JP5634087B2 - Endothelin-1 mRNA expression increase inhibitor, stem cell growth factor mRNA expression increase inhibitor, basic fibroblast growth factor mRNA expression increase inhibitor, and proopiomelanocortin mRNA expression increase inhibitor - Google Patents
Endothelin-1 mRNA expression increase inhibitor, stem cell growth factor mRNA expression increase inhibitor, basic fibroblast growth factor mRNA expression increase inhibitor, and proopiomelanocortin mRNA expression increase inhibitor Download PDFInfo
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- JP5634087B2 JP5634087B2 JP2010057264A JP2010057264A JP5634087B2 JP 5634087 B2 JP5634087 B2 JP 5634087B2 JP 2010057264 A JP2010057264 A JP 2010057264A JP 2010057264 A JP2010057264 A JP 2010057264A JP 5634087 B2 JP5634087 B2 JP 5634087B2
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Description
本発明は、エンドセリン−１ｍＲＮＡ発現上昇抑制剤、幹細胞増殖因子ｍＲＮＡ発現上昇抑制剤、塩基性線維芽細胞増殖因子ｍＲＮＡ発現上昇抑制剤及びプロオピオメラノコルチンｍＲＮＡ発現上昇抑制剤に関する。 The present invention relates to an endothelin-1 mRNA expression increase inhibitor, a stem cell growth factor mRNA expression increase inhibitor, a basic fibroblast growth factor mRNA expression increase inhibitor, and a proopiomelanocortin mRNA expression increase inhibitor.
シミ、ソバカス、日焼け後の皮膚色素沈着症等は、皮膚内に存在する色素細胞（メラノサイト）が活性化することにより、メラニンの産生が著しく亢進した結果として生じるものであり、中高年齢層における肌の悩みの一つになっている。 Spots, buckwheat, skin pigmentation after sunburn, etc. occur as a result of markedly increased production of melanin due to activation of pigment cells (melanocytes) present in the skin. It has become one of the troubles.
従来の美白剤開発は、メラニン生成の律速酵素であるチロシナーゼに注力して進められてきたが、近年、紫外線ＵＶ−Ｂ照射後に表皮ケラチノサイトから産生され、メラノサイトを活性化するサイトカインとして、α−メラノサイト刺激ホルモン（α−ＭＳＨ）、エンドセリン−１、一酸化窒素（ＮＯ）等が知られており、これらが関与する情報伝達系を遮断することによりメラニンの産生を抑制して美白効果を導く各種作用剤の開発が盛んに行われてきている。 The development of conventional whitening agents has been focused on tyrosinase, which is the rate-limiting enzyme for melanin production. Recently, α-melanocyte is produced as a cytokine that is produced from epidermal keratinocytes after UV-B irradiation and activates melanocytes. Stimulating hormone (α-MSH), endothelin-1, nitric oxide (NO), etc. are known, and various actions leading to the whitening effect by inhibiting the production of melanin by blocking the information transmission system involved. The development of agents has been actively conducted.
また、循環器系疾患（例えば、心筋梗塞、高血圧、虚血性心疾患等）や腎疾患（例えば、急性腎不全等）の患者において、血中エンドセリン−１濃度が上昇していることが知られているため、エンドセリン−１の過剰分泌を抑制すること、すなわちエンドセリン−１ｍＲＮＡの発現上昇を抑制することによって、これらの疾患の予防・治療効果が期待できると考えられる。 It is also known that blood endothelin-1 levels are elevated in patients with cardiovascular diseases (eg, myocardial infarction, hypertension, ischemic heart disease, etc.) and renal diseases (eg, acute renal failure). Therefore, it is considered that the prevention / treatment effect of these diseases can be expected by suppressing the excessive secretion of endothelin-1, that is, suppressing the increase in the expression of endothelin-1 mRNA.
このような考えに基づき、エンドセリン−１のメラノサイトへの作用を阻害する生薬としては、カミツレ抽出物・アルテア抽出物（非特許文献１参照）等が知られており、表皮ケラチノサイトからのエンドセリン−１産生を抑制する生薬としては、ジユ抽出物（非特許文献１参照）、β−グリチルレチン酸ステアリル（特許文献１参照）等が知られている。 Based on this idea, chamomile extract, artea extract (see Non-Patent Document 1) and the like are known as herbal medicines that inhibit the action of endothelin-1 on melanocytes, and endothelin-1 from epidermal keratinocytes is known. Known herbal medicines that suppress the production include citrus extract (see Non-patent Document 1), β-glycyrrhetinic acid stearyl (see Patent Document 1), and the like.
幹細胞増殖因子（Stem Cell Factor，ＳＣＦ）は、Mast Cell Growth Factor、C-Kit Ligand、Steel Factor等とも呼ばれ、角化細胞、線維芽細胞、血管内皮細胞、骨髄ストローマ細胞等から産生されるタンパク質である。ＳＣＦは、多能性造血幹細胞、生殖細胞、肥満細胞、巨核球系前駆細胞、顆粒球・マクロファージ系前駆細胞、色素細胞等の増殖や分化を促進する作用を有することが知られている。また、ＳＣＦは、シミ部位や紫外線照射等によって発現が亢進することが知られている（非特許文献２参照）。 Stem cell factor (SCF), also called Mast Cell Growth Factor, C-Kit Ligand, Steel Factor, etc., is a protein produced from keratinocytes, fibroblasts, vascular endothelial cells, bone marrow stromal cells, etc. It is. SCF is known to have an action of promoting proliferation and differentiation of pluripotent hematopoietic stem cells, germ cells, mast cells, megakaryocyte precursor cells, granulocyte / macrophage precursor cells, pigment cells and the like. Moreover, it is known that the expression of SCF is enhanced by a spot site, ultraviolet irradiation or the like (see Non-Patent Document 2).
ＳＣＦとしては、２７３のアミノ酸残基からなる膜結合型ＳＣＦと、タンパク質分解酵素の作用により切断され、膜から遊離する分泌型ＳＣＦとが知られている。膜結合型ＳＣＦは、角化細胞等に結合したまま色素細胞のＳＣＦレセプターに結合し、色素細胞の増殖を促進する。また、分泌型ＳＣＦは、その結合部位にて切断され、細胞膜から遊離し、色素細胞のＳＣＦレセプターに結合することによって、色素細胞の増殖を促進する。さらに、ＳＣＦは、急性骨髄性白血病患者において、インターロイキン−３（Interleukin-3，ＩＬ−３）や顆粒球・マクロファージ・コロニー刺激因子（Granulocyte Macrophage Colony Stimulating Factor，ＧＭ−ＣＳＦ）の共存下で骨髄芽球の増殖を促進することが知られている（非特許文献３参照）。 As SCF, a membrane-bound SCF composed of 273 amino acid residues and a secreted SCF that is cleaved by the action of a proteolytic enzyme and released from the membrane are known. Membrane-bound SCF binds to the SCF receptor of the pigment cell while bound to keratinocytes and promotes the proliferation of the pigment cell. Secreted SCF is cleaved at the binding site, released from the cell membrane, and bound to the SCF receptor of the pigment cell, thereby promoting proliferation of the pigment cell. Furthermore, SCF is a bone marrow in patients with acute myeloid leukemia in the presence of interleukin-3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF). It is known to promote the proliferation of blasts (see Non-Patent Document 3).
塩基性線維芽細胞増殖因子（basic Fibroblast Growth Factor，ｂＦＧＦ）は、ＦＧＦ−２とも呼ばれ、紫外線照射により角化細胞からの遊離が促進され、遊離されたｂＦＧＦが色素細胞に作用してメラニン合成を促進し、かつ色素細胞の細胞分裂をも促進すると考えられている（非特許文献４参照）。また、ｂＦＧＦは、血管新生促進因子として知られており、腫瘍細胞（特に、悪性腫瘍細胞）における血管新生を促進すること等が知られている。 Basic fibroblast growth factor (bFGF), also called FGF-2, is released from keratinocytes by UV irradiation, and the released bFGF acts on pigment cells to synthesize melanin. It is thought that this also promotes cell division of pigment cells (see Non-Patent Document 4). Further, bFGF is known as an angiogenesis promoting factor, and is known to promote angiogenesis in tumor cells (particularly malignant tumor cells).
そのため、ＳＣＦ及びｂＦＧＦの異常産生は、色素細胞の異常増殖につながり、メラニン産生を亢進させ、シミ、ソバカス、くすみ等の原因となると考えられる。また、ＳＣＦの異常産生は、骨髄芽球の異常増殖につながり、それにより骨髄異形成症候群、急性骨髄性白血病（ＡＭＬ）等の疾患を引き起こすものと考えられ、ｂＦＧＦの異常産生は、腫瘍細胞における血管新生を促進し、それにより腫瘍細胞の増殖につながるものと考えられる。 Therefore, abnormal production of SCF and bFGF may lead to abnormal proliferation of pigment cells, increase melanin production, and cause stains, freckles, dullness, and the like. In addition, abnormal production of SCF is thought to lead to abnormal proliferation of myeloblasts, thereby causing diseases such as myelodysplastic syndrome, acute myeloid leukemia (AML), and abnormal production of bFGF occurs in tumor cells. It is thought to promote angiogenesis, thereby leading to the growth of tumor cells.
したがって、ＳＣＦｍＲＮＡ及びｂＦＧＦｍＲＮＡの発現上昇を抑制することは、色素細胞の増殖を抑制し、皮膚におけるメラニンの過剰産生を抑制し、日焼け後の色素沈着、シミ、ソバカス等の予防又は抑制に有用であると考えられる。また、ＳＣＦの発現上昇を抑制することは、骨髄芽球の異常増殖を抑制し、骨髄異形成症候群、急性骨髄性白血病等の予防又は治療に有用であると考えられ、ｂＦＧＦの発現上昇を抑制することは、腫瘍細胞における血管新生を抑制し、腫瘍細胞の増殖を抑制することで、がん治療等に有用であると考えられる。 Therefore, suppressing the increase in expression of SCF mRNA and bFGF mRNA suppresses pigment cell proliferation, suppresses excessive production of melanin in the skin, and is useful for prevention or suppression of pigmentation, sun spots, buckwheat etc. after sunburn. it is conceivable that. Moreover, suppressing the increase in the expression of SCF suppresses the abnormal proliferation of myeloblasts and is considered useful for the prevention or treatment of myelodysplastic syndrome, acute myeloid leukemia, etc., and suppresses the increase in the expression of bFGF. This is considered to be useful for cancer treatment and the like by suppressing angiogenesis in tumor cells and suppressing the growth of tumor cells.
このような考えに基づき、ＳＣＦの産生・放出を抑制する作用を有するものとして、例えば、バラエキスローズ水、チャエキス、ホップエキス、サンザシエキス、アズキ末、シラカバエキス、ケイヒエキス、チョウジエキス、アルニカエキス、ボタンエキス、ボダイジュ、クロレラエキス、ローマカミツレエキス、紅茶エキス、ユーカリエキス、ソウジュツエキス末、ビャクジュツエキス末、ウーロン茶エキス末、オノニスエキス、アセンヤクエキス、ブドウ葉エキス、ボウフウエキス、クワエキス、パリエタリアエキス、アンソッコウエキス、ステビアエキス、ヒノキ、ショウブ根エキス、ダイズエキス、カギカズラ、サボンソウエキス、アルテアエキス、オトギリソウエキス及びヨモギエキス等が知られている（特許文献２参照）。また、ｂＦＧＦの作用を抑制し得るものとして、例えば、オノニスエキス等が知られている（特許文献３参照）。 Based on such an idea, for example, rose extract water, tea extract, hop extract, hawthorn extract, azuki bean powder, birch extract, caihi extract, clove extract, arnica extract, Button extract, Bodaiju, Chlorella extract, Roman chamomile extract, Black tea extract, Eucalyptus extract, Sojutsu extract powder, Peony extract powder, Oolong tea extract powder, Onionis extract, Asenya extract, Grape leaf extract, Bowfish extract, Mulberry extract, Parietaria extract, Anne Known are persica extract, stevia extract, cypress, ginger root extract, soybean extract, scallop extract, bonito extract, altea extract, hypericum extract, and mugwort extract (see Patent Document 2). Moreover, as a thing which can suppress the effect | action of bFGF, onony extract etc. are known, for example (refer patent document 3).
皮膚表皮細胞から分泌されてメラニンの生成に関与するホルモンであるＡＣＴＨやメラノサイトを活性化するサイトカインとしてのα−ＭＳＨは、プロオピオメラノコルチン（ＰＯＭＣ）を前駆体として産生されることが知られている。そのため、ＰＯＭＣの産生を抑制すること、すなわちＰＯＭＣｍＲＮＡの発現上昇を抑制することで、結果的にメラニンの生成を抑制することができ、日焼け後の色素沈着、シミ、ソバカス等を予防又は改善することができると考えられる。 It is known that α-MSH as a cytokine that activates ACTH and melanocytes, which are secreted from skin epidermis cells and are involved in the production of melanin, is produced using proopiomelanocortin (POMC) as a precursor. . Therefore, by suppressing the production of POMC, that is, by suppressing the increase in the expression of POMC mRNA, it is possible to suppress the production of melanin as a result, and to prevent or improve pigmentation, blemishes, freckles, etc. after sunburn It is thought that you can.
従来、ＰＯＭＣの発現を抑制する作用を有するものとしては、例えば、パンテノール、塩酸ピリドキシン及びニコチン酸アミド等が知られている（特許文献４参照）。 Conventionally, what has the effect | action which suppresses the expression of POMC is known, for example, panthenol, pyridoxine hydrochloride, nicotinamide, and the like (see Patent Document 4).
本発明は、エンドセリン−１ｍＲＮＡ発現上昇抑制作用、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用及びＰＯＭＣｍＲＮＡ発現上昇抑制作用を有する物質を見出し、それを有効成分とするエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＰＯＭＣｍＲＮＡ発現上昇抑制剤、エンドセリン−１ｍＲＮＡ発現上昇に起因する疾患の予防・治療剤、ＳＣＦｍＲＮＡ発現上昇に起因する疾患の予防・治療剤、ｂＦＧＦｍＲＮＡ発現上昇に起因する疾患の予防・治療剤及びＰＯＭＣｍＲＮＡ発現上昇に起因する疾患の予防・治療剤を提供することを目的とする。 The present invention finds a substance having an endothelin-1 mRNA expression increase inhibitory action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action, and a POMC mRNA expression increase suppressive action, and an endothelin-1 mRNA expression increase suppressor comprising the same as an active ingredient, SCF mRNA Expression increase inhibitor, bFGF mRNA expression increase inhibitor, POMC mRNA expression increase inhibitor, disease prevention / treatment agent caused by endothelin-1 mRNA expression increase, disease prevention / treatment agent caused by SCF mRNA expression increase, bFGF mRNA expression increase It is an object of the present invention to provide a preventive / therapeutic agent for a disease to be prevented and a preventive / therapeutic agent for a disease caused by increased expression of POMC mRNA.
本発明者らは、スターフルーツ抽出物からエンドセリン−１ｍＲＮＡ発現上昇抑制作用、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用及びＰＯＭＣｍＲＮＡ発現上昇抑制作用を有するフラボン化合物を単離・同定ことに成功し、本発明を完成するに至った。 The present inventors succeeded in isolating and identifying a flavone compound having an endothelin-1 mRNA expression increase inhibitory action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action and a POMC mRNA expression increase suppressive action from a star fruit extract, The present invention has been completed.
すなわち、本発明は、下記式（Ｉ）で表されるフラボン化合物を有効成分として含有するエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＰＯＭＣｍＲＮＡ発現上昇抑制剤、エンドセリン−１ｍＲＮＡ発現上昇に起因する疾患の予防・治療剤、ＳＣＦｍＲＮＡ発現上昇に起因する疾患の予防・治療剤、ｂＦＧＦｍＲＮＡ発現上昇に起因する疾患の予防・治療剤及びＰＯＭＣｍＲＮＡ発現上昇に起因する疾患の予防・治療剤を提供する。 That is, the present invention relates to an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, a POMC mRNA expression increase inhibitor, endothelin, which contains a flavone compound represented by the following formula (I) as an active ingredient. -1 Preventive / Therapeutic Agent for Disease Caused by Increased Expression of mRNA, Preventive / Therapeutic Agent for Disease Caused by Increased Expression of SCF mRNA, Preventive / Therapeutic Agent for Disease Caused by Increased Expression of bFGF mRNA, and Preventing / Provide a therapeutic agent.
安全性の高い天然物であるスターフルーツから得られる上記式（Ｉ）で表されるフラボン化合物は、エンドセリン−１ｍＲＮＡ発現上昇抑制作用、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用及びＰＯＭＣｍＲＮＡ発現上昇抑制作用を示すため、これをエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤及びＰＯＭＣｍＲＮＡ発現上昇抑制剤として利用することにより、シミ、ソバカス、皮膚の色黒（皮膚色素沈着症）等を予防、治療又は改善することができる。 The flavone compound represented by the above formula (I) obtained from star fruit, which is a highly safe natural product, suppresses endothelin-1 mRNA expression increase, suppresses SCF mRNA expression, suppresses bFGF mRNA expression, and POMC mRNA expression. In order to show the action, this is used as an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, and a POMC mRNA expression increase inhibitor, thereby causing spots, freckles, and skin darkness (skin pigmentation). Can be prevented, treated or ameliorated.
以下、本発明について説明する。
本発明のフラボン化合物は、下記式（Ｉ）で表されるものである。以下、下記式（Ｉ）で表されるフラボン化合物を「カランボラフラボン」と称する。
The present invention will be described below.
The flavone compound of the present invention is represented by the following formula (I). Hereinafter, the flavone compound represented by the following formula (I) is referred to as “carambola flavone”.
カランボラフラボンは、スターフルーツから単離することができるが、スターフルーツ以外の植物にも存在する可能性がある。したがって、カランボラフラボンの単離源は、スターフルーツに限定されるものではなく、カランボラフラボンを含有するいずれの植物を用いてもよい。 Carambola flavones can be isolated from star fruit, but may also be present in plants other than star fruit. Therefore, the source of isolation of carambola flavones is not limited to star fruit, and any plant containing carambola flavones may be used.
カランボラフラボンは、例えば、スターフルーツ抽出物に、液−液分配抽出、各種クロマトグラフィー、膜分離等のフラボン化合物を濃縮するのに有効な精製操作を施した後、ポリビニルアルコール系ポリマー等を固定相として用いた液体クロマトグラフィーにより処理することで得ることができる。 Carambola flavones are, for example, fixed to polyvinyl alcohol polymers after being subjected to purification operations effective for concentrating flavone compounds such as liquid-liquid partition extraction, various types of chromatography, and membrane separation. It can be obtained by processing by liquid chromatography used as a phase.
具体的には、まず、スターフルーツを水、親水性有機溶媒又はこれらの混合溶媒による抽出処理に付し、スターフルーツ抽出物を得る。 Specifically, first, the star fruit is subjected to an extraction treatment with water, a hydrophilic organic solvent or a mixed solvent thereof to obtain a star fruit extract.
抽出原料として使用するスターフルーツの構成部位は、特に限定されるものではなく、例えば、葉部、茎部、果実部、根部等が挙げられる。これらのうち、特に葉部を抽出原料として使用することが好ましく、これらを単独で使用してもよいし、併用してもよい。ここで「葉部」には、完全葉の他、葉の一部（例えば、葉身、葉柄、托葉、葉鞘等）も含まれる。なお、スターフルーツ（Averrhoa carambola L.）は、カタバミ科に属し、新鮮な果実は食用にされており、その果実は断面が星形であることからスターフルーツと呼ばれている。スターフルーツは、沖縄、中国東南部や雲南その他熱帯各地で栽培されており、これらの地域から容易に入手することができる。 The constituent part of the star fruit used as the extraction raw material is not particularly limited, and examples thereof include a leaf part, a stem part, a fruit part, and a root part. Among these, it is preferable to use a leaf part as an extraction raw material especially, and these may be used independently and may be used together. Here, the “leaf part” includes not only a complete leaf but also a part of the leaf (eg, leaf blade, petiole, cocoon leaf, leaf sheath, etc.). Star fruit (Averrhoa carambola L.) belongs to the family Oxalis, fresh fruit is edible, and the fruit is called star fruit because its cross section is star-shaped. Star fruit is cultivated in Okinawa, southeastern China, Yunnan and other tropical regions, and can be easily obtained from these regions.
スターフルーツ抽出物は、抽出原料を乾燥した後、そのまま又は粗砕機を用いて粉砕し、抽出溶媒による抽出に供することにより得ることができる。乾燥は天日で行ってもよいし、通常使用される乾燥機を用いて行ってもよい。また、ヘキサン等の非極性溶媒によって脱脂等の前処理を施してから抽出原料として使用してもよい。脱脂等の前処理を行うことにより、スターフルーツの極性溶媒による抽出処理を効率よく行うことができる。 The star fruit extract can be obtained by drying an extraction raw material, pulverizing the raw material as it is or using a crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, the extraction treatment of the star fruit with a polar solvent can be performed efficiently.
抽出溶媒としては、極性溶媒を使用するのが好ましく、例えば、水、親水性有機溶媒等が挙げられ、これらを単独で又は２種以上を組み合わせて、室温又は溶媒の沸点以下の温度で使用するのが好ましい。 As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferred.
抽出溶媒として使用し得る水としては、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等のほか、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧調整、緩衝化等が含まれる。したがって、本発明において抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。 Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
抽出溶媒として使用し得る親水性有機溶媒としては、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級脂肪族アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３−ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２〜５の多価アルコール等が挙げられる。 Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.
２種以上の極性溶媒の混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には、水１０容量部に対して低級脂肪族アルコール１〜９０容量部を混合することが好ましく、水と低級脂肪族ケトンとの混合液を使用する場合には、水１０容量部に対して低級脂肪族ケトン１〜４０容量部を混合することが好ましく、水と多価アルコールとの混合液を使用する場合には、水１０容量部に対して多価アルコール１０〜９０容量部を混合することが好ましい。 When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a mixed solution of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with respect to 10 parts by volume of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by volume of a lower aliphatic ketone with 10 parts by volume of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by volume of a polyhydric alcohol with respect to the volume part.
抽出処理は、少なくとも抽出原料に含まれるカランボラフラボンを抽出溶媒に溶出させ得る限り特に限定はされず、常法に従って行うことができる。例えば、抽出原料の５〜１５倍量（質量比）の抽出溶媒に、抽出原料を浸漬し、常温又は還流加熱下で可溶性成分を抽出させた後、濾過して抽出残渣を除去することにより抽出液を得ることができる。得られた抽出液から溶媒を留去するとペースト状の濃縮物が得られ、この濃縮物をさらに乾燥すると乾燥物が得られる。 The extraction treatment is not particularly limited as long as at least carambola flavone contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.
次に、得られたスターフルーツ抽出物を吸着剤に吸着させた後、水、水溶性溶媒又はこれらの混合溶媒で溶出させて、フラボン化合物を含有する画分を分画する。 Next, after the obtained star fruit extract is adsorbed on an adsorbent, it is eluted with water, a water-soluble solvent or a mixed solvent thereof to fractionate a fraction containing a flavone compound.
スターフルーツ抽出物に含まれ得る親水性有機溶媒は、吸着剤に吸着させる前に、必要に応じて留去する。親水性有機溶媒を留去した抽出物は、例えば、水、水溶性溶媒又はこれらの混合溶媒に溶解又は懸濁させた後、吸着剤に吸着させる。 The hydrophilic organic solvent that can be contained in the star fruit extract is distilled off as necessary before adsorbing to the adsorbent. The extract obtained by distilling off the hydrophilic organic solvent is dissolved or suspended in, for example, water, a water-soluble solvent or a mixed solvent thereof, and then adsorbed on an adsorbent.
溶解、懸濁又は溶出に用いられる水溶性溶媒として、上記親水性有機溶媒を用いることができ、上記炭素数１〜５の低級アルコール、この中でも特にメタノール、エタノールを用いるのが好ましい。水及び水溶性溶媒の混合溶媒を用いる場合、その混合比は適宜調整することができる。例えば、水とメタノールとの混合溶媒を使用する場合、水とメタノールとの混合比を０：１００〜１００：０（容量比）、好ましくは４０：６０〜６０：４０（容量比）とすることができる。 As the water-soluble solvent used for dissolution, suspension or elution, the above-mentioned hydrophilic organic solvent can be used, and it is preferable to use the lower alcohol having 1 to 5 carbon atoms, particularly methanol and ethanol among these. When a mixed solvent of water and a water-soluble solvent is used, the mixing ratio can be adjusted as appropriate. For example, when using a mixed solvent of water and methanol, the mixing ratio of water and methanol should be 0: 100 to 100: 0 (volume ratio), preferably 40:60 to 60:40 (volume ratio). Can do.
吸着剤は、フラボン化合物を吸着し得る限り特に限定されるものではないが、イオン交換樹脂、合成吸着樹脂、活性炭、キレート樹脂、シリカゲル、アルミナゲル系吸着剤、多孔質ガラス等の公知の吸着剤を単独で又は組み合わせて用いることができる。好ましくは、多孔質性合成吸着樹脂であるダイヤイオンＨＰ−２０（いずれも三菱化学社製）等の多孔性合成吸着剤を用いたカラムクロマトグラフィーと、オクタデシルシリル化シリカゲル（ＯＤＳ）であるクロマトレックスＯＤＳ ＤＭ１０２０Ｔ（富士シリシア化学社製）等を充填剤として用いた逆相カラムクロマトグラフィーとを併用する。 The adsorbent is not particularly limited as long as it can adsorb the flavone compound, but known adsorbents such as ion exchange resin, synthetic adsorption resin, activated carbon, chelate resin, silica gel, alumina gel-based adsorbent, porous glass, etc. Can be used alone or in combination. Preferably, column chromatography using a porous synthetic adsorbent such as Diaion HP-20 (all manufactured by Mitsubishi Chemical Corporation), which is a porous synthetic adsorption resin, and Chromatrex, which is octadecylsilylated silica gel (ODS). Combined with reverse phase column chromatography using ODS DM1020T (manufactured by Fuji Silysia Chemical Ltd.) as a filler.
最後に、得られた溶出液を、移動相として水、水溶性溶媒又はこれらの混合溶媒を用いた液体カラムクロマトグラフィーにより分画し、精製されたフラボン化合物を得る。 Finally, the obtained eluate is fractionated by liquid column chromatography using water, a water-soluble solvent or a mixed solvent thereof as a mobile phase to obtain a purified flavone compound.
液体クロマトグラフィーによる分画方法は、特に制限されるものではなく、通常の方法で行うことができる。例えば、固定相としては、シリカゲル（例えば、シリカゲル６０（製品名），メルク社製）、オクタデシルシリル（Octa Decyl Silyl，Ｃ１８Ｈ３７Ｓｉ）基で表面が修飾された化学結合型多孔性球状シリカゲル（例えば、クロマトレックスODS DM1020T（製品名），富士シリシア化学社製）、ヒドロキシプロピル化したデキストラン等のゲル（例えば、SEPHADEX LH-20（製品名），ＧＥヘルスケア・ジャパン社製）等を単独で又は組み合わせて用いることができる。 The fractionation method by liquid chromatography is not particularly limited, and can be performed by a usual method. For example, as the stationary phase, a chemically bonded porous spherical silica gel whose surface is modified with a silica gel (for example, silica gel 60 (product name), manufactured by Merck & Co., Inc.) or an octadecyl silyl (C 18 H 37 Si) group. (For example, Chromatorex ODS DM1020T (product name), manufactured by Fuji Silysia Chemical Ltd.), gels such as hydroxypropylated dextran (for example, SEPHADEX LH-20 (product name), manufactured by GE Healthcare Japan), etc. Or in combination.
水溶性溶媒としては、上記親水性有機溶媒を用いることができ、特にメタノール、エタノール、アセトニトリル等を用いることが好ましい。水及び水溶性溶媒を用いる場合、その混合比は適宜調整することができる。例えば、水とアセトニトリルとの混合溶媒を使用する場合、水とアセトニトリルとの混合比を２０：８０〜８０：２０（容量比）、好ましくは６０：４０（容量比）とすることができる。 As the water-soluble solvent, the above hydrophilic organic solvent can be used, and methanol, ethanol, acetonitrile and the like are particularly preferable. When water and a water-soluble solvent are used, the mixing ratio can be adjusted as appropriate. For example, when a mixed solvent of water and acetonitrile is used, the mixing ratio of water and acetonitrile can be 20:80 to 80:20 (volume ratio), preferably 60:40 (volume ratio).
このようにして、液体クロマトグラフィーを利用してカランボラフラボンを含有する画分を分画することにより、精製されたカランボラフラボンを得ることができる。 In this way, a purified carambola flavone can be obtained by fractionating a fraction containing carambola flavone using liquid chromatography.
以上のようにして得られるカランボラフラボンは、エンドセリン−１ｍＲＮＡ発現上昇抑制作用、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用又はＰＯＭＣｍＲＮＡ発現上昇抑制作用を有しているため、それらの作用を利用してエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤の有効成分として使用することができる。 The carambola flavones obtained as described above have an endothelin-1 mRNA expression increase suppressive action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action, or a POMC mRNA expression increase suppressive action. Thus, it can be used as an active ingredient of an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, or a POMC mRNA expression increase inhibitor.
また、カランボラフラボンは、そのエンドセリン−１ｍＲＮＡ発現上昇抑制作用を利用して、エンドセリン−１ｍＲＮＡの発現上昇に起因する疾患の予防・治療剤（例えば、心筋梗塞治療剤、高血圧治療剤、虚血性心疾患治療剤、急性腎不全治療剤等）の有効成分として用いることもできる。 Also, carambola flavone uses its endothelin-1 mRNA expression increase inhibitory action to prevent or treat diseases caused by increased endothelin-1 mRNA expression (for example, myocardial infarction therapeutic agent, hypertensive therapeutic agent, ischemic heart It can also be used as an active ingredient of disease therapeutic agents, acute renal failure therapeutic agents, etc.).
さらに、カランボラフラボンは、そのＳＣＦｍＲＮＡ発現上昇抑制作用を利用して、ＳＣＦｍＲＮＡの発現上昇に起因する疾患の予防・治療剤（例えば、骨髄異形成症候群予防・治療剤、急性骨髄性白血病予防・治療剤、抗腫瘍剤等）の有効成分として用いることもできる。 Furthermore, carambola flavone uses its SCF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased expression of SCF mRNA (for example, prevention and treatment of myelodysplastic syndrome, prevention and treatment of acute myeloid leukemia) Agent, antitumor agent, etc.).
さらにまた、カランボラフラボンは、そのｂＦＧＦｍＲＮＡ発現上昇抑制作用を利用して、ｂＦＧＦｍＲＮＡの発現上昇に起因する疾患の予防・治療剤（例えば、血管新生抑制剤、抗がん剤、抗腫瘍剤、がん細胞の転移を抑制する医薬組成物等）の有効成分として用いることもできる。 Furthermore, carambola flavone uses its bFGF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased bFGF mRNA expression (for example, angiogenesis inhibitors, anticancer agents, antitumor agents, It can also be used as an active ingredient of a pharmaceutical composition or the like that suppresses metastasis of cancer cells.
また、カランボラフラボンは、そのＰＯＭＣｍＲＮＡ発現上昇抑制作用を利用して、ＰＯＭＣｍＲＮＡの発現上昇に起因する疾患（例えば、ストレス性の皮膚掻痒症等）の予防・治療剤の有効成分として用いることもできる。 Carambola flavone can also be used as an active ingredient of a prophylactic / therapeutic agent for diseases caused by increased expression of POMC mRNA (for example, stress pruritus etc.) by utilizing its POMC mRNA expression increase suppressing action. .
本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤は、カランボラフラボンのみからなるものでもよいし、カランボラフラボンを製剤化したものでもよい。 The endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention may be composed solely of carambola flavone, or may be formulated with carambola flavone. Good.
カランボラフラボンは、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、液状等の任意の剤形に製剤化することができる。この際、助剤としては、例えば、賦形剤、安定剤、矯臭剤等を用いることができる。カランボラフラボンを製剤化したエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤の形態としては、例えば、軟膏剤、外用液剤、貼付剤等が挙げられる。 Carambola flavone can be formulated into any dosage form such as powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin and any other auxiliary agent. it can. At this time, as an auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent and the like can be used. Examples of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor formulated with carambola flavone include ointments, liquids for external use, patches and the like. It is done.
なお、本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤は、必要に応じて、エンドセリン−１ｍＲＮＡ発現上昇抑制作用、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用又はＰＯＭＣｍＲＮＡ発現上昇抑制作用を有する天然抽出物等を、カランボラフラボンとともに配合して有効成分として用いることができる。 In addition, the endothelin-1 mRNA expression increase suppressor, the SCF mRNA expression increase suppressor, the bFGF mRNA expression increase suppressor, or the POMC mRNA expression increase suppressor of the present invention are, as necessary, an endothelin-1 mRNA expression increase suppressive action or an SCF mRNA expression increase suppressive action. A natural extract or the like having an inhibitory effect on bFGF mRNA expression or POMC mRNA expression can be blended with carambola flavone and used as an active ingredient.
本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤の患者に対する投与方法としては、皮下組織内投与、筋肉内投与、静脈内投与、経口投与、経皮投与等が挙げられるが、疾患の種類に応じて、その予防・治療等に好適な方法を適宜選択すればよい。また、本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤の投与量も、疾患の種類、重症度、患者の個人差、投与方法、投与期間等によって適宜増減すればよい。 As an administration method to the patient of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention, subcutaneous tissue administration, intramuscular administration, intravenous administration, oral administration Administration, transdermal administration, and the like can be mentioned, and a suitable method for the prevention / treatment or the like may be appropriately selected according to the type of disease. In addition, the dosage of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention is also the type of disease, severity, individual differences among patients, administration methods, What is necessary is just to increase / decrease suitably according to an administration period etc.
本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するエンドセリン−１ｍＲＮＡ発現上昇抑制作用を通じて、エンドセリン−１の産生を抑制し、シミ、ソバカス、皮膚の色黒（皮膚色素沈着症）等を予防・改善することができる。また、本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するエンドセリン−１ｍＲＮＡ発現上昇抑制作用を通じて、心筋梗塞、高血圧、虚血性心疾患、急性腎不全等の疾患を予防、治療又は改善することができる。ただし、本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤は、これらの用途以外にもエンドセリン−１ｍＲＮＡ発現上昇抑制作用を発揮することに意義のあるすべての用途に用いることができる。 The endothelin-1 mRNA expression increase inhibitor of the present invention suppresses endothelin-1 production through an inhibitory effect on the increase in endothelin-1 mRNA expression possessed by carambola flavones, stains, buckwheat, skin darkness (skin pigmentation), etc. Can be prevented and improved. Moreover, the endothelin-1 mRNA expression increase inhibitor of the present invention prevents, treats, or improves diseases such as myocardial infarction, hypertension, ischemic heart disease, and acute renal failure through an inhibitory effect on the increase in endothelin-1 mRNA expression of carambolaflavone. can do. However, the endothelin-1 mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful in exhibiting the endothelin-1 mRNA expression increase suppressive action in addition to these uses.
本発明のＳＣＦｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するＳＣＦｍＲＮＡ発現上昇抑制作用を通じて、ＳＣＦの発現の上昇を抑制することができ、これにより色素細胞の増殖やメラニンの産生を抑制し、シミ、ソバカス、皮膚色素沈着症等を予防又は改善することができ、美白効果を得ることができる。また、本発明のＳＣＦｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するＳＣＦｍＲＮＡ発現上昇抑制作用を通じて、骨髄芽球の異常増殖を抑制することができ、これにより骨髄異形成症候群、急性骨髄性白血病等の疾患を予防、治療又は改善することができる。ただし、本発明のＳＣＦｍＲＮＡ発現上昇抑制剤は、これらの用途以外にもＳＣＦｍＲＮＡ発現上昇抑制作用を発揮することに意義のあるすべての用途に用いることができる。 The SCF mRNA expression increase inhibitor of the present invention can suppress the increase in SCF expression through the SCF mRNA expression increase suppressive action of carambola flavone, thereby suppressing pigment cell proliferation and melanin production, Buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. In addition, the SCF mRNA expression increase inhibitor of the present invention can suppress abnormal proliferation of myeloblasts through the suppressive action of carambola flavone on SCF mRNA expression, and thereby, such as myelodysplastic syndrome, acute myeloid leukemia, etc. The disease can be prevented, treated or ameliorated. However, the SCF mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful for exhibiting the SCF mRNA expression increase suppression action in addition to these applications.
本発明のｂＦＧＦｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するｂＦＧＦｍＲＮＡ発現上昇抑制作用を通じて、ｂＦＧＦの発現の上昇を抑制することができ、これにより色素細胞の増殖やメラニンの産生を抑制し、シミ、ソバカス、皮膚色素沈着症等を予防又は改善することができ、美白効果を得ることができる。また、本発明のｂＦＧＦｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するｂＦＧＦｍＲＮＡ発現上昇抑制作用を通じて、腫瘍細胞における異常な血管新生を抑制し、がん等の疾患を予防、治療又は改善をすることができる。ただし、本発明のｂＦＧＦｍＲＮＡ発現上昇抑制剤は、これらの用途以外にもｂＦＧＦｍＲＮＡ発現上昇抑制作用を発揮することに意義のあるすべての用途に用いることができる。 The bFGF mRNA expression increase inhibitor of the present invention can suppress the increase in bFGF expression through the bFGF mRNA expression increase suppressive action possessed by carambola flavone, thereby suppressing the proliferation of pigment cells and the production of melanin, Buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. In addition, the bFGF mRNA expression increase inhibitor of the present invention may suppress abnormal angiogenesis in tumor cells through the bFGF mRNA expression increase suppression action of carambola flavone, and prevent, treat or improve diseases such as cancer. it can. However, the bFGF mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful for exhibiting the bFGF mRNA expression increase suppression action in addition to these applications.
本発明のＰＯＭＣｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するＰＯＭＣｍＲＮＡ発現上昇抑制作用を通じて、ＰＯＭＣの発現の上昇を抑制することができ、これによりメラノサイトを活性化するサイトカインとしてのα−ＭＳＨの生合成を抑制し、シミ、ソバカス、皮膚色素沈着症等を予防又は改善することができ、美白効果を得ることができる。また、本発明のＰＯＭＣｍＲＮＡ発現上昇抑制剤は、カランボラフラボンが有するＰＯＭＣｍＲＮＡ発現上昇抑制作用を通じて、ストレス性の皮膚掻痒症等を予防、治療又は改善することができる。ただし、本発明のＰＯＭＣｍＲＮＡ発現上昇抑制剤は、これらの用途以外にもＰＯＭＣｍＲＮＡ発現上昇抑制作用を発揮することに意義のあるすべての用途に用いることができる。 The POMC mRNA expression increase inhibitor of the present invention can suppress the increase in POMC expression through the inhibitory action of POMC mRNA expression increase possessed by carambola flavone, thereby biosynthesis of α-MSH as a cytokine that activates melanocytes. Can be suppressed, and spots, buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. Moreover, the POMC mRNA expression increase inhibitor of the present invention can prevent, treat or improve stress-related pruritus and the like through the POMC mRNA expression increase suppression action of carambola flavone. However, the POMC mRNA expression increase inhibitor of the present invention can be used for all purposes other than these applications that are meaningful for exhibiting the POMC mRNA expression increase suppression action.
なお、本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤又はＰＯＭＣｍＲＮＡ発現上昇抑制剤は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物に対して適用することもできる。 In addition, although the endothelin-1 mRNA expression increase inhibitor, the SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor or the POMC mRNA expression increase inhibitor of the present invention are suitably applied to humans, their effects are as follows. Can be applied to animals other than humans as long as
以下、製造例及び試験例を示し、本発明を具体的に説明するが、本発明は下記の各例に何ら制限されるものではない。 Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.
〔製造例１〕カランボラフラボンの製造
抽出原料としてのスターフルーツの葉部の粉砕物５ｋｇを５０容量％エタノール２０Ｌに投入し、穏やかに攪拌しながら３時間、８０℃にて加熱抽出した後、熱時濾過した。残渣について同様の抽出操作を繰り返し、得られた濾液を合わせ、４０℃で減圧下に濃縮し、さらに減圧乾燥機で乾燥して粉末状のスターフルーツ抽出物１８１５ｇを得た（収率３６．３％）。
[Production Example 1] Production of carambola flavone 5 kg of pulverized star fruit leaf as an extraction raw material was placed in 20 L of 50% ethanol by volume and heated and extracted at 80 ° C for 3 hours with gentle stirring. Filtered hot. The same extraction operation was repeated for the residue, and the obtained filtrates were combined, concentrated under reduced pressure at 40 ° C., and further dried with a vacuum dryer to obtain 1815 g of a powdery star fruit extract (yield 36.3). %).
得られたスターフルーツ抽出物１８１５ｇを、水１０００ｍＬを加えて懸濁させ、多孔性樹脂（ダイアイオンＨＰ−２０，６ｋｇ）に付し、水３０Ｌ、４０容量％メタノール３０Ｌ、８０容量％メタノール３０Ｌ、メタノール３０Ｌの順で溶出させた。次いで、８０容量％メタノール３０Ｌで溶出させた画分に含まれる８０容量％メタノールを留去して、８０容量％メタノール溶出画分１６９．８ｇを得た。得られた８０容量％メタノール溶出画分１６９．８ｇを、クロロホルム：メタノール：水＝１０：５：１（容量比）の混合溶媒に溶解し、シリカゲル（商品名：シリカゲル６０，メルク社製）を充填したガラス製のカラム上部から注入して、シリカゲルに吸着させた。次いで、移動相としてクロロホルム：メタノール：水＝１０：５：１（容量比）を流し、その溶出液を集め、脱溶媒して、フラボン化合物濃縮物４２．８ｇを得た。 1815 g of the obtained star fruit extract was suspended by adding 1000 mL of water, attached to a porous resin (Diaion HP-20, 6 kg), 30 L of water, 30 L of 40 volume% methanol, 30 L of 80 volume% methanol, Elution was carried out in the order of 30 L of methanol. Subsequently, 80 volume% methanol contained in the fraction eluted with 30 L of 80 volume% methanol was distilled off to obtain 169.8 g of an 80 volume% methanol eluted fraction. 169.8 g of the 80 volume% methanol elution fraction obtained was dissolved in a mixed solvent of chloroform: methanol: water = 10: 5: 1 (volume ratio), and silica gel (trade name: silica gel 60, manufactured by Merck & Co., Inc.) was used. It was injected from the top of a packed glass column and adsorbed onto silica gel. Subsequently, chloroform: methanol: water = 10: 5: 1 (volume ratio) was passed as the mobile phase, and the eluate was collected and desolvated to obtain 42.8 g of a flavone compound concentrate.
得られたフラボン化合物濃縮物４２．８ｇを、アセトニトリル：水＝２：５（容量比）の混合溶媒に溶解し、ＯＤＳ（商品名：クロマトレックスＯＤＳ ＤＭ１０２０Ｔ，富士シリシア化学社製）を充填したガラス製のカラム上部から注入して、ＯＤＳに吸着させた。次いで、移動相としてアセトニトリル：水＝２：５（容量比）を流し、その溶出液を集め、脱溶媒して、フラボン化合物濃縮物２２．０ｇを得た。 42.8 g of the obtained flavone compound concentrate was dissolved in a mixed solvent of acetonitrile: water = 2: 5 (volume ratio) and filled with ODS (trade name: Chromatorex ODS DM1020T, manufactured by Fuji Silysia Chemical Ltd.). It was injected from the top of the manufactured column and adsorbed on ODS. Next, acetonitrile: water = 2: 5 (volume ratio) was passed as a mobile phase, and the eluate was collected and desolvated to obtain 22.0 g of a flavone compound concentrate.
得られたフラボン化合物濃縮物２２．０ｇを、メタノールに溶解し、疎水性基を有するゲル（Sephadex LH-20，ＧＥヘルスケア・ジャパン社製）を充填したガラス製のカラム上部から注入して、吸着させた。次いで、移動相としてメタノールを流し、その溶出液を集め、脱溶媒して、フラボン化合物濃縮物１６．２ｇを得た。 22.0 g of the obtained flavone compound concentrate was dissolved in methanol and injected from the top of a glass column filled with a gel having a hydrophobic group (Sephadex LH-20, manufactured by GE Healthcare Japan), Adsorbed. Subsequently, methanol was passed as a mobile phase, and the eluate was collected and desolvated to obtain 16.2 g of a flavone compound concentrate.
得られたフラボン化合物濃縮物１６．２ｇを下記の条件で高速液体クロマトグラフィーを用いて分画し、カランボラフラボン（１．２ｇ）を単離した。 The obtained flavone compound concentrate (16.2 g) was fractionated using high performance liquid chromatography under the following conditions to isolate carambola flavone (1.2 g).
＜高速液体クロマトグラフィー条件１＞
固定相：ＪＡＩＧＥＬ ＧＳ−３１０（日本分析工業社製）を２本連結
カラム径：２０ｍｍ
カラム長：５００ｍｍ
移動相：メタノール
移動相流量：５ｍＬ／ｍｉｎ
検出：ＲＩ
<High performance liquid chromatography condition 1>
Stationary phase: 2 JAIGEL GS-310 (manufactured by Nippon Analytical Industrial Co., Ltd.) Connection column diameter: 20 mm
Column length: 500mm
Mobile phase: Methanol Mobile phase flow rate: 5 mL / min
Detection: RI
＜高速液体クロマトグラフィー条件２＞
固定相：Ｄｅｖｅｌｏｓｉｌ ＲＰＡＱＵＥＯＵＳ−ＡＲ−５（野村化学社製）
カラム径：２０ｍｍ
カラム長：２５０ｍｍ
移動相：アセトニトリル：水＝３：７（容量比）
移動相流量：９ｍＬ／ｍｉｎ
検出：ＲＩ
<High performance liquid chromatography condition 2>
Stationary phase: Develosil RPAQUEOUS-AR-5 (manufactured by Nomura Chemical Co., Ltd.)
Column diameter: 20mm
Column length: 250mm
Mobile phase: acetonitrile: water = 3: 7 (volume ratio)
Mobile phase flow rate: 9 mL / min
Detection: RI
単離したカランボラフラボンについて１Ｈ−ＮＭＲ及び１３Ｃ−ＮＭＲ分析を行なった。結果を下記に示す。 The isolated carambola flavone was subjected to 1 H-NMR and 13 C-NMR analysis. The results are shown below.
＜１Ｈ−ＮＭＲケミカルシフトδ（帰属水素）：＞
0.54(3H, d, J=5.6Hz, Rha-6-H), 1.14(3H, d, J=6.0H1, Fuc-6-H), 2.38(1H, m, Rha-5-H), 2.92(1H, br.s, Rha-4-H), 3.17(1H, overlapped, Rha-3-H), 3.54(1H, br.s, Fuc-4-H), 3.62(1H, overlapped, Rha-2-H), 3.62(1H, overlapped, Fuc-3-H), 3.72(1H, br.q, J=6.0Hz, Fuc-5-H), 4.16(1H, br.t, J=8.8Hz, Fuc-2-H), 4.39(1H, d, J=9.6Hz, Fuc-1-H), 5.01(1H, s, Rha-1-H), 6.51(1H, s, 8-H), 6.74(1H, s, 3-H), 6.92(2H, d, J=8.8Hz, 3', 5'-H), 7.90(2H, d, J=8.8Hz, 2', 6'-H）
< 1 H-NMR chemical shift δ (assigned hydrogen):>
0.54 (3H, d, J = 5.6Hz, Rha-6-H), 1.14 (3H, d, J = 6.0H1, Fuc-6-H), 2.38 (1H, m, Rha-5-H), 2.92 (1H, br.s, Rha-4-H), 3.17 (1H, overlapped, Rha-3-H), 3.54 (1H, br.s, Fuc-4-H), 3.62 (1H, overlapped, Rha- 2-H), 3.62 (1H, overlapped, Fuc-3-H), 3.72 (1H, br.q, J = 6.0Hz, Fuc-5-H), 4.16 (1H, br.t, J = 8.8Hz , Fuc-2-H), 4.39 (1H, d, J = 9.6Hz, Fuc-1-H), 5.01 (1H, s, Rha-1-H), 6.51 (1H, s, 8-H), 6.74 (1H, s, 3-H), 6.92 (2H, d, J = 8.8Hz, 3 ', 5'-H), 7.90 (2H, d, J = 8.8Hz, 2', 6'-H)
＜１３Ｃ−ＮＭＲケミカルシフトδ（帰属水素）：＞
16.8(Fuc-6-C), 17.5(Rha-6-C), 68.0(Rha-5-C), 70.3(Rha-3-C), 70.4(Rha-2-C), 71.2(Fuc-1-C), 71.3(Rha-4-C), 72.0(Fuc-4-C), 73.6(Fuc-2- C), 73.6(Fuc-5-C), 75.7(Fuc-3-C), 94.4(8-C), 100.3(Rha-1-C), 102.7(3-C), 103.4(10-C), 108.8(6-C), 115.8(3', 5'-C), 120.9(1'-C), 128.1(2', 6'-C), 156.3(9-C), 158.9(5-C), 160.9(4'-C), 162.5(7-C), 163.3(2-C), 181.5(4-C)
< 13 C-NMR chemical shift δ (assigned hydrogen):>
16.8 (Fuc-6-C), 17.5 (Rha-6-C), 68.0 (Rha-5-C), 70.3 (Rha-3-C), 70.4 (Rha-2-C), 71.2 (Fuc-1 -C), 71.3 (Rha-4-C), 72.0 (Fuc-4-C), 73.6 (Fuc-2-C), 73.6 (Fuc-5-C), 75.7 (Fuc-3-C), 94.4 (8-C), 100.3 (Rha-1-C), 102.7 (3-C), 103.4 (10-C), 108.8 (6-C), 115.8 (3 ', 5'-C), 120.9 (1 '-C), 128.1 (2', 6'-C), 156.3 (9-C), 158.9 (5-C), 160.9 (4'-C), 162.5 (7-C), 163.3 (2-C ), 181.5 (4-C)
以上の結果から、スターフルーツ抽出物から単離されたカランボラフラボンが、下記式（Ｉ）で表されるフラボン化合物であることが確認された。 From the above results, it was confirmed that the carambola flavone isolated from the star fruit extract was a flavone compound represented by the following formula (I).
〔試験例１〕エンドセリン−１ｍＲＮＡ発現上昇抑制作用試験
上記カランボラフラボン（試料１）について、以下のようにしてエンドセリン−１ｍＲＮＡ発現上昇抑制作用を試験した。
[Test Example 1] Endothelin-1 mRNA expression increase inhibitory action test The above-mentioned carambola flavone (sample 1) was tested for the endothelin-1 mRNA expression increase suppressive action as follows.
正常ヒト新生児包皮表皮角化細胞（normal human epidermal keratinocyte，ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（EpiLife-KG2）において、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で前培養し、トリプシン処理により細胞を集めた。 Normal human epidermal keratinocyte (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm 2 flask at 37 ° C., 5% CO 2 -95% air The cells were precultured under the above conditions and cells were collected by trypsin treatment.
EpiLife-KG2を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で一晩培養した。２４時間後に培養液を捨て、ＨＥＰＥＳ緩衝液１ｍＬを加えてＵＶ−Ｂ照射（５０ｍＪ／ｃｍ２）を行い、その後EpiLife-KG2で必要濃度に溶解した試験試料（試料１，試料濃度は下記表１を参照）を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で２４時間培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ社製，Cat.no．311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるように総ＲＮＡを調製した。 EpiLife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 -95% air. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm 2 ) was performed, and then the test sample dissolved in EpiLife-KG2 to the required concentration (sample 1, sample concentration is shown in Table 1 below) 2 mL) was added to each petri dish and cultured at 37 ° C. under 5% CO 2 -95% air for 24 hours. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、エンドセリン−１及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社）を用いて、TaKaRa SYBR PrimeScript RT-PCR Kit（Perfect Real Time，code No．RR063A）によるリアルタイム2 Step RT-PCR反応により行った。エンドセリン−１のｍＲＮＡの発現量は、紫外線未照射・試料無添加、紫外線照射・試料無添加及び紫外線照射・試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに紫外線未照射・試料無添加の補正値を１００とした時の紫外線照射・試料無添加及び紫外線照射・試料添加の補正値を算出した。得られた結果から、下記式によりエンドセリン−１ｍＲＮＡ発現上昇抑制率（％）を算出した。 Using this total RNA as a template, the expression levels of mRNA for endothelin-1 and GAPDH as an internal standard were measured. The detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of endothelin-1 mRNA is the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Then, the correction values were calculated, and the correction values for UV irradiation / no sample addition and UV irradiation / sample addition when the correction value for UV non-irradiation / no sample addition was 100 were calculated. From the obtained results, the endothelin-1 mRNA expression increase suppression rate (%) was calculated by the following formula.
ｍＲＮＡ発現上昇抑制率(％)＝{(Ａ−Ｂ)−(Ａ−Ｃ)}／(Ａ−Ｂ)×１００
式中Ａは「紫外線未照射・試料無添加時の補正値」を表し、Ｂは「紫外線照射・試料無添加時の補正値」を表し、Ｃは「紫外線照射・試料添加時の補正値」を表す。
結果を表１に示す。
mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 1.
表１に示すように、カランボラフラボンは、優れたエンドセリン−１ｍＲＮＡ発現上昇抑制作用を有することが確認された。 As shown in Table 1, it was confirmed that carambola flavone has an excellent inhibitory effect on endothelin-1 mRNA expression increase.
〔試験例２〕ＳＣＦｍＲＮＡ発現上昇抑制作用試験
上記カランボラフラボン（試料１）について、以下のようにしてＳＣＦｍＲＮＡ発現上昇抑制作用を試験した。
[Test Example 2] SCF mRNA expression increase inhibitory action test The carambola flavone (sample 1) was tested for SCF mRNA expression increase inhibitory action as follows.
正常ヒト新生児包皮表皮角化細胞（ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（EpiLife-KG2）において、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で前培養し、トリプシン処理により細胞を集めた。 Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm 2 flask under conditions of 37 ° C., 5% CO 2 -95% air. The cells were cultured and collected by trypsinization.
EpiLife-KG2を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で一晩培養した。２４時間後に培養液を捨て、ＨＥＰＥＳ緩衝液１ｍＬを加えてＵＶ−Ｂ照射（５０ｍＪ／ｃｍ２）を行い、その後EpiLife-KG2で必要濃度に溶解した試験試料（試料１，試料濃度は下記表２を参照）を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で２４時間培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ社製，Cat.no．311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるように総ＲＮＡを調製した。 EpiLife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 -95% air. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm 2 ) was performed, and then the test sample dissolved in EpiLife-KG2 to the required concentration (sample 1, sample concentration is shown in Table 2 below) 2 mL) was added to each petri dish and cultured at 37 ° C. under 5% CO 2 -95% air for 24 hours. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、ＳＣＦ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社）を用いて、TaKaRa SYBR PrimeScript RT-PCR Kit（Perfect Real Time，code No．RR063A）によるリアルタイム2 Step RT-PCR反応により行った。ＳＣＦのｍＲＮＡの発現量は、紫外線未照射・試料無添加、紫外線照射・試料無添加及び紫外線照射・試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに紫外線未照射・試料無添加の補正値を１００とした時の紫外線照射・試料無添加および紫外線照射・試料添加の補正値を算出した。得られた結果から、下記式によりＳＣＦｍＲＮＡ発現上昇抑制率（％）を算出した。 Using this total RNA as a template, the expression level of mRNA of SCF and GAPDH as an internal standard was measured. The detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of SCF mRNA was corrected with the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the SCF mRNA expression increase suppression rate (%) was calculated by the following formula.
ｍＲＮＡ発現上昇抑制率(％)＝{(Ａ−Ｂ)−(Ａ−Ｃ)}／(Ａ−Ｂ)×１００
式中Ａは「紫外線未照射・試料無添加時の補正値」を表し、Ｂは「紫外線照射・試料無添加時の補正値」を表し、Ｃは「紫外線照射・試料添加時の補正値」を表す。
結果を表２に示す。
mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 2.
表２に示すように、カランボラフラボンは、優れたＳＣＦｍＲＮＡ発現上昇抑制作用を有することが確認された。 As shown in Table 2, it was confirmed that carambola flavone has an excellent SCF mRNA expression increase inhibitory action.
〔試験例３〕ｂＦＧＦｍＲＮＡ発現上昇抑制作用試験
上記カランボラフラボン（試料１）について、以下のようにしてｂＦＧＦｍＲＮＡ発現上昇抑制作用を試験した。
[Test Example 3] bFGF mRNA expression increase inhibitory action test The carambola flavone (Sample 1) was tested for bFGF mRNA expression increase suppressive action as follows.
正常ヒト新生児包皮表皮角化細胞（ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（EpiLife-KG2）において、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で前培養し、トリプシン処理により細胞を集めた。 Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm 2 flask under conditions of 37 ° C., 5% CO 2 -95% air. The cells were cultured and collected by trypsinization.
EpiLife-KG2を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で一晩培養した。２４時間後に培養液を捨て、ＨＥＰＥＳ緩衝液１ｍＬを加えてＵＶ−Ｂ照射（５０ｍＪ／ｃｍ２）を行い、その後EpiLife-KG2で必要濃度に溶解した試験試料（試料１，試料濃度は下記表３を参照）を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で２４時間培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ社製，Cat.no．311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるように総ＲＮＡを調製した。 EpiLife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 -95% air. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm 2 ) was performed, and then the test sample dissolved in EpiLife-KG2 to the required concentration (sample 1, sample concentration is shown in Table 3 below) 2 mL) was added to each petri dish and cultured at 37 ° C. under 5% CO 2 -95% air for 24 hours. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、ｂＦＧＦ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社）を用いて、TaKaRa SYBR PrimeScript RT-PCR Kit（Perfect Real Time，code No．RR063A）によるリアルタイム2 Step RT-PCR反応により行った。ｂＦＧＦのｍＲＮＡの発現量は、紫外線未照射・試料無添加、紫外線照射・試料無添加及び紫外線照射・試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに紫外線未照射・試料無添加の補正値を１００とした時の紫外線照射・試料無添加および紫外線照射・試料添加の補正値を算出した。得られた結果から、下記式によりｂＦＧＦｍＲＮＡ発現上昇抑制率（％）を算出した。 Using this total RNA as a template, the expression level of bFGF and mRNA of GAPDH as an internal standard was measured. The detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of bFGF mRNA is corrected with the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the bFGF mRNA expression increase suppression rate (%) was calculated by the following formula.
ｍＲＮＡ発現上昇抑制率(％)＝{(Ａ−Ｂ)−(Ａ−Ｃ)}／(Ａ−Ｂ)×１００
式中Ａは「紫外線未照射・試料無添加時の補正値」を表し、Ｂは「紫外線照射・試料無添加時の補正値」を表し、Ｃは「紫外線照射・試料添加時の補正値」を表す。
結果を表３に示す。
mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 3.
表３に示すように、カランボラフラボンは、優れたｂＦＧＦｍＲＮＡ発現上昇抑制作用を有することが確認された。 As shown in Table 3, carambola flavone was confirmed to have an excellent inhibitory effect on the increase in bFGF mRNA expression.
〔試験例４〕ＰＯＭＣｍＲＮＡ発現上昇抑制作用試験
上記カランボラフラボン（試料１）について、以下のようにしてＰＯＭＣｍＲＮＡ発現上昇抑制作用を試験した。
[Test Example 4] POMC mRNA expression increase inhibitory action test The above-mentioned carambola flavone (sample 1) was tested for POMC mRNA expression increase suppressive action as follows.
正常ヒト新生児包皮表皮角化細胞（ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（EpiLife-KG2）において、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で前培養し、トリプシン処理により細胞を集めた。 Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm 2 flask under conditions of 37 ° C., 5% CO 2 -95% air. The cells were cultured and collected by trypsinization.
EpiLife-KG2を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で一晩培養した。２４時間後に培養液を捨て、ＨＥＰＥＳ緩衝液１ｍＬを加えてＵＶ−Ｂ照射（５０ｍＪ／ｃｍ２）を行い、その後EpiLife-KG2で必要濃度に溶解した試験試料（試料１，試料濃度は下記表４を参照）を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下で２４時間培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ社製，Cat.no．311-02501）にて総ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるように総ＲＮＡを調製した。 EpiLife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 -95% air. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm 2 ) was performed, and then the test sample dissolved in EpiLife-KG2 to the required concentration (Sample 1, Sample concentration is shown in Table 4 below) 2 mL) was added to each petri dish and cultured at 37 ° C. under 5% CO 2 -95% air for 24 hours. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.
この総ＲＮＡを鋳型とし、ＰＯＭＣ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Smart Cycler（Cepheid社）を用いて、TaKaRa SYBR PrimeScript RT-PCR Kit（Perfect Real Time，code No．RR063A）によるリアルタイム2 Step RT-PCR反応により行った。ＰＯＭＣのｍＲＮＡの発現量は、紫外線未照射・試料無添加、紫外線照射・試料無添加及び紫外線照射・試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに紫外線未照射・試料無添加の補正値を１００とした時の紫外線照射・試料無添加および紫外線照射・試料添加の補正値を算出した。得られた結果から、下記式によりＰＯＭＣｍＲＮＡ発現上昇抑制率（％）を算出した。 Using this total RNA as a template, the expression level of POMC and the internal standard GAPDH mRNA was measured. The detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). POMC mRNA expression level was corrected with GAPDH values based on total RNA preparations prepared from cells that were cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the POMC mRNA expression increase suppression rate (%) was calculated by the following formula.
ｍＲＮＡ発現上昇抑制率(％)＝{(Ａ−Ｂ)−(Ａ−Ｃ)}／(Ａ−Ｂ)×１００
式中Ａは「紫外線未照射・試料無添加時の補正値」を表し、Ｂは「紫外線照射・試料無添加時の補正値」を表し、Ｃは「紫外線照射・試料添加時の補正値」を表す。
結果を表４に示す。
mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 4.
表４に示すように、カランボラフラボンは、優れたＰＯＭＣｍＲＮＡ発現上昇抑制作用を有することが確認された。 As shown in Table 4, it was confirmed that carambola flavone has an excellent POMC mRNA expression increase inhibitory action.
本発明のエンドセリン−１ｍＲＮＡ発現上昇抑制剤、ＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤及びＰＯＭＣｍＲＮＡ発現上昇抑制剤は、シミ、ソバカス、皮膚の色黒（皮膚色素沈着症）等の予防・改善に大きく貢献できる。 The endothelin-1 mRNA expression increase inhibitor, the SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, and the POMC mRNA expression increase inhibitor of the present invention are useful for preventing and improving spots, freckles, skin darkness (skin pigmentation) and the like. It can contribute greatly.
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