JP2010202588A - UP-REGULATION SUPPRESSANT OF STEM CELL FACTOR (SCF) mRNA, UP-REGULATION SUPPRESSANT OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) mRNA, EXPRESSION ENHANCER OF SERINE-PALMITOYL TRANSFERASE (SPT) mRNA, AND EXPRESSION ENHANCER OF AQUAPORIN 3 (AQP3) mRNA - Google Patents
UP-REGULATION SUPPRESSANT OF STEM CELL FACTOR (SCF) mRNA, UP-REGULATION SUPPRESSANT OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) mRNA, EXPRESSION ENHANCER OF SERINE-PALMITOYL TRANSFERASE (SPT) mRNA, AND EXPRESSION ENHANCER OF AQUAPORIN 3 (AQP3) mRNA Download PDFInfo
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本発明は、幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制剤、塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ発現上昇抑制剤、セリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進剤及びアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進剤に関する。 The present invention relates to an inhibitor of stem cell growth factor (SCF) mRNA expression increase, a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor, a serine palmitoyltransferase (SPT) mRNA expression promoter, and aquaporin 3 (AQP3) mRNA expression. Relates to accelerators.
幹細胞増殖因子（Stem Cell Factor，ＳＣＦ）は、Must 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１参照）。 Stem Cell Factor (SCF), also called Must 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 progenitor cells, granulocyte / macrophage progenitor 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 1).
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２参照）。 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 2).
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３参照）。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 3). 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１参照）。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２参照）。 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, Japanese cypress, camphor root extract, soybean extract, scallop extract, bonito extract, altea extract, hypericum extract and mugwort extract (see Patent Document 1). Moreover, as a thing which can suppress the effect | action of bFGF, onony extract etc. are known, for example (refer patent document 2).
セラミドは、表皮細胞の角化の過程においてセリンとパルミトイル−ＣｏＡとを基に、セラミド合成の律速酵素として知られるセリンパルミトイルトランスフェラーゼ（ＳＰＴ）をはじめとする酵素の働きにより生成される。セラミドは、皮膚最外層を覆う角質細胞間脂質の主成分として特異的に存在し、皮膚本来が持つ生体と外界とのバリア膜としての機能維持に重要な役割を果たしている。 Ceramide is produced by the action of an enzyme such as serine palmitoyltransferase (SPT) known as a rate-limiting enzyme for ceramide synthesis based on serine and palmitoyl-CoA in the process of keratinization of epidermal cells. Ceramide exists specifically as the main component of the keratinocyte lipid covering the outermost layer of the skin, and plays an important role in maintaining the function of the skin itself as a barrier film between the living body and the outside world.
角層の構造は、レンガとモルタルとに例えられ、１５層ほどに積み重なった角層細胞を細胞間脂質が繋ぎ止める形で強固なバリア膜を形成している。角層細胞は、アミノ酸を主成分とする天然保湿因子を細胞内に含有することによって水分を保持し、一方、角質細胞間脂質は、約５０％のセラミドを主成分とし、コレステロール、脂肪酸等の両親媒性脂質から構成されており、疎水性部分と親水性部分とが交互に繰り返される層板構造、いわゆるラメラ構造を特徴としている。 The stratum corneum structure is compared to brick and mortar, and a strong barrier membrane is formed in such a way that intercellular lipids connect the stratum corneum cells stacked in about 15 layers. The stratum corneum retains moisture by containing a natural moisturizing factor mainly composed of amino acids in the cell, while stratum corneum lipids are mainly composed of about 50% ceramide, such as cholesterol and fatty acids. It is composed of amphipathic lipids and is characterized by a so-called lamellar structure in which hydrophobic portions and hydrophilic portions are alternately repeated.
様々な内的・外的要因による皮膚のバリア機能の低下は、経表皮水分蒸散量を増加させ、皮膚のかさつき、落屑、掻痒感等を惹き起こし、いわゆる乾燥肌に陥る。また、皮膚のバリア機能の低下は、皮膚の炎症を増大させ、外界からの様々な刺激に対する防御機能が低下するという悪循環に陥る。最近の研究において、加齢により、又はバリア障害として知られるアトピー性皮膚炎患者において、角層セラミド成分（いわゆる細胞間脂質）の減少や組成変化が報告されており（非特許文献４参照）、皮膚のバリア機能の維持、改善にセラミドが重要であることが広く知られるようになっている。このような考え方に基づいて、皮膚のバリア機能を改善する方法として、セラミドを外部から補う方法（非特許文献５参照）や皮膚内部においてセラミド産生能を高める方法（非特許文献６参照）等が知られている。 The decrease in the skin barrier function due to various internal and external factors increases the transepidermal water transpiration, causing skin roughness, desquamation, pruritus, etc., resulting in so-called dry skin. Moreover, the decrease in the barrier function of the skin increases the inflammation of the skin and falls into a vicious circle in which the protective function against various stimuli from the outside world is reduced. In recent studies, in patients with atopic dermatitis known as aging or barrier disorder, a decrease in the stratum corneum ceramide component (so-called intercellular lipid) and a composition change have been reported (see Non-Patent Document 4), It is widely known that ceramide is important for maintaining and improving the barrier function of the skin. Based on such an idea, methods for improving the barrier function of the skin include a method of supplementing ceramide from the outside (see Non-Patent Document 5), a method of increasing the ceramide production ability inside the skin (see Non-Patent Document 6), and the like. Are known.
皮膚細胞では、水チャンネルとして知られるアクアポリンが、細胞膜上に発現して、細胞間隙の水をはじめとする低分子物質を細胞内へ取り込む役割を担っていることが知られている。 In skin cells, it is known that aquaporins, known as water channels, are expressed on the cell membrane and play a role of taking in low-molecular substances such as interstitial water into cells.
ヒトでは、１３種類のアクアポリン（ＡＱＰ０〜ＡＱＰ１２）の存在が知られている。表皮細胞においては、主としてＡＱＰ３が存在しており、水に加えて、水分保持作用に関与するグリセロールや尿素等の低分子化合物をも取り込む役割を担っていると考えられている。 In humans, the presence of 13 types of aquaporins (AQP0 to AQP12) is known. In epidermal cells, AQP3 is mainly present, and it is considered to play a role of taking in low-molecular compounds such as glycerol and urea involved in water retention action in addition to water.
しかしながら、ＡＱＰ３は加齢とともに減少し、このことが水分保持機能の低下の一因であることが示唆されているため、ＡＱＰ３の発現を促進することにより、加齢による水分保持能やバリア機能等を制御することが可能であると考えられる（非特許文献７参照）。このような考えに基づき、ＡＱＰ３発現促進作用を有するものとして、例えば、トコフェリルレチノエート（特許文献３参照）、ノウゼンハレン科植物より得られる抽出物（特許文献４参照）等が知られている。 However, AQP3 decreases with aging, and it has been suggested that this contributes to a decrease in water retention function. Therefore, by promoting the expression of AQP3, the water retention capacity and barrier function due to aging, etc. Can be controlled (see Non-Patent Document 7). Based on such an idea, for example, tocopheryl retinoate (see Patent Document 3), an extract obtained from a Nozenhalen plant (see Patent Document 4) and the like are known as having AQP3 expression promoting action.
本発明は、幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制作用、塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ発現上昇抑制作用、セリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進作用又はアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進作用を有するものを見出し、それを有効成分とする幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制剤、塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ発現上昇抑制剤、セリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進剤及びアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進剤を提供することを目的とする。 The present invention relates to an inhibitory effect on stem cell growth factor (SCF) mRNA expression increase, an inhibitory effect on basic fibroblast growth factor (bFGF) mRNA expression, a serine palmitoyltransferase (SPT) mRNA expression promoting action, or an aquaporin 3 (AQP3) mRNA expression. Finding an agent having a promoting action, and using it as an active ingredient, an inhibitor of stem cell growth factor (SCF) mRNA expression increase, a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor, serine palmitoyltransferase (SPT) mRNA expression An object is to provide a promoter and an aquaporin 3 (AQP3) mRNA expression promoter.
上記課題を解決するために、本発明の幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制剤、塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ発現上昇抑制剤、セリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進剤又はアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進は、アップルミント熱水抽出物を有効成分として含有することを特徴とする。 In order to solve the above-mentioned problems, a stem cell growth factor (SCF) mRNA expression increase inhibitor, a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor, a serine palmitoyltransferase (SPT) mRNA expression promoter of the present invention, or Aquaporin 3 (AQP3) mRNA expression promotion includes an apple mint hot water extract as an active ingredient.
本発明によれば、安全性の高い天然抽出物を有効成分として含有する幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制剤、塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ発現上昇抑制剤、セリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進剤及びアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進を提供することができる。 According to the present invention, a stem cell growth factor (SCF) mRNA expression increase inhibitor, a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor, serine palmitoyltransferase, which contains a highly safe natural extract as an active ingredient. (SPT) mRNA expression promoter and aquaporin 3 (AQP3) mRNA expression promotion can be provided.
以下、本発明について説明する。
本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤及びＡＱＰ３ｍＲＮＡ発現促進剤は、アップルミント熱水抽出物を有効成分として含有する。
The present invention will be described below.
The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the SPT mRNA expression promoter, and the AQP3 mRNA expression promoter of the present invention contain apple mint hot water extract as an active ingredient.
本発明において「アップルミント熱水抽出物」には、アップルミントを抽出原料として用い、熱水を抽出溶媒として用いて得られる抽出液、当該抽出液の希釈液若しくは濃縮液、当該抽出液を乾燥して得られる乾燥物、又はこれらの粗精製物若しくは精製物のいずれもが含まれる。 In the present invention, the “apple mint hot water extract” is an extract obtained using apple mint as an extraction raw material and hot water as an extraction solvent, a diluted or concentrated solution of the extract, and the extract is dried. The dried product obtained in this way, or any of these crudely purified products or purified products.
本発明において使用する抽出原料は、アップルミント（学名：Mentha suaveolens）である。アップルミントは、地中海沿岸からヨーロッパが原産地であるシソ科ハッカ属に属する多年草であり、これらの地域から容易に入手することができる。抽出原料として使用し得るアップルミントの構成部位としては、例えば、葉部、枝部、茎部、果実部、種子部、花部、根部又はこれらの部位の混合物等が挙げられるが、好ましくは葉部である。 The extraction raw material used in the present invention is apple mint (scientific name: Mentha suaveolens). Apple mint is a perennial belonging to the genus Labiatae, which originates in Europe from the Mediterranean coast, and can be easily obtained from these regions. Examples of the constituent parts of apple mint that can be used as an extraction raw material include leaf parts, branch parts, stem parts, fruit parts, seed parts, flower parts, root parts, or a mixture of these parts, preferably leaves. Part.
アップルミント熱水抽出物に含有されるＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用、ＳＰＴｍＲＮＡ発現促進作用又はＡＱＰ３ｍＲＮＡ発現促進作用を有する物質の詳細は不明であるが、植物の抽出に一般に用いられている抽出方法によって、アップルミントからこれらの作用を有する抽出物を得ることができる。 Details of the substance having SCF mRNA expression increase inhibitory action, bFGF mRNA expression increase inhibitory action, SPT mRNA expression promotion action or AQP3 mRNA expression promotion action contained in the apple mint hot water extract are unknown, but it is generally used for plant extraction. Extracts having these actions can be obtained from apple mint by the extraction method used.
アップルミント熱水抽出物は、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、抽出溶媒としての熱水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、アップルミントの熱水による抽出処理を効率よく行うことができる。 The apple mint hot water 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 hot water as 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, extraction treatment of apple mint with hot water can be performed efficiently.
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.
抽出処理は、抽出原料に含まれる可溶性成分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５〜２５倍量（質量比）の抽出溶媒である水に抽出原料を浸漬させ、６０〜１００℃の温度条件下で加熱して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 the soluble component 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 water that is an extraction solvent 5 to 25 times the mass of the extraction raw material (mass ratio), heated under a temperature condition of 60 to 100 ° C. to extract soluble components, and then filtered. An extraction liquid can be obtained by removing the extraction residue. 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.
以上のようにして得られるアップルミント熱水抽出物は、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用、ＳＰＴｍＲＮＡ発現促進作用又はＡＱＰ３ｍＲＮＡ発現促進作用を有しているため、それらの作用を利用してＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤の有効成分として使用することができる。 The apple mint hot water extract obtained as described above has an SCF mRNA expression increase suppressing action, a bFGF mRNA expression increase suppressing action, an SPT mRNA expression promoting action or an AQP3 mRNA expression promoting action. It can be used as an active ingredient of an SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, SPT mRNA expression promoter, or AQP3 mRNA expression promoter.
また、アップルミント熱水抽出物は、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。 In addition, apple mint hot water extract uses its SCF mRNA expression increase inhibitory action to prevent or treat a disease caused by increased expression of SCF mRNA (for example, myelodysplastic syndrome preventive or therapeutic agent, acute myeloid leukemia). Prophylactic / therapeutic agents, antitumor agents, etc.)
さらに、アップルミント熱水抽出物は、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, the apple mint hot water extract uses its bFGF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased expression of bFGF mRNA (for example, angiogenesis inhibitors, anticancer agents, antitumor agents). It can also be used as an active ingredient of a pharmaceutical composition that suppresses cancer cell metastasis.
さらにまた、アップルミント熱水抽出物は、そのＳＰＴｍＲＮＡ発現促進作用を通じて、セラミドの合成を促進し、皮膚バリア機能を改善することができるため、皮膚バリア機能改善剤の有効成分として用いることもできる。具体的にはアトピー性皮膚炎等の炎症性疾患による皮膚バリア機能障害の予防・治療剤等の有効成分として用いることもできる。また、アップルミント熱水抽出物は、そのＳＰＴｍＲＮＡ発現促進作用を通じて、セラミドの合成を促進することができるため、セラミドの合成障害に起因する疾患（例えば、アトピー性皮膚炎等）の予防・治療剤の有効成分として用いることもできる。 Furthermore, since the apple mint hot water extract can promote the synthesis of ceramide and improve the skin barrier function through its SPT mRNA expression promoting action, it can also be used as an active ingredient of a skin barrier function improving agent. Specifically, it can also be used as an active ingredient such as a prophylactic / therapeutic agent for skin barrier dysfunction due to inflammatory diseases such as atopic dermatitis. Moreover, since the apple mint hot water extract can promote the synthesis of ceramide through its SPT mRNA expression promoting action, it prevents or treats diseases caused by ceramide synthesis disorders (for example, atopic dermatitis, etc.) It can also be used as an active ingredient.
本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤は、アップルミント熱水抽出物のみからなるものでもよいし、アップルミント熱水抽出物を製剤化したものでもよい。 The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the SPT mRNA expression promoter or the AQP3 mRNA expression promoter of the present invention may consist only of apple mint hot water extract, or an apple mint hot water extract was formulated. It may be a thing.
アップルミント熱水抽出物は、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。アップルミント熱水抽出物を製剤化したＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤の形態としては、例えば、軟膏剤、外用液剤、貼付剤等が挙げられる。 Applemint hot water extract is formulated into any dosage form such as powder, granule, liquid, etc. according to conventional methods using pharmaceutically acceptable carriers such as dextrin and cyclodextrin and other optional auxiliaries. can do. In this case, as an auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent, and the like can be used. Examples of the SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, SPT mRNA expression promoter or AQP3 mRNA expression promoter formulated with apple mint hot water extract include ointments, liquids for external use, patches and the like. .
なお、本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤は、必要に応じて、ＳＣＦｍＲＮＡ発現上昇抑制作用、ｂＦＧＦｍＲＮＡ発現上昇抑制作用、ＳＰＴｍＲＮＡ発現促進作用又はＡＱＰ３ｍＲＮＡ発現促進作用を有する天然抽出物等を、アップルミント熱水抽出物とともに配合して有効成分として用いることができる。 The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the SPT mRNA expression promoter or the AQP3 mRNA expression promoter of the present invention is, as necessary, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action, an SPT mRNA expression promoting action, or A natural extract or the like having AQP3 mRNA expression promoting action can be blended with an apple mint hot water extract and used as an active ingredient.
本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進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。 Examples of the method of administration of the SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, SPT mRNA expression promoter or AQP3 mRNA expression promoter of the present invention to patients include subcutaneous tissue administration, intramuscular administration, intravenous administration, oral administration, and transdermal. Administration and the like can be mentioned, but a suitable method for the prevention / treatment or the like may be appropriately selected according to the type of disease.
また、本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤の投与量も、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。 In addition, the dose of the SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, SPT mRNA expression promoter or AQP3 mRNA expression promoter of the present invention depends on the type of disease, severity, individual differences among patients, administration method, administration period, etc. What is necessary is just to increase / decrease suitably.
本発明のＳＣＦｍＲＮＡ発現上昇抑制剤は、アップルミント熱水抽出物が有する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、アップルミント熱水抽出物が有する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 suppression action of the apple mint hot water extract, thereby suppressing the proliferation of pigment cells and the production of melanin. , Spots, freckles, 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 growth of myeloblasts through the SCF mRNA expression increase suppressive action of the apple mint hot water extract. Diseases such as leukemia 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 in 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、アップルミント熱水抽出物が有する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、アップルミント熱水抽出物が有する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 of the apple mint hot water extract, thereby suppressing the proliferation of pigment cells and the production of melanin. , Spots, freckles, skin pigmentation and the like can be prevented or improved, and a whitening effect can be obtained. In addition, the bFGF mRNA expression increase inhibitor of the present invention suppresses abnormal angiogenesis in tumor cells through the bFGF mRNA expression increase suppressive action of apple mint hot water extract, and prevents, treats or improves diseases such as cancer. can do. However, the bFGF mRNA expression increase inhibitor of the present invention can be used for all purposes other than these applications that are meaningful for exhibiting the bFGF mRNA expression increase suppression action.
本発明のＳＰＴｍＲＮＡ発現促進剤は、アップルミント熱水抽出物が有するＳＰＴｍＲＮＡ発現促進作用を通じて、セラミドの合成を促進することができ、これにより、しわ、弾力性の低下等の皮膚の老化症状を予防又は改善することができるとともに、アトピー性皮膚炎等の炎症性疾患による皮膚バリア機能障害やアトピー性皮膚炎等のセラミド合成障害に起因する疾患を予防、治療又は改善することができる。ただし、本発明のＳＰＴｍＲＮＡ発現促進剤は、これらの用途以外にもＳＰＴｍＲＮＡ発現促進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 SPT mRNA expression promoter of the present invention can promote the synthesis of ceramide through the SPT mRNA expression promoting action of apple mint hot water extract, thereby preventing skin aging symptoms such as wrinkles and reduced elasticity. In addition to being able to improve, it is possible to prevent, treat or ameliorate diseases caused by skin barrier dysfunction due to inflammatory diseases such as atopic dermatitis and ceramide synthesis disorders such as atopic dermatitis. However, the SPT mRNA expression promoting agent of the present invention can be used for all uses that are meaningful for exerting the SPT mRNA expression promoting action in addition to these uses.
本発明のＡＱＰ３ｍＲＮＡ発現促進剤は、アップルミント熱水抽出物が有するＡＱＰ３ｍＲＮＡ発現促進作用を通じて、アクアポリン３（ＡＱＰ３）の発現を促進することができるため、皮膚の水分保持能やバリア機能を改善することができ、それにより皮膚の乾燥、しわの形成、弾力性の低下等の皮膚の老化症状を予防又は改善することができる。ただし、本発明のＡＱＰ３ｍＲＮＡ発現促進剤は、これらの用途以外にもＡＱＰ３ｍＲＮＡ発現促進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 AQP3 mRNA expression promoter of the present invention can promote the expression of aquaporin 3 (AQP3) through the AQP3 mRNA expression promoting action of the apple mint hot water extract, thereby improving the water retention capacity and barrier function of the skin. It is possible to prevent or improve skin aging symptoms such as skin dryness, wrinkle formation, and reduced elasticity. However, the AQP3 mRNA expression promoter of the present invention can be used for all purposes other than these uses that are meaningful for exerting the AQP3 mRNA expression promoting action.
なお、本発明のＳＣＦｍＲＮＡ発現上昇抑制剤、ｂＦＧＦｍＲＮＡ発現上昇抑制剤、ＳＰＴｍＲＮＡ発現促進剤又はＡＱＰ３ｍＲＮＡ発現促進剤は、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。 The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the SPT mRNA expression promoter or the AQP3 mRNA expression promoter of the present invention are suitably applied to humans, and each of the functions and effects is exerted. As long as it is applicable to animals other than humans.
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〕アップルミント熱水抽出物の製造
アップルミントの葉部１００ｇに抽出溶媒として水２０００ｍＬを加え、９０℃で１時間加熱抽出した。得られた抽出液を減圧下に濃縮し、さらに乾燥してアップルミント熱水抽出物３１ｇ（試料１）を得た。
[Production Example 1] Production of apple mint hot water extract To 100 g of apple mint leaves, 2000 mL of water was added as an extraction solvent, and the mixture was extracted by heating at 90 ° C for 1 hour. The obtained extract was concentrated under reduced pressure and further dried to obtain 31 g of apple mint hot water extract (sample 1).
〔試験例１〕ＳＣＦｍＲＮＡ発現上昇抑制作用試験
アップルミント熱水抽出物（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 1] SCF mRNA expression increase inhibitory action test The apple mint hot water extract (sample 1) was tested for SCF 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回収した。 Normal human epidermal keratinocyte (NHEK) in an 80 cm 2 flask in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes (EpiLife-KG2) 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：１μｇ／ｍＬ）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: 1 μg / mL) 2 mL was added to each petri dish, and cultured for 24 hours under conditions of 37 ° C., 5% CO 2 -95% air. 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（Stem Cell Factor）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ＳＣＦｍＲＮＡ発現上昇抑制率（％）を算出した。 Using this total RNA as a template, the expression level of SCF (Stem Cell Factor) and GAPDH mRNA 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 taken as 100. From the obtained results, the SCF mRNA expression increase suppression rate (%) was calculated by the following formula.
ＳＣＦｍＲＮＡ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。
SCF mRNA expression increase suppression rate (%) = {(A−B) − (A−C)} / (A−B) × 100
In the formula, A represents “correction value when UV irradiation is not performed and no sample is added”, B is “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”. ".
以上のようにして算出した結果、アップルミント熱水抽出物のＳＣＦｍＲＮＡ発現上昇抑制率は６５．８％であった。この結果から、アップルミント熱水抽出物は、優れたＳＣＦｍＲＮＡ発現上昇抑制作用を有することが判明した。 As a result of the above calculation, the SCF mRNA expression increase suppression rate of the apple mint hot water extract was 65.8%. From this result, it was found that the apple mint hot water extract has an excellent inhibitory effect on the increase in SCF mRNA expression.
〔試験例２〕ｂＦＧＦｍＲＮＡ発現上昇抑制作用試験
アップルミント熱水抽出物（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 2] bFGF mRNA expression increase inhibitory action test The apple mint hot water extract (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（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回収した。 Normal human epidermal keratinocyte (NHEK) in an 80 cm 2 flask in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes (EpiLife-KG2) 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：１μｇ／ｍＬ）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: 1 μg / mL) 2 mL was added to each petri dish, and cultured for 24 hours under conditions of 37 ° C., 5% CO 2 -95% air. 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（basic Fibroblast Growth Factor）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 (basic fibroblast growth factor) 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). 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 taken as 100. From the obtained results, the bFGF mRNA expression increase suppression rate (%) was calculated by the following formula.
ｂＦＧＦｍＲＮＡ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。
bFGF mRNA expression increase suppression rate (%) = {(A−B) − (A−C)} / (A−B) × 100
In the formula, A represents “correction value when UV irradiation is not performed and no sample is added”, B is “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”. ".
以上のようにして算出した結果、アップルミント熱水抽出物のｂＦＧＦｍＲＮＡ発現上昇抑制率は６３．８％であった。この結果から、アップルミント熱水抽出物は、優れたｂＦＧＦｍＲＮＡ発現上昇抑制作用を有することが判明した。 As a result of the calculation as described above, the suppression rate of bFGF mRNA expression increase of the apple mint hot water extract was 63.8%. From this result, it was found that the apple mint hot water extract has an excellent inhibitory effect on the increase in bFGF mRNA expression.
〔試験例３〕ＳＰＴｍＲＮＡ発現促進作用試験
アップルミント熱水抽出物（試料１）について、以下のようにしてＳＰＴｍＲＮＡ発現促進作用を試験した。
[Test Example 3] SPT mRNA expression promoting action test The apple mint hot water extract (sample 1) was tested for SPT mRNA expression promoting 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回収した。 Normal human epidermal keratinocyte (NHEK) in an 80 cm 2 flask in growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes (EpiLife-KG2) 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、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, and 2 mL of a test sample (sample 1, refer to Table 1 below for sample concentration) dissolved in EpiLife-KG2 was added to each petri dish at 37 ° C., 5% CO 2 -95. The cells were cultured for 24 hours under the condition of% air. 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、ＳＰＴ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。ＳＰＴのｍＲＮＡの発現量は、試料無添加及び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、さらに試料無添加の補正値を１００とした時の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ＳＰＴｍＲＮＡ発現促進率（％）を算出した。 Using this total RNA as a template, the expression level of SPT 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 SPT mRNA was determined based on the total RNA preparation prepared from cells cultured with and without addition of the sample, and the correction value was calculated with the value of GAPDH. The correction value of the sample addition at that time was calculated. From the obtained results, the SPT mRNA expression promotion rate (%) was calculated by the following formula.
ＳＰＴｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表１に示す。
SPT mRNA expression promotion rate (%) = A / B × 100
In the formula, A represents “correction value when sample is added” and B represents “correction value when sample is not added”.
The results are shown in Table 1.
表１に示すように、アップルミント熱水抽出物は、優れたＳＰＴｍＲＮＡ発現促進作用を有することが判明した。 As shown in Table 1, it was found that the apple mint hot water extract has an excellent SPT mRNA expression promoting action.
〔試験例４〕ＡＱＰ３ｍＲＮＡ発現促進作用試験
アップルミント熱水抽出物（試料１）について、以下のようにしてＡＱＰ３ｍＲＮＡ発現促進作用を試験した。
[Test Example 4] AQP3 mRNA expression promoting action test The apple mint hot water extract (sample 1) was tested for AQP3 mRNA expression promoting 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回収した。 Normal human epidermal keratinocyte (NHEK) in an 80 cm 2 flask in growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes (EpiLife-KG2) 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、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, and 2 mL of a test sample dissolved in EpiLife-KG2 to a required concentration (Sample 1, see Table 2 below for the sample concentration) was added to each dish, and 37 ° C., 5% CO 2 -95. The cells were cultured for 24 hours under the condition of% air. 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、ＡＱＰ３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。ＡＱＰ３のｍＲＮＡの発現量は、試料無添加及び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、さらに試料無添加の補正値を１００とした時の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ＡＱＰ３ｍＲＮＡ発現促進率（％）を算出した。 Using this total RNA as a template, the expression levels of AQP3 and the internal standard GAPDH mRNA 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 AQP3 mRNA was determined based on the total RNA preparation prepared from cells cultured with and without addition of the sample, and the correction value was calculated with the value of GAPDH. The correction value of the sample addition at that time was calculated. From the obtained results, the AQP3 mRNA expression promotion rate (%) was calculated by the following formula.
ＡＱＰ３ｍＲＮＡ発現促進率(％)＝Ａ／Ｂ×１００
式中、Ａは「試料添加時の補正値」を表し、Ｂは「試料無添加時の補正値」を表す。
結果を表２に示す。
AQP3 mRNA expression promotion rate (%) = A / B × 100
In the formula, A represents “correction value when sample is added” and B represents “correction value when sample is not added”.
The results are shown in Table 2.
表２に示すように、アップルミント熱水抽出物は、優れたＡＱＰ３ｍＲＮＡ発現促進作用を有することが判明した。 As shown in Table 2, it was found that the apple mint hot water extract has an excellent AQP3 mRNA expression promoting action.
本発明の幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制剤及び塩基性線維芽細胞増殖因子（ｂＦＧＦ）ｍＲＮＡ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、本発明のセリンパルミトイルトランスフェラーゼ（ＳＰＴ）ｍＲＮＡ発現促進剤及びアクアポリン３（ＡＱＰ３）ｍＲＮＡ発現促進剤は、加齢による水分保持能や皮膚バリア機能の低下等の予防・改善に大きく貢献できる。 The stem cell growth factor (SCF) mRNA expression increase inhibitor and the basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor of the present invention are used to prevent spots, buckwheat, skin darkness (skin pigmentation), etc. For the improvement, the serine palmitoyltransferase (SPT) mRNA expression promoter and aquaporin 3 (AQP3) mRNA expression promoter of the present invention can greatly contribute to the prevention and improvement of water retention ability and skin barrier function due to aging.
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| JP2009050236A JP2010202588A (en) | 2009-03-04 | 2009-03-04 | UP-REGULATION SUPPRESSANT OF STEM CELL FACTOR (SCF) mRNA, UP-REGULATION SUPPRESSANT OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) mRNA, EXPRESSION ENHANCER OF SERINE-PALMITOYL TRANSFERASE (SPT) mRNA, AND EXPRESSION ENHANCER OF AQUAPORIN 3 (AQP3) mRNA |
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| JP2014047145A (en) * | 2012-08-30 | 2014-03-17 | Honda Hideo | Silica microcapsules of lipid component having lamella structure, and production method thereof |
| JP2015203008A (en) * | 2014-04-11 | 2015-11-16 | 富士化学工業株式会社 | Aquaporin production improver |
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| JP2015203008A (en) * | 2014-04-11 | 2015-11-16 | 富士化学工業株式会社 | Aquaporin production improver |
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