JP2015178493A - Tropoelastin expression promoter and elastin production promoter - Google Patents
Tropoelastin expression promoter and elastin production promoter Download PDFInfo
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- JP2015178493A JP2015178493A JP2015038676A JP2015038676A JP2015178493A JP 2015178493 A JP2015178493 A JP 2015178493A JP 2015038676 A JP2015038676 A JP 2015038676A JP 2015038676 A JP2015038676 A JP 2015038676A JP 2015178493 A JP2015178493 A JP 2015178493A
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- Prior art keywords
- tropoelastin
- elastin
- expression
- chondroitin disaccharide
- trisulfate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Landscapes
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Abstract
Description
本発明は、細胞、特に皮膚線維芽細胞におけるトロポエラスチンの発現を促進し、ひいてはエラスチンの産生を促進する、トロポエラスチン発現促進剤およびエラスチン産生促進剤に関する。 The present invention relates to a tropoelastin expression promoter and an elastin production promoter that promote the expression of tropoelastin in cells, particularly skin fibroblasts, and thus promote the production of elastin.
加齢や紫外線等による皮膚の老化には、皮膚真皮の細胞外マトリックス成分の減少や変性が関与することが知られている。
主な細胞外マトリックス成分としては、コラーゲン、エラスチン、フィブロネクチン、ラミニン等の線維状タンパク質、およびヒアルロン酸等のムコ多糖類が知られている。
It is known that the aging of skin due to aging, ultraviolet rays, etc. involves the reduction or degeneration of extracellular matrix components of the dermis.
As main extracellular matrix components, fibrous proteins such as collagen, elastin, fibronectin and laminin, and mucopolysaccharides such as hyaluronic acid are known.
上記細胞外マトリックス成分のうち、コラーゲンまたはヒアルロン酸に対し、軟骨や関節の滑液に含まれるムコ多糖類であるコンドロイチン硫酸が産生促進作用を有することが知られ、コンドロイチン硫酸を有効成分として含有するコラーゲン産生促進剤、およびヒアルロン酸産生促進剤が開示されている（特許文献１、２）。
一方、同じ皮膚真皮の細胞外マトリックス成分であるエラスチンに対しては、ラット新生児の肺線維芽細胞において、不溶性エラスチンの沈着が減少すること、ケロイド線維芽細胞において、コンドロイチン硫酸により、細胞外マトリックスへのエラスチンの蓄積が減少することが報告されている（非特許文献１、２）。
Among the extracellular matrix components described above, chondroitin sulfate, a mucopolysaccharide contained in cartilage and joint synovial fluid, is known to have a production promoting action on collagen or hyaluronic acid, and contains chondroitin sulfate as an active ingredient. Collagen production promoters and hyaluronic acid production promoters are disclosed (Patent Documents 1 and 2).
On the other hand, elastin, an extracellular matrix component of the same dermal dermis, reduces the deposition of insoluble elastin in rat neonatal lung fibroblasts. In keloid fibroblasts, chondroitin sulphate leads to the extracellular matrix. It has been reported that the accumulation of elastin is reduced (Non-patent Documents 1 and 2).
エラスチンは、細胞外マトリックスを構成する弾性線維の主成分であり、組織に弾力性および柔軟性を付加する。特に、皮膚真皮においては、皮膚に弾力性を与え、皮膚のはりを保持する機能を有する。加齢によるエラスチン線維の減少は、皮膚のたるみやしわといった老化症状の大きな要因となるといわれている。また、紫外線による光老化では、真皮網状層のエラスチン線維が変性沈着して、弾力性がなく硬化したしわの多い光老化皮膚の原因となるといわれている。 Elastin is the main component of the elastic fibers that make up the extracellular matrix and adds elasticity and flexibility to the tissue. In particular, the skin dermis has a function of imparting elasticity to the skin and holding the skin beam. It is said that a decrease in elastin fibers due to aging is a major factor in aging symptoms such as skin sagging and wrinkles. In addition, it is said that photoaging with ultraviolet rays causes elastin fibers in the dermal reticulate layer to degenerate and cause photo-aging skin that is hard and wrinkled without elasticity.
皮膚の老化の進行に伴う上記のようなエラスチンの減少や変性を防止すべく、エラスチンを分解する酵素であるエラスターゼの活性を阻害し、また、エラスターゼの産生を抑制する物質のスクリーニングが行われてきた。また、細胞外マトリックスにおけるエラスチンの生成を促進する物質の探求も行われている。 In order to prevent the above-mentioned decrease and degeneration of elastin due to the progress of skin aging, screening of substances that inhibit the activity of elastase, an enzyme that degrades elastin, and suppresses the production of elastase has been performed. It was. There is also a search for substances that promote the production of elastin in the extracellular matrix.
しかしながら、エラスターゼ活性阻害作用またはエラスターゼ産生抑制作用、あるいはエラスチンの産生促進作用を有する成分は、多くが植物抽出物等の天然由来成分に求められており、その有効性、安定性、経皮吸収性等の観点から、十分満足できるものが得られているとは言い難い。 However, many components having an elastase activity inhibitory effect, an elastase production inhibitory effect, or an elastin production promoting effect are required for naturally derived ingredients such as plant extracts, and their effectiveness, stability, and transdermal absorbability. From such viewpoints, it is hard to say that satisfactory products are obtained.
本発明は、細胞外マトリックスにおけるエラスチンの産生を促進するため、エラスチンの前駆体であるトロポエラスチンの発現を促進し、ひいてはエラスチンの産生を促進し得るトロポエラスチン発現促進剤およびエラスチン産生促進剤を提供することを目的とする。 The present invention relates to a tropoelastin expression promoter and an elastin production promoter capable of promoting the expression of tropoelastin, which is a precursor of elastin, and thus promoting the production of elastin in order to promote the production of elastin in the extracellular matrix. The purpose is to provide.
本発明者は、上記課題を解決すべく鋭意検討した結果、コンドロイチン二糖、すなわちコンドロイチンの構成単位であるグルクロン酸とＮ−アセチルガラクトサミンよりなる二糖、のトリ硫酸エステルまたはその塩が、トロポエラスチンの発現を促進することで細胞外マトリックスにおけるエラスチンの産生を促進することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a trisulfate ester or a salt of chondroitin disaccharide, that is, a disaccharide composed of glucuronic acid and N-acetylgalactosamine, which are constituent units of chondroitin, is tropotropin. The inventors have found that elastin expression is promoted to promote the production of elastin in the extracellular matrix, and the present invention has been completed.
すなわち、本発明は、次の［１］〜［２１］に関する。
［１］コンドロイチン二糖のトリ硫酸エステルまたはその塩を含有する、トロポエラスチン発現促進剤。
［２］コンドロイチン二糖が、下記式（１）で示されるＮ−アセチルコンドロシンまたは下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体である、上記［１］に記載のトロポエラスチン発現促進剤。
That is, the present invention relates to the following [1] to [21].
[1] A tropoelastin expression promoter containing a chondroitin disaccharide trisulfate or a salt thereof.
[2] The toro according to the above [1], wherein the chondroitin disaccharide is a chemical modification of N-acetylchondrosin represented by the following formula (1) or N-acetylchondrosin represented by the following formula (2): Poelastin expression promoter.
［３］コンドロイチン二糖のトリ硫酸エステルが、コンドロイチン二糖のグルクロン酸残基の２位の水酸基、ならびにＮ−アセチルガラクトサミン残基の４位および６位の水酸基に硫酸基がエステル結合したものである、上記［１］または［２］に記載のトロポエラスチン発現促進剤。
［４］皮膚線維芽細胞のトロポエラスチンの発現を促進する、上記［１］〜［３］のいずれかに記載のトロポエラスチン発現促進剤。
［５］皮膚の老化の防止または改善用である、上記［１］〜［４］のいずれかに記載のトロポエラスチン発現促進剤。
［６］皮膚外用医薬品、皮膚外用医薬部外品または皮膚用化粧品である、上記［１］〜［５］のいずれかに記載のトロポエラスチン発現促進剤。
［７］コンドロイチン二糖のトリ硫酸エステルまたはその塩を含有する、エラスチン産生促進剤。
［８］コンドロイチン二糖が、下記式（１）で示されるＮ−アセチルコンドロシンまたは下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体である、上記［７］に記載のエラスチン産生促進剤。
[3] A trisulfate ester of chondroitin disaccharide is obtained by esterifying a sulfate group to the hydroxyl group at the 2-position of the glucuronic acid residue of the chondroitin disaccharide and the hydroxyl groups at the 4-position and 6-position of the N-acetylgalactosamine residue. The tropoelastin expression promoter according to [1] or [2] above.
[4] The tropoelastin expression promoter according to any one of the above [1] to [3], which promotes the expression of tropoelastin in skin fibroblasts.
[5] The tropoelastin expression promoter according to any one of the above [1] to [4], which is for preventing or improving skin aging.
[6] The tropoelastin expression promoter according to any one of the above [1] to [5], which is a skin external pharmaceutical, a skin external quasi-drug, or a skin cosmetic.
[7] An elastin production promoter comprising a trisulfate ester of chondroitin disaccharide or a salt thereof.
[8] The elastin according to [7] above, wherein the chondroitin disaccharide is an N-acetylchondrosin represented by the following formula (1) or a chemical modification of the N-acetylchondrosin represented by the following formula (2): Production promoter.
［９］コンドロイチン二糖のトリ硫酸エステルが、コンドロイチン二糖のグルクロン酸残基の２位の水酸基、ならびにＮ−アセチルガラクトサミン残基の４位および６位の水酸基に硫酸基がエステル結合したものである、上記［７］または［８］に記載のエラスチン産生促進剤。
［１０］皮膚真皮の細胞外マトリックスにおけるエラスチンの産生を促進する、上記［７］〜［９］のいずれかに記載のエラスチン産生促進剤。
［１１］皮膚の老化の防止または改善用である、上記［７］〜［１０］のいずれかに記載のエラスチン産生促進剤。
［１２］皮膚外用医薬品、皮膚外用医薬部外品または皮膚用化粧品である、上記［７］〜［１１］のいずれかに記載のエラスチン産生促進剤。
［１３］細胞にコンドロイチン二糖のトリ硫酸エステルまたはその塩を作用させることによる、トロポエラスチンの発現の促進方法。
［１４］コンドロイチン二糖が、下記式（１）で示されるＮ−アセチルコンドロシンまたは下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体である、上記［１３］に記載の促進方法。
[9] A trisulfate ester of chondroitin disaccharide is obtained by esterifying a sulfate group to the hydroxyl group at the 2-position of the glucuronic acid residue of the chondroitin disaccharide and the hydroxyl groups at the 4-position and 6-position of the N-acetylgalactosamine residue. The elastin production promoter according to [7] or [8] above.
[10] The elastin production promoter according to any one of [7] to [9], which promotes the production of elastin in the extracellular matrix of the skin dermis.
[11] The elastin production promoter according to any one of the above [7] to [10], which is used for preventing or improving skin aging.
[12] The elastin production promoter according to any one of the above [7] to [11], which is a skin external medicine, a skin external quasi-drug, or a skin cosmetic.
[13] A method of promoting the expression of tropoelastin by allowing a cell to act on a trisulfate ester of chondroitin disaccharide or a salt thereof.
[14] The promotion according to [13] above, wherein the chondroitin disaccharide is an N-acetylchondrosin represented by the following formula (1) or a chemical modification of the N-acetylchondrosin represented by the following formula (2): Method.
［１５］コンドロイチン二糖のトリ硫酸エステルが、コンドロイチン二糖のグルクロン酸残基の２位の水酸基、ならびにＮ−アセチルガラクトサミン残基の４位および６位の水酸基に硫酸基がエステル結合したものである、上記［１３］または［１４］に記載の促進方法。
［１６］細胞が皮膚線維芽細胞である、上記［１３］〜［１５］のいずれかに記載の促進方法。
［１７］細胞にコンドロイチン二糖のトリ硫酸エステルまたはその塩を作用させ、トロポエラスチンの発現を促進することによる、エラスチン産生の促進方法。
［１８］コンドロイチン二糖が、下記式（１）で示されるＮ−アセチルコンドロシンまたは下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体である、上記［１７］に記載の促進方法。
[15] A trisulfate ester of chondroitin disaccharide is obtained by esterifying a sulfate group to the hydroxyl group at the 2-position of the glucuronic acid residue of the chondroitin disaccharide and the hydroxyl groups at the 4-position and 6-position of the N-acetylgalactosamine residue. The promotion method according to [13] or [14] above.
[16] The promotion method according to any one of [13] to [15], wherein the cell is a dermal fibroblast.
[17] A method for promoting elastin production by causing chondroitin disaccharide trisulfate or a salt thereof to act on cells to promote the expression of tropoelastin.
[18] The promotion according to [17] above, wherein the chondroitin disaccharide is a chemical modification of N-acetylchondrosin represented by the following formula (1) or N-acetylchondrosin represented by the following formula (2): Method.
［１９］コンドロイチン二糖のトリ硫酸エステルが、コンドロイチン二糖のグルクロン酸残基の２位の水酸基、ならびにＮ−アセチルガラクトサミン残基の４位および６位の水酸基に硫酸基がエステル結合したものである、上記［１７］または［１８］に記載の促進方法。
［２０］細胞が皮膚線維芽細胞である、上記［１７］〜［１９］のいずれかに記載の促進方法。
［２１］上記［１７］〜［２０］のいずれかに記載の促進方法によって得られたエラスチン。
[19] The trisulfate ester of chondroitin disaccharide is an ester bond of a sulfate group to the hydroxyl group at the 2-position of the glucuronic acid residue of the chondroitin disaccharide and the hydroxyl groups at the 4-position and 6-position of the N-acetylgalactosamine residue. The promotion method according to [17] or [18] above.
[20] The promotion method according to any one of [17] to [19], wherein the cells are dermal fibroblasts.
[21] Elastin obtained by the promotion method according to any one of [17] to [20].
本発明に係るトロポエラスチン発現促進剤およびエラスチン産生促進剤は、トロポエラスチン遺伝子の発現を増加させて、エラスチンの前駆体であるトロポエラスチンタンパク質の発現を増加させることができ、皮膚真皮の細胞外マトリックス等において、エラスチン線維の産生を促進することができる。
また、本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、経皮吸収性に優れ、エラスチンの減少、変性による皮膚の老化を有効に防止し、または改善することができる。
さらに、本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、皮膚線維芽細胞等の細胞に作用させて、該細胞のトロポエラスチンの発現を促進してエラスチンの産生を促進することができ、皮膚シートの生成等、再生医療分野においても有用である。
The tropoelastin expression promoter and elastin production promoter according to the present invention can increase the expression of the tropoelastin gene and increase the expression of the tropoelastin protein, which is a precursor of elastin. Production of elastin fibers can be promoted in the extracellular matrix or the like.
Moreover, the tropoelastin expression promoter and elastin production promoter of the present invention are excellent in transdermal absorbability, and can effectively prevent or improve skin aging due to a decrease in elastin and degeneration.
Furthermore, the tropoelastin expression promoter and elastin production promoter of the present invention can act on cells such as dermal fibroblasts to promote the expression of tropoelastin in the cells and promote the production of elastin. It is also useful in the field of regenerative medicine, such as production of skin sheets.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、コンドロイチン二糖のトリ硫酸エステルまたはその塩を有効成分として含有する。 The tropoelastin expression promoter and elastin production promoter of the present invention contain chondroitin disaccharide trisulfate or a salt thereof as an active ingredient.
コンドロイチン二糖は、グルクロン酸とＮ−アセチルガラクトサミンがβ−１，３−結合してなる二糖であり、コンドロイチンの構成単位である。
本発明において、コンドロイチン二糖のトリ硫酸エステルを構成するコンドロイチン二糖としては、下記式（１）で示されるＮ−アセチルコンドロシン［３−Ｏ−β−Ｄ−グルコピラヌロノシル−２−（アセチルアミノ）−２−デオキシ−β−Ｄ−ガラクトピラノース］、および下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体が挙げられる。下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体は、Ｎ−アセチルコンドロシンのグルクロン酸残基の４位水酸基が水素に置換され、４位と５位の間の結合が二重結合となった化合物である。
Chondroitin disaccharide is a disaccharide formed by glucuronic acid and N-acetylgalactosamine in a β-1,3-linkage, and is a constituent unit of chondroitin.
In the present invention, the chondroitin disaccharide constituting the trisulfate ester of chondroitin disaccharide is N-acetylchondrosin represented by the following formula (1) [3-O-β-D-glucopyranuronosyl-2- (Acetylamino) -2-deoxy-β-D-galactopyranose], and a chemically modified form of N-acetylchondrosin represented by the following formula (2). In the chemically modified form of N-acetylchondrosin represented by the following formula (2), the 4-position hydroxyl group of the glucuronic acid residue of N-acetylchondrosin is substituted with hydrogen, and the bond between the 4-position and 5-position is two. It is a compound that has become a double bond.
本発明においては、上記Ｎ−アセチルコンドロシンまたはその化学修飾体のトリ硫酸エステルを好適に用いることができる。 In the present invention, the above-described trisulfate ester of N-acetylchondrosin or a chemically modified product thereof can be suitably used.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤に含有されるコンドロイチン二糖のトリ硫酸エステルとしては、コンドロイチン二糖のグルクロン酸残基の２位の水酸基、ならびにＮ−アセチルガラクトサミン残基の４位および６位の水酸基に硫酸基がエステル結合したものが好ましく用いられる。
また、本発明においては、コンドロイチン二糖のトリ硫酸エステルの塩としては、薬学的に許容される塩であれば、特に限定されることなく用いることができるが、ナトリウム塩、カリウム塩等のアルカリ金属塩、マグネシウム塩等のアルカリ土類金属塩、アンモニウム塩等が、好ましい塩として例示される。
Examples of the trisulfate ester of chondroitin disaccharide contained in the tropoelastin expression promoter and elastin production promoter of the present invention include the hydroxyl group at the 2-position of the glucuronic acid residue of the chondroitin disaccharide and the N-acetylgalactosamine residue. Those in which a sulfate group is ester-bonded to the hydroxyl groups at the 4-position and the 6-position are preferably used.
In the present invention, the trisulfate ester salt of chondroitin disaccharide can be used without particular limitation as long as it is a pharmaceutically acceptable salt, but an alkali such as sodium salt or potassium salt can be used. Examples of preferable salts include alkaline earth metal salts such as metal salts and magnesium salts, and ammonium salts.
コンドロイチン二糖のトリ硫酸エステルは、コンドロイチナーゼＡＢＣ等、コンドロイチン硫酸分解酵素を用いて、種々のタイプのコンドロイチン硫酸を分解する方法等、自体公知の方法によって製造することができるが、デキストラ ラボラトリーズ（ＤＥＸＴＲＡ ＬＡＢＯＲＡＴＯＲＩＥＳ）社等より提供されている市販の製品を用いることが便利である。 Chondroitin disaccharide trisulfate can be produced by a method known per se such as chondroitinase ABC or a method of degrading various types of chondroitin sulfate using a chondroitin sulfate degrading enzyme. It is convenient to use a commercially available product provided by DEXTRA LABORATORIES.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤には、上記したコンドロイチン二糖のトリ硫酸エステル、およびその塩からなる群より１種を選択し、単独で含有させてもよく、２種以上を選択し、組み合わせて含有させてもよい。
本発明のトロポエラスチン発現促進剤またはエラスチン産生促進剤におけるコンドロイチン二糖のトリ硫酸エステルまたはその塩の含有量は、その剤形の種類等にもよるが、トロポエラスチン発現促進効果またはエラスチン産生促進効果および製剤安定性等を考慮すると、トロポエラスチン発現促進剤またはエラスチン産生促進剤の全量に対し、コンドロイチン二糖のトリ硫酸エステルの量として０．００１重量％〜１０重量％とすることが好ましく、０．０１重量％〜５重量％とすることがより好ましく、０．１重量％〜３重量％とすることがさらに好ましい。
The tropoelastin expression promoter and elastin production promoter of the present invention may be selected from the group consisting of the above-described chondroitin disaccharide trisulfate and its salt, and may be contained alone. The above may be selected and contained in combination.
The content of the trisulfate ester of chondroitin disaccharide or a salt thereof in the tropoelastin expression promoter or elastin production promoter of the present invention depends on the type of dosage form, etc., but the tropoelastin expression promoting effect or elastin production In consideration of the promoting effect and formulation stability, the amount of chondroitin disaccharide trisulfate may be 0.001 wt% to 10 wt% with respect to the total amount of the tropoelastin expression promoter or elastin production promoter. Preferably, it is more preferable to set it as 0.01 weight%-5 weight%, and it is further more preferable to set it as 0.1 weight%-3 weight%.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、一般的な製剤化方法、たとえば第１６改正日本薬局方製剤総則に記載された方法に従い、製剤化に際して一般的に用いられる溶剤または基剤に、有効成分であるコンドロイチン二糖のトリ硫酸エステルまたはその塩を加え、必要に応じて賦形剤、乳化剤、溶解補助剤、懸濁化剤等の添加剤等を加えて混合し、均質に溶解、分散または懸濁して調製することができ、液剤、ゲル剤、クリーム剤等の半固形剤、固形剤、スプレー剤等、種々の剤形で提供することができる。 The tropoelastin expression promoter and elastin production promoter of the present invention are prepared by a general formulation method, for example, a solvent or a group generally used for formulation according to the method described in the 16th revised Japanese Pharmacopoeia Formulation General Rules. Add the active ingredient, chondroitin disaccharide trisulfate or its salt, and add additives such as excipients, emulsifiers, solubilizers, suspending agents, etc. It can be prepared by dissolving, dispersing or suspending in various forms such as a liquid agent, a gel agent, a semi-solid agent such as a cream agent, a solid agent, a spray agent and the like.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、エラスチンの前駆体であるトロポエラスチンの遺伝子の発現を促進し、トロポエラスチンタンパク質の生成を促進することにより、エラスチンの産生を促進する。それゆえ、エラスチン線維の再構築効果が期待される。
特に、皮膚線維芽細胞のトロポエラスチン発現の促進に有効であり、皮膚真皮の細胞外マトリックスにおけるエラスチン産生の促進、エラスチン線維の再構築に有用である。
従って、本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、しわ、たるみ等、エラスチンの減少または変性に起因する皮膚の老化症状の発現、進行を防止し、または前記老化症状を改善するために好ましく用いられる。
The tropoelastin expression promoter and elastin production promoter of the present invention promote the production of elastin by promoting the expression of the tropoelastin gene, which is a precursor of elastin, and promoting the production of tropoelastin protein. To do. Therefore, an elastin fiber reconstruction effect is expected.
In particular, it is effective for promoting the expression of tropoelastin in dermal fibroblasts, and is useful for promoting the production of elastin in the extracellular matrix of skin dermis and reconstructing elastin fibers.
Therefore, the tropoelastin expression promoter and elastin production promoter of the present invention prevent the development and progression of skin aging symptoms caused by the decrease or degeneration of elastin, such as wrinkles and sagging, or improve the aging symptoms. Therefore, it is preferably used.
よって、本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、皮膚外用医薬品、皮膚外用医薬部外品または皮膚用化粧品として、好ましく提供することができる。 Therefore, the tropoelastin expression promoter and elastin production promoter of the present invention can be preferably provided as a skin external medicine, a skin external quasi-drug, or a skin cosmetic.
皮膚外用医薬品としては、外用散剤等の外用固形剤；ローション剤、リニメント剤等の外用液剤；外用エアゾール剤、ポンプスプレー剤等のスプレー剤；油脂性軟膏剤、水溶性軟膏剤等の軟膏剤；水中油型または油中水型のクリーム剤；水性または油性ゲル剤；テープ剤またはパップ剤等の貼付剤等が挙げられる。これらは、一般的な溶剤または基剤を用い、必要に応じて一般的な添加剤を加え、また、必要に応じて支持体、剥離ライナー等を用いて、一般的な皮膚外用医薬品の製造方法、たとえば第１６改正日本薬局方製剤総則「皮膚などに適用する製剤」に記載された方法に準じて調製される。 As a skin external medicine, an external solid preparation such as an external powder; an external liquid such as a lotion or a liniment; a spray such as an external aerosol or a pump spray; an ointment such as an oily ointment or a water-soluble ointment; Examples include oil-in-water or water-in-oil creams; aqueous or oily gels; patches such as tapes or poultices. These use a general solvent or base, add general additives as necessary, and use a support, release liner, etc., as necessary, to produce a general skin external medicine. For example, it is prepared according to the method described in the 16th revised Japanese Pharmacopoeia General Rules for Preparations Applied to Skin and the like.
上記溶剤としては、精製水、エタノール、イソプロパノール、ジプロピレングリコール等が挙げられ、これらより１種または２種以上を選択して用いることができる。
上記基剤としては、オリーブ油、ダイズ油、ツバキ油、ゴマ油、落花生油、カカオ脂、牛脂、豚脂等の動植物性油脂；カルナウバロウ、ミツロウ等のロウ；オクチルドデカノール、セタノール、ステアリルアルコール等の脂肪族アルコール；オレイン酸、パルミチン酸、ステアリン酸等の脂肪酸；スクワラン、白色ワセリン、流動パラフィン、セレシン、マイクロクリスタリンワックス等の炭化水素といった油脂性基剤、ゼラチン、マクロゴール等の親水性基剤が挙げられ、これらより１種または２種以上を選択して用いることができる。
Examples of the solvent include purified water, ethanol, isopropanol, dipropylene glycol, and the like, and one or more of them can be selected and used.
As the above-mentioned base, animal and vegetable oils such as olive oil, soybean oil, camellia oil, sesame oil, peanut oil, cacao butter, beef tallow and pork fat; wax such as carnauba wax and beeswax; fat such as octyldodecanol, cetanol and stearyl alcohol Aliphatic alcohols; fatty acids such as oleic acid, palmitic acid, stearic acid; and oily bases such as squalane, white petrolatum, liquid paraffin, ceresin, and microcrystalline wax, and hydrophilic bases such as gelatin and macrogol From these, one or more can be selected and used.
また、上記添加剤としては、軽質無水ケイ酸、結晶セルロース、デキストリン等の賦形剤；アジピン酸ジイソプロピル、カプリン酸、クロタミトン、炭酸プロピレン等の溶解補助剤；アルギン酸プロピレングリコールエステル、ジオクチルスルホコハク酸ナトリウム、大豆レシチン、ポビドン等の懸濁化剤；ポリオキシエチレン硬化ヒマシ油、ポリオキシエチレンソルビタンモノラウレート、ポリオキシエチレンソルビタンモノステアレート、ポリオキシエチレンソルビタンモノオレエート、グリセリンモノステアレート、ショ糖脂肪酸エステル等の界面活性剤または乳化剤；カルボキシビニルポリマー、キサンタンガム、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ポリビニルアルコール（部分ケン化物）等の粘稠剤；トリアセチン、ミリスチン酸イソプロピル等の可塑剤；グリセリン、１，３−ブチレングリコール、ＤＬ−ピロリドンカルボン酸ナトリウム、ヒアルロン酸ナトリウム等の保湿剤；エデト酸ナトリウム、ソルビトール、チモール、ポリオキシエチレンポリオキシプロピレングリコール等の安定化剤；アスコルビン酸、エリソルビン酸ナトリウム、酢酸トコフェロール、ジブチルヒドロキシトルエン等の抗酸化剤；ソルビン酸、デヒドロ酢酸ナトリウム、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸ブチル等の保存剤；塩酸、クエン酸、クエン酸ナトリウム、酢酸、酢酸ナトリウム、水酸化ナトリウム、リン酸水素ナトリウム等のｐＨ調整剤などが挙げられ、必要に応じて、１種または２種以上を選択して用いる。 Examples of the additive include light anhydrous silicic acid, crystalline cellulose, dextrin and the like; solubilization aids such as diisopropyl adipate, capric acid, crotamiton, propylene carbonate; propylene glycol alginate, sodium dioctylsulfosuccinate, Suspending agents such as soybean lecithin and povidone; polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, glycerin monostearate, sucrose fatty acid Surfactants or emulsifiers such as esters; thickeners such as carboxyvinyl polymer, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, polyvinyl alcohol (partially saponified product); Plasticizers such as riacetin and isopropyl myristate; humectants such as glycerin, 1,3-butylene glycol, sodium DL-pyrrolidonecarboxylate and sodium hyaluronate; sodium edetate, sorbitol, thymol, polyoxyethylene polyoxypropylene glycol, etc. Stabilizers such as ascorbic acid, sodium erythorbate, tocopherol acetate, dibutylhydroxytoluene, etc .; preservatives such as sorbic acid, sodium dehydroacetate, methyl parahydroxybenzoate, ethyl paraoxybenzoate, butyl paraoxybenzoate; Examples include pH adjusters such as hydrochloric acid, citric acid, sodium citrate, acetic acid, sodium acetate, sodium hydroxide, sodium hydrogen phosphate, etc. .
なお、本発明のトロポエラスチン発現促進剤またはエラスチン産生促進剤が皮膚外用医薬品として提供される場合には、本発明の特徴を損なわない範囲で、コエンザイムＱ１０、デオキシリボ核酸ナトリウム、アシタバ抽出物、クロレラ抽出物、酵母抽出物、オタネニンジン抽出物、ヒバマタ抽出物、ロイヤルゼリー抽出物等の細胞賦活剤；加水分解シルク、カッコン抽出物、シャクヤク抽出物、卵殻膜タンパク質、レチノール等の抗シワ・抗老化剤；アズレン、アラントイン、カミツレ抽出物、カンゾウ抽出物、キダチアロエ抽出物、グリチルリチン酸等の抗炎症・肌荒れ防止剤等を含有させることができる。 In addition, when the tropoelastin expression promoter or elastin production promoter of the present invention is provided as a skin external medicine, coenzyme Q 10 , deoxyribonucleic acid sodium, acitaba extract, as long as the characteristics of the present invention are not impaired, Cell activators such as chlorella extract, yeast extract, ginseng extract, hibamata extract, royal jelly extract; hydrolyzed silk, cuckoo extract, peonies extract, eggshell membrane protein, retinol and other anti-wrinkle / anti-aging Agents: Azulene, allantoin, chamomile extract, licorice extract, Kidachi aloe extract, anti-inflammatory / rough skin prevention agent such as glycyrrhizic acid and the like can be contained.
本発明のトロポエラスチン発現促進剤またはエラスチン産生促進剤が皮膚外用医薬品として提供される場合において、その適用量は、トロポエラスチン発現促進剤またはエラスチン産生促進剤の剤形、対象となる患者の性別、年齢、皮膚の老化症状やその程度等により異なるが、コンドロイチン二糖のトリ硫酸エステルの量として、好ましくは１日当たり０．１μｇ／ｃｍ２〜２００μｇ／ｃｍ２であり、より好ましくは１μｇ／ｃｍ２〜５０μｇ／ｃｍ２である。前記の量は、１回で適用してもよく、数回に分けて適用してもよい。 In the case where the tropoelastin expression promoter or elastin production promoter of the present invention is provided as a skin external medicine, the amount applied is the dosage form of the tropoelastin expression promoter or elastin production promoter, the target patient sex, age, it varies by aging symptoms and the degree or the like of the skin, as the amount of tri-sulfate of chondroitin disaccharide, preferably 1 day 0.1μg / cm 2 ~200μg / cm 2 , more preferably 1 [mu] g / cm 2 to 50 μg / cm 2 . The above amount may be applied once, or may be applied in several times.
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、皮膚外用医薬部外品または皮膚用化粧品としても提供することができる。かかる医薬部外品または化粧品は、皮膚外用医薬品に準じて製造することができ、化粧水、美容液、乳液、クリーム、パック等の形態で提供され得る。 The tropoelastin expression promoter and elastin production promoter of the present invention can also be provided as a skin quasi-drug or a skin cosmetic. Such quasi-drugs or cosmetics can be produced according to skin external medicines and can be provided in the form of lotion, cosmetic liquid, milky lotion, cream, pack and the like.
また、本発明は、上記コンドロイチン二糖のトリ硫酸エステルまたはその塩を細胞に作用させて、トロポエラスチンの発現を促進する方法を提供する。
さらに、本発明は、上記コンドロイチン二糖のトリ硫酸エステルまたはその塩を細胞に作用させ、トロポエラスチンの発現を促進することにより、エラスチン産生を促進する方法を提供する。
コンドロイチン二糖のトリ硫酸エステルまたはその塩を作用させる細胞としては、皮膚線維芽細胞が好ましく、皮膚線維芽細胞にコンドロイチン二糖のトリ硫酸エステルまたはその塩を作用させることにより、皮膚線維芽細胞において、エラスチンの前駆体であるトロポエラスチンの発現を促進することができるため、皮膚真皮の細胞外マトリックスにおけるエラスチン産生を促進し、前記細胞外マトリックスを構成する弾性線維の主成分であるエラスチン線維の構築を図ることができる。
The present invention also provides a method of promoting the expression of tropoelastin by allowing the above-mentioned chondroitin disaccharide trisulfate or a salt thereof to act on cells.
Furthermore, the present invention provides a method of promoting elastin production by causing the above-described chondroitin disaccharide trisulfate or a salt thereof to act on cells to promote the expression of tropoelastin.
Skin fibroblasts are preferred as the cells that act on chondroitin disaccharide trisulfate or its salt. In skin fibroblasts, chondroitin disaccharide trisulfate or its salt acts on skin fibroblasts. Since it can promote the expression of tropoelastin, a precursor of elastin, it promotes the production of elastin in the extracellular matrix of skin dermis, and the elastin fibers that are the main components of the elastic fibers that constitute the extracellular matrix Can be built.
皮膚線維芽細胞におけるトロポエラスチンの発現を促進させるためには、コンドロイチン二糖のトリ硫酸エステルまたはその塩は、コンドロイチン二糖のトリ硫酸エステルの量として、皮膚線維芽細胞５×１０４個に対し、０．０２μｇ〜３μｇ作用させることが好ましく、０．０３μｇ〜１μｇ作用させることがより好ましい。 In order to promote the expression of tropoelastin in dermal fibroblasts, chondroitin disaccharide trisulfate or its salt is added to 5 × 10 4 skin fibroblasts as the amount of chondroitin disaccharide trisulfate. On the other hand, it is preferably 0.02 μg to 3 μg, more preferably 0.03 μg to 1 μg.
本発明の上記トロポエラスチンの発現促進方法およびエラスチン産生の促進方法は、医療用皮膚シートの調製等、再生医療分野において有用である。 The tropoelastin expression promoting method and elastin production promoting method of the present invention are useful in the field of regenerative medicine such as preparation of medical skin sheets.
以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.
まず、以下の試験例にて用いた皮膚線維芽細胞の培養方法、および各評価に用いた試料について示す。
なお、以下の試験例にて、特に製品名および提供元を記載しない試薬等は、和光純薬工業株式会社より入手した。
First, the skin fibroblast culture method used in the following test examples and the samples used for each evaluation are shown.
In the following test examples, reagents and the like that do not particularly describe the product name and the provider were obtained from Wako Pure Chemical Industries, Ltd.
（１）皮膚線維芽細胞の培養方法
１０（ｗ／ｖ）％牛胎仔血清添加ダルベッコ修正基礎培地（ＤＭＥＭ−１０％ＦＢＳ）３ｍＬに正常ヒト皮膚線維芽細胞（胎児由来）を５×１０４個播種し、３７℃、５％ＣＯ２にて２４時間インキュベートした。
（２）試料
（ｉ）コンドロイチン二糖トリ硫酸エステル四ナトリウム（ＣＤｉ−ＴｒｉＳ・Ｎａ４）：コンドロイチン二糖（下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体）のグルクロン酸残基の２位、Ｎ−アセチルガラクトサミン残基の４位および６位に硫酸基が結合したトリ硫酸エステルの四ナトリウム塩（「Ｃｈｏｎｄｒｏｉｔｉｎ ｄｉｓａｃｃｈａｒｉｄｅ ΔＤｉ−ｔｒｉＳ，ｓｏｄｉｕｍ ｓａｌｔ」（カタログＮｏ．Ｃ３２０７）、デキストラ ラボラトリーズ（ＤＥＸＴＲＡ ＬＡＢＯＲＡＴＯＲＩＥＳ）社）を用いた。
（ｉｉ）コンドロイチン二糖硫酸エステル二ナトリウム（ＣＤｉ−Ｓ・Ｎａ２）：コンドロイチン二糖（下記式（２）で示されるＮ−アセチルコンドロシンの化学修飾体）のＮ−アセチルガラクトサミン残基の６位に硫酸基が結合した硫酸エステルの二ナトリウム塩（「Ｃｈｏｎｄｒｏｉｔｉｎ dｉｓａｃｃｈａｒｉｄｅ ΔＤｉ−６Ｓ，ｓｏｄｉｕｍ ｓａｌｔ」（カタログＮｏ．Ｃ３２０３）、デキストラ ラボラトリーズ（ＤＥＸＴＲＡ ＬＡＢＯＲＡＴＯＲＩＥＳ）社）を用いた。
(1) Culture method of dermal fibroblasts 5 × 10 4 normal human dermal fibroblasts (fetal origin) in 3 mL of Dulbecco's modified basal medium (DMEM-10% FBS) supplemented with 10 (w / v)% fetal calf serum Seeding and incubating at 37 ° C., 5% CO 2 for 24 hours.
(2) Sample (i) Chondroitin disaccharide trisulfate tetrasodium salt (CDi-TriS · Na 4 ): Glucuronic acid residue of chondroitin disaccharide (chemically modified form of N-acetylchondrosin represented by the following formula (2)) Tetrasodium salt of a trisulfate ester having a sulfate group bonded to the 2nd position of the group, the 4th position and the 6th position of the N-acetylgalactosamine residue (“Chondroitin disaccharide ΔDi-triS, sodium salt” (Catalog No. C3207), (DEXTRA LABORATORIES) was used.
(Ii) Chondroitin disaccharide sulfate disodium (CDi-S · Na 2 ): 6 of the N-acetylgalactosamine residue of chondroitin disaccharide (chemical modification of N-acetylchondrosin represented by the following formula (2)) A disodium salt of a sulfate ester having a sulfate group bonded to the position (“Chondroitin disaccharide ΔDi-6S, sodium salt” (catalog No. C3203), DEXTRA LABORATORIES) was used.
（ｉｉｉ）低分子量の高硫酸化コンドロイチン硫酸（Ｌ−ＣＳ）：グルクロン酸およびＮ−アセチルガラクトサミンよりなる二糖単位がトリ硫酸エステル化されたコンドロイチン硫酸（重量平均分子量＝３，７００Ｄａ）は、特開２０１２−１５７２７１号公報および特開２０１０−０７７２５６号公報に記載された方法に従って調製した。重量平均分子量は、マルトペンタオースおよびプルランを標準試料として用い、ゲル浸透クロマトグラフィー（ＧＰＣ）−示差屈折計（ＲＩ）法により求めた。
（ｉｖ）高分子量の高硫酸化コンドロイチン硫酸（Ｈ−ＣＳ）：グルクロン酸およびＮ−アセチルガラクトサミンよりなる二糖単位がトリ硫酸エステル化されたコンドロイチン硫酸（重量平均分子量＝４，７００Ｄａ）は、特開２０１２−１５７２７１号公報に記載された方法に従って調製した。重量平均分子量は、マルトペンタオースおよびプルランを標準試料として用い、ゲル浸透クロマトグラフィー（ＧＰＣ）−示差屈折計（ＲＩ）法により求めた。
(Iii) Low molecular weight highly sulfated chondroitin sulfate (L-CS): Chondroitin sulfate (weight average molecular weight = 3,700 Da) in which a disaccharide unit consisting of glucuronic acid and N-acetylgalactosamine is trisulfate esterified is It was prepared according to the methods described in Kai 2012-157271 and JP 2010-077256. The weight average molecular weight was determined by gel permeation chromatography (GPC) -differential refractometer (RI) method using maltopentaose and pullulan as standard samples.
(Iv) High-molecular-weight highly sulfated chondroitin sulfate (H-CS): Chondroitin sulfate (weight average molecular weight = 4,700 Da) in which a disaccharide unit composed of glucuronic acid and N-acetylgalactosamine is trisulfate esterified is It was prepared according to the method described in Kai 2012-157271. The weight average molecular weight was determined by gel permeation chromatography (GPC) -differential refractometer (RI) method using maltopentaose and pullulan as standard samples.
［試験例１］コンドロイチン二糖トリ硫酸エステルがトロポエラスチン遺伝子発現に及ぼす効果の評価
（１）トロポエラスチン遺伝子発現に対するＣＤｉ−ＴｒｉＳ・Ｎａ４、ＣＤｉ−Ｓ・Ｎａ２およびＨ−ＣＳの影響
（ｉ）６０ｍｍディッシュにて、培養した正常ヒト皮膚線維芽細胞（培地３ｍＬ；細胞数＝５×１０４個／ｍＬ）に各試料の水溶液（１０μｇ／ｍＬ）を３０μＬ添加（各試料の最終濃度＝０．１μｇ／ｍＬ）し、３７℃、５％ＣＯ２にて２４時間インキュベートした後培地を除去し、３ｍＬのリン酸緩衝生理食塩水（ＰＢＳ）で洗浄した。なお、試料を添加せずに同様に処理したものを対照とした。
（ｉｉ）ＰＢＳを除去し、ＲＮＡ抽出試薬（ＲＮＡ Ｉｓｏ Ｐｌｕｓ、タカラバイオ株式会社）を１ｍＬ添加し、細胞ＲＮＡ抽出液を得た。
（ｉｉｉ）細胞ＲＮＡ抽出液１ｍＬとクロロホルム２００μＬを混和し、１２，０００×ｇ、４℃の条件で１５分間遠心分離した後、上清を回収し、クロロホルム２００μＬを加えて混和し、１２，０００×ｇ、４℃の条件で１５分間遠心分離した。
（ｉｖ）上清を回収し、イソプロパノール５００μＬと混和し、−３０℃で一夜静置し、１２，０００×ｇ、４℃の条件で１５分間遠心分離した。
（ｖ）上清を除去し、ＲＮＡ沈殿物に冷７０（ｖ／ｖ）％エタノール５００μＬを添加し、１２，０００×ｇ、４℃の条件で１５分間遠心分離した。
（ｖｉ）再度上清を除去し、ＲＮＡ沈殿物に冷７０（ｖ／ｖ）％エタノール５００μＬを添加し、１２，０００×ｇ、４℃の条件で１５分間遠心分離し、ＲＮＡ沈殿物をジエチルピロカーボネート（ＤＥＰＣ）処理水（ＤＥＰＣ ｔｒｅａｔｅｄ ｗａｔｅｒ、株式会社ニッポンジーン）で溶解して、マイクロプレートリーダー（日本モレキュラーデバイス株式会社）により吸光度を測定し、ＲＮＡの濃度および純度を算出した（２６０ｎｍ／２８０ｎｍ）。
（ｖｉｉ）逆転写キット（ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ ＫＩＴ（Ｐ
ｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ、タカラバイオ株式会社））および逆転写装置（Ｖｅｒｉｔｉ ９６ ｗｅｌｌ Ｔｈｅｒｍａｌ ｃｙｃｌａｒ（アプライド バイオシステムズ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）社）を用いて、ＲＮＡを逆転写し、Ｒｅａｌ Ｔｉｍｅ ＰＣＲキット（ＳＹＢＲ Ｐｒｅｍｉｘ ＥＸ ｔａｑ（Ｔｌｉ ＲＮａｓｅ Ｐｌｕｓ、タカラバイオ株式会社））を用いてポリメラーゼ連鎖反応（ＰＣＲ）を行い（ＰＣＲ装置：Ｓｔｅｐ Ｏｎｅ Ｐｌｕｓ リアルタイムＰＣＲシステム、（アプライド バイオシステムズ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）社）、トロポエラスチン遺伝子の発現量を求めた。
結果は、対照におけるトロポエラスチン遺伝子発現量を１とした場合の相対値にて、表１および図１に示した。
[Test Example 1] Evaluation of the effect of chondroitin disaccharide trisulfate on tropoelastin gene expression (1) Effects of CDi-TriS · Na 4 , CDi-S · Na 2 and H-CS on tropoelastin gene expression (I) 30 μL of each sample aqueous solution (10 μg / mL) was added to normal human dermal fibroblasts cultured in a 60 mm dish (medium 3 mL; number of cells = 5 × 10 4 cells / mL) (final concentration of each sample) = 0.1 μg / mL) and incubated at 37 ° C. and 5% CO 2 for 24 hours, and then the medium was removed and washed with 3 mL of phosphate buffered saline (PBS). In addition, what was processed similarly without adding a sample was used as a control.
(Ii) PBS was removed, and 1 mL of RNA extraction reagent (RNA Iso Plus, Takara Bio Inc.) was added to obtain a cellular RNA extract.
(Iii) 1 mL of cellular RNA extract and 200 μL of chloroform are mixed and centrifuged at 12,000 × g and 4 ° C. for 15 minutes, then the supernatant is recovered, 200 μL of chloroform is added and mixed, and 12,000 is mixed. Centrifuged for 15 minutes under the conditions of xg and 4 ° C.
(Iv) The supernatant was collected, mixed with 500 μL of isopropanol, allowed to stand at −30 ° C. overnight, and centrifuged at 12,000 × g and 4 ° C. for 15 minutes.
(V) The supernatant was removed, 500 μL of cold 70 (v / v)% ethanol was added to the RNA precipitate, and centrifuged at 12,000 × g and 4 ° C. for 15 minutes.
(Vi) Remove the supernatant again, add 500 μL of cold 70 (v / v)% ethanol to the RNA precipitate, centrifuge for 15 minutes at 12,000 × g, 4 ° C. Dissolved in pyrocarbonate (DEPC) -treated water (DEPC treated water, Nippon Gene Co., Ltd.), the absorbance was measured with a microplate reader (Nippon Molecular Devices Co., Ltd.), and the RNA concentration and purity were calculated (260 nm / 280 nm) .
(Vii) Reverse transcription kit (PrimeScript RT reagent KIT (P
RNA using reverse Real Time (Takara Bio Inc.)) and reverse transcription apparatus (Veriti 96 well Thermal cycler (Applied Biosystems)), Real Time PCR kit (SYBR Premix E) RNase Plus (Takara Bio Inc.))) (PCR apparatus: Step One Plus real-time PCR system, (Applied Biosystems), and the expression level of tropoelastin gene Asked.
The results are shown in Table 1 and FIG. 1 as relative values when the tropoelastin gene expression level in the control is 1.
表１および図１に示されるように、ＣＤｉ−ＴｒｉＳ・Ｎａ４を正常ヒト線維芽細胞に２４時間作用させた場合には、対照の３倍近いトロポエラスチン遺伝子の発現が見られたが、Ｈ−ＣＳ、ＣＤｉ−Ｓ・Ｎａ２によっては、トロポエラスチン遺伝子発現の有意な増加は見られなかった。 As shown in Table 1 and FIG. 1, when CDi-TriS · Na 4 was allowed to act on normal human fibroblasts for 24 hours, expression of the tropoelastin gene nearly three times that of the control was observed. No significant increase in tropoelastin gene expression was observed with H-CS and CDi-S · Na 2 .
（２）トロポエラスチン遺伝子発現に対するＣＤｉ−ＴｒｉＳ・Ｎａ４、Ｌ−ＣＳおよびＨ−ＣＳの影響
試料として、ＣＤｉ−Ｓ・Ｎａ２の代わりにＬ−ＣＳを用い、試料添加後のインキュベーション時間を８時間および２４時間とした他は、上記（１）と同様に処理または操作し、各試料を８時間または２４時間作用させた後のトロポエラスチンの発現量を求めた。
結果は、対照におけるトロポエラスチン遺伝子発現量を１とした場合の相対値にて、表２および図２に示した。
(2) Influence of CDi-TriS · Na 4 , L-CS and H-CS on tropoelastin gene expression L-CS was used instead of CDi-S · Na 2 as a sample, and the incubation time after addition of the sample was determined. The expression level of tropoelastin was determined after each sample was treated or operated in the same manner as (1) above except that the time was 8 hours and 24 hours, and each sample was allowed to act for 8 hours or 24 hours.
The results are shown in Table 2 and FIG. 2 as relative values when the tropoelastin gene expression level in the control is 1.
表２および図２に示されるように、各試料を８時間作用させた後には、いずれの試料によっても、トロポエラスチン遺伝子発現の増加が見られた。しかし、２４時間作用させた後では、Ｌ−ＣＳ、Ｈ−ＣＳによるトロポエラスチン遺伝子発現量は、それぞれ対照の１．４２倍および対照の１．０５倍にまで減少したのに対し、ＣＤｉ−ＴｒｉＳ・Ｎａ４を作用させた場合の遺伝子発現量は対照の２．７５倍であり、ＣＤｉ−ＴｒｉＳ・Ｎａ４によるトロポエラスチン遺伝子発現の増加は、長時間（２４時間作用後）まで持続することが認められた。 As shown in Table 2 and FIG. 2, after each sample was allowed to act for 8 hours, an increase in tropoelastin gene expression was observed in any sample. However, after 24 hours of action, tropoelastin gene expression by L-CS and H-CS decreased to 1.42 times that of the control and 1.05 times that of the control, respectively, whereas CDi- The gene expression level when TriS · Na 4 is allowed to act is 2.75 times that of the control, and the increase in tropoelastin gene expression by CDi-TriS · Na 4 lasts for a long time (after 24 hours of action). It was recognized that
［試験例２］トロポエラスチンタンパク質発現に対するＣＤｉ−ＴｒｉＳ・Ｎａ４、Ｌ−ＣＳおよびＨ−ＣＳの影響の評価
（１）培養した正常ヒト皮膚線維芽細胞（培地３ｍＬ；細胞数＝５×１０４個／ｍＬ）に各試料の水溶液（３０μｇ／ｍＬ）３０μＬを添加（各試料の最終濃度＝０．３μｇ／ｍＬ）し、３７℃、５％ＣＯ２にて２４時間インキュベートした後、培地を除去し、３ｍＬのＰＢＳで洗浄した。なお、試料を添加せずに同様に処理したものを対照とした。
（２）ＰＢＳを除去し、タンパク質抽出用溶液（２（ｗ／ｖ）％ ドデシル硫酸ナトリウム（ＳＤＳ）、１０（ｖ／ｖ）％グリセロール、１０ｍＭオルトバナジン酸ナトリウム、６２．５ｍＭトリス−塩酸緩衝液、ｐＨ＝６．８）４００μＬを添加し、タンパク質を抽出した。
（３）ローリー法によりタンパク質量を定量し、対照および各試料添加群のタンパク質抽出液について、タンパク質含有量が同量になるように調整して、以下の通り、ウェスタンブロット法により、トロポエラスチンタンパク質を検出、定量した。
（４）（３）でタンパク質含有量を調整した各群のタンパク質抽出液と、ＳＤＳ−ＰＡＧＥサンプルバッファー（６２．５ｍＭトリス−塩酸緩衝液（ｐＨ＝６．８）、２（ｗ／ｖ）％ＳＤＳ、１０（ｖ／ｖ）％グリセロール、１（ｗ／ｖ）％ブロモフェノールブルー）を１：１で混和し、９５℃で３分間加温した後、７（ｗ／ｖ）％ポリアクリルアミドゲル（トロポエラスチン検出用）または１０（ｗ／ｖ）％ポリアクリルアミドゲル（β−アクチン検出用）を用いて、ミニプロティアンＴｅｔｒａセル（バイオ−ラッド（ＢＩＯ−ＲＡＤ）社）にてＳＤＳ−ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）を行った（泳動用バッファー：２．５ｍＭトリス、１９０ｍＭグリシン、０．１（ｗ／ｖ）％ＳＤＳ水溶液）。
（５）ＳＤＳ−ＰＡＧＥにより分離したタンパク質をゲルからポリフッ化ビニリデン（ＰＶＤＦ）膜（Ａｍｅｒｓｈａｍ Ｈｙ ｂｏｎｄ −Ｐ、ポアサイズ＝０．４５μｍ、ジーイー ヘルスケア（ＧＥ Ｈｅａｌｔｈｃａｒｅ）社）へ転写（転写装置：ｃｒｉｔｅｒｉｏｎ トランスブロットセル（バイオ−ラッド（ＢＩＯ−ＲＡＤ）社）、転写用バッファー：２．５ｍＭトリス、１９０ｍＭグリシン、１５（ｖ／ｖ）％メタノール水溶液、転写条件：４５分間、３Ａ）し、ブロッキングバッファー（５（ｗ／ｖ）％スキムミルク、１（ｗ／ｖ）％ Ｔｗｅｅｎ２０、０．１３Ｍ塩化ナトリウム、０．０２Ｍトリス−塩酸緩衝液、ｐＨ＝７．２）中にて９０分間振とうした。
（６）Ｔｗｅｅｎ ２０添加トリス緩衝生理食塩水（ＴＢＳ−Ｔ；１（ｗ／ｖ）％ Ｔｗｅｅｎ ２０、０．１３Ｍ塩化ナトリウム、０．０２Ｍトリス−塩酸緩衝液、ｐＨ＝７．２）で５分間振とうする操作を３回繰り返した後、１次抗体（トロポエラスチン検出用：Ｒａｂｂｉｔ Ａｎｔｉ−Ｅｌａｓｔｉｎ ｐｏｌｙｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ、Ｕｎｃｏｎｊｕｇａｔｅｄ（バイオス（Ｂｉｏｓｓ）社、β−アクチン検出用：β‐ａｃｔｉｎ（１３Ｅ５）Ｒａｂｂｉｔ ｍＡｂ（セルシグナリングテクノロジー（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ ｔｅｃｈｎｏｌｏｇｙ）社）反応溶液に一夜浸漬した。
（７）ＴＢＳ−Ｔで２０分間振とうする操作を３回繰り返した後、２次抗体（Ａｍｅｒｓｈａｍ ＥＣＬ Ｒａｂｂｉｔ ＩｇＧ、Ｈｏｒｓｅｒａｄｉｓｈ Ｐｅｒｏｘｉｄａｓｅ ｌｉｎｋｅｄ ｗｈｏｌｅ ａｎｔｉｂｏｄｙ（ジーイー ヘルスケア（ＧＥ Ｈｅａｌｔｈｃａｒｅ）社）反応溶液中で１時間振とうした。
（８）ＴＢＳ−Ｔで１０分間振とうする操作を３回繰り返した後、化学発光試薬（Ａｍｅｒｓｈａｍ ＥＣＬ Ｐｒｉｍｅ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｒｅａｇｅｎｔ（ジーイー ヘルスケア（ＧＥ Ｈｅａｌｔｈｃａｒｅ）社）を添加し、高感度化学発光標識画像入力装置（ＬＡＳ−１０００ Ｐｌｕｓ、富士フィルム株式会社）にて画像を解析した。
[Test Example 2] Evaluation of effects of CDi-TriS · Na 4 , L-CS and H-CS on tropoelastin protein expression (1) Normal human skin fibroblasts cultured (3 mL of medium; number of cells = 5 × 10 4 samples / mL) was added 30 μL of each sample aqueous solution (30 μg / mL) (final concentration of each sample = 0.3 μg / mL) and incubated at 37 ° C. and 5% CO 2 for 24 hours. Removed and washed with 3 mL PBS. In addition, what was processed similarly without adding a sample was used as a control.
(2) PBS is removed, and a protein extraction solution (2 (w / v)% sodium dodecyl sulfate (SDS), 10 (v / v)% glycerol, 10 mM sodium orthovanadate, 62.5 mM Tris-HCl buffer , PH = 6.8) 400 μL was added to extract the protein.
(3) The amount of protein is quantified by the Raleigh method, and the protein extract of the control and each sample addition group is adjusted so that the protein content is the same, and tropoelastin is obtained by Western blotting as follows. Protein was detected and quantified.
(4) Protein extract of each group whose protein content was adjusted in (3), and SDS-PAGE sample buffer (62.5 mM Tris-HCl buffer (pH = 6.8), 2 (w / v)% SDS, 10 (v / v)% glycerol, 1 (w / v)% bromophenol blue) was mixed 1: 1, heated at 95 ° C. for 3 minutes, and then 7 (w / v)% polyacrylamide gel. SDS-polyacrylamide (for Bio-Rad (BIO-RAD)) using 10% (w / v)% polyacrylamide gel (for β-actin detection) or 10% (w / v) polyacrylamide gel (for detection of β-actin). Gel electrophoresis (SDS-PAGE) was performed (electrophoresis buffer: 2.5 mM Tris, 190 mM glycine, 0.1 (w / v)% SDS aqueous solution).
(5) Transfer the protein separated by SDS-PAGE from the gel to a polyvinylidene fluoride (PVDF) membrane (Amersham Hy bond-P, pore size = 0.45 μm, GE Healthcare) (transfer apparatus: criterion transformer) Blot cell (Bio-Rad (BIO-RAD)), transfer buffer: 2.5 mM Tris, 190 mM glycine, 15 (v / v)% methanol aqueous solution, transfer conditions: 45 minutes, 3 A) and blocking buffer (5 The mixture was shaken in (w / v)% skim milk, 1 (w / v)% Tween 20, 0.13M sodium chloride, 0.02M Tris-HCl buffer, pH = 7.2) for 90 minutes.
(6) Tween 20-added Tris-buffered saline (TBS-T; 1 (w / v)% Tween 20, 0.13 M sodium chloride, 0.02 M Tris-HCl buffer, pH = 7.2) for 5 minutes After repeating the shaking operation three times, the primary antibody (for tropoelastin detection: Rabbit Anti-Elastin polyclonal Antibody, Unconjugated (Bioss, for β-actin detection: β-actin (13E5) Rabbit mAb (Cell Signaling Technology Co., Ltd.) The reaction solution was immersed overnight.
(7) The operation of shaking with TBS-T for 20 minutes was repeated 3 times, and then the reaction solution for secondary antibody (Amersham ECL Rabbit IgG, Horseradish Peroxidase linked whole antibody (GE Healthcare) for 1 hour) Shake.
(8) After repeating the operation of shaking for 10 minutes with TBS-T three times, a chemiluminescent reagent (Amersham ECL Prime Western Detection Reagent (GE Healthcare)) was added, and a highly sensitive chemiluminescent label Images were analyzed with an image input device (LAS-1000 Plus, Fuji Film Co., Ltd.).
ウェスタンブロット法による解析の結果、６０ｋＤａ付近にトロポエラスチンのバンドが検出された。トロポエラスチンタンパク質の発現量は、β−アクチンタンパク質の発現量に対する量として求め、対照における前記発現量を１とした場合の相対値により、表３および図３に示した。 As a result of Western blot analysis, a tropoelastin band was detected in the vicinity of 60 kDa. The expression level of tropoelastin protein was determined as an amount relative to the expression level of β-actin protein, and is shown in Table 3 and FIG. 3 as relative values when the expression level in the control is 1.
表３および図３に示されるように、ＣＤｉ−ＴｒｉＳ・Ｎａ４を２４時間作用させた後において、トロポエラスチンタンパク質の発現が対照の約１．５倍に増加したことが認められた。一方、Ｌ−ＣＳ、Ｈ−ＣＳを２４時間作用させた後には、トロポエラスチンタンパク質の発現の増加は認められなかった。 As shown in Table 3 and FIG. 3, it was observed that the expression of tropoelastin protein increased about 1.5 times that of the control after CDi-TriS · Na 4 was allowed to act for 24 hours. On the other hand, after L-CS and H-CS were allowed to act for 24 hours, no increase in the expression of tropoelastin protein was observed.
［試験例３］トロポエラスチンタンパク質発現に対するＣＤｉ−ＴｒｉＳ・Ｎａ４の影響の評価
培養した正常ヒト皮膚線維芽細胞におけるトロポエラスチンタンパク質発現に対するＣＤｉ−ＴｒｉＳ・Ｎａ４の影響について、ＣＤｉ−ＴｒｉＳ・Ｎａ４のインキュベーション時間を４８時間とした他は、上記試験例２と同様にタンパク質を回収してウェスタンブロット法により解析し、ＣＤｉ−ＴｒｉＳ・Ｎａ４を４８時間作用させた後におけるトロポエラスチンタンパク質発現に対する影響を評価した。
結果は、β−アクチンタンパク質の発現量に対するトロポエラスチンタンパク質の発現量について、対照における前記発現量を１とした場合の相対値により、表４および図４に示した。
Effect of Test Example 3 tropoelastin CDi-TriS · Na 4 for tropoelastin protein expression in normal human dermal fibroblasts were evaluated culture of the effect of CDi-TriS · Na 4 for protein expression, CDi-Tris · The protein was collected and analyzed by Western blotting in the same manner as in Test Example 2 except that the incubation time of Na 4 was 48 hours, and tropoelastin protein after CDi-TriS · Na 4 was allowed to act for 48 hours. The effect on expression was evaluated.
The results are shown in Table 4 and FIG. 4 with respect to the expression level of tropoelastin protein relative to the expression level of β-actin protein, with the relative value when the expression level in the control is 1.
表４および図４より、ＣＤｉ−ＴｒｉＳ・Ｎａ４を４８時間作用させた後において、トロポエラスチンタンパク質の発現は対照の約４倍にまで増加することが認められた。 From Table 4 and FIG. 4, it was recognized that the expression of tropoelastin protein increased to about 4 times that of the control after CDi-TriS · Na 4 was allowed to act for 48 hours.
［試験例４］エラスチン産生に対するＣＤｉ−ＴｒｉＳ・Ｎａ４の影響の評価
（１）培養した正常ヒト皮膚線維芽細胞（培地３ｍＬ；細胞数＝５×１０４個／ｍＬ）にＣＤｉ−ＴｒｉＳ・Ｎａ４水溶液（３０μｇ／ｍＬ）３０μＬを添加（ＣＤｉ−ＴｒｉＳ・Ｎａ４の最終濃度＝０．３μｇ／ｍＬ）し、３７℃、５％ＣＯ２にて２４時間インキュベートした後、培地を除去し、３ｍＬのＰＢＳで洗浄した。なお、ＣＤｉ−ＴｒｉＳ・Ｎａ４を添加せずに同様に処理したものを対照とした。
（２）ＰＢＳを除去し、タンパク質抽出用溶液（２（ｗ／ｖ）％ ドデシル硫酸ナトリウム（ＳＤＳ）、１０（ｖ／ｖ）％グリセロール、１０ｍＭオルトバナジン酸ナトリウム、６．２７ｍＭトリス−塩酸緩衝液、ｐＨ＝６．８）４００μＬを添加し、タンパク質を抽出した。
（３）ローリー法によりタンパク質量を定量し、対照およびＣＤｉ−ＴｒｉＳ・Ｎａ４添加群のタンパク質抽出液について、タンパク質含有量が同量になるように調整して、以下の通り、ウェスタンブロット法により、エラスチンタンパク質を検出、定量した。
（４）（３）でタンパク質含有量を調整した両群のタンパク質抽出液と、ＳＤＳ−ＰＡＧＥサンプルバッファー（２．５ｍＭ トリス−塩酸緩衝液（ｐＨ＝６．８）、２（ｗ／ｖ）％ＳＤＳ、１０（ｖ／ｖ）％グリセロール、１（ｗ／ｖ）％ブロモフェノールブルー）を１：１で混和し、９５℃で３分間加温した後、７（ｗ／ｖ）％ポリアクリルアミドゲル（エラスチン検出用）または１０（ｗ／ｖ）％ポリアクリルアミドゲル（β−アクチン検出用）を用いて、ミニプロティアンＴｅｔｒａセル（バイオ−ラッド（ＢＩＯ−ＲＡＤ）社）にてＳＤＳ−ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）を行った（泳動用バッファー：２．５ｍＭ トリス、１９０ｍＭグリシン、０．１（ｗ／ｖ）％ ＳＤＳ水溶液）。
（５）ＳＤＳ−ＰＡＧＥにより分離したタンパク質をゲルからポリフッ化ビニリデン（ＰＶＤＦ）膜（Ａｍｅｒｓｈａｍ Ｈｙ ｂｏｎｄ −Ｐ、ポアサイズ＝０．４５μｍ、ジーイー ヘルスケア（ＧＥ Ｈｅａｌｔｈｃａｒｅ）社）へ転写（転写装置：ｃｒｉｔｅｒｉｏｎ トランスブロットセル（バイオ−ラッド（ＢＩＯ−ＲＡＤ）社）、転写用バッファー：２．５ｍＭ トリス、１９０ｍＭグリシン、１５（ｖ／ｖ）％メタノール水溶液、転写条件：４０分間、１００Ｖ、３Ａ）し、ブロッキングバッファー（５（ｗ／ｖ）％スキムミルク、１（ｗ／ｖ）％Ｔｗｅｅｎ２０、０．１３Ｍ塩化ナトリウム、０．０２Ｍトリス−塩酸緩衝液、ｐＨ＝７．２）中にて６０分間振とうした。
（６）Ｔｗｅｅｎ ２０添加トリス緩衝生理食塩水（ＴＢＳ−Ｔ；１（ｗ／ｖ）％ Ｔｗｅｅｎ ２０、０．１３Ｍ塩化ナトリウム、０．０２Ｍトリス−塩酸緩衝液、ｐＨ＝７．２）で１５分間振とうする操作を３回繰り返した後、１次抗体（エラスチン検出用：Ｒａｂｂｉｔ Ａｎｔｉ−Ｅｌａｓｔｉｎ ｐｏｌｙｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ、Ｕｎｃｏｎｊｕｇａｔｅｄ（バイオス（Ｂｉｏｓｓ）社、β−アクチン検出用：β−ａｃｔｉｎ（１３Ｅ５）Ｒａｂｂｉｔ ｍＡｂ（セルシグナリングテクノロジー（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ ｔｅｃｈｎｏｌｏｇｙ）社）反応溶液に一夜浸漬した。
（７）ＴＢＳ−Ｔで１５分間振とうする操作を３回繰り返した後、２次抗体（Ａｍｅｒｓｈａｍ ＥＣＬ Ｒａｂｂｉｔ ＩｇＧ、Ｈｏｒｓｅｒａｄｉｓｈ Ｐｅｒｏｘｉｄａｓｅ ｌｉｎｋｅｄ ｗｈｏｌｅ ａｎｔｉｂｏｄｙ（ジーイー ヘルスケア（ＧＥ Ｈｅａｌｔｈｃａｒｅ）社）反応溶液中で１時間振とうした。
（８）ＴＢＳ−Ｔで１０分間振とうする操作を３回繰り返した後、化学発光試薬（ＥＣＬ Ｐｒｉｍｅ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ （ＢＩＯ−ＲＡＤ社）を添加し、高感度化学発光標識画像入力装置（ＬＡＳ−１０００ Ｐｌｕｓ、富士フィルム株式会社）にて画像を解析した。
[Test Example 4] Evaluation of the effect of CDi-TriS · Na 4 on elastin production (1) CDi-TriS · Na was added to cultured normal human skin fibroblasts (medium 3 mL; number of cells = 5 × 10 4 cells / mL). 4 30 μL of aqueous solution (30 μg / mL) was added (final concentration of CDi-TriS · Na 4 = 0.3 μg / mL) and incubated at 37 ° C., 5% CO 2 for 24 hours, after which the medium was removed and 3 mL Washed with PBS. Incidentally, as a control for those treated similarly without adding CDi-TriS · Na 4.
(2) PBS is removed, and a protein extraction solution (2 (w / v)% sodium dodecyl sulfate (SDS), 10 (v / v)% glycerol, 10 mM sodium orthovanadate, 6.27 mM Tris-HCl buffer , PH = 6.8) 400 μL was added to extract the protein.
(3) The amount of protein was quantified by the Raleigh method, and the protein extract of the control and CDi-TriS • Na 4 added group was adjusted so that the protein content would be the same amount. The elastin protein was detected and quantified.
(4) Protein extracts of both groups whose protein content was adjusted in (3), and SDS-PAGE sample buffer (2.5 mM Tris-HCl buffer (pH = 6.8), 2 (w / v)% SDS, 10 (v / v)% glycerol, 1 (w / v)% bromophenol blue) was mixed 1: 1, heated at 95 ° C. for 3 minutes, and then 7 (w / v)% polyacrylamide gel. (Elastin detection) or 10 (w / v)% polyacrylamide gel (for β-actin detection), SDS-polyacrylamide gel electricity in mini-Protean Tetra cell (Bio-Rad (BIO-RAD)) Electrophoresis (SDS-PAGE) was performed (electrophoresis buffer: 2.5 mM Tris, 190 mM glycine, 0.1 (w / v)% SDS aqueous solution).
(5) Transfer the protein separated by SDS-PAGE from the gel to a polyvinylidene fluoride (PVDF) membrane (Amersham Hy bond-P, pore size = 0.45 μm, GE Healthcare) (transfer apparatus: criterion transformer) Blot cell (Bio-Rad (BIO-RAD)), transfer buffer: 2.5 mM Tris, 190 mM glycine, 15 (v / v)% aqueous methanol, transfer conditions: 40 minutes, 100 V, 3 A), blocking buffer The mixture was shaken in (5 (w / v)% skim milk, 1 (w / v)% Tween 20, 0.13 M sodium chloride, 0.02 M Tris-HCl buffer, pH = 7.2) for 60 minutes.
(6) Tween 20-added Tris-buffered saline (TBS-T; 1 (w / v)% Tween 20, 0.13 M sodium chloride, 0.02 M Tris-HCl buffer, pH = 7.2) for 15 minutes After the shaking operation was repeated 3 times, the primary antibody (for elastin detection: Rabbit Anti-Elastin polyclonal antibody, Unconjugated (Bioss, for β-actin detection: β-actin (13E5) Rabbit mAb ( It was immersed overnight in a reaction solution of Signaling Technology (Cell signaling technology).
(7) The operation of shaking with TBS-T for 15 minutes was repeated 3 times, and then the reaction was carried out for 1 hour in a secondary antibody (Amersham ECL Rabbit IgG, Horseradish Peroxidase linked whole antibody) solution in GE Healthcare Shake.
(8) After repeating the operation of shaking for 10 minutes with TBS-T three times, a chemiluminescence reagent (ECL Prime Western Blotting Detection (BIO-RAD)) was added, and a high-sensitivity chemiluminescent label image input device (LAS-) 1000 Plus, Fuji Film Co., Ltd.).
ウェスタンブロット法による解析の結果、７５ｋＤａ付近にエラスチンのバンドを検出した。エラスチンの産生量は、β−アクチンタンパク質の発現量に対する量として求め、対照における前記産生量を１とした場合の相対値により、表５および図５に示した。 As a result of analysis by Western blotting, an elastin band was detected in the vicinity of 75 kDa. The production amount of elastin was determined as an amount relative to the expression amount of β-actin protein, and is shown in Table 5 and FIG. 5 as relative values when the production amount in the control is 1.
表５および図５に示されるように、ＣＤｉ−ＴｒｉＳ・Ｎａ４を２４時間作用させた後において、エラスチンの産生が対照の約１．４倍に増加したことが認められた。 As shown in Table 5 and FIG. 5, it was observed that elastin production increased about 1.4 times that of the control after CDi-TriS · Na 4 was allowed to act for 24 hours.
［実施例１］水中油型乳剤性ローション剤
表６に示す処方に従い、水中油型乳剤性ローション剤を調製する。すなわち、表６中のスクワラン以下、モノステアリン酸グリセリンまでの成分を容器に秤量し、約７５℃に加温し、撹拌溶解させて油相とする。別容器に、表６中のカルボキシビニルポリマー以下、精製水までの成分を秤量し、約７５℃に加温し、撹拌溶解させて水相とする。前記油相に前記水相を加え、ホモミキサーで均一に乳化した後、室温まで冷却して水中油型乳剤性ローション剤を得る。
[Example 1] Oil-in-water emulsion lotion According to the formulation shown in Table 6, an oil-in-water emulsion lotion is prepared. That is, the components up to squalane in Table 6 and up to glyceryl monostearate are weighed in a container, heated to about 75 ° C., dissolved with stirring to obtain an oil phase. In a separate container, the components up to carboxyvinyl polymer in Table 6 and up to purified water are weighed, heated to about 75 ° C., dissolved with stirring to obtain an aqueous phase. The aqueous phase is added to the oil phase and uniformly emulsified with a homomixer, and then cooled to room temperature to obtain an oil-in-water emulsion lotion.
以上詳述したように、本発明により、トロポエラスチン遺伝子の発現を増加させて、エラスチンの前駆体であるトロポエラスチンタンパク質の発現を増加させることができ、特に皮膚真皮の細胞外マトリックスにおいて、エラスチン線維の生成を促進させ得るトロポエラスチン発現促進剤およびエラスチン産生促進剤を提供することができる。
本発明のトロポエラスチン発現促進剤およびエラスチン産生促進剤は、エラスチンの減少、変性による皮膚の老化の防止または改善に有用で、皮膚外用医薬品、皮膚外用医薬部外品または皮膚用化粧品として提供することができる。
さらに、本発明により、皮膚シートの生成等、再生医療分野において有用な皮膚線維芽細胞等のトロポエラスチンの発現促進方法、およびエラスチン産生の促進方法を提供することができる。
As described in detail above, according to the present invention, it is possible to increase the expression of tropoelastin gene and increase the expression of tropoelastin protein which is a precursor of elastin, particularly in the extracellular matrix of skin dermis. A tropoelastin expression promoter and an elastin production promoter capable of promoting the generation of elastin fibers can be provided.
The tropoelastin expression promoter and elastin production promoter of the present invention are useful for prevention or improvement of skin aging due to reduction or degeneration of elastin, and are provided as a skin external medicine, a skin external quasi-drug, or a skin cosmetic. be able to.
Furthermore, the present invention can provide a method for promoting the expression of tropoelastin such as skin fibroblasts and the like, and a method for promoting elastin production, which are useful in the field of regenerative medicine, such as the production of skin sheets.
本出願は、わが国で出願された特願２０１４−０３８４７５を基礎とするものであり、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2014-038475 for which it applied in Japan, The content is altogether included in this specification.
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Cited By (3)
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| JP2018158898A (en) * | 2017-03-22 | 2018-10-11 | 大阪瓦斯株式会社 | Tropoelastin expression promoter |
| WO2019175364A1 (en) * | 2018-03-16 | 2019-09-19 | Institute of Technology, Tralee | A treatment composition |
| JP2021054723A (en) * | 2019-09-27 | 2021-04-08 | 株式会社ファンケル | Agent for improving stereostructure of elastin fiber and agent for straightening the same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003503436A (en) * | 1999-06-30 | 2003-01-28 | ルーイ・ジェイ・ユ | Oligosaccharide aldonic acids and their topical use |
| JP2006143605A (en) * | 2004-11-16 | 2006-06-08 | Miyao Shunsuke | Method for producing percutaneously administrative agent and orally administrative agent each promoting fibroblast proliferation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003503436A (en) * | 1999-06-30 | 2003-01-28 | ルーイ・ジェイ・ユ | Oligosaccharide aldonic acids and their topical use |
| JP2006143605A (en) * | 2004-11-16 | 2006-06-08 | Miyao Shunsuke | Method for producing percutaneously administrative agent and orally administrative agent each promoting fibroblast proliferation |
Non-Patent Citations (2)
| Title |
|---|
| BIOL.PHARM.BULL.(2004),VOL.27,NO.1,P.47-51, JPN6018047587 * |
| 日本香粧品学会誌(2015.03.31),VOL.39,NO.1,P.1-6, JPN6018047586 * |
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| JP2018158898A (en) * | 2017-03-22 | 2018-10-11 | 大阪瓦斯株式会社 | Tropoelastin expression promoter |
| WO2019175364A1 (en) * | 2018-03-16 | 2019-09-19 | Institute of Technology, Tralee | A treatment composition |
| JP2021054723A (en) * | 2019-09-27 | 2021-04-08 | 株式会社ファンケル | Agent for improving stereostructure of elastin fiber and agent for straightening the same |
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