JP2021054778A - BRG-1 expression promoter - Google Patents
BRG-1 expression promoter Download PDFInfo
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- JP2021054778A JP2021054778A JP2019182227A JP2019182227A JP2021054778A JP 2021054778 A JP2021054778 A JP 2021054778A JP 2019182227 A JP2019182227 A JP 2019182227A JP 2019182227 A JP2019182227 A JP 2019182227A JP 2021054778 A JP2021054778 A JP 2021054778A
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- JP
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- Prior art keywords
- glycyrrhetinic acid
- salt
- brg
- expression
- skin
- Prior art date
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Abstract
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 elasticity as it ages, resulting in decreased firmness, wrinkles, and sagging. Especially on the skin of the face, neck, shoulders and other areas exposed to sunlight, the formation of wrinkles and sagging, or the formation of stains and freckles is more pronounced than other areas not exposed to light due to the effects of chronic ultraviolet rays. Become. Skin aging that occurs in these UV-exposed areas is called photoaging. Photoaging is a change in the skin caused by chronic sun exposure, especially exposure to ultraviolet light, which accelerates the internal aging process of the skin.
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 constituents 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 or 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。 Since eukaryotic cell DNA is in a highly folded chromatin structure, in order for the gene to be transcribed and exert its function, this chromatin structure is unraveled and transcription factors and transcriptional activators can access the DNA. It is important to be. The protein population having the function of solving the chromatin structure is called a chromatin remodeling complex, and one of them is a 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). The fact that BRG-1 is considered to be involved in the repair of DNA damaged by UV irradiation (Non-Patent Documents 2 and 3) and that the cell proliferation of melanoma knocked down by BRG-1 decreased (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, there is a relationship between the expression level of BRG-1 in human skin and skin elasticity, and the expression of collagen and elastic fiber constituent factors, which are components of ECM, is expressed by the decrease in the expression of BRG-1 in fibroblasts. It was clarified that the amount was reduced, and the control of the expression or activity of BRG-1 was used as an index to control the formation of collagen fibers, elastic fibers, ECM, etc., improve the elasticity of the skin, and reduce the wrinkles, sagging, and firmness of the skin. It has been reported that materials capable of preventing or improving collagen can be screened (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 various anti-inflammatory effects, alveolar bone resorption inhibitory effects due to periodontal disease, histamine release inhibitory effects, and the like. It is incorporated into the oral composition.
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 an extract of licorice containing a large amount of glycyrrhizic acid has an adipose stem cell attracting action, suppresses dermis cavitation, and is effective in improving skin sagging (Patent Document 2).
However, it is not known that glycyrrhetinic acid has an action of promoting the expression of BRG-1.
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, reduction of firmness, and the like.
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 components that can be applied over a long period of time, the present inventors have found that glycyrrhetinic acid has an excellent BRG-1 expression promoting action and is useful for promoting the formation of elastic fibers. It was.
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 a salt thereof as an active ingredient.
2) An elastic fiber formation promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
3) A collagen fiber formation promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
4) A hyaluronic acid production promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
5) A hyaluronic acid decomposition inhibitor containing glycyrrhetinic acid or a salt thereof as an active ingredient.
6) An agent for preventing or improving skin wrinkles containing glycyrrhetinic acid or a salt thereof as an active ingredient.
7) An agent for preventing or improving skin sagging containing glycyrrhetinic acid or a salt thereof as an active ingredient.
8) A preventive or ameliorating agent for reducing skin firmness containing glycyrrhetinic acid or a salt thereof 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, and the wrinkles, sagging, and firmness of the skin are prevented or improved. 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。 The "glycyrrhetinic acid" in the present invention specifically refers to 3β-hydroxy-11-oxooleana-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 salt of glycyrrhetinic acid include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as magnesium and calcium.
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。 Glycyrrhetinic acid can be obtained by hydrolyzing glycyrrhizic acid obtained from licorice (licorice, Glycyrrhiza glabra, Glycyrrhiza uralensis or other similar plants) (Bioengineering Vol. 89, No. 11, 2011 656-659), In addition, a commercially available product, for example, "Agritinone" (Joban Plant Chemistry 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 Examples below, glycyrrhetinic acid has the effect of promoting the expression of BRG-1 at the gene and protein levels. As described above, since the expression level of collagen and elastic fiber constituent factors, which are components of ECM, decreases due to the decrease in the expression of BRG-1, the expression of BRG-1 is promoted to reduce the expression of collagen fibers. It is considered that the formation of collagen and elastic fibers can be promoted to improve skin elasticity, and wrinkles, sagging, and reduction of firmness of the skin can be prevented or improved. In fact, glycyrrhetinic acid has an effect of promoting the deposition of elastin on microfibrils and an effect of promoting collagen fiber formation, promotes the production of hyaluronic acid, which is one of the constituents of ECM, and decomposes hyaluronic acid. It was confirmed that it has an inhibitory 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 a salt thereof is a BRG-1 expression promoter, an elastic fibrosis promoter, a collagen fibrosis promoter, a hyaluronic acid production promoter, a hyaluronic acid decomposition inhibitor, a skin wrinkle prevention or ameliorating agent, and a skin sagging prevention agent. Alternatively, it can be an improving agent (hereinafter, also referred to as "BRG-1 expression promoter, etc."), to promote the expression of BRG-1, to promote the formation of elastic fibers, to promote the formation of collagen fibers, and to to promote hyaluronic acid. It can be used to promote production, suppress hyaluronic acid degradation, prevent or improve skin wrinkles, prevent or improve skin sagging, prevent or improve skin firmness, and also. It can be used to produce a BRG-1 expression promoter 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" can be administration or ingestion to a human or non-human animal, and may be therapeutic or non-therapeutic use. The term "non-therapeutic" means a concept that does not include medical practice, that is, a concept that does not include a method of surgery, treatment, or diagnosis of a human being, more specifically, a doctor or a person who has been instructed by a doctor to treat a human being. It is a concept that does not include a method 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, promotion of BRG-1 expression includes promotion of expression at the gene level and promotion of 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 stabilization of mRNA, suppression of mRNA degradation, and promotion of expression at the protein level of mRNA. Includes promotion in translation, stabilization promotion and degradation suppression of synthesized BRG-1 protein. Of these, in the present invention, promotion of mRNA expression at the gene level is preferable, and promotion of transcription into mRNA is more preferable. Here, "BRG-1" is also known as SMARCA4, and refers to a human BRG-1 protein represented by AAI3645.1 (GenBank), or a homolog or ortholog thereof.
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。
Further, in the present invention, the elastic fiber refers to an elastin fiber, and it is considered that elastin is deposited and crosslinked around a microfibril. Therefore, "promotion of elastic fiber formation" means an increase in the amount of elastic fibers, in particular, an increase in the amount of elastic fibers due to the promotion of the process of depositing and cross-linking elastin around microfibrils.
Further, "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, "decrease in firmness" means a decrease in skin elasticity due to qualitative and quantitative decrease in collagen fibers, elastin fibers, and hyaluronic acid in the skin caused by aging, and "wrinkle" means a decrease in firmness. It means folds and muscles that occur on the surface of the skin due to such factors, and "sag" means a state in which the skin morphology hangs down due to gravity that occurs due to a decrease in elasticity.
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, the term "prevention" means 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.
In addition, "improvement" means improvement of disease, symptom or condition, prevention or delay of deterioration of disease, symptom or condition, or reversal, prevention or delay of progression of disease or symptom.
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 and the like of the present invention, when ingested or administered to animals including humans, have a BRG-1 expression promoting effect, an elastic fibrosis promoting effect, a collagen fibrosis promoting effect, a hyaluronic acid production promoting effect, and the like. Hyaluronic acid decomposition inhibitory effect, skin wrinkle prevention or improvement effect, skin sagging prevention or improvement effect, skin firmness reduction prevention or improvement effect. It can be a material or formulation. The drug, quasi-drug, cosmetic, or food may contain other active ingredients for preventing or improving skin wrinkles, sagging, and firmness.
In addition, the foods include functional foods, foods for specified health uses, supplements, etc., which are based on the concept of preventing or improving skin wrinkles, sagging, and firmness, and indicating that fact 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 drug (including quasi-drugs) containing glycyrrhetinic acid or a salt thereof is arbitrary, and may be oral administration or parenteral administration. Dosage forms for oral administration include solid formulations such as tablets, granules, powders and capsules, as well as liquid formulations such as elixir, syrup and suspension. Dosage forms for parenteral administration include topical skin, transdermal, transmucosal, nasal, intestinal, injection, suppository, injection, inhalation, and patch. Of these, a suitable formulation form is an external preparation for skin, and specific examples thereof include forms such as ointments, emulsions, creams, emulsions, 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 a salt thereof include face and body cosmetics (for example, lotions, gels, creams, facial masks, etc.), makeup cosmetics, facial or body cleansers, and the like. 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 the food containing glycyrrhetinic acid or a salt thereof may be solid, semi-solid or liquid (for example, a beverage). Examples of the food include soft drinks, tea-based beverages, coffee beverages, fruit juice beverages, carbonated beverages, jelly-like beverages, near-water beverages, and foods and drinks such as jelly, wafers, biscuits, bread, noodles, and sausages. Various foods such as goods and nutritional foods, and their raw materials can be mentioned.
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。 Such pharmaceuticals (including non-pharmaceutical products), cosmetics, and food preparations contain glycyrrhetinic acid or a salt thereof, as required, as a carrier or food material that is pharmaceutically or cosmetically acceptable, and the other active ingredients described above. It can be produced according to a conventional method in combination with an ingredient, other medicinal ingredients, cosmetic ingredients, food additives and the like. Pharmaceutically or cosmetically acceptable carriers include, for example, various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, etc. Examples thereof include an ultraviolet absorber, an emulsion stabilizer, a pH adjuster, a pigment, and a fragrance. Examples of the other medicinal and cosmetic ingredients include plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, refreshing agents, anti-fat leaking agents, cleaning agents, makeup ingredients, etc. Food additives include, for example, solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bitterness agents, pH regulators, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, etc. Examples thereof include thickeners, fixing agents, dispersants, fluidity improvers, moisturizers, fragrances, seasonings, flavor adjusters 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 a salt thereof in the above-mentioned pharmaceutical products (including quasi-drugs), cosmetics and foods varies depending on the form of the agent used for administration, and therefore cannot be unconditionally stated, but for example, 0.0001 mass. % Or more, more preferably 0.0005% by mass or more, further preferably 0.001% by mass or more, and preferably 50% by mass or less, more preferably 20% by mass or less, still more preferable. 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 more preferably 0.001 to 10% by mass. When the active ingredient is in the form of a salt, the content thereof shall be converted into the content of the corresponding free substance.
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 pharmaceutical products (including quasi-drugs), cosmetics and foods may be an amount capable of achieving the effects of the present invention. The dose may vary depending on the subject's condition, body weight, sex, age or other factors, but per adult (60 kg) per day, in terms of glycyrrhetinic acid, for example, preferably 0.0001 mg or more, more preferably. It 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, 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 further 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。 Targets to which the BRG-1 expression promoter of the present invention is applied include humans and non-human animals having a decrease in skin elasticity or a risk thereof, and humans who desire prevention or improvement of skin wrinkles and sagging. Examples of non-human animals include non-human mammals, and examples of non-human mammals include apes, other primates, mice, rats, hamsters, horses, cows, pigs, sheep, goats, dogs, cats, etc. And companion animals and the like.
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 above-described embodiment, the following aspects are further disclosed in the present invention.
<1> A BRG-1 expression promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<2> An elastic fiber formation promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<3> A collagen fiber formation promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<4> A hyaluronic acid production promoter containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<5> A hyaluronic acid decomposition inhibitor containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<6> An agent for preventing or improving skin wrinkles containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<7> An agent for preventing or improving skin sagging containing glycyrrhetinic acid or a salt thereof as an active ingredient.
<8> A preventive or ameliorating agent for reducing skin firmness 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 a salt thereof 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 a salt thereof for producing a skin wrinkle prevention or improving agent.
<15> Use of glycyrrhetinic acid or a salt thereof for producing a skin sagging preventive or ameliorating agent.
<16> Use of glycyrrhetinic acid or a salt thereof for producing a preventive or ameliorating agent for reducing skin firmness.
＜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 suppressing 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 the decrease in 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 a salt thereof for promoting elastic fiber formation.
<27> Use of glycyrrhetinic acid or a salt thereof for promoting collagen fibrosis.
<28> Use of glycyrrhetinic acid or a salt thereof for promoting hyaluronic acid production.
<29> Use of glycyrrhetinic acid or a salt thereof for suppressing hyaluronic acid decomposition.
<30> Use of glycyrrhetinic acid or a salt thereof for prevention or improvement of skin wrinkles.
<31> Use of glycyrrhetinic acid or a salt thereof for prevention or improvement of skin sagging.
<32> Use of glycyrrhetinic acid or a salt thereof for prevention or improvement of reduction of skin firmness.
<33> In the above <25> to <32>, the above 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 the expression of BRG-1, which comprises 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 fibrosis, which comprises 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 fibrosis, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject who needs it.
<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 who needs it.
<38> A method for suppressing hyaluronic acid decomposition, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject who needs it.
<39> A method for preventing or improving skin wrinkles, which comprises 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 who needs it.
<41> A method for preventing or ameliorating a decrease in skin firmness, which comprises administering or ingesting an effective amount of glycyrrhetinic acid or a salt thereof to a subject who needs it.
<42> In <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 action (1)
Normal human dermal fibroblasts (Krabou) were seeded in 24-well plates at a cell density of 5 × 10 4 cells / well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 1 μM or 5 μM. Only ethanol, which is a solvent, was added to the control. Two days later, total RNA was recovered from the cells using the RNeasy mini kit (QIAGEN) according to the attached protocol. For the obtained RNA, total RNA was cDNAd according to the attached protocol using a High capacity RNA to cDNA kit (Applied Biosystems). Then, using the prepared cDNA as a template, the expression level of the BRG-1 gene was quantified using TaqMan gene expression responses (Applied Biosystems). The obtained value was corrected by the gene expression level of glyceraldehyde-3-phosphate dehydogenase (GAPDH). The result of the gene expression level when the control is set to 1 is shown in FIG.
From FIG. 1, a concentration-dependent and significant BRG-1 gene expression-promoting effect was confirmed for glycyrrhetinic acid.
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 action (2)
Normal human dermal fibroblasts (Krabou) were seeded in 12-well plates at a cell density of 2 × 10 5 cells / well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 5 μM. Only ethanol, which is a solvent, was added to the control. After 4 days, proteins were extracted from cells using Cell Lysis buffer (Cell signaling technologies) and used for Western blotting analysis. After separation using 5 to 15% SDS-gel, it was transferred to a PVDF membrane according to a conventional method and incubated with an anti-human BRG-1 antibody (Santa Cruz) or an anti-human β-actin antibody (Cell Signaling Technology). After that, the band was visualized using a peroxidase-labeled secondary antibody and an Enhanced Chemiluminescence Plus color-developing reagent (GE Healthcare). The band concentration was quantified with Amersham Imager 600 (GE Healthcare). The results are shown in FIG.
From FIG. 2, a significant BRG-1 protein expression promoting action was confirmed for glycyrrhetinic acid.
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 fibrosis promoting action A cover glass is placed in each well of a 24-well plate, and normal human dermal fibroblasts (Kurabou) are placed on the cover glass at a cell density of 3 × 10 5 cells / well in DMEM medium (Invitrogen). Was sown using. The next day, the medium was replaced with 2% FBS-added DMEM / F12 medium (Sigma-Aldrich), and glycyrrhetinic acid dissolved in ethanol (Agritinone, Joban Plant Chemistry Laboratory Co., Ltd.) was added so that the final concentration was 3 μM. Only ethanol, which is a solvent, was added to the control. After 14 days after the medium was changed every 3 to 4 days, the cells were immobilized 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。
Using mouse anti-human Elastin antibody (Millipore) and rabbit anti-human Fiberlin-1 antibody (Elastin Product Company), Alexa Fluo 546-labeled anti-mouse IgG antibody (Invitrogen), and Alexa Fluo 488-labeled anti-rabbit IgG antibody (Invitrogen). Immunostaining was performed. The cells adhered to the cover glass were mounted on a slide glass using ProLong Gold antigen fact with DAPI (Invitrogen), and using a fluorescence microscope, fibrillin-1, which is a constituent molecule of elastic fibers (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) were observed. The results are shown in FIG.
In the cells to which glycyrrhetinic acid was added as compared with the control, there was no difference in the number of cells (DAPI staining) and Fibrillin-1 staining, but the intensity of Elastin staining was increased, and Fibrillin-1 and Elastin were overlapped. 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 fibrils.
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 fibrosis promoting action A cover glass is placed in each well of a 24-well plate, and normal human dermal fibroblasts (Kurabou) are placed on the cover glass at a cell density of 3 × 10 5 cells / well in DMEM medium (Invitrogen). Was sown using. The next day, the medium was replaced with 2% FBS-added DMEM / F12 medium (Sigma-Aldrich), and glycyrrhetinic acid dissolved in ethanol (Agritinone, Joban Plant Chemistry Laboratory Co., Ltd.) was added. Only ethanol, which is a solvent, was added to the control. After 7 days, the cells were immobilized 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). The cells adhered to the cover glass were mounted on a slide glass using ProLong Gold antigen fact with DAPI (Invitrogen), and Collagen I (excitation wavelength: 488 nm, observation wavelength: 525 nm) and nucleus (excitation wavelength: 525 nm) and nuclei (excitation wavelength:: 350 nm, observation wavelength: 470 nm) was observed. The results are shown in FIG.
There was no difference in the number of cells (DAPI staining) in the cells to which glycyrrhetinic acid was added as compared with the control, but since the staining intensity of Collagen I was increased, glycyrrhetinic acid promoted collagen fibrosis. It was confirmed that.
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 Action on hyaluronic acid-related factors (1) Normal human dermal fibroblasts (Kurabou) were seeded in 24-well plates using DMEM medium (Invitrogen) at a cell density of 5 × 10 4 cells / well. The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 5 μM. Only ethanol, which is a solvent, was added to the control. The next day, total RNA was recovered from the cells using the RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was cDNAd as 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 synthesis 2), and HYBID (KIAA1199) were quantified using TaqMan gene expression responses (Applied Biosystems). The obtained value was corrected by the gene expression level of glyceraldehyde-3-phosphate dehydogenase (GAPDH). The results of each gene expression level when the control is 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 hyaluronan synthase, and suppresses the expression of the HYBID gene, which is important for hyaluronan degradation. Therefore, it is considered that glycyrrhetinic acid has a hyaluronic acid production promoting action and a hyaluronic acid decomposition suppressing action.
（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 (Krabou) were seeded on a 12-well plate at a cell density of 2 × 10 5 cells / well using DMEM medium (Invitrogen). The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 5 μM. Only ethanol, which is a solvent, was added to the control. After 4 days, proteins were extracted from cells using Cell Lysis buffer (Cell signaling technologies) and used for Western blotting analysis. After separation using 5 to 15% SDS-gel, it is transferred to a PVDF membrane according to a conventional method to obtain an anti-human BRG-1 antibody (Santa Cruz) or an anti-human HYBID antibody (Proc Natl Acad Sci US A. 2013 Apr. 2; 110 (14): Prepared according to 5612-7.), Incubated with anti-human β-actin antibody (Cell Signaling Technology), and then banded with peroxidase-labeled secondary antibody and Enhanced Chemiluminesis Plus color-developing reagent (GE Healthcare). Was visualized. The results are shown in FIG. 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 decomposition inhibiting effect 24-well plates were seeded with MEM medium (Sigma) at a cell density of normal human dermal fibroblasts (Kurabo) to 1.5 × 10 5 cells / well. The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 3 μM. After 3 days, a fluorescently labeled polymer HA (FAHA-H1; PG Research, final concentration: 10 mg / ml) was added. After 3 days, the culture supernatant was collected and subjected to a hyaluronic acid decomposition suppression evaluation test.
The HPLC system used LaChrom Elite (Hitachi High-Technologies Corporation), and the detection was performed at an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm. 0.2M NaCl was used as a solvent, and the flow rate was 0.5 ml / min. A SEC column (Tosoh TSK-GL G5000 PW-XL) was used as the column, the column temperature was 25 ° C., and the injection amount was 5 ml. The hyaluronic acid decomposition rate was calculated by the following formula as the amount of decrease in high molecular weight hyaluronic acid.
Hyaluronic acid decomposition rate (%) = (peak height of high molecular weight hyaluronic acid before addition to cells-cells to which glycyrrhetinic acid was added (control is cells to which ethanol, which is a solvent for glycyrrhetinic acid, was added at the same concentration) and after incubation High molecular weight hyaluronic acid peak height) / (High molecular weight hyaluronic acid peak height before addition to cells-Cells without glycyrrhetinic acid added and high molecular weight hyaluronic acid peak height after incubation) × 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 Action In a 12-well plate, normal human dermal fibroblasts (Kurabo) were seeded using DMEM medium (Invitrogen) at a cell density of 2 × 10 5 cells / well. The next day, glycyrrhetinic acid dissolved in ethanol (Agritinone, Tokiwa Phytochemical Research Institute Co., Ltd.) was added so that the final concentration was 1 μM. Only ethanol, which is a solvent, was added to the control. After 4 days, the culture supernatant was collected, and the amount of hyaluronic acid was quantified according to the attached protocol using the QnE Hyaluronic Acid ELISA Assay Kit (Biotech Trading Partners). 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 promoting action by promoting the expression of the HAS2 gene by glycyrrhetinic acid and the degradation suppressing action based on the suppression of 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 action of dipotassium glycyrrhizinate and stearyl glycyrrhetinate In a 24-well plate, normal human dermal fibroblasts (Kurabou) were used in DMEM medium (Invitrogen) at a cell density of 5 × 10 4 cells / well. Was sown. The next day, glycyrrhetinic acid (Joban Phytochemical Research Institute Co., Ltd., agritinone, solvent: ethanol), dipotassium glycyrrhizinate (Alps Pharmaceutical Industry Co., Ltd., solvent: DMSO) or stearyl glycyrrhetinate (Joban Phytochemical Research Institute Co., Ltd. , Solvent ethanol) was added so that the final concentration was 5 μM. In addition, only each solvent was added to the control at the same concentration. Two days later, total RNA was recovered from the cells using the RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was cDNAd as total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Then, using the prepared cDNA as a template, the expression level of the BRG-1 gene was quantified using TaqMan gene expression responses (Applied Biosystems). The obtained value was corrected by the gene expression level of glyceraldehyde-3-phosphate dehydogenase (GAPDH). The results are shown in FIG.
From FIG. 8, it was confirmed that dipotassium glycyrrhizinate and stearyl glycyrrhetinate did not show a BRG-1 expression promoting action, and only glycyrrhetinic acid showed an excellent BRG-1 expression promoting action.
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 action of licorice extract Normal human dermal fibroblasts (Kurabo) were seeded in a 24-well plate using DMEM medium (Invitrogen) at a cell density of 5 × 10 4 cells / well. The next day, licorice (Glycyrrhiza grabra) extract BG-J (Maruzen Pharmaceuticals, extraction site: root / rhizome, 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 as a control. Two days later, total RNA was recovered from the cells using the RNeasy mini kit (QIAGEN) according to the attached protocol. The obtained RNA was cDNAd as total RNA using a High capacity RNA to cDNA kit (Applied Biosystems) according to the attached protocol. Then, using the prepared cDNA as a template, the expression level of the BRG-1 gene was quantified using TaqMan gene expression responses (Applied Biosystems). The obtained value was corrected by the gene expression level of glyceraldehyde-3-phosphate dehydogenase (GAPDH). The result of the BRG-1 gene expression level of the licorice extract when the control is set to 1 is shown in FIG.
From FIG. 9, no significant BRG-1 expression promoting effect was observed in the licorice extract at any added concentration.
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