JP7421298B2 - BRG-1 expression promoter - Google Patents
BRG-1 expression promoter Download PDFInfo
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- JP7421298B2 JP7421298B2 JP2019182227A JP2019182227A JP7421298B2 JP 7421298 B2 JP7421298 B2 JP 7421298B2 JP 2019182227 A JP2019182227 A JP 2019182227A JP 2019182227 A JP2019182227 A JP 2019182227A JP 7421298 B2 JP7421298 B2 JP 7421298B2
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- glycyrrhetinic acid
- brg
- salt
- expression
- promoting
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Description
本発明は、皮膚弾性の改善に有用なＢＲＧ－１発現促進剤に関する。 The present invention relates to a BRG-1 expression promoter useful for improving skin elasticity.
人間の皮膚は、加齢と共に老化して弾力を喪失し、ハリの低下や、シワ、たるみの発生を生じる。特に顔面や首筋、肩など太陽光のあたる部分の皮膚では、慢性的な紫外線の影響により、シワやたるみの形成、又はしみやそばかすの形成が、他の光のあたらない部分と比較し顕著となる。これら紫外線暴露部で起こる皮膚老化は光老化と呼ばれる。光老化とは、慢性的な日光照射、特に紫外線への曝露によって皮膚の内的な老化プロセスが加速されて起こる皮膚の変化である。 Human skin ages and loses its elasticity with age, resulting in a decrease in firmness, wrinkles, and sagging. Particularly on the skin in areas exposed to sunlight such as the face, neck, and shoulders, the formation of wrinkles and sagging, as well as the formation of spots and freckles, is more pronounced than in other areas that are not exposed to sunlight due to the effects of chronic ultraviolet rays. Become. Skin aging that occurs in areas exposed to UV rays is called photoaging. Photoaging is a change in the skin that occurs when the skin's internal aging process is accelerated by chronic sunlight exposure, particularly ultraviolet light.
真皮の弾性の維持には、細胞外マトリクス（ＥＣＭ）の構成成分である弾性線維やコラーゲン線維が重要な役割を担っている。弾性線維は、トロポエラスチン分子と、フィブリリン－１から主に構成されるミクロフィブリルとの凝集体が重合して構築されている。皮膚の光老化は、コラーゲン線維や弾性線維の減少や分解を伴う。 Elastic fibers and collagen fibers, which are components of the extracellular matrix (ECM), play an important role in maintaining the elasticity of the dermis. Elastic fibers are constructed by polymerizing aggregates of tropoelastin molecules and microfibrils mainly composed of fibrillin-1. Photoaging of the skin is accompanied by a decrease and decomposition of collagen fibers and elastic fibers.
真核細胞のＤＮＡは高度に折りたたまれたクロマチン構造にあるため、遺伝子が転写されその機能を発揮するためには、このクロマチン構造が解かれ、転写因子や転写活性化因子がＤＮＡにアクセス可能になることが重要である。クロマチン構造を解く機能を持ったタンパク質集団はクロマチンリモデリング複合体と呼ばれ、それらの１つがＳＷＩ／ＳＮＦ複合体である。ＢＲＧ－１（別名：ＳＭＡＲＣＡ４）は、ＳＷＩ／ＳＮＦ複合体のコアサブユニットとして機能するタンパク質である（非特許文献１）。ＢＲＧ－１がＵＶ照射により傷ついたＤＮＡの修復に関与すると考えられること（非特許文献２、３）や、ＢＲＧ－１をノックダウンしたメラノーマの細胞増殖が低下したこと（非特許文献４）が報告されている。 DNA in eukaryotic cells has a highly folded chromatin structure, so in order for genes to be transcribed and perform their functions, this chromatin structure must be unwound to allow transcription factors and transcription activators to access the DNA. It is important to become A group of proteins that have the function of unraveling the chromatin structure are called chromatin remodeling complexes, and one of them is the SWI/SNF complex. BRG-1 (also known as SMARCA4) is a protein that functions as a core subunit of the SWI/SNF complex (Non-Patent Document 1). BRG-1 is thought to be involved in the repair of DNA damaged by UV irradiation (Non-Patent Documents 2, 3), and cell proliferation of melanomas in which BRG-1 was knocked down was reduced (Non-Patent Document 4). It has been reported.
そして、最近、ヒトの皮膚におけるＢＲＧ－１の発現量と皮膚弾性との間に関連性があり、線維芽細胞におけるＢＲＧ－１の発現低下によりＥＣＭの成分であるコラーゲンや弾性線維構成因子の発現量が低下することが明らかにされ、ＢＲＧ－１の発現又は活性の制御を指標として、コラーゲン線維、弾性線維、ＥＣＭ等の形成制御、皮膚弾力性の改善、皮膚のシワ、たるみ、ハリの低下の予防又は改善等が可能な素材をスクリーニングできることが報告されている（特許文献１）。 Recently, a relationship has been found between the expression level of BRG-1 in human skin and skin elasticity. It has been shown that the expression or activity of BRG-1 is controlled to control the formation of collagen fibers, elastic fibers, ECM, etc., improve skin elasticity, and reduce skin wrinkles, sagging, and firmness. It has been reported that it is possible to screen for materials that can prevent or improve (Patent Document 1).
一方、グリチルレチン酸は、甘草から得られるグリチルリチン酸を加水分解することにより得られる物質であり、抗炎症作用、歯周病による歯槽骨吸収抑制作用及びヒスタミン遊離抑制作用等を有することから、種々の口腔用組成物に配合されている。 On the other hand, glycyrrhetinic acid is a substance obtained by hydrolyzing glycyrrhizic acid obtained from licorice, and has anti-inflammatory effects, inhibitory effects on alveolar bone resorption caused by periodontal disease, and inhibitory effects on histamine release, so it is used in various ways. It is blended into oral compositions.
グリチルリチン酸を多く含む甘草の抽出物には、脂肪幹細胞誘引作用があり、真皮空洞化を抑制し、皮膚のたるみ改善に効果があることが報告されている（特許文献２）。
しかしながら、グリチルレチン酸にＢＲＧ－１の発現を促進する作用があることは知られていない。
It has been reported that a licorice extract containing a large amount of glycyrrhizic acid has an adipose stem cell attracting effect, suppresses dermal cavitation, and is effective in improving skin sagging (Patent Document 2).
However, it is not known that glycyrrhetinic acid has an effect of promoting BRG-1 expression.
本発明は、ＢＲＧ－１の発現を促進することにより皮膚弾性を改善し、皮膚のシワ、たるみ、ハリの低下等を予防又は改善する素材を提供することに関する。 The present invention relates to providing a material that improves skin elasticity by promoting the expression of BRG-1 and prevents or improves skin wrinkles, sagging, loss of firmness, etc.
本発明者らは、長期的に適用可能な安全性の高い成分について、種々検討した結果、グリチルレチン酸に優れたＢＲＧ－１発現促進作用があり、弾性線維の形成促進に有用であることを見出した。 As a result of various studies on highly safe ingredients that can be applied over the long term, the present inventors found that glycyrrhetinic acid has an excellent effect of promoting BRG-1 expression and is useful for promoting the formation of elastic fibers. Ta.
本発明は以下の１）～８）に係るものである。
１）グリチルレチン酸又はその塩を有効成分とするＢＲＧ－１発現促進剤。
２）グリチルレチン酸又はその塩を有効成分とする弾性線維形成促進剤。
３）グリチルレチン酸又はその塩を有効成分とするコラーゲン線維形成促進剤。
４）グリチルレチン酸又はその塩を有効成分とするヒアルロン酸産生促進剤。
５）グリチルレチン酸又はその塩を有効成分とするヒアルロン酸分解抑制剤。
６）グリチルレチン酸又はその塩を有効成分とする皮膚シワ予防又は改善剤。
７）グリチルレチン酸又はその塩を有効成分とする皮膚たるみ予防又は改善剤。
８）グリチルレチン酸又はその塩を有効成分とする皮膚ハリの低下予防又は改善剤。
The present invention relates to the following 1) to 8).
1) A BRG-1 expression promoter containing glycyrrhetinic acid or its salt as an active ingredient.
2) An elastic fiber formation promoter containing glycyrrhetinic acid or its salt as an active ingredient.
3) A collagen fiber formation promoter containing glycyrrhetinic acid or its salt as an active ingredient.
4) Hyaluronic acid production promoter containing glycyrrhetinic acid or its salt as an active ingredient.
5) Hyaluronic acid decomposition inhibitor containing glycyrrhetinic acid or its salt as an active ingredient.
6) A skin wrinkle prevention or improvement agent containing glycyrrhetinic acid or its salt as an active ingredient.
7) A skin sagging prevention or improvement agent containing glycyrrhetinic acid or its salt as an active ingredient.
8) An agent for preventing or improving skin elasticity containing glycyrrhetinic acid or its salt as an active ingredient.
本発明によれば、ＢＲＧ－１の発現を促進することにより、コラーゲン線維や弾性線維の形成を促進させて皮膚弾力性を改善し、皮膚のシワ、たるみ、ハリの低下等を予防又は改善することができる。 According to the present invention, by promoting the expression of BRG-1, the formation of collagen fibers and elastic fibers is promoted to improve skin elasticity, thereby preventing or improving skin wrinkles, sagging, loss of firmness, etc. be able to.
本発明における「グリチルレチン酸」は、具体的には３β－ヒドロキシ－１１－オキソオレアナ－１２－エン－３０－カルボン酸（β－グリチルレチン酸）を指す。 "Glycyrrhetinic acid" in the present invention specifically refers to 3β-hydroxy-11-oxoolean-12-ene-30-carboxylic acid (β-glycyrrhetinic acid).
また、グリチルレチン酸の塩としては、例えばナトリウム、カリウム等のアルカリ金属塩、マグネシウム、カルシウム等のアルカリ土類金属塩等が挙げられる。 Examples of the salts of glycyrrhetinic acid include salts of alkali metals such as sodium and potassium, salts of alkaline earth metals such as magnesium and calcium, and the like.
グリチルレチン酸は、甘草（カンゾウ、Glycyrrhiza glabra、Glycyrrhiza uralensis又はその他同属植物）等から得られるグリチルリチン酸を加水分解することにより得ることができ（生物工学 第89巻 第11号 2011年 656-659）、また、市販品、例えば「アグリチノン」（株式会社常磐植物化学研究所））を用いることもできる。 Glycyrrhizic acid can be obtained by hydrolyzing glycyrrhizic acid obtained from licorice (licorice, Glycyrrhiza glabra, Glycyrrhiza uralensis, or other congenerous plants) (Biotechnology Vol. 89, No. 11, 2011, 656-659), Furthermore, commercially available products such as "Agritinone" (Tokiwa Phytochemical Research Institute Co., Ltd.) can also be used.
後記実施例に示すように、グリチルレチン酸は、ＢＲＧ－１の発現を遺伝子及びタンパク質レベルで促進する作用を有する。前述したとおり、ＢＲＧ－１の発現低下によりＥＣＭの成分であるコラーゲンや弾性線維構成因子の発現量が低下することから（前記特許文献１）、ＢＲＧ－１の発現を促進することにより、コラーゲン線維や弾性線維の形成を促進させて皮膚弾力性を改善し、皮膚のシワ、たるみ、ハリの低下等を予防又は改善することができると考えられる。実際にグリチルレチン酸は、ミクロフィブリルへのエラスチンの沈着促進作用、コラーゲン線維形成促進作用を有し、また、ＥＣＭの構成成分の一つであるヒアルロン酸の産生を促進すると共に、ヒアルロン酸の分解を抑制する作用を有することが確認された。 As shown in the Examples below, glycyrrhetinic acid has the effect of promoting BRG-1 expression at the gene and protein levels. As mentioned above, a decrease in the expression of BRG-1 causes a decrease in the expression levels of collagen and elastic fiber constituent factors, which are components of the ECM (Patent Document 1); therefore, by promoting the expression of BRG-1, collagen fibers It is thought that it is possible to improve skin elasticity by promoting the formation of elastic fibers and to prevent or improve skin wrinkles, sagging, loss of firmness, etc. In fact, glycyrrhetinic acid has the effect of promoting elastin deposition in microfibrils and collagen fiber formation, and also promotes the production of hyaluronic acid, which is one of the constituent components of ECM, and inhibits the decomposition of hyaluronic acid. It was confirmed that it has a suppressive effect.
従って、グリチルレチン酸又はその塩は、ＢＲＧ－１発現促進剤、弾性線維形成促進剤、コラーゲン線維形成促進剤、ヒアルロン酸産生促進剤、ヒアルロン酸分解抑制剤、皮膚シワ予防又は改善剤、皮膚たるみ予防又は改善剤（以下、「ＢＲＧ－１発現促進剤等」とも称す）となり得、ＢＲＧ－１の発現を促進するため、弾性線維形成を促進するため、コラーゲン線維の形成を促進するため、ヒアルロン酸産生を促進するため、ヒアルロン酸分解を抑制するため、皮膚シワを予防又は改善するため、皮膚たるみを予防又は改善するため、皮膚ハリの低下を予防又は改善するために使用することができ、またＢＲＧ－１発現促進剤等を製造するために使用することができる。 Therefore, glycyrrhetinic acid or its salt is a BRG-1 expression promoter, an elastic fiber formation promoter, a collagen fiber formation promoter, a hyaluronic acid production promoter, a hyaluronic acid decomposition inhibitor, a skin wrinkle prevention or improvement agent, and a skin sagging prevention agent. or an improving agent (hereinafter also referred to as "BRG-1 expression promoter, etc."), in order to promote the expression of BRG-1, to promote elastic fiber formation, to promote collagen fiber formation, It can be used to promote production, inhibit hyaluronic acid decomposition, prevent or improve skin wrinkles, prevent or improve skin sagging, prevent or improve skin firmness, and It can be used to produce BRG-1 expression promoters and the like.
ここで、「使用」は、ヒト若しくは非ヒト動物への投与又は摂取であり得、また治療的使用であっても非治療的使用であってもよい。尚、「非治療的」とは、医療行為を含まない概念、すなわち人間を手術、治療又は診断する方法を含まない概念、より具体的には医師又は医師の指示を受けた者が人間に対して手術、治療又は診断を実施する方法を含まない概念である。 Here, "use" may be administration or ingestion to humans or non-human animals, and may be therapeutic or non-therapeutic use. Furthermore, "non-therapeutic" is a concept that does not include medical treatment, that is, a method that does not include methods of surgery, treatment, or diagnosis of humans, and more specifically, "non-therapeutic" is a concept that does not include medical procedures, that is, methods of surgery, treatment, or diagnosis of humans. This concept does not include methods of performing surgery, treatment, or diagnosis.
本発明において、ＢＲＧ－１発現促進には、遺伝子レベルでの発現促進及びタンパク質レベルでの発現促進が包含される。遺伝子レベルでの発現促進には、ＢＲＧ－１をコードするｍＲＮＡの発現におけるｍＲＮＡへの転写促進、ｍＲＮＡの安定化促進、ｍＲＮＡの分解抑制が挙げられ、タンパク質レベルでの発現促進には、ｍＲＮＡの翻訳における促進、合成されたＢＲＧ－１タンパク質の安定化促進や分解抑制が含まれる。これらのうち、本発明では遺伝子レベルでのｍＲＮＡの発現促進が好ましく、ｍＲＮＡへの転写促進がより好ましい。ここで「ＢＲＧ－１」は、別名ＳＭＡＲＣＡ４といい、ＡＡＩ３６６４５．１（ＧｅｎＢａｎｋ）で表されるヒトＢＲＧ－１タンパク質、又はそのホモログ、オーソログをいう。 In the present invention, promoting BRG-1 expression includes promoting expression at the gene level and promoting expression at the protein level. Promotion of expression at the gene level includes promotion of transcription into mRNA in the expression of mRNA encoding BRG-1, promotion of mRNA stabilization, and suppression of mRNA degradation. Promotion of expression at the protein level includes promotion of transcription of mRNA encoding BRG-1. This includes promotion of translation, promotion of stabilization of synthesized BRG-1 protein, and inhibition of degradation. Among these, in the present invention, promotion of mRNA expression at the gene level is preferred, and promotion of transcription into mRNA is more preferred. Here, "BRG-1" is also known as SMARCA4 and refers to the human BRG-1 protein represented by AAI36645.1 (GenBank), or its homolog or ortholog.
また、本発明において、弾性線維とは、エラスチン線維を指し、ミクロフィブリルの周りにエラスチンが沈着・架橋してできると考えられている。したがって、「弾性線維形成促進」とは、弾性線維量の増加、特にミクロフィブリルの周りにエラスチンが沈着・架橋する工程が促進されることによる弾性線維量の増加を意味する。
また、「コラーゲン線維形成促進」とは、コラーゲン線維量の増加、特に皮膚中のコラーゲン線維の主成分であるコラーゲンＩ量の増加を意味する。
Furthermore, in the present invention, elastic fibers refer to elastin fibers, which are thought to be formed by depositing and crosslinking elastin around microfibrils. Therefore, "promotion of elastic fiber formation" means an increase in the amount of elastic fibers, particularly an increase in the amount of elastic fibers due to promotion of the process of depositing and crosslinking elastin around microfibrils.
Moreover, "promotion of collagen fiber formation" means an increase in the amount of collagen fibers, particularly an increase in the amount of collagen I, which is the main component of collagen fibers in the skin.
本発明において、「ハリの低下」とは老化によって生じる皮膚中のコラーゲン線維、エラスチン線維、ヒアルロン酸の質的・量的低下に伴う皮膚弾性の低下を意味し、「シワ」とはハリの低下等に伴って皮膚表面に生じるひだや筋を意味し、「たるみ」とはハリの低下等に伴って生じる重力により皮膚形態が下垂した状態を意味する。 In the present invention, "decreased firmness" refers to a decrease in skin elasticity due to a qualitative and quantitative decrease in collagen fibers, elastin fibers, and hyaluronic acid in the skin caused by aging, and "wrinkles" refers to a decrease in firmness. ``Sagging'' refers to the folds and lines that occur on the skin surface as a result of sagging, etc., and ``sagging'' refers to a state in which the skin shape sag due to gravity, which occurs as a result of a decrease in firmness, etc.
本明細書において、「予防」とは、個体における疾患若しくは症状の発症の防止又は遅延、あるいは個体の疾患若しくは症状の発症の危険性を低下させることをいう。
また、「改善」とは、疾患、症状又は状態の好転、疾患、症状又は状態の悪化の防止又は遅延、あるいは疾患又は症状の進行の逆転、防止又は遅延をいう。
As used herein, "prevention" refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing a disease or symptom in an individual.
Furthermore, "improvement" refers to improvement of a disease, symptom or condition, prevention or delay of deterioration of a disease, symptom or condition, or reversal, prevention or delay of progression of a disease or condition.
本発明のＢＲＧ－１発現促進剤等は、ヒトを含む動物に摂取又は投与した場合に、ＢＲＧ－１の発現促進効果、弾性線維形成促進効果、コラーゲン線維形成促進効果、ヒアルロン酸産生促進効果、ヒアルロン酸分解抑制効果、皮膚シワ予防又は改善効果、皮膚たるみ予防又は改善効果、皮膚ハリの低下予防又は改善効果を発揮する医薬品、医薬部外品、化粧品、食品となり、或いはこれらへ配合するための素材又は製剤となり得る。当該医薬品、医薬部外品、化粧品、食品には、皮膚シワやたるみやハリの低下予防又は改善のための他の有効成分などが含有されていてもよい。
また、当該食品には、皮膚シワやたるみやハリの低下予防又は改善をコンセプトとし、必要に応じてその旨を表示した機能性食品、特定保健用食品、サプリメント等が包含される。
The BRG-1 expression promoter of the present invention, when ingested or administered to animals including humans, has an effect of promoting BRG-1 expression, an effect of promoting elastic fiber formation, an effect of promoting collagen fiber formation, an effect of promoting hyaluronic acid production, To be used as pharmaceuticals, quasi-drugs, cosmetics, and foods that exhibit the effect of inhibiting hyaluronic acid decomposition, preventing or improving skin wrinkles, preventing or improving skin sagging, or preventing or improving skin firmness, or to be incorporated into these. It can be a material or a preparation. The pharmaceuticals, quasi-drugs, cosmetics, and foods may contain other active ingredients for preventing or improving skin wrinkles, sagging, and a decrease in firmness.
In addition, such foods include functional foods, foods for specified health uses, supplements, etc. that are designed to prevent or improve skin wrinkles, sagging, and loss of firmness, and are labeled to that effect as necessary.
グリチルレチン酸又はその塩を含む上記医薬品（医薬部外品を含む）の投与形態は任意であり、経口投与及び非経口投与の何れであってもよい。経口投与のための剤型としては、錠剤、顆粒剤、散剤、カプセル剤のような固形製剤、ならびにエリキシル、シロップ、懸濁液のような液体製剤が挙げられる。非経口投与のための剤型としては、皮膚外用、経皮、経粘膜、経鼻、経腸、注射、坐剤、注射、吸入、貼付等の各製剤が挙げられる。このうち、好適な製剤形態は皮膚外用剤であり、具体的には、軟膏、乳化液、クリーム、乳液、ローション、ジェル、エアゾール等の形態が挙げられる。 The administration form of the above-mentioned pharmaceuticals (including quasi-drugs) containing glycyrrhetinic acid or its salts is arbitrary, and may be administered either orally or parenterally. Dosage forms for oral administration include solid preparations such as tablets, granules, powders, and capsules, and liquid preparations such as elixirs, syrups, and suspensions. Dosage forms for parenteral administration include external skin, transdermal, transmucosal, nasal, rectal, injection, suppository, injection, inhalation, and patch formulations. Among these, preferred formulations are external preparations for the skin, and specific examples include ointments, emulsions, creams, milky lotions, lotions, gels, and aerosols.
グリチルレチン酸又はその塩を含む化粧品の好ましい例としては、顔、ボディ用の化粧料（例えば、ローション、ゲル、クリーム、パックなど）、メークアップ用化粧料、顔又はボディ用の洗浄料などが挙げられる。 Preferred examples of cosmetics containing glycyrrhetinic acid or its salts include face and body cosmetics (e.g., lotions, gels, creams, packs, etc.), makeup cosmetics, and face or body cleansers. It will be done.
グリチルレチン酸又はその塩を含む食品の形態としては、固形、半固形又は液状（例えば飲料）であり得る。該食品の例としては、例えば、清涼飲料水、茶系飲料、コーヒー飲料、果汁飲料、炭酸飲料、ゼリー状飲料、ニアウォーター等の飲料、ゼリー、ウエハース、ビスケット、パン、麺、ソーセージ等の飲食品や栄養食等の各種食品、ならびにそれらの原料が挙げられる。 The form of food containing glycyrrhetinic acid or its salt may be solid, semi-solid, or liquid (for example, a drink). Examples of such foods include beverages such as soft drinks, tea drinks, coffee drinks, fruit juice drinks, carbonated drinks, jelly drinks, near water, etc., foods and drinks such as jelly, wafers, biscuits, bread, noodles, and sausages. Examples include various foods such as food products and nutritional foods, as well as their raw materials.
斯かる医薬品（医薬部外品を含む）や化粧品や食品の各製剤は、グリチルレチン酸又はその塩を、必要に応じて薬学的に又は化粧品に許容される担体又は食品材料、上述した他の有効成分、その他の薬効成分、化粧成分もしくは食品添加物等と組みあわせて、常法に従って製造することができる。当該薬学的に又は化粧料に許容される担体としては、例えば、各種油剤、界面活性剤、ゲル化剤、緩衝剤、防腐剤、酸化防止剤、溶剤、分散剤、キレート剤、増粘剤、紫外線吸収剤、乳化安定剤、ｐＨ調整剤、色素、香料等が挙げられる。当該その他の薬効成分、化粧成分としては、例えば、植物抽出物、殺菌剤、保湿剤、抗炎症剤、抗菌剤、角質溶解剤、清涼剤、抗脂漏剤、洗浄剤、メークアップ成分等、食品添加物としては、例えば、溶剤、軟化剤、油、乳化剤、防腐剤、酸味料、甘味料、苦味料、ｐＨ調整剤、安定剤、着色剤、紫外線吸収剤、酸化防止剤、保湿剤、増粘剤、固着剤、分散剤、流動性改善剤、湿潤剤、香科、調味料、風味調整剤等が挙げられる。 Each of these pharmaceutical (including quasi-drugs), cosmetics, and food preparations contains glycyrrhetinic acid or its salt, as necessary, in a pharmaceutically or cosmetically acceptable carrier or food material, or in combination with the other effective agents mentioned above. It can be manufactured according to conventional methods by combining the ingredients with other medicinal ingredients, cosmetic ingredients, food additives, etc. Examples of pharmaceutically or cosmetically acceptable carriers include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, Examples include ultraviolet absorbers, emulsion stabilizers, pH adjusters, pigments, fragrances, and the like. Examples of other medicinal and cosmetic ingredients include plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, refreshing agents, antiseborrheic agents, cleaning agents, makeup ingredients, etc. Examples of food additives include solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bitterants, pH adjusters, stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants, Examples include thickeners, fixing agents, dispersants, fluidity improvers, wetting agents, aromatics, seasonings, flavor modifiers, and the like.
上記の医薬品（医薬部外品を含む）や化粧品や食品中のグリチルレチン酸又はその塩の含有量は、投与に用いる剤の形態に応じて異なるため一概には言えないが、例えば０．０００１質量％以上が好ましく、より好ましくは０．０００５質量％以上であり、さらに好ましくは０．００１質量％以上であり、かつ、５０質量％以下が好ましく、より好ましくは２０質量％以下であり、さらに好ましくは１０質量％以下である。また、好ましくは０．０００１～５０質量％、より好ましくは０．０００５～２０質量％、より好ましくは０．００１～１０質量％である。なお、有効成分が塩の形態である場合、その含有量は、対応する遊離体の含有量に換算するものとする。 The content of glycyrrhetinic acid or its salt in the above-mentioned pharmaceuticals (including quasi-drugs), cosmetics, and foods varies depending on the form of the agent used for administration, so it cannot be stated unconditionally, but for example, 0.0001 mass % or more, more preferably 0.0005% by mass or more, even more preferably 0.001% by mass or more, and preferably 50% by mass or less, more preferably 20% by mass or less, and even more preferably is 10% by mass or less. Further, it is preferably 0.0001 to 50% by mass, more preferably 0.0005 to 20% by mass, and even more preferably 0.001 to 10% by mass. In addition, when the active ingredient is in the form of a salt, its content shall be converted to the content of the corresponding educt.
上記の医薬品（医薬部外品を含む）や化粧品や食品の投与量は、本発明の効果を達成できる量であり得る。当該投与量は、対象者の状態、体重、性別、年齢又はその他の要因に従って変動し得るが、成人（６０ｋｇ）１人当たり１日、グリチルレチン酸換算で、例えば好ましくは０．０００１ｍｇ以上、より好ましくは０．００１ｍｇ以上、さらに好ましくは０．０１ｍｇ以上であり、且つ好ましくは１０,０００ｍｇ以下、より好ましくは１，０００ｍｇ以下、さらに好ましくは１００ｍｇ以下である。また、好ましくは０．０００１～１０,０００ｍｇ、より好ましくは０．００１～１，０００ｍｇ、さらに好ましくは０．０１～１００ｍｇである。 The dosage of the above-mentioned pharmaceuticals (including quasi-drugs), cosmetics, and foods may be such that the effects of the present invention can be achieved. The dosage may vary depending on the subject's condition, weight, sex, age, or other factors, but it is preferably 0.0001 mg or more, more preferably 0.0001 mg or more in terms of glycyrrhetinic acid per adult (60 kg) per day. The amount is 0.001 mg or more, more preferably 0.01 mg or more, and preferably 10,000 mg or less, more preferably 1,000 mg or less, and still more preferably 100 mg or less. Further, it is preferably 0.0001 to 10,000 mg, more preferably 0.001 to 1,000 mg, and still more preferably 0.01 to 100 mg.
本発明のＢＲＧ－１発現促進剤等を適用する対象としては、皮膚弾力性の低下又はそのおそれのあるヒト及び非ヒト動物、皮膚シワやたるみの予防又は改善を所望するヒトが挙げられる。非ヒト動物としては、非ヒト哺乳動物などが挙げられ、非ヒト哺乳動物としては、例えば、類人猿、その他霊長類、マウス、ラット、ハムスター、ウマ、ウシ、ブタ、ヒツジ、ヤギ、イヌ、ネコ、およびコンパニオン動物などが挙げられる。 Subjects to whom the BRG-1 expression promoter and the like of the present invention are applied include humans and non-human animals who have or are at risk of having decreased skin elasticity, and humans who desire to prevent or improve skin wrinkles and sagging. Examples of non-human animals include non-human mammals, such as apes, other primates, mice, rats, hamsters, horses, cows, pigs, sheep, goats, dogs, cats, and companion animals.
上述した実施形態に関し、本発明においては更に以下の態様が開示される。
＜１＞グリチルレチン酸又はその塩を有効成分とするＢＲＧ－１発現促進剤。
＜２＞グリチルレチン酸又はその塩を有効成分とする弾性線維形成促進剤。
＜３＞グリチルレチン酸又はその塩を有効成分とするコラーゲン線維形成促進剤。
＜４＞グリチルレチン酸又はその塩を有効成分とするヒアルロン酸産生促進剤。
＜５＞グリチルレチン酸又はその塩を有効成分とするヒアルロン酸分解抑制剤。
＜６＞グリチルレチン酸又はその塩を有効成分とする皮膚シワ予防又は改善剤。
＜７＞グリチルレチン酸又はその塩を有効成分とする皮膚たるみ予防又は改善剤。
＜８＞グリチルレチン酸又はその塩を有効成分とする皮膚ハリの低下予防又は改善剤。
Regarding the embodiments described above, the present invention further discloses the following aspects.
<1> A BRG-1 expression promoter containing glycyrrhetinic acid or its salt as an active ingredient.
<2> Elastic fiber formation promoter containing glycyrrhetinic acid or its salt as an active ingredient.
<3> A collagen fiber formation promoter containing glycyrrhetinic acid or its salt as an active ingredient.
<4> Hyaluronic acid production promoter containing glycyrrhetinic acid or its salt as an active ingredient.
<5> Hyaluronic acid decomposition inhibitor containing glycyrrhetinic acid or its salt as an active ingredient.
<6> Skin wrinkle prevention or improvement agent containing glycyrrhetinic acid or its salt as an active ingredient.
<7> A skin sagging prevention or improvement agent containing glycyrrhetinic acid or its salt as an active ingredient.
<8> An agent for preventing or improving skin elasticity containing glycyrrhetinic acid or a salt thereof as an active ingredient.
＜９＞ＢＲＧ－１発現促進剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１０＞弾性線維形成促進剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１１＞コラーゲン線維形成促進剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１２＞ヒアルロン酸産生促進剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１３＞ヒアルロン酸分解抑制剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１４＞皮膚シワ予防又は改善剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１５＞皮膚たるみ予防又は改善剤を製造するための、グリチルレチン酸又はその塩の使用。
＜１６＞皮膚ハリの低下予防又は改善剤を製造するための、グリチルレチン酸又はその塩の使用。
<9> Use of glycyrrhetinic acid or a salt thereof for producing a BRG-1 expression promoter.
<10> Use of glycyrrhetinic acid or a salt thereof for producing an elastic fiber formation promoter.
<11> Use of glycyrrhetinic acid or its salt for producing a collagen fiber formation promoter.
<12> Use of glycyrrhetinic acid or a salt thereof for producing a hyaluronic acid production promoter.
<13> Use of glycyrrhetinic acid or a salt thereof for producing a hyaluronic acid decomposition inhibitor.
<14> Use of glycyrrhetinic acid or its salt for producing a skin wrinkle prevention or improvement agent.
<15> Use of glycyrrhetinic acid or a salt thereof for producing an agent for preventing or improving skin sagging.
<16> Use of glycyrrhetinic acid or a salt thereof for producing an agent for preventing or improving skin elasticity.
＜１７＞ＢＲＧ－１発現促進に使用するための、グリチルレチン酸又はその塩。
＜１８＞弾性線維形成促進に使用するための、グリチルレチン酸又はその塩。
＜１９＞コラーゲン線維形成促進に使用するための、グリチルレチン酸又はその塩。
＜２０＞ヒアルロン酸産生促進に使用するための、グリチルレチン酸又はその塩。
＜２１＞ヒアルロン酸分解抑制に使用するための、グリチルレチン酸又はその塩。
＜２２＞皮膚シワ予防又は改善に使用するための、グリチルレチン酸又はその塩。
＜２３＞皮膚たるみ予防又は改善に使用するための、グリチルレチン酸又はその塩。
＜２４＞皮膚ハリの低下予防又は改善に使用するための、グリチルレチン酸又はその塩。
<17> Glycyrrhetinic acid or a salt thereof for use in promoting BRG-1 expression.
<18> Glycyrrhetinic acid or a salt thereof for use in promoting elastic fiber formation.
<19> Glycyrrhetinic acid or a salt thereof for use in promoting collagen fiber formation.
<20> Glycyrrhetinic acid or a salt thereof for use in promoting hyaluronic acid production.
<21> Glycyrrhetinic acid or a salt thereof for use in inhibiting hyaluronic acid decomposition.
<22> Glycyrrhetinic acid or a salt thereof for use in preventing or improving skin wrinkles.
<23> Glycyrrhetinic acid or a salt thereof for use in preventing or improving skin sagging.
<24> Glycyrrhetinic acid or a salt thereof for use in preventing or improving skin firmness.
＜２５＞ＢＲＧ－１発現促進のための、グリチルレチン酸又はその塩の使用。
＜２６＞弾性線維形成促進のための、グリチルレチン酸又はその塩の使用。
＜２７＞コラーゲン線維形成促進のための、グリチルレチン酸又はその塩の使用。
＜２８＞ヒアルロン酸産生促進のための、グリチルレチン酸又はその塩の使用。
＜２９＞ヒアルロン酸分解抑制のための、グリチルレチン酸又はその塩の使用。
＜３０＞皮膚シワ予防又は改善のための、グリチルレチン酸又はその塩の使用。
＜３１＞皮膚たるみ予防又は改善のための、グリチルレチン酸又はその塩の使用。
＜３２＞皮膚ハリの低下予防又は改善のための、グリチルレチン酸又はその塩の使用。
＜３３＞上記＜２５＞～＜３２＞において、上記使用は、好ましくは、美容的又は審美的な目的での非治療的な使用である。
<25> Use of glycyrrhetinic acid or a salt thereof for promoting BRG-1 expression.
<26> Use of glycyrrhetinic acid or its salt for promoting elastic fiber formation.
<27> Use of glycyrrhetinic acid or its salt for promoting collagen fiber formation.
<28> Use of glycyrrhetinic acid or its salt for promoting hyaluronic acid production.
<29> Use of glycyrrhetinic acid or its salt for inhibiting hyaluronic acid decomposition.
<30> Use of glycyrrhetinic acid or its salt for preventing or improving skin wrinkles.
<31> Use of glycyrrhetinic acid or its salt for preventing or improving skin sagging.
<32> Use of glycyrrhetinic acid or its salt for preventing or improving skin firmness.
<33> In the above <25> to <32>, the use is preferably a non-therapeutic use for cosmetic or aesthetic purposes.
＜３４＞ＢＲＧ－１発現促進方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜３５＞弾性線維形成促進方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜３６＞コラーゲン線維形成促進方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜３７＞ヒアルロン酸産生促進方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜３８＞ヒアルロン酸分解抑制方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜３９＞皮膚シワ予防又は改善方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜４０＞皮膚たるみ予防又は改善方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜４１＞皮膚ハリの低下予防又は改善方法であって、それを必要とする対象にグリチルレチン酸又はその塩を有効量で投与又は摂取させることを含む、方法。
＜４２＞上記＜３４＞～＜４１＞において、上記方法は、好ましくは、美容的又は審美的な目的での非治療的な方法である。
<34> A method for promoting BRG-1 expression, the method comprising administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<35> A method for promoting elastic fiber formation, the method comprising administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<36> A method for promoting collagen fibril formation, the method comprising administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<37> A method for promoting hyaluronic acid production, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<38> A method for inhibiting hyaluronic acid degradation, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<39> A method for preventing or improving skin wrinkles, the method comprising administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<40> A method for preventing or improving skin sagging, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<41> A method for preventing or improving skin elasticity, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject in need thereof.
<42> In the above <34> to <41>, the method is preferably a non-therapeutic method for cosmetic or aesthetic purposes.
実施例１ ＢＲＧ－１発現促進作用（１）
２４ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を５×１０４ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が１μＭまたは５μＭとなるように添加した。なお、コントールには溶媒であるエタノールのみを添加した。２日後、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（ＱＩＡＧＥＮ）を用い、添付のプロトコールに従って細胞からトータルＲＮＡを回収した。得られたＲＮＡは、Ｈｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ ｔｏ ｃＤＮＡ ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用い、添付のプロトコールに従ってトータルＲＮＡのｃＤＮＡ化を行った。次いで、調製したｃＤＮＡを鋳型として、ＴａｑＭａｎ ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＢＲＧ－１遺伝子発現量の定量化を行った。なお、得られた値は、ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）の遺伝子発現量で補正した。コントロールを１とした場合の遺伝子発現量の結果を図１に示す。
図１より、グリチルレチン酸に濃度依存的で有意なＢＲＧ－１遺伝子発現促進作用が確認された。
Example 1 BRG-1 expression promoting effect (1)
Normal human dermal fibroblasts (Kurabo Industries) were seeded in a 24-well plate at a cell density of 5×10 4 cells/well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid (Agritinon, Tokiwa Phytochemical Research Institute, Inc.) dissolved in ethanol was added to a final concentration of 1 μM or 5 μM. Note that only ethanol as a solvent was added as a control. Two days later, total RNA was collected from the cells using an RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was converted into cDNA of total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Next, the BRG-1 gene expression level was quantified using TaqMan gene expression assays (Applied Biosystems) using the prepared cDNA as a template. Note that the obtained values were corrected by the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of the gene expression level when the control was set as 1 are shown in FIG.
From FIG. 1, it was confirmed that glycyrrhetinic acid had a significant concentration-dependent promoting effect on BRG-1 gene expression.
実施例２ ＢＲＧ－１発現促進作用（２）
１２ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を２×１０５ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が５μＭとなるように添加した。なお、コントロールには溶媒であるエタノールのみを添加した。４日後、Ｃｅｌｌ ｌｙｓｉｓ ｂｕｆｆｅｒ（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて細胞からタンパク質を抽出し、ウェスタンブロッティング解析に用いた。５～１５％のＳＤＳ－ｇｅｌを用いて分離した後、定法に従ってＰＶＤＦメンブレンに転写し、抗ヒトＢＲＧ－１抗体（Ｓａｎｔａ Ｃｒｕｚ）、または、抗ヒトβ－ａｃｔｉｎ抗体（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ）とインキュベートした後、ペルオキシダーゼ標識二次抗体及びＥｎｈａｎｃｅｄ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ Ｐｌｕｓ 発色試薬（ＧＥ Ｈｅａｌｔｈｃａｒｅ）を用いてバンドを可視化した。バンド濃度はＡｍｅｒｓｈａｍ Ｉｍａｇｅｒ ６００（ＧＥ Ｈｅａｌｔｈｃａｒｅ）で定量した。結果を図２に示す。
図２より、グリチルレチン酸に有意なＢＲＧ－１タンパク質発現促進作用が確認された。
Example 2 BRG-1 expression promoting effect (2)
Normal human dermal fibroblasts (Kurabo Industries) were seeded in a 12-well plate at a cell density of 2×10 5 cells/well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., Agritinon) dissolved in ethanol was added at a final concentration of 5 μM. Note that only ethanol as a solvent was added to the control. After 4 days, proteins were extracted from the cells using Cell lysis buffer (Cell signaling technologies) and used for Western blotting analysis. After separation using 5-15% SDS-gel, it was transferred to a PVDF membrane according to a standard method and incubated with anti-human BRG-1 antibody (Santa Cruz) or anti-human β-actin antibody (Cell Signaling Technology). Thereafter, bands were visualized using a peroxidase-labeled secondary antibody and Enhanced Chemiluminescence Plus coloring reagent (GE Healthcare). Band concentrations were quantified using an Amersham Imager 600 (GE Healthcare). The results are shown in Figure 2.
From FIG. 2, it was confirmed that glycyrrhetinic acid had a significant effect of promoting BRG-1 protein expression.
実施例３ エラスチン線維形成促進作用
２４ウェルプレートの各ウェルにカバーガラスを設置し、その上に３×１０5ｃｅｌｌｓ／ウェルの細胞密度で正常ヒト真皮線維芽細胞（クラボウ）をＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、２％ＦＢＳ添加ＤＭＥＭ／Ｆ１２培地（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）に交換し、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が３μＭとなるように添加した。なお、コントロールには溶媒であるエタノールのみを添加した。３～４日おきに培地交換をして１４日後、細胞を氷冷メタノールで固定化した後、弾性線維の免疫染色を下記手順で行った。
Example 3 Elastin fiber formation promoting effect A cover glass was placed in each well of a 24-well plate, and normal human dermal fibroblasts (Kurabo) were placed on it at a cell density of 3 x 10 5 cells/well in DMEM medium (Invitrogen). It was sown using. The next day, the medium was changed to DMEM/F12 medium supplemented with 2% FBS (Sigma-Aldrich), and glycyrrhetinic acid (Tokiwa Phytochemical Research Institute, Inc., Agritinon) dissolved in ethanol was added to a final concentration of 3 μM. Note that only ethanol as a solvent was added to the control. After 14 days after exchanging the medium every 3 to 4 days, the cells were fixed with ice-cold methanol, and then immunostaining of elastic fibers was performed according to the following procedure.
マウス抗ヒトＥｌａｓｔｉｎ抗体（Ｍｉｌｌｉｐｏｒｅ）及びウサギ抗ヒトＦｉｂｒｉｌｌｉｎ－１抗体（Ｅｌａｓｔｉｎ Ｐｒｏｄｕｃｔ Ｃｏｍｐａｎｙ）と、Ａｌｅｘａ Ｆｌｕｏ ５４６標識抗マウスＩｇＧ抗体（Ｉｎｖｉｔｒｏｇｅｎ）及びＡｌｅｘａ Ｆｌｕｏ ４８８標識抗ウサギＩｇＧ抗体（Ｉｎｖｉｔｒｏｇｅｎ）を用いて細胞の免疫染色を行った。カバーガラスに接着した細胞をＰｒｏＬｏｎｇ Ｇｏｌｄ ａｎｔｉｆａｄｅ ｒｅａｇｅｎｔ ｗｉｔｈ ＤＡＰＩ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてスライドガラス上にマウントし、蛍光顕微鏡を用いて弾性線維の構成分子であるフィブリリン－１（励起波長：４８８ｎｍ、観察波長：５２５ｎｍ）、エラスチン（励起波長：５４６ｎｍ、観察波長：５７３ｎｍ）及び核（励起波長：３５０ｎｍ、観察波長：４７０ｎｍ）を観察した。結果を図３に示す。
コントロールと比較してグリチルレチン酸を添加した細胞において、細胞数(ＤＡＰＩ染色)とＦｉｂｒｉｌｌｉｎ－１染色に差は認められなかったが、Ｅｌａｓｔｉｎ染色の強度が増加しており、Ｆｉｂｒｉｌｌｉｎ－１とＥｌａｓｔｉｎを重ね合わせたＭｅｒｇｅ画像の強度の増加が認められた。これらのことより、グリチルレチン酸はミクロフィブリルへのエラスチンの沈着を促進し、成熟したエラスチン線維形成を促進することが確認された。
Mouse anti-human Elastin antibody (Millipore) and rabbit anti-human Fibrillin-1 antibody (Elastin Product Company), Alexa Fluo 546-labeled anti-mouse IgG antibody (Invitrogen) and Alexa Fluo 488-labeled anti-rabbit IgG antibody (Invitrogen) cells using vitrogen) immunostaining was performed. Cells adhered to the cover glass were mounted on a slide glass using ProLong Gold antifade reagent with DAPI (Invitrogen), and fibrillin-1, a constituent molecule of elastic fibers, was detected using a fluorescence microscope (excitation wavelength: 488 nm, observation wavelength: 525 nm), elastin (excitation wavelength: 546 nm, observation wavelength: 573 nm), and nuclei (excitation wavelength: 350 nm, observation wavelength: 470 nm). The results are shown in Figure 3.
There was no difference in cell number (DAPI staining) and Fibrillin-1 staining in cells to which glycyrrhetinic acid was added compared to controls, but the intensity of Elastin staining increased, and Fibrillin-1 and Elastin were superimposed. An increase in the intensity of the combined Merge image was observed. From these results, it was confirmed that glycyrrhetinic acid promotes the deposition of elastin on microfibrils and promotes the formation of mature elastin fibers.
実施例４ コラーゲン線維形成促進作用
２４ウェルプレートの各ウェルにカバーガラスを設置し、その上に３×１０５ｃｅｌｌｓ／ウェルの細胞密度で正常ヒト真皮線維芽細胞（クラボウ）をＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、２％ＦＢＳ添加ＤＭＥＭ／Ｆ１２培地（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）に交換し、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を添加した。なお、コントロールには溶媒であるエタノールのみを添加した。７日後、細胞を氷冷メタノールで固定化した後、コラーゲン線維の免疫染色を下記手順で行った。
Example 4 Collagen fiber formation promoting effect A cover glass was placed in each well of a 24-well plate, and normal human dermal fibroblasts (Kurabo) were placed on it at a cell density of 3 x 10 5 cells/well in DMEM medium (Invitrogen). It was sown using. The next day, the medium was changed to DMEM/F12 medium (Sigma-Aldrich) supplemented with 2% FBS, and glycyrrhetinic acid (Agritinon, Tokiwa Phytochemical Research Institute, Inc.) dissolved in ethanol was added. Note that only ethanol as a solvent was added to the control. Seven days later, the cells were fixed with ice-cold methanol, and then immunostaining of collagen fibers was performed according to the following procedure.
ウサギ抗ヒトＣｏｌｌａｇｅｎ Ｉ抗体（Ｃｅｄａｒｌａｎｅ）と、Ａｌｅｘａ Ｆｌｕｏ ４８８標識抗ウサギＩｇＧ抗体（Ｉｎｖｉｔｒｏｇｅｎ）を用いて細胞の免疫染色を行った。カバーガラスに接着した細胞をＰｒｏＬｏｎｇ Ｇｏｌｄ ａｎｔｉｆａｄｅ ｒｅａｇｅｎｔ ｗｉｔｈ ＤＡＰＩ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてスライドガラス上にマウントし、蛍光顕微鏡を用いてＣｏｌｌａｇｅｎ Ｉ（励起波長：４８８ｎｍ、観察波長：５２５ｎｍ）及び核（励起波長：３５０ｎｍ、観察波長：４７０ｎｍ）を観察した。結果を図４に示す。
コントロールと比較してグリチルレチン酸を添加した細胞において、細胞数(ＤＡＰＩ染色)に差は認められなかったが、Ｃｏｌｌａｇｅｎ Ｉの染色強度が増加していたことから、グリチルレチン酸はコラーゲン線維形成を促進することが確認された。
Cells were immunostained using rabbit anti-human Collagen I antibody (Cedarlane) and Alexa Fluo 488-labeled anti-rabbit IgG antibody (Invitrogen). Cells adhered to the cover glass were mounted on a slide glass using ProLong Gold antifade reagent with DAPI (Invitrogen), and Collagen I (excitation wavelength: 488 nm, observation wavelength: 525 nm) and nuclei (excitation wavelength: 350 nm, observation wavelength: 470 nm). The results are shown in Figure 4.
Although there was no difference in the number of cells (DAPI staining) in the cells to which glycyrrhetinic acid was added compared to the control, the staining intensity of Collagen I increased, suggesting that glycyrrhetinic acid promotes collagen fibril formation. This was confirmed.
実施例５ ヒアルロン酸関連因子への作用
（１）２４ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を５×１０４ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が５μＭとなるように添加した。なお、コントロールには溶媒であるエタノールのみを添加した。翌日、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（ＱＩＡＧＥＮ）を用い添付のプロトコールに従って細胞からトータルＲＮＡを回収した。得られたＲＮＡはＨｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ ｔｏ ｃＤＮＡ ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用い添付のプロトコールに従ってトータルＲＮＡのｃＤＮＡ化を行った。次いで、調製したｃＤＮＡを鋳型として、ＴａｑＭａｎ ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＢＲＧ－１、ＨＡＳ２（Ｈｙａｌｕｒｏｎａｎ ｓｙｎｔｈａｓｅ ２）、ＨＹＢＩＤ（ＫＩＡＡ１１９９）遺伝子発現量の定量化を行った。なお、得られた値は、ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）の遺伝子発現量で補正した。コントールを１とした場合の各遺伝子発現量の結果を図５Ａに示す。
図５Ａより、グリチルレチン酸はヒアルロン酸合成酵素であるＨＡＳ２遺伝子発現を促進し、ヒアルロン酸分解に重要なＨＹＢＩＤ遺伝子発現を抑制することが確認された。従って、グリチルレチン酸にはヒアルロン酸産生促進作用とヒアルロン酸分解抑制作用があると考えられる。
Example 5 Effect on hyaluronic acid-related factors (1) Normal human dermal fibroblasts (Kurabo) were seeded in a 24-well plate at a cell density of 5×10 4 cells/well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., Agritinon) dissolved in ethanol was added at a final concentration of 5 μM. Note that only ethanol as a solvent was added to the control. The next day, total RNA was collected from the cells using an RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was converted into cDNA of total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Next, using the prepared cDNA as a template, the expression levels of BRG-1, HAS2 (Hyaluronan synthase 2), and HYBID (KIAA1199) genes were quantified using TaqMan gene expression assays (Applied Biosystems). I did it. Note that the obtained values were corrected by the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of the expression level of each gene when the control was set to 1 are shown in FIG. 5A.
From FIG. 5A, it was confirmed that glycyrrhetinic acid promotes the expression of the HAS2 gene, which is a hyaluronic acid synthase, and suppresses the expression of the HYBID gene, which is important for hyaluronic acid decomposition. Therefore, glycyrrhetinic acid is considered to have an effect of promoting hyaluronic acid production and an effect of suppressing hyaluronic acid decomposition.
（２）１２ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を２×１０５ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が５μＭとなるように添加した。なお、コントロールには溶媒であるエタノールのみを添加した。４日後、Ｃｅｌｌ ｌｙｓｉｓ ｂｕｆｆｅｒ（Ｃｅｌｌ ｓｉｇｎａｌｉｎｇ ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて細胞からタンパクを抽出し、ウェスタンブロッティング解析に用いた。５～１５％のＳＤＳ－ｇｅｌを用いて分離した後、定法に従ってＰＶＤＦメンブレンに転写し、抗ヒトＢＲＧ－１抗体（Ｓａｎｔａ Ｃｒｕｚ）、または、抗ヒトＨＹＢＩＤ抗体（Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5612-7.に従い調製）、抗ヒトβ－ａｃｔｉｎ抗体（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ）とインキュベートした後、ペルオキシダーゼ標識二次抗体及びＥｎｈａｎｃｅｄ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ Ｐｌｕｓ発色試薬（ＧＥ Ｈｅａｌｔｈｃａｒｅ）を用いてバンドを可視化した。結果を図５Ｂに示す。
図５Ｂより、グリチルレチン酸はＨＹＢＩＤタンパク発現を抑制することが確認された。
(2) Normal human dermal fibroblasts (Kurabo Industries) were seeded in a 12-well plate at a cell density of 2×10 5 cells/well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., Agritinon) dissolved in ethanol was added at a final concentration of 5 μM. Note that only ethanol as a solvent was added to the control. After 4 days, proteins were extracted from the cells using Cell lysis buffer (Cell signaling technologies) and used for Western blotting analysis. After separation using 5-15% SDS-gel, it was transferred to a PVDF membrane according to a standard method, and anti-human BRG-1 antibody (Santa Cruz) or anti-human HYBID antibody (Proc Natl Acad Sci US A. 2013 Apr. 2;110(14):5612-7), and incubated with anti-human β-actin antibody (Cell Signaling Technology), followed by peroxidase-labeled secondary antibody and Enhanced Chemiluminescence Plus coloring reagent (GE Healthcare). band using was visualized. The results are shown in Figure 5B.
From FIG. 5B, it was confirmed that glycyrrhetinic acid suppresses HYBID protein expression.
実施例６ ヒアルロン分解抑制作用
２４ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を１．５×１０５ｃｅｌｌｓ／ウェルの細胞密度でＭＥＭ培地（Ｓｉｇｍａ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が３μＭとなるように添加した。３日後、蛍光標識高分子ＨＡ（ＦＡＨＡ－Ｈ１；ＰＧリサーチ、終濃度：１０ｍｇ／ｍｌ）を添加した。３日後、培養上清を回収してヒアルロン酸分解抑制評価試験に供した。
ＨＰＬＣシステムはＬａＣｈｒｏｍ Ｅｌｉｔｅ（日立ハイテクノロジーズ）を用い、検出は励起波長４９５ｎｍ、蛍光波長５２０ｎｍで行った。溶媒には０．２Ｍ ＮａＣｌを用い、流速は０．５ｍｌ／ｍｉｎにて実施した。カラムはＳＥＣカラム（東ソー ＴＳＫ－ＧＬ Ｇ５０００ ＰＷ－ＸＬ）を用い、カラム温度は２５℃、インジェクション量は５ｍｌとした。ヒアルロン酸分解率は高分子ヒアルロン酸の減少量とし、次の式にて算出した。
ヒアルロン酸分解率（％）＝（細胞に添加する前の高分子ヒアルロン酸のピーク高－グリチルレチン酸を添加した細胞（コントロールはグリチルレチン酸の溶媒であるエタノールを同濃度添加した細胞）とインキュベーション後の高分子ヒアルロン酸のピーク高）／（細胞に添加する前の高分子ヒアルロン酸のピーク高－グリチルレチン酸を添加しない細胞とインキュベーション後の高分子ヒアルロン酸のピーク高）×１００。
結果を図６に示す。図６より、グリチルレチン酸はヒアルロン酸の分解を抑制することが確認された。
Example 6 Hyaluronic Degradation Inhibition Effect Normal human dermal fibroblasts (Kurabo) were seeded at a cell density of 1.5×10 5 cells/well in a 24-well plate using MEM medium (Sigma). The next day, glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., Agritinon) dissolved in ethanol was added at a final concentration of 3 μM. Three days later, fluorescently labeled polymeric HA (FAHA-H1; PG Research, final concentration: 10 mg/ml) was added. Three days later, the culture supernatant was collected and subjected to a hyaluronic acid degradation inhibition evaluation test.
The HPLC system used was LaChrom Elite (Hitachi High Technologies), and detection was performed at an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm. 0.2 M NaCl was used as the solvent, and the flow rate was 0.5 ml/min. The column used was an SEC column (Tosoh TSK-GL G5000 PW-XL), the column temperature was 25° C., and the injection volume was 5 ml. The hyaluronic acid decomposition rate was calculated using the following formula as the amount of decrease in polymeric hyaluronic acid.
Hyaluronic acid degradation rate (%) = (Peak height of polymeric hyaluronic acid before addition to cells - cells to which glycyrrhetinic acid was added (control is cells to which the same concentration of ethanol, a solvent for glycyrrhetinic acid, was added) and after incubation Peak height of polymeric hyaluronic acid)/(peak height of polymeric hyaluronic acid before addition to cells - peak height of polymeric hyaluronic acid after incubation with cells without addition of glycyrrhetinic acid)×100.
The results are shown in FIG. From FIG. 6, it was confirmed that glycyrrhetinic acid suppresses the decomposition of hyaluronic acid.
実施例７ ヒアルロン酸産生促進作用
１２ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を２×１０５ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、エタノールに溶解したグリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン）を、終濃度が１μＭとなるように添加した。なお、コントロールには溶媒であるエタノールのみを添加した。４日後、培養上清を回収し、ＱｎＥ Ｈｙａｌｕｒｏｎｉｃ Ａｃｉｄ ＥＬＩＳＡ Ａｓｓａｙ Ｋｉｔ（Ｂｉｏｔｅｃｈ Ｔｒａｄｉｎｇ Ｐａｒｔｎｅｒｓ）を用い、添付のプロトコールにしたがってヒアルロン酸量の定量を行った。結果を図７に示す。
図７より、グリチルレチン酸はヒアルロン酸量を増加させることが確認された。この結果は、グリチルレチン酸によるＨＡＳ２遺伝子発現の促進による産生促進作用とＨＹＢＩＤ遺伝子の抑制に基く分解抑制作用によるものと推察される。
Example 7 Hyaluronic acid production promoting effect Normal human dermal fibroblasts (Kurabo) were seeded at a cell density of 2×10 5 cells/well in a 12-well plate using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., Agritinon) dissolved in ethanol was added to a final concentration of 1 μM. Note that only ethanol as a solvent was added to the control. Four days later, the culture supernatant was collected, and the amount of hyaluronic acid was quantified using QnE Hyaluronic Acid ELISA Assay Kit (Biotech Trading Partners) according to the attached protocol. The results are shown in FIG.
From FIG. 7, it was confirmed that glycyrrhetinic acid increases the amount of hyaluronic acid. This result is presumed to be due to the production-enhancing effect of glycyrrhetinic acid by promoting HAS2 gene expression and the decomposition-inhibiting effect by suppressing the HYBID gene.
比較例１ グリチルリチン酸ジカリウム及びグリチルレチン酸ステアリルのＢＲＧ－１発現促進作用
２４ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を５×１０４ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、グリチルレチン酸（株式会社常磐植物化学研究所、アグリチノン、溶媒：エタノール）、グリチルリチン酸ジカリウム（アルプス薬品工業株式会社、溶媒：ＤＭＳＯ）またはグリチルレチン酸ステアリル（株式会社常磐植物化学研究所、アグリチノンステアリル、溶媒エタノール）を、終濃度が５μＭとなるように添加した。なお、コントロールには各溶媒のみを同濃度添加した。２日後、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（ＱＩＡＧＥＮ）を用い添付のプロトコールに従って細胞からトータルＲＮＡを回収した。得られたＲＮＡはＨｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ ｔｏ ｃＤＮＡ ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用い添付のプロトコールに従ってトータルＲＮＡのｃＤＮＡ化を行った。次いで、調製したｃＤＮＡを鋳型として、ＴａｑＭａｎ ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＢＲＧ－１遺伝子発現量の定量化を行った。なお、得られた値は、ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）の遺伝子発現量で補正した。結果を図８に示す。
図８より、グリチルリチン酸ジカリウム及びグリチルレチン酸ステアリルはＢＲＧ－１発現促進作用を示さず、グリチルレチン酸のみが優れたＢＲＧ－１発現促進作用を示すことが確認された。
Comparative Example 1 BRG-1 expression promoting effect of dipotassium glycyrrhizinate and stearyl glycyrrhetinate Normal human dermal fibroblasts (Kurabo) were cultured in a 24-well plate at a cell density of 5 x 10 4 cells/well using DMEM medium (Invitrogen). The seeds were sown. The next day, use glycyrrhetinic acid (Tokiwa Phytochemical Research Institute Co., Ltd., agritinone, solvent: ethanol), dipotassium glycyrrhizinate (Alps Pharmaceutical Industries Co., Ltd., solvent: DMSO) or stearyl glycyrrhetinate (Tokiwa Phytochemical Research Institute Co., Ltd., agritinone stearyl). , solvent ethanol) was added to a final concentration of 5 μM. Note that each solvent alone at the same concentration was added to the control. Two days later, total RNA was collected from the cells using an RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was converted into cDNA of total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Next, the BRG-1 gene expression level was quantified using TaqMan gene expression assays (Applied Biosystems) using the prepared cDNA as a template. Note that the obtained values were corrected by the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results are shown in FIG.
From FIG. 8, it was confirmed that dipotassium glycyrrhizinate and stearyl glycyrrhetinate did not show an effect of promoting BRG-1 expression, and only glycyrrhetinic acid showed an excellent effect of promoting BRG-1 expression.
比較例２ カンゾウ抽出液のＢＲＧ－１発現促進作用
２４ウェルプレートに、正常ヒト真皮線維芽細胞（クラボウ）を５×１０４ｃｅｌｌｓ／ウェルの細胞密度でＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて播種した。翌日、カンゾウ（Ｇｌｙｃｙｒｒｈｉｚａ ｇｌａｂｒａ）抽出液ＢＧ－Ｊ（丸善製薬、抽出部位：根・根茎、抽出溶媒：５０％（ｖ／ｖ）ブチレングリコール）を各種濃度で添加した。なお、いずれの場合もブチレングリコールの終濃度０.５％となるように調整し、コントロールにはブチレングリコールのみを添加した。２日後、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（ＱＩＡＧＥＮ）を用い添付のプロトコールに従って細胞からトータルＲＮＡを回収した。得られたＲＮＡはＨｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ ｔｏ ｃＤＮＡ ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用い添付のプロトコールに従ってトータルＲＮＡのｃＤＮＡ化を行った。次いで、調製したｃＤＮＡを鋳型として、ＴａｑＭａｎ ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＢＲＧ－１遺伝子発現量の定量化を行った。なお、得られた値は、ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－３－ｐｈｏｓｐｈａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＧＡＰＤＨ）の遺伝子発現量で補正した。コントロールを１とした場合のカンゾウ抽出液のＢＲＧ－１遺伝子発現量の結果を図９に示す。
図９より、カンゾウ抽出液には何れの添加濃度でも有意なＢＲＧ－１発現促進作用は認められなかった。
Comparative Example 2 BRG-1 Expression Promoting Effect of Licorice Extract Normal human dermal fibroblasts (Kurabo) were seeded in a 24-well plate at a cell density of 5×10 4 cells/well using DMEM medium (Invitrogen). The next day, licorice (Glycyrrhiza glabra) extract BG-J (Maruzen Pharmaceutical, extraction site: roots and rhizomes, extraction solvent: 50% (v/v) butylene glycol) was added at various concentrations. In each case, the final concentration of butylene glycol was adjusted to 0.5%, and only butylene glycol was added to the control. Two days later, total RNA was collected from the cells using an RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was converted into cDNA of total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Next, the BRG-1 gene expression level was quantified using TaqMan gene expression assays (Applied Biosystems) using the prepared cDNA as a template. Note that the obtained values were corrected by the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). FIG. 9 shows the results of the BRG-1 gene expression level in the liquorice extract when the control was set as 1.
From FIG. 9, no significant BRG-1 expression promoting effect was observed in the licorice extract at any concentration added.
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| JP2012041302A (en) | 2010-08-20 | 2012-03-01 | Kracie Home Products Ltd | Skin cosmetic |
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| WO2019078370A1 (en) | 2017-10-20 | 2019-04-25 | ロート製薬株式会社 | Composition for ameliorating skin disorders |
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