production, comprising the composition according to (1).
(3) A composition for enhancing resistance to external stimulation of skin cells, comprising the composition according to (1).
(4) The composition according to (3), wherein the external stimulus is an ultraviolet ray.
(5) A resistance improver against redness (redness) generation induced by ultraviolet exposure.
本発明により、ＤＪ−１タンパク質の産生を促進する組成物が提供される。本発明の組成物は皮膚外用剤として有用である。またあらかじめ、皮膚細胞、特に角層細胞におけるＤＪ−１タンパク質を増加させておくことで、外部刺激に対する細胞の抵抗性が増強され、紫外線によって誘発される炎症反応などを抑制することができる。このため紫外線曝露によって誘発される炎症や発赤に対する抵抗性を向上させることができる。 The present invention provides a composition that promotes the production of DJ-1 protein. The composition of the present invention is useful as a skin external preparation. Further, by increasing the DJ-1 protein in skin cells, particularly stratum corneum cells, the resistance of the cells to external stimuli is enhanced, and the inflammatory reaction induced by ultraviolet rays can be suppressed. For this reason, resistance to inflammation and redness induced by exposure to ultraviolet rays can be improved.
以下、本発明について詳細に説明する。
本願はクルクミン類及び／又はレスベラトロール類を有効成分として含有するＤＪ−１タンパク質産生促進用組成物に係る発明である。
Hereinafter, the present invention will be described in detail.
The present application is an invention relating to a composition for promoting production of DJ-1 protein containing curcumin and / or resveratrol as an active ingredient.
ヒトＤＪ−１タンパク質は、前記したとおり、ヒト皮膚細胞や神経細胞において多く発現されるタンパク質である。
皮膚のＤＪ−１タンパク質産生を促進することにより、皮膚内のＤＪ−１タンパク質量を増加させ、皮膚の外部刺激に対する抵抗性を増加させることが期待できる。
As described above, human DJ-1 protein is a protein that is highly expressed in human skin cells and nerve cells.
By promoting the production of DJ-1 protein in the skin, it can be expected that the amount of DJ-1 protein in the skin is increased and the resistance to external stimulation of the skin is increased.
本発明のＤＪ−１タンパク質産生促進用組成物は、クルクミン類及び／又はレスベラトロール類をＤＪ−１タンパク質産生促進の有効成分として含有している。 The composition for promoting production of DJ-1 protein of the present invention contains curcumin and / or resveratrol as an active ingredient for promoting production of DJ-1 protein.
本発明の組成物の有効成分であるクルクミン (ｃｕｒｃｕｍｉｎ) 類は、ウコン（ターメリック、学名Ｃｕｒｃｕｍａ ｌｏｎｇａ）などに含まれる黄色の、公知のポリフェノール化合物の総称である。スパイスや食品領域の着色剤として利用され、安全性も熟知されている。
本発明に用いるクルクミン類は、クルクミンまたはクルクミン誘導体として分類される化合物をいい、クルクミン、デメトキシクルクミン、ビスデメトキシクルクミン、シス−トランス−クルクミン、およびシクロクルクミンを含むが、これに限定されない。なかでもクルクミンが特に好ましい。また、植物ウコンの根茎から抽出されたウコン抽出物であってもよい。ウコン抽出物は健康食品やサプリメントの原料として市販されており、このような市販品を用いることもできる。
Curcumin, which is an active ingredient of the composition of the present invention, is a generic name for yellow known polyphenol compounds contained in turmeric (turmeric, scientific name Curcuma longa) and the like. It is used as a colorant for spices and foods and is well known for its safety.
Curcumins used in the present invention refer to compounds classified as curcumin or curcumin derivatives, including but not limited to curcumin, demethoxycurcumin, bisdemethoxycurcumin, cis-trans-curcumin, and cyclocurcumin. Of these, curcumin is particularly preferable. Moreover, the turmeric extract extracted from the rhizome of the plant turmeric may be sufficient. Turmeric extract is commercially available as a raw material for health foods and supplements, and such commercially available products can also be used.
レスベラトロール（ｒｅｓｖｅｒａｔｒｏｌ）はスチルベノイド（スチルベン誘導体）ポリフェノールの一種であって、系統名は３，５，４’−トリヒドロキシ−ｔｒａｎｓ−スチルベンとも呼ばれる。レスベラトロールは、いくつかの植物でファイトアレキシンとして機能しており、またブドウの果皮などにも含まれる抗酸化物質として知られている。レスベラトロールは二量体を形成し、ε−ビニフェリンとも呼ばれる。また配糖体も存在しており、本発明においては、これらを総称してレスベラトロール類と呼ぶ。レスベラトロール類の中でも、単量体のレスベラトロールが好ましい。レスベラトロール類は、ブドウの果皮や赤ワイン、ピーナッツの赤皮、メリンジョ（グネツム）、イタドリの根から得ることができる。健康食品の原料として販売されており、これを使用することもできる。 Resveratrol is a kind of stilbenoid (stilbene derivative) polyphenol, and the systematic name is also called 3,5,4'-trihydroxy-trans-stilbene. Resveratrol functions as a phytoalexin in some plants and is also known as an antioxidant contained in grape skins. Resveratrol forms a dimer and is also called ε-viniferin. Glycosides also exist, and in the present invention, these are collectively referred to as resveratrols. Among the resveratrols, monomeric resveratrol is preferable. Resveratrols can be obtained from grape skins, red wine, peanut red skin, merinjo (Gnetsum) and itadori roots. It is sold as a raw material for health foods and can be used.
本発明のＤＪ−１タンパク質産生促進用組成物として医薬品、医薬部外品、化粧品、食品、飲料などを例示できる。その投与経路は、経口投与、経皮投与のいずれも可能である。なかでも経皮投与が特に好ましく、化粧品や皮膚外用医薬などの経皮組成物の形態を採用することが好ましい。経口投与などにより、有効成分を皮膚に到達させることもできる。
ＤＪ−１タンパク質産生促進用組成物とするためには、例えば外用剤とする場合、クルクミン類及び／又はレスベラトロール類を、クルクミン換算で、外用剤質量当たり０．００１〜０．１質量％、あるいはレスベラトロール換算で外用剤質量当たり０．０１〜１質量％含有させることで、皮膚に塗布等の方法で外用すると皮膚細胞のＤＪ−１タンパク質を産生促進できる。なおＤＪ−１タンパク質の産生促進効果を確認するためには、テープストリッピング法などによって皮膚角層試料を採取して、これを特許文献２に記載された操作方法に従って抽出し、ＥＬＩＳＡ法などで測定する。
Examples of the composition for promoting production of DJ-1 protein of the present invention include pharmaceuticals, quasi drugs, cosmetics, foods, and beverages. The administration route can be either oral administration or transdermal administration. Of these, transdermal administration is particularly preferable, and it is preferable to adopt the form of a transdermal composition such as cosmetics and external medicine for skin. The active ingredient can also reach the skin by oral administration or the like.
In order to obtain a composition for promoting the production of DJ-1 protein, for example, when it is used as an external preparation, curcumin and / or resveratrol is converted into curcumin in an amount of 0.001 to 0.1% by mass per external preparation mass. Alternatively, by containing 0.01 to 1% by mass of the external preparation mass in terms of resveratrol, the production of the skin cell DJ-1 protein can be promoted by external application by a method such as application to the skin. In order to confirm the production promotion effect of DJ-1 protein, a skin stratum corneum sample is collected by a tape stripping method or the like, extracted according to the operation method described in Patent Document 2, and measured by an ELISA method or the like. To do.
本発明のクルクミン類及び／又はレスベラトロール類を含むＤＪ−１タンパク質産生促進用組成物を人に投与する製剤とするために、通常の食品、医薬品、化粧料などの製剤化で使用される任意成分を含有することができる。このような任意成分としては、経口投与組成物であれば、例えば、乳糖や白糖などの賦形剤、デンプン、セルロース、アラビアガム、ヒドロキシプロピルセルロースなどの結合剤、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロースカルシウムなどの崩壊剤、大豆レシチン、ショ糖脂肪酸エステルなどの界面活性剤、マルチトールやソルビトールなどの甘味剤、クエン酸などの酸味剤、リン酸塩などの緩衝剤、シェラックやツェインなどの皮膜形成剤、タルク、ロウ類などの滑沢剤、軽質無水ケイ酸、乾燥水酸化アルミニウムゲルなどの流動促進剤、生理食塩水、ブドウ糖水溶液などの希釈剤、矯味矯臭剤、着色剤、殺菌剤、防腐剤、香料などが好適に例示できる。経皮投与組成物であれば、スクワラン、ワセリン、マイクロクリスタリンワックスなどの炭化水素類、ホホバ油、カルナウバワックス、オレイン酸オクチルドデシルなどのエステル類、オリーブ油、牛脂、椰子油などのトリグリセライド類、ステアリン酸、オレイン酸、レチノイン酸などの脂肪酸、エタノール、イソプロパノール等の低級アルコール、オレイルアルコール、ステアリルアルコール、オクチルドデカノール等の高級アルコール、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、アルキルベタイン塩等の両性界面活性剤類、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ソルビタン脂肪酸エステル、脂肪酸モノグリセライド、これらのポリオキシエチレン付加物、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン脂肪酸エステル、ポリオキシエチレンヒマシ油、ポリオキシエチレン硬化ヒマシ油等の非イオン界面活性剤類、ジプロピレングリコール、ポリエチレングリコール、グリセリン、１，３−ブタンジオール、１，２−ペンタンジオール等の多価アルコール類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等を含有することができる。投与製剤としては、化粧料や塗布剤、貼付剤などの外用剤の形態が好ましい。 In order to obtain a preparation for administering a composition for promoting the production of DJ-1 protein containing curcumin and / or resveratrol of the present invention to humans, it is used in the preparation of ordinary foods, pharmaceuticals, cosmetics and the like. Optional ingredients can be included. As such an optional component, if it is a composition for oral administration, for example, excipients such as lactose and sucrose, binders such as starch, cellulose, gum arabic and hydroxypropyl cellulose, sodium carboxymethylcellulose, carboxymethylcellulose calcium and the like Disintegrants, soy lecithin, surfactants such as sucrose fatty acid esters, sweeteners such as maltitol and sorbitol, sour agents such as citric acid, buffering agents such as phosphate, film forming agents such as shellac and zein, Lubricants such as talc and waxes, light anhydrous silicic acid, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline and aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, A fragrance | flavor etc. can illustrate suitably. For transdermal administration compositions, hydrocarbons such as squalane, petrolatum, microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow, coconut oil, stearin Acids, fatty acids such as oleic acid and retinoic acid, lower alcohols such as ethanol and isopropanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate , Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, poly Nonionic surfactants such as xylethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, dipropylene glycol, polyethylene glycol, glycerin, 1,3-butanediol, 1, Polyhydric alcohols such as 2-pentanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders and the like can be contained. As an administration formulation, the form of external preparations, such as cosmetics, a coating agent, and a patch, is preferable.
以下にクルクミンとレスベラトロールのＤＪ−１タンパク質の産生促進効果を確認した試験例を示し、本発明の組成物の示す効果について具体的に説明する。 The test example which confirmed the production promotion effect of DJ-1 protein of curcumin and resveratrol below is shown, and the effect which the composition of this invention shows is demonstrated concretely.
＜培養ヒト皮膚角層細胞を用いたＤＪ−１タンパク質産生促進試験＞
１．試験例１
（１）試験方法
６ｗｅｌｌ ｐｌａｔｅにHaCaT細胞（ヒト表皮角化細胞株）を播種して培養し、２日後にクルクミンを０、５、１０μＭになるように添加し、３時間培養した。また同様にレスベラトロールを０、２０、４０μＭになるように添加し、３時間培養した。
培養終了後細胞を回収し、常法によって細胞を破壊して細胞内のＤＪ−１タンパク質量をウエスタンブロット法により検出した。また細胞内のアクチンタンパク質を同様にウエスタンブロット法で検出し、アクチンタンパク質量に対するＤＪ−１タンパク質量を計算し、これをＤＪ−１タンパク質産生量とした。
<DJ-1 protein production promotion test using cultured human skin horny layer cells>
1. Test example 1
(1) Test method HaCaT cells (human epidermal keratinocyte cell line) were seeded on 6-well plates and cultured, and after 2 days, curcumin was added to 0, 5, 10 μM and cultured for 3 hours. Similarly, resveratrol was added to 0, 20, and 40 μM and cultured for 3 hours.
After completion of the culture, the cells were collected, disrupted by a conventional method, and the amount of DJ-1 protein in the cells was detected by Western blotting. Similarly, intracellular actin protein was detected by Western blotting, and the amount of DJ-1 protein relative to the amount of actin protein was calculated, and this was used as the amount of DJ-1 protein produced.
（２）結果
クルクミンによる産生促進試験結果を図１、レスベラトロールによる産生促進試験結果を図２に示す。
クルクミン、レスベラトロールは、HaCaT細胞に対して作用し、ＤＪ−１タンパク質産生を促進することが確認できた。
(2) Results FIG. 1 shows the results of the production promotion test using curcumin, and FIG. 2 shows the results of the production promotion test using resveratrol.
It was confirmed that curcumin and resveratrol act on HaCaT cells and promote DJ-1 protein production.
２．試験例２（細胞への処理時間を６時間に延長）
（１）試験方法
６ｗｅｌｌ ｐｌａｔｅに正常ヒト表皮角化細胞を播種して培養し、２日後にクルクミンを０、１、３、１０、３０μＭになるように添加し、６時間培養した。また同様にレスベラトロールを０、０．００１、０．０１％になるように添加し、さらに６時間培養した。
培養終了後細胞を回収し、常法によって細胞を破壊して細胞内のＤＪ−１タンパク質量をウエスタンブロット法により検出した。サンプルを添加していない細胞のＤＪ−１タンパク質発現量を１として、それに対する比率で表記した。
2. Test Example 2 (Processing time for cells extended to 6 hours)
(1) Test method Normal human epidermal keratinocytes were seeded on a 6-well plate and cultured, and after 2 days, curcumin was added to 0, 1, 3, 10, and 30 μM and cultured for 6 hours. Similarly, resveratrol was added to 0, 0.001, and 0.01%, and further cultured for 6 hours.
After completion of the culture, the cells were collected, disrupted by a conventional method, and the amount of DJ-1 protein in the cells was detected by Western blotting. The expression level of DJ-1 protein in the cells to which no sample was added was defined as 1, and the ratio was expressed as a ratio.
（２）結果
クルクミンによる産生促進試験結果を図３、レスベラトロールによる産生促進試験結果を図４に示す。
試験例１で用いたHaCaT細胞と異なり正常ヒト表皮角化細胞の結果であるが、ともに表皮角化細胞であり、クルクミン、レスベラトロールの処理時間を延長した場合も、ヒト皮膚細胞に対して作用し、ＤＪ−１タンパク質産生を促進することが確認できた。
またレスベラトロール及びクルクミンは、紫外線などの外部刺激に対する抵抗性を向上させるため、紫外線などの外部刺激に対する抵抗性向上剤として使用できる。
(2) Results FIG. 3 shows the results of the production promotion test using curcumin, and FIG. 4 shows the results of the production promotion test using resveratrol.
Unlike the HaCaT cells used in Test Example 1, these are the results of normal human epidermal keratinocytes, but both are epidermal keratinocytes, and even when the treatment time of curcumin and resveratrol is extended, It was confirmed that it acts and promotes the production of DJ-1 protein.
Resveratrol and curcumin can be used as a resistance improver for external stimuli such as ultraviolet rays because they improve resistance to external stimuli such as ultraviolet rays.
３．試験例３（細胞の生存率に対する効果）
（１）試験方法
３５ｍｍディッシュに正常ヒト表皮角化細胞を播種し、１日後にクルクミンもしくはレスベラトロールを添加した。添加量は、クルクミンを０、１、３μＭ、レスベラトロールを０、０．０００１、０．００１％になるよう添加した。２４時間後に細胞をＰＢＳ（−）で洗浄し、ＨＡＮＫＳ液に置換してＵＶＢ１５ｍＪ／ｃｍ２を照射した。なお、細胞の生存率は、照射２４時間後にＡｌａｍａｒＢｌｕｅ ａｓｓａｙを行い、ＵＶＢ無照射、サンプル無添加細胞の蛍光強度を１として、それに対する比率で表記した。
3. Test Example 3 (Effect on cell viability)
(1) Test method Normal human epidermal keratinocytes were seeded in a 35 mm dish, and curcumin or resveratrol was added one day later. The addition amount was 0, 1, 3 μM for curcumin and 0, 0.0001, 0.001% for resveratrol. After 24 hours, the cells were washed with PBS (−), replaced with HANKS solution, and irradiated with UVB 15 mJ / cm 2 . In addition, the survival rate of the cells was expressed as a ratio with respect to the case where AlamarBlue assay was performed 24 hours after irradiation and the fluorescence intensity of the cells without UVB irradiation and without sample addition was 1.
（２）結果
クルクミンによる細胞生存率測定結果を図５、レスベラトロールによる細胞生存率測定結果を図６に示す。
クルクミン、レスベラトロールとも、紫外線照射後の細胞生存率が向上した。特にレスベラトロール０．００１質量％を添加した場合の細胞生存率は、ＵＶＢ無照射を上回った。
したがってクルクミン及びレスベラトロールは皮膚表皮細胞に対する紫外線などの外部刺激に対する抵抗性を改善することが明らかとなった。
(2) Results FIG. 5 shows the cell viability measurement results with curcumin, and FIG. 6 shows the cell viability measurement results with resveratrol.
Both curcumin and resveratrol improved cell viability after UV irradiation. In particular, the cell viability when resveratrol was added at 0.001% by mass exceeded that without UVB irradiation.
Therefore, it was revealed that curcumin and resveratrol improve the resistance to external stimuli such as ultraviolet rays on skin epidermal cells.
Claims (5)
Resistance improver against redness (redness) induced by UV exposure.
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