JP7100351B2 - CXCL12 expression promoter - Google Patents
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本発明は、ＣＸＣＬ１２発現促進剤、及び色素幹細胞の分化抑制剤に関する。 The present invention relates to a CXCL12 expression promoter and a dye stem cell differentiation inhibitor.
皮膚や毛髪の色は、メラノサイトによって産生されるメラニンにより、大きく左右される。生体内のメラノサイトの起源は、毛包内バルジ領域に存在する色素幹細胞であることが知られている。色素幹細胞から分化したメラノブラストは、表皮や毛球へと移動し、Ｔｙｒｏｓｉｎａｓｅ（ＴＹＲ）、ＴＹＲ－ｒｅｌａｔｅｄ ｐｒｏｔｅｉｎ－１（ＴＹＲＰ１）及びＤｏｐａｃｈｒｏｍｅ ｔａｕｔｏｍｅｒａｓｅ（ＤＣＴ）などの一連のメラニン合成関連酵素の発現を獲得し、メラニン合成能を持った成熟したメラノサイトへ分化することで、それぞれ皮膚や毛髪の色素産生に関わる（非特許文献１）。したがって、色素幹細胞の維持や分化における異常は、白髪の原因となる。例えば、白髪化のメカニズムの一つとして、バルジ領域において色素幹細胞がメラノサイトへ異所性に分化してしまうことで、色素幹細胞が枯渇し、メラノサイトの供給源がなくなってしまうことが明らかになっている（非特許文献２）。 The color of skin and hair is greatly influenced by the melanin produced by melanocytes. The origin of melanocytes in vivo is known to be pigment stem cells present in the bulge region within hair follicles. Melanoblasts differentiated from pigmented stem cells migrate to the epidermis and hair bulbs and acquire the expression of a series of melanin synthesis-related enzymes such as Tyrosinase (TYR), TYR-related protein-1 (TYRP1) and Dopachrome tautomerase (DCT). However, by differentiating into mature melanocytes with melanin synthesizing ability, they are involved in the production of pigments in the skin and hair, respectively (Non-Patent Document 1). Therefore, abnormalities in the maintenance and differentiation of pigment stem cells cause gray hair. For example, as one of the mechanisms of whitening, it has become clear that pigment stem cells ectopically differentiate into melanocytes in the bulge region, resulting in depletion of pigment stem cells and loss of the source of melanocytes. (Non-Patent Document 2).
従来、白髪の改善方法は、毛包内に存在するメラノサイトを活性化し、メラニン生成やメラノサイトの増殖を促進する方法が主流であった。しかしながら、かかる方法は、メラノサイトの起源となる色素幹細胞を制御するものでなく、限定的又は対処療法的であり、根本的な白髪の改善は望めない。 Conventionally, the main method for improving white hair has been to activate melanocytes existing in hair follicles and promote melanin production and melanocyte proliferation. However, such a method does not control the pigment stem cells that are the origin of melanocytes, is limited or symptomatic, and cannot be expected to improve the fundamental white hair.
一般的に、幹細胞は、ニッチと呼ばれる幹細胞の周囲を取り巻く微小環境によって制御されている（非特許文献３）。毛包内バルジ領域において、毛包幹細胞は色素幹細胞の周囲を取り囲むように存在し、色素幹細胞のニッチ細胞として機能している。よって、毛包幹細胞の性質に着目し、色素幹細胞のニッチを適切に制御できれば、従来のメラノサイトの活性化や増殖促進による方法と比べて、白髪を根本的に改善する方法として極めて有効といえる。 In general, stem cells are controlled by a microenvironment surrounding the stem cells, which is called a niche (Non-Patent Document 3). In the bulge region within the hair follicle, the hair follicle stem cell exists so as to surround the pigment stem cell and functions as a niche cell of the pigment stem cell. Therefore, if attention is paid to the properties of hair follicle stem cells and the niche of pigment stem cells can be appropriately controlled, it can be said that it is extremely effective as a method for fundamentally improving white hair as compared with the conventional method of activating melanocytes and promoting proliferation.
一方、本発明者らによるこれまでの研究で、ケモカインの一種であるＣＸＣＬ１２（別名ｓｔｒｏｍａｌ ｃｅｌｌ－ｄｅｒｉｖｅｄ ｆａｃｔｏｒ－１：ＳＤＦ－１）が、毛包内バルジ領域の毛包幹細胞において特異的に発現し、その発現量は黒髪の毛根部よりも白髪の毛根部で低いこと、毛包幹細胞により産生されたＣＸＣＬ１２が色素幹細胞の異所性の分化を抑制するとともに、メラノサイトの分化関連遺伝子の発現を抑制することが明らかとなっている（特許文献１）。よって、このメラノサイトの起源となる色素幹細胞のニッチを構成するＣＸＣＬ１２を制御することができれば、メラノサイトのみをターゲットとする従来のアプローチよりも、根本的かつ効果的な白髪の予防及び／又は改善効果が期待できる。また、ＣＸＣＬ１２のような因子を生体内で制御することは困難であるため、外部から制御できる薬剤の開発が待たれている。 On the other hand, in previous studies by the present inventors, CXCL12 (also known as stromal cell-derived factor-1: SDF-1), which is a kind of chemokine, is specifically expressed in hair follicle stem cells in the bulge region in hair follicles. The expression level is lower in the hair root of white hair than in the hair root of black hair, and CXCL12 produced by hair follicle stem cells suppresses ectopic differentiation of pigment stem cells and suppresses the expression of melanosite differentiation-related genes. Has been clarified (Patent Document 1). Therefore, if CXCL12, which constitutes the niche of pigment stem cells that are the origin of melanocytes, can be controlled, a fundamental and effective prevention and / or improvement effect on white hair can be achieved as compared with the conventional approach targeting only melanocytes. You can expect it. Further, since it is difficult to control a factor such as CXCL12 in vivo, the development of a drug that can be controlled from the outside is awaited.
従って、本発明は、色素幹細胞のニッチを構成し、色素幹細胞の分化抑制に関与するＣＸＣＬ１２を生体の外部から制御できる物質を見出し、これを、白髪を根本的にかつ効率的に予防及び／又は改善するための薬剤として提供することを課題とする。 Therefore, the present invention has found a substance that constitutes a niche of pigment stem cells and can control CXCL12 involved in the suppression of differentiation of pigment stem cells from the outside of the living body, and prevents and / or prevents white hair fundamentally and efficiently. The challenge is to provide it as a drug for improvement.
本発明者らは上記課題を解決すべく鋭意研究をした結果、トリュフの抽出物、所定の前処理をしたオタネニンジンの抽出物、及びこれらの混合物が、毛包幹細胞におけるＣＸＣＬ１２の発現を有意に増加させることを見出し、本発明を完成させるに至った。 As a result of diligent research to solve the above-mentioned problems, the present inventors significantly increased the expression of CXCL12 in hair follicle stem cells by the extract of truffle, the extract of Otaneninjin which had been subjected to a predetermined pretreatment, and a mixture thereof. It was found that the present invention was completed.
すなわち、本発明は、以下の発明を包含する。
（１）トリュフの抽出物、及び蒸気加熱処理後、加熱乾燥処理されたオタネニンジンの抽出物から選ばれる１種又は２種の抽出物を有効成分として含有することを特徴とする、ＣＸＣＬ１２（ｓｔｒｏｍａｌ ｃｅｌｌ-ｄｅｒｉｖｅｄ ｆａｃｔｏｒ－１）の発現促進剤。
（２）トリュフの抽出物、及び蒸気加熱処理後、加熱乾燥処理されたオタネニンジンの抽出物から選ばれる１種又は２種の抽出物を有効成分として含有することを特徴とする、色素幹細胞の分化抑制剤。
（３）前記オタネニンジンが、乾燥又は生のオタネニンジンである、（１）又は（２）に記載の剤。
That is, the present invention includes the following inventions.
(1) CXCL12 (stromal cell) containing one or two kinds of extracts selected from the extract of truffle and the extract of Panax ginseng heat-dried after steam heat treatment as an active ingredient. -An expression promoter of dried factor-1).
(2) Differentiation of pigment stem cells, which comprises one or two kinds of extracts selected from the extract of truffle and the extract of Panax ginseng heat-dried after steam heat treatment as an active ingredient. Inhibitor.
(3) The agent according to (1) or (2), wherein the ginseng is dried or raw ginseng.
本発明のＣＸＣＬ１２発現促進剤は、色素幹細胞の分化抑制に関与するＣＸＣＬ１２の発現を有意に増加させることができるので、白髪の根本的な予防及び／又は改善に有効である。 Since the CXCL12 expression promoter of the present invention can significantly increase the expression of CXCL12 involved in the suppression of differentiation of pigment stem cells, it is effective for the fundamental prevention and / or improvement of white hair.
本発明のＣＸＣＬ１２発現促進剤は、トリュフの抽出物、及び蒸気加熱処理後、加熱乾燥処理されたオタネニンジンの抽出物から選ばれる１種又は２種の抽出物を有効成分として含有することを特徴とする。本発明における「ＣＸＣＬ１２発現促進」とは、生体レベルまたは培養レベルでＣＸＣＬ１２の発現を促進することをいう。 The CXCL12 expression promoter of the present invention is characterized by containing one or two kinds of extracts selected from the extract of truffle and the extract of Panax ginseng heat-dried after steam heat treatment as an active ingredient. do. "Promotion of CXCL12 expression" in the present invention means promoting the expression of CXCL12 at the biological level or the culture level.
ＣＸＣＬ１２（別名ｓｔｒｏｍａｌ ｃｅｌｌ－ｄｅｒｉｖｅｄ ｆａｃｔｏｒ－１）は、Ｇタンパク質共役受容体（ＧＰＣＲ）であるケモカイン受容体ＣＸＣＲ４，７のリガンドである。前述のように、ＣＸＣＬ１２は、色素幹細胞のニッチ（幹細胞の生態的適所）である毛包内バルジ領域の毛包幹細胞において特異的に発現し、毛包幹細胞により産生されたＣＸＣＬ１２は毛包内バルジ領域における色素幹細胞の分化を抑制するとともに、メラノサイトの分化関連遺伝子の発現を抑制する作用を有することが知られている（特開２０１８－０２７９２０号公報）。後述の実施例に示されるように、トリュフの抽出物、蒸気加熱処理後、加熱乾燥処理されたオタネニンジン（以下、「修治オタネニンジン」と記載する場合がある）の抽出物、及びこれらの混合物は、ＣＸＣＬ１２発現促進効果を有する。よって、トリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物は、ＣＸＣＬ１２の発現を促進することで、色素幹細胞のニッチである毛包内バルジ領域の色素幹細胞の分化抑制が可能となるので、色素幹細胞の分化抑制剤の有効成分とすることができる。本発明において「色素幹細胞の分化抑制」とは、本来未分化な状態で維持される毛包内バルジ領域（ニッチ）における色素幹細胞の異所性分化の抑制、単離された色素幹細胞の生体外での分化の抑制、及び生体レベル又は培養レベルでの未分化性の維持を意味する。よって、本発明の色素幹細胞の分化抑制剤は、色素幹細胞の未分化状態維持剤、機能維持剤ともいうことができる。 CXCL12 (also known as stromal cell-developed factor-1) is a ligand for the chemokine receptor CXCR4,7, which is a G protein-coupled receptor (GPCR). As described above, CXCL12 is specifically expressed in hair follicle stem cells in the bulge region within hair follicles, which is a niche of pigment stem cells (the ecological location of stem cells), and CXCL12 produced by hair follicle stem cells is bulge in hair follicles. It is known to have an action of suppressing the differentiation of pigment stem cells in the region and the expression of a gene related to the differentiation of melanosite (Japanese Patent Laid-Open No. 2018-0279220). As shown in Examples described later, an extract of truffles, an extract of Panax ginseng that has been heat-dried after steam heat treatment (hereinafter, may be referred to as “Shuji Panax ginseng”), and a mixture thereof are used. It has the effect of promoting the expression of CXCL12. Therefore, one or two extracts selected from the truffle extract and the Shuji Otaneninjin extract promote the expression of CXCL12 to differentiate the pigment stem cells in the bulge region in the hair follicle, which is a niche of pigment stem cells. Since it can be suppressed, it can be used as an active ingredient of an agent for suppressing differentiation of pigment stem cells. In the present invention, "inhibition of differentiation of pigmented stem cells" means suppression of ectopic differentiation of pigmented stem cells in the bulge region (nitch) in hair follicles, which is originally maintained in an undifferentiated state, and in vitro isolated pigmented stem cells. It means the suppression of differentiation in the body and the maintenance of undifferentiated state at the biological level or the culture level. Therefore, the agent for suppressing the differentiation of pigmented stem cells of the present invention can also be said to be an agent for maintaining the undifferentiated state of the pigmented stem cells and an agent for maintaining the function.
また、ＣＸＣＬ１２は、毛包内バルジ領域における色素幹細胞の異所性分化の抑制により、メラノサイトの供給源となる色素幹細胞の減少又は枯渇を阻止できるので、トリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物は、白髪の予防及び／又は改善剤の有効成分としても利用できる。本発明において、「白髪の予防及び／又は改善」には、白髪発生の阻止、白髪の程度（本数や範囲）の改善、白髪化の進行速度の低下、白髪から黒髪への変化、白髪に伴う脱毛の抑制、白髪に伴う毛髪の光沢や弾性の減少の抑制などが含まれる。また、白髪の予防及び／又は改善効果は、頭髪に直接な作用機序を示す場合と頭部における経皮的な作用機序を示す場合の両方を含む。 In addition, CXCL12 can prevent the decrease or depletion of pigment stem cells, which are the source of melanocytes, by suppressing the ectopic differentiation of pigment stem cells in the bulge region in the hair follicle. The selected one or two extracts can also be used as an active ingredient of a preventive and / or ameliorating agent for white hair. In the present invention, "prevention and / or improvement of gray hair" includes prevention of the occurrence of gray hair, improvement of the degree (number and range) of gray hair, decrease in the rate of progression of gray hair, change from gray hair to black hair, and accompanying gray hair. This includes suppressing hair loss and suppressing the decrease in hair gloss and elasticity associated with white hair. In addition, the preventive and / or ameliorating effect of white hair includes both the case of showing a direct mechanism of action on the hair and the case of showing a percutaneous mechanism of action on the head.
本発明に用いるトリュフ（学名：Tuber spp.)は、セイヨウショウロ目（Tuberales)、セイヨウショウロ科（Tuberaceae)に属する子嚢菌であり、塊状で地中に発生し、子実層は外に開いていない。子実体の多くは強い香りを持ち、リスやウサギのような動物が掘り出して食用とする。トリュフは、世界３大珍味のひとつであり高級フランス料理に使用される。本発明に用いるトリュフの種類としては、白トリュフ（Tuber magnatum Pico）、黒トリュフ（Tuber melanosporum Vitt）が好ましい。 The truffle (scientific name: Tuber spp.) Used in the present invention is an ascomycete belonging to the order Tuberaceae and Tuberaceae, which occurs in the ground in the form of a mass and the grain layer is open to the outside. do not have. Many fruiting bodies have a strong scent and are excavated by animals such as squirrels and rabbits for food. Truffles are one of the world's three major delicacies and are used in fine French cuisine. As the type of truffle used in the present invention, white truffle (Tuber magnatum Pico) and black truffle (Tuber melanosporum Vitt) are preferable.
本発明において、トリュフの抽出物は、子実体または菌糸体の抽出物をいうが、子実体の抽出物が好ましい。また、抽出には、子実体または菌糸体をそのまま使用してもよく、乾燥、粉砕、細切等の処理を行ってもよい。 In the present invention, the extract of truffle refers to the extract of fruiting body or mycelium, but the extract of fruiting body is preferable. Further, for extraction, fruiting bodies or mycelium may be used as they are, or may be subjected to treatments such as drying, crushing, and shredding.
抽出方法は、特に限定されないが、水もしくは熱水、または水と有機溶媒の混合溶媒を用い、攪拌またはカラム抽出する方法により行うことができる。有機溶媒としては、低級アルコール類（メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール等）、液状多価アルコール類（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）、ケトン類（アセトン、メチルエチルケトン等）、アセトニトリル、エステル類（酢酸エチル、酢酸ブチル等）、炭化水素類（ヘキサン、ヘプタン、流動パラフィン等）、エーテル類（エチルエーテル、テトラヒドロフラン、プロピルエーテル等）が挙げられる。なかでも、低級アルコール、液状多価アルコール等の極性溶媒が好ましく、エタノール、１，３－ブチレングリコール、プロピレングリコール等の水溶性有機溶媒がより好ましく、これらの一種又は二種以上を用いてもよい。特に好ましい抽出溶媒としては、水、または水－エタノール系の混合極性溶媒が挙げられる。溶媒の使用量については、特に限定はなく、例えば上記トリュフ（乾燥重量）に対し、１０倍以上、好ましくは２０倍以上であればよいが、抽出後に濃縮を行なったり、単離したりする場合の操作の便宜上１００倍以下であることが好ましい。また、抽出温度や時間は、用いる溶媒の種類によるが、例えば、１０～１００℃、好ましくは３０～９０℃で、３０分～２４時間、好ましくは１～１０時間を例示することができる。また、抽出物は、抽出した溶液のまま用いてもよいが、必要に応じて、その効果に影響のない範囲で、濃縮（有機溶媒、減圧濃縮、膜濃縮などによる濃縮）、希釈、濾過、活性炭等による脱色、脱臭、エタノール沈殿等の処理を行ってから用いてもよい。さらには、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理を行い、乾燥物として用いてもよい。 The extraction method is not particularly limited, but can be carried out by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the organic solvent include lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) and liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.). , Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Be done. Among them, polar solvents such as lower alcohols and liquid polyhydric alcohols are preferable, water-soluble organic solvents such as ethanol, 1,3-butylene glycol and propylene glycol are more preferable, and one or more of these may be used. .. Particularly preferred extraction solvents include water or water-ethanol mixed polar solvents. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more the amount of the above truffle (dry weight), but when it is concentrated or isolated after extraction. For convenience of operation, it is preferably 100 times or less. The extraction temperature and time may be, for example, 10 to 100 ° C., preferably 30 to 90 ° C., 30 minutes to 24 hours, preferably 1 to 10 hours, depending on the type of solvent used. The extract may be used as it is in the extracted solution, but if necessary, concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, etc., is performed within a range that does not affect the effect. It may be used after being subjected to treatments such as decolorization, deodorization, and ethanol precipitation with activated carbon or the like. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze-drying and the like, and used as a dried product.
本発明において用いるオタネニンジン（学名：Panax ginseng C.A.Mey、別名：高麗人参、朝鮮人参、薬用人参）は、ウコギ科(Araliaceae)トチバニンジン属（Panax）に属する多年草で、生薬の「ニンジン」（和名：人参、学名：Ginseng Radix)の基原植物である。オタネニンジンの根を乾燥させたものが生薬として用いられているが、その製造方法の違いから、根の皮を剥ぎ、乾燥させた「白参」と、皮を付けたまま蒸して乾燥させた「紅参」に大別される。 Panax ginseng (scientific name: Panax ginseng C.A.Mey, also known as Korean ginseng, ginseng, medicated ginseng) is a perennial herb belonging to the ginseng family (Araliaceae) and ginseng (Panax). Ginseng, scientific name: Ginseng Radix) is the original plant. Dried roots of Panax ginseng are used as crude drugs, but due to the difference in the manufacturing method, the roots are peeled and dried "white ginseng" and steamed and dried with the skin attached. It is roughly divided into "red ginseng".
本発明において、オタネニンジンは、その葉、茎、果実、果皮、花、花芽、種子、全草、根、根茎等の植物体の一部又は植物体全体、それらの混合物のいずれも用いることができるが、根(特に、側根）、葉、茎が好ましい。 In the present invention, Otaneninjin can be used as a part of a plant such as leaves, stems, fruits, peels, flowers, flower buds, seeds, whole plants, roots, rhizomes, or the whole plant, or a mixture thereof. However, roots (particularly lateral roots), leaves and stems are preferred.
本発明においてオタネニンジンは、乾燥オタネニンジン又は生のオタネニンジンのいずれも用いることができるが、乾燥オタネニンジンが好ましい。乾燥オタネニンジンの場合、水分含量が２０％以下、好ましくは１０％以下となるまで乾燥させたものが好ましい。水分含量は、日本薬局方の乾燥減量などの方法を用いて測定することができる。乾燥方法としては、植物体の乾燥方法として通常用いられ、水分含量が上記の範囲となる方法であれば特に限定はされないが、例えば、自然乾燥（風乾）、天日乾燥、通風乾燥、熱風乾燥、噴霧乾燥、減圧乾燥、真空乾燥等が挙げられる。 As the Panax ginseng, either dried Panax ginseng or raw Panax ginseng can be used in the present invention, but dried Panax ginseng is preferable. In the case of dried ginseng, it is preferable that the ginseng is dried until the water content is 20% or less, preferably 10% or less. The water content can be measured using a method such as drying weight loss according to the Japanese Pharmacopoeia. The drying method is usually used as a method for drying a plant, and is not particularly limited as long as the water content is within the above range. For example, natural drying (air drying), sun drying, ventilation drying, hot air drying, etc. , Spray drying, vacuum drying, vacuum drying and the like.
本発明において、上記の乾燥オタネニンジン又は生のオタネニンジンを抽出する前に、生薬の加工の際に行われる修治処理（蒸して乾かすという加工処理）に相当する蒸気加熱処理と加熱乾燥処理を行う。蒸気加熱処理は、熱水、飽和水蒸気、過熱蒸気、減圧（真空）蒸気等を熱媒体とし、高湿度雰囲気下、例えば、湿度８０％以上の雰囲気下で対象物を加熱する処理をいう。蒸気加熱は熱媒体を対象物に直接接触させることによって行ってもよく、または対象物を、熱交換機を通じて間接的に加熱してもよい。また加熱は常圧下および加圧下のいずれで行ってもよい。蒸気加熱処理の条件としては、温度は、７０～１８０℃が好ましく、１００～１５０℃がより好ましく、１００～１２０℃がさらに好ましい。時間は、温度によって異なるが、１～１５時間が好ましく、２～１０時間がより好ましく、４～８時間がさらに好ましい。これらの温度及び時間の条件はあくまで例示であり、温度及び時間の相互の関係で適宜変更できる。また、本発明における蒸気加熱処理は、連続式またはバッチ式のスチーマー（蒸し機）やオートクレーブなどを用いて行えばよい。 In the present invention, before extracting the above-mentioned dried ginseng or raw ginseng, a steam heat treatment and a heat-drying treatment corresponding to a repair treatment (a processing treatment of steaming and drying) performed at the time of processing a crude drug are performed. The steam heat treatment refers to a treatment in which hot water, saturated steam, superheated steam, reduced pressure (vacuum) steam, or the like is used as a heat medium to heat an object in a high humidity atmosphere, for example, an atmosphere having a humidity of 80% or more. Steam heating may be performed by bringing the heat medium into direct contact with the object, or the object may be indirectly heated through a heat exchanger. Further, heating may be performed under normal pressure or pressure. As the conditions for the steam heat treatment, the temperature is preferably 70 to 180 ° C, more preferably 100 to 150 ° C, still more preferably 100 to 120 ° C. The time varies depending on the temperature, but is preferably 1 to 15 hours, more preferably 2 to 10 hours, still more preferably 4 to 8 hours. These temperature and time conditions are merely examples, and can be appropriately changed depending on the mutual relationship between temperature and time. Further, the steam heat treatment in the present invention may be carried out by using a continuous or batch type steamer (steamer), an autoclave, or the like.
蒸気加熱処理されたオタネニンジンは、水分を含んでいるため、加熱乾燥処理を行なう。加熱乾燥の温度としては、４０～８５℃が好ましく、５０～７０℃がより好ましい。乾燥方法としては、通風乾燥、熱風乾燥、マイクロ波乾燥等を用いることができる。乾燥時間（加熱時間）は、加熱温度、蒸気加熱処理後のオタネニンジンの水分含量、乾燥する総量によって異なり、特定はできないが、約６～２４時間の範囲である。 Since the steam-heat-treated Panax ginseng contains water, it is heat-dried. The temperature for heating and drying is preferably 40 to 85 ° C, more preferably 50 to 70 ° C. As the drying method, ventilation drying, hot air drying, microwave drying and the like can be used. The drying time (heating time) varies depending on the heating temperature, the water content of Panax ginseng after the steam heat treatment, and the total amount of drying, and is not specified, but is in the range of about 6 to 24 hours.
上記のようにして蒸気加熱処理後、加熱乾燥処理を施したオタネニンジン（修治オタネニンジン）を抽出する。抽出方法は、特に限定されないが、水もしくは熱水、または水と有機溶媒の混合溶媒を用い、攪拌またはカラム抽出する方法により行うことができる。有機溶媒としては、低級アルコール類（メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール等）、液状多価アルコール類（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）、ケトン類（アセトン、メチルエチルケトン等）、アセトニトリル、エステル類（酢酸エチル、酢酸ブチル等）、炭化水素類（ヘキサン、ヘプタン、流動パラフィン等）、エーテル類（エチルエーテル、テトラヒドロフラン、プロピルエーテル等）が挙げられる。なかでも、低級アルコール、液状多価アルコール等の極性溶媒が好ましく、エタノール、１，３－ブチレングリコール、プロピレングリコール等の水溶性有機溶媒がより好ましく、これらの一種又は二種以上を用いてもよい。特に好ましい抽出溶媒としては、水、または水－エタノール系の混合極性溶媒、１，３－ブチレングリコールが挙げられる。溶媒の使用量については、特に限定はなく、例えば上記オタネニンジン（乾燥重量）に対し、１０倍以上、好ましくは２０倍以上であればよいが、抽出後に濃縮を行なったり、単離したりする場合の操作の便宜上１００倍以下であることが好ましい。また、抽出温度や時間は、用いる溶媒の種類によるが、例えば、１０～１００℃、好ましくは３０～９０℃で、３０分～２４時間、好ましくは１～１０時間を例示することができる。また、抽出物は、抽出した溶液のまま用いてもよいが、必要に応じて、その効果に影響のない範囲で、濃縮（有機溶媒、減圧濃縮、膜濃縮などによる濃縮）、希釈、濾過、活性炭等による脱色、脱臭、エタノール沈殿等の処理を行ってから用いてもよい。さらには、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理を行い、乾燥物として用いてもよい。 After the steam heat treatment as described above, the ginseng (Ginseng Shuji) that has been heat-dried is extracted. The extraction method is not particularly limited, but can be carried out by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the organic solvent include lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) and liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.). , Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Be done. Among them, polar solvents such as lower alcohols and liquid polyhydric alcohols are preferable, water-soluble organic solvents such as ethanol, 1,3-butylene glycol and propylene glycol are more preferable, and one or more of these may be used. .. Particularly preferable extraction solvents include water, a water-ethanol mixed polar solvent, and 1,3-butylene glycol. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more the amount of the ginseng (dry weight), but when it is concentrated or isolated after extraction. For convenience of operation, it is preferably 100 times or less. The extraction temperature and time may be, for example, 10 to 100 ° C., preferably 30 to 90 ° C., 30 minutes to 24 hours, preferably 1 to 10 hours, depending on the type of solvent used. The extract may be used as it is in the extracted solution, but if necessary, concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, etc., is performed within a range that does not affect the effect. It may be used after being subjected to treatments such as decolorization, deodorization, and ethanol precipitation with activated carbon or the like. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze-drying and the like, and used as a dried product.
本発明において、トリュフの抽出物及び修治オタネニンジンの抽出物は、いずれか１種を用いてもよいが、両者を併用するとＣＸＣＬ１２発現促進効果が増強するので好ましい。トリュフの抽出物と修治オタネニンジンの抽出物を併用する場合、混合比率は限定されないが、好ましくは１：１０～１０：１であり、より好ましくは１：５～５：１であり、さらに好ましくは１：２～２：１であり、最も好ましくは１：１である。 In the present invention, either one of the truffle extract and the rehabilitated ginseng extract may be used, but it is preferable to use both of them in combination because the CXCL12 expression promoting effect is enhanced. When the extract of truffle and the extract of Panax ginseng are used in combination, the mixing ratio is not limited, but is preferably 1:10 to 10: 1, more preferably 1: 5 to 5: 1, and even more preferably. It is 1: 2 to 2: 1, most preferably 1: 1.
トリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物を、白髪の予防及び／又は改善剤として用いる場合は、そのまま使用することも可能であるが、本発明の効果を損なわない範囲で適当な添加物等と混合し、白髪の予防及び／又は改善用の化粧品、医薬部外品、医薬品などの組成物の形態とすることができる。なかでも、頭皮や毛髪に使用するのに適した製剤形態に製剤化した毛髪用組成物が好ましい。 When one or two kinds of extracts selected from the extract of truffle and the extract of Panax ginseng are used as a preventive and / or ameliorating agent for white hair, they can be used as they are, but the effect of the present invention. It can be mixed with an appropriate additive or the like as long as it does not impair the above, and can be in the form of a composition such as cosmetics, quasi-drugs, and pharmaceuticals for preventing and / or improving gray hair. Among them, a hair composition formulated in a formulation form suitable for use on the scalp and hair is preferable.
毛髪用組成物は、皮膚外用組成物において通常使用されている各種の成分、添加剤、基剤等をその種類に応じて選択し、適宜配合し、当分野で公知の手法に従って製造することができる。配合する成分、添加剤、基剤としては、例えば、希釈剤（精製水、エタノール等）、油脂類（オリーブ油、ヤシ油、月見草油、ホホバ油、ヒマシ油、硬化ヒマシ油等）、ロウ類（ラノリン、ミツロウ、カルナウバロウ等）、炭化水素類（流動パラフィン、スクワレン、スクワラン、ワセリン等）、脂肪酸類（ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、ベヘニン酸等）、高級アルコール類（ミリスチルアルコール、セタノール、セトステアリルアルコール、ステアリルアルコール、ベヘニルアルコール等）、エステル類（ミリスチン酸イソプロピル、パルミチン酸イソプロピル、オクタン酸セチル、トリオクタン酸グリセリン、ミリスチン酸オクチルドデシル、ステアリン酸オクチル、ステアリン酸ステアリル等）、有機酸類（クエン酸、乳酸、α-ヒドロキシ酢酸、ピロリドンカルボン酸等）、糖類（マルチトール、ソルビトール、キシロビオース、N-アセチル-D-グルコサミン等）、界面活性剤、シリコーン油、保湿剤、増粘剤、紫外線吸収剤、金属イオン封鎖剤、清涼化剤、抗酸化剤、安定化剤、防腐剤、消炎剤、殺菌剤、香料、着色料等が挙げられる。 The hair composition can be produced according to a method known in the art by selecting various components, additives, bases and the like usually used in the external composition for skin according to the type and appropriately blending them. can. Ingredients, additives, and bases to be blended include, for example, diluents (refined water, ethanol, etc.), fats and oils (olive oil, palm oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes ( Lanolin, honeydew, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalane, squalane, vaseline, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearyl acid, behenic acid, etc.), higher alcohols (myristyl alcohol, etc.) Setanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate, cetyl octanate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids ( Citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (martitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), surfactants, silicone oils, moisturizers, thickeners, ultraviolet rays Examples thereof include absorbents, metal ion sequestering agents, cooling agents, antioxidants, stabilizers, preservatives, anti-inflammatory agents, bactericides, fragrances, coloring agents and the like.
また、上記毛髪用組成物には、本発明の効果に悪影響を及ぼさない限り、育毛・養毛に効果的な成分として従来より知られている成分を含めてもよい。例えば、センブリエキス、柑橘類エキス等の植物抽出エキス、ビタミンＢ６、ビタミンＥ及びその誘導体、ビオチン等のビタミン類、パントテン酸及びその誘導体、グリチルリチン酸及びその誘導体、ニコチン酸エステル、セリン、メチオニン等のアミノ酸類、セフォランチン、塩化カルプロニウム、ミノキシジル、ニコランジル、アセチルコリン誘導体、サイクロスポリン類、及びエストラジオール等の女性ホルモン剤等、ならびにこれらの混合物が挙げられる。 In addition, the above-mentioned hair composition may contain components conventionally known as effective components for hair growth and hair growth, as long as the effects of the present invention are not adversely affected. For example, plant extract such as assembly extract and citrus extract, vitamin B 6 , vitamin E and its derivative, vitamins such as biotin, pantothenic acid and its derivative, glycyrrhizinic acid and its derivative, nicotinic acid ester, serine, methionine and the like. Examples thereof include amino acids, cephoranthin, carpronium chloride, minoxidyl, nicolangyl, acetylcholine derivatives, cyclosporins, female hormones such as estradiol, and mixtures thereof.
本発明において、毛髪用組成物は、頭皮や毛髪に使用するものを広く指し、頭皮や毛髪に適用可能なものであればいずれでもよく、剤型は特に問わない。例えば、液状、乳液状、クリーム状、ゲル状、ペースト状、スプレー状等のいずれであってもよい。具体的な製品形態としては、クリーム、ローション、乳剤、軟膏、ゲル、ヘアシャンプー、ヘアリンス、ヘアトリートメント、ヘアコンディショナー、スカルプトリートメント、ヘアスプレー、ヘアパック、ヘアエッセンス、ヘアトニック、ヘアリキッド、ヘアムースなどが挙げられる。 In the present invention, the composition for hair broadly refers to those used for scalp and hair, and may be any composition applicable to scalp and hair, and the dosage form is not particularly limited. For example, it may be liquid, milky, creamy, gelled, pasty, sprayed or the like. Specific product forms include creams, lotions, emulsions, ointments, gels, hair shampoos, hair rinses, hair treatments, hair conditioners, scalp treatments, hair sprays, hair packs, hair essences, hair tonics, hair liquids, hair mousses, etc. Can be mentioned.
これらの製品形態をとる毛髪用組成物中のトリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物の含有量は、形態に応じて異なるので特定することはできないが、一般に、当該組成物の総重量に対し、０．０００１～２０重量％（ｗ／ｗ）、好ましくは０．００１～１０重量％（ｗ／ｗ）である。トリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物の添加の方法については、予め加えておいても、製造途中で添加しても良く、作業性を考えて適宜選択すれば良い。 The content of one or two extracts selected from the extract of truffle and the extract of Panax ginseng in the hair composition taking these product forms varies depending on the form and cannot be specified. Generally, it is 0.0001 to 20% by weight (w / w), preferably 0.001 to 10% by weight (w / w), based on the total weight of the composition. Regarding the method of adding one or two kinds of extracts selected from the extract of truffle and the extract of Panax ginseng, it may be added in advance or added in the middle of production, as appropriate in consideration of workability. Just choose.
トリュフの抽出物及び修治オタネニンジンの抽出物から選ばれる１種又は２種の抽出物はまた、色素幹細胞を再生医療に使用するために培養する際に、当該細胞の未分化状態を維持するための培地添加剤としても使用することができる。当該培地添加剤を添加した培地で培養した色素幹細胞は、未分化性を保持しつつ、一定期間生存可能である。細胞の未分化状態維持期間は、培養目的、基礎培地の種類、培養温度などに応じて、適宜変更することができる。 One or two extracts selected from the extract of truffle and the extract of Panax ginseng are also used to maintain the undifferentiated state of the pigment stem cells when they are cultured for use in regenerative medicine. It can also be used as a medium additive. The pigment stem cells cultured in the medium to which the medium additive is added can survive for a certain period of time while maintaining undifferentiated state. The period for maintaining the undifferentiated state of cells can be appropriately changed depending on the purpose of culture, the type of basal medium, the culture temperature, and the like.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these.
[実施例１] トリュフ、オタネニンジンの抽出物の製造例
トリュフ、オタネニンジンの抽出物を以下のとおり製造した。
[Example 1] Example of production of extract of truffle and Panax ginseng An extract of truffle and Panax ginseng was produced as follows.
（製造例１）トリュフの熱水抽出物の調製
トリュフの粉砕物１００ｇに精製水１Ｌを加え、９０～１００℃で２時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥してトリュフの熱水抽出物を５．１ｇ得た。
(Production Example 1) Preparation of hot water extract of truffle Add 1 L of purified water to 100 g of crushed truffle, extract at 90-100 ° C for 2 hours, filter, concentrate the filtrate, freeze-dry and truffle. 5.1 g of the hot water extract of the above was obtained.
（製造例２）生オタネニンジンの根（修治処理／蒸気加熱：１０５℃、加熱乾燥：５０℃）の熱水抽出物の調製
収穫後の生のオタネニンジン（水分量８０％）を１０５℃で８時間蒸した後、５０℃で乾燥させた（日本薬局方の「紅参」に適合）。このオタネニンジンの乾燥物４０ｇに精製水８００ｍＬを加え、９５～１００℃で２時間抽出した後、濾過し、その濾液を濃縮乾固して、蒸気加熱処理（１０５℃）された生オタネニンジンの根の熱水抽出物を１７．５ｇ得た。
(Production Example 2) Preparation of hot water extract of raw ginseng root (repair treatment / steam heating: 105 ° C, heat drying: 50 ° C) Raw ginseng (water content 80%) after harvesting at 105 ° C for 8 hours After steaming, it was dried at 50 ° C (compatible with "Red Ginseng" of the Japanese Pharmacy). 800 mL of purified water was added to 40 g of the dried product of Otaneninjin, extracted at 95 to 100 ° C. for 2 hours, filtered, and the filtrate was concentrated to dryness, and the roots of raw Otaneninjin were steam-heat-treated (105 ° C.). 17.5 g of hot water extract was obtained.
（製造例３）乾燥オタネニンジンの根（修治処理／蒸気加熱：１０５℃、加熱乾燥：５０℃）の熱水抽出物の調製
乾燥したオタネニンジンの根（水分量９％）を１０５℃で８時間蒸した後、５０℃で乾燥させた。このオタネニンジンの乾燥物４０ｇに精製水８００ｍＬを加え、９５～１００℃で２時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して蒸気加熱処理（１０５℃）された乾燥オタネニンジンの根の熱水抽出物を１８．７ｇ得た。
(Production Example 3) Preparation of hot water extract of dried ginseng root (repair treatment / steam heating: 105 ° C, heat drying: 50 ° C) Dried ginseng root (water content 9%) is steamed at 105 ° C for 8 hours. After that, it was dried at 50 ° C. 800 mL of purified water is added to 40 g of the dried product of Otaneninjin, extracted at 95 to 100 ° C. for 2 hours, filtered, the filtrate is concentrated, freeze-dried and steam-heated (105 ° C.). 18.7 g of hot water extract was obtained.
（製造例４）乾燥オタネニンジンの根（修治処理／蒸気加熱：１０５℃、加熱乾燥：７０℃）の５０％エタノール抽出物の調製
乾燥したオタネニンジンの根（水分量９％）を１０５℃で８時間蒸した後、７０℃で乾燥させた。このオタネニンジンの乾燥物１００ｇに精製水５００ｍＬ及びエタノール５００ｍＬを加え、常温で７日間抽出した後、濾過し、その濾液を濃縮乾固して、蒸気加熱処理（１０５℃）された乾燥オタネニンジンの根の５０％エタノール抽出物を４３ｇ得た。
(Production Example 4) Preparation of 50% ethanol extract of dried ginseng root (repair treatment / steam heating: 105 ° C, heat drying: 70 ° C) Dried ginseng root (water content 9%) at 105 ° C for 8 hours. After steaming, it was dried at 70 ° C. 500 mL of purified water and 500 mL of ethanol were added to 100 g of this dried ginseng, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness, and the roots of dried ginseng were steam-heat-treated (105 ° C). 43 g of 50% ethanol extract was obtained.
（製造例５）乾燥オタネニンジンの葉及び茎（修治処理／蒸気加熱：１００℃、加熱乾燥：６０℃）の１，３－ブチレングリコール抽出物の調製
乾燥したオタネニンジンの葉及び茎（水分量１０％）を１００℃で４時間蒸した後、６０℃で乾燥させた。このオタネニンジンの乾燥物２０ｇに１，３－ブチレングリコール２００ｍＬを加え、常温で７日間抽出した後、濾過し、蒸気加熱処理（１００℃）された乾燥オタネニンジンの根及び茎の１，３－ブチレングリコール抽出物を１４１ｇ得た。
(Production Example 5) Preparation of 1,3-butylene glycol extract of dried ginseng leaves and stems (repair treatment / steam heating: 100 ° C., heat drying: 60 ° C.) Dried ginseng leaves and stems (water content 10%) ) Was steamed at 100 ° C. for 4 hours and then dried at 60 ° C. 200 mL of 1,3-butylene glycol was added to 20 g of this dried ginseng product, extracted at room temperature for 7 days, filtered, and steam-heat-treated (100 ° C.) for dried ginseng root and stem 1,3-butylene glycol. 141 g of extract was obtained.
（比較製造例１）生オタネニンジンの根（未修治／蒸気加熱処理なし）の熱水抽出物の調製
収穫後の生のオタネニンジンの根を６５℃で乾燥させたもの（白参）４０ｇに精製水８００ｍＬを加え、９５～１００℃で２時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して生オタネニンジンの根の熱水抽出物を１２．５ｇ得た。
(Comparative Production Example 1) Preparation of Hot Water Extract of Raw Otaneninjin Root (Unrepaired / No Steam Heat Treatment) Purified water to 40 g of raw Otaneninjin Root after harvest dried at 65 ° C (white ginseng). 800 mL was added, and the mixture was extracted at 95 to 100 ° C. for 2 hours, filtered, and the filtrate was concentrated and lyophilized to obtain 12.5 g of a hot water extract of raw Otaneninjin root.
[実施例２] トリュフ、オタネニンジンＣＸＣＬ１２発現促進効果
実施例１の各製造剤で製造したトリュフの抽出物、オタネニンジンの各抽出物が、色素幹細胞のニッチを構成し、維持を担う、毛包幹細胞のＣＸＣＬ１２にどのような影響を与えるかを解析するために、毛包幹細胞にこれらの抽出物を作用させ、その際のＣＸＣＬ１２の遺伝子発現量について解析を行った。
[Example 2] Truffle, Otaneninjin CXCL12 expression promoting effect A hair follicle stem cell in which each of the truffle extract and Otaneninjin extract produced by each of the manufacturing agents of Example 1 constitutes and maintains a niche of pigment stem cells. In order to analyze the effect on CXCL12, these extracts were allowed to act on hair follicle stem cells, and the gene expression level of CXCL12 at that time was analyzed.
（１）ヒト毛包幹細胞の培養
ヒトの毛髪を毛抜きで採取し、メス等を用いて毛包組織のバルジ領域を含む組織を回収した。ＰＢＳ（－）にて洗浄した後、トリプシン(ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製)処理を行った。その後、セルストレイナー(ＦＡＬＣＯＮ社製)を用いて、細胞を単離し、回収した。回収した細胞を培養プレートに播種し、ＫＧ２培地(ＫＵＲＡＢＯ社製)を用いてコンフルエントになるまで維持した。コンフルエントになった細胞を回収し、培養プレートに再び播種し、その後生着し、増殖している細胞を毛包幹細胞として以下の試験に用いた。
(1) Cultivation of human hair follicle stem cells Human hair was collected by tweezers, and the tissue containing the bulge region of the hair follicle tissue was collected using a scalpel or the like. After washing with PBS (-), it was treated with trypsin (manufactured by BD Biosciences). Then, cells were isolated and recovered using a cell strainer (manufactured by FALCON). The collected cells were seeded on a culture plate and maintained using KG2 medium (manufactured by KURABO) until they became confluent. The cells that became confluent were collected, seeded again on the culture plate, then engrafted, and the proliferating cells were used as hair follicle stem cells in the following tests.
（２）毛包幹細胞におけるＣＸＣＬ１２発現促進効果
毛包幹細胞に対して上記製造例で得られた各抽出物を最終濃度が１００μｇ／ｍＬとなるように添加した。ただし、トリュフの抽出物とオタネニンジンの抽出物の混合抽出物については２種の抽出物を５０μｇ／ｍＬずつ添加し、混合抽出物の最終濃度が１００μｇ／ｍＬとなるようにした。抽出物を添加して、４８時間後の細胞を回収し、毛包幹細胞におけるＣＸＣＬ１２の遺伝子発現量を解析した。
(2) Effect of promoting CXCL12 expression on hair follicle stem cells Each extract obtained in the above production example was added to hair follicle stem cells so that the final concentration was 100 μg / mL. However, for the mixed extract of the truffle extract and the ginseng extract, two kinds of extracts were added at 50 μg / mL each so that the final concentration of the mixed extract was 100 μg / mL. The extract was added, and the cells were collected 48 hours later, and the gene expression level of CXCL12 in hair follicle stem cells was analyzed.
ＣＸＣＬ１２遺伝子発現解析は次の通り行った。抽出物添加後の毛包幹細胞をＰＢＳ（－）にて２回洗浄した後、Trizol Reagent(Invitrogen社製)によって細胞からＲＮＡを抽出した。２－ＳＴＥＰリアルタイムＰＣＲキット(Applied Biosystems社製)を用いて、抽出したＲＮＡをｃＤＮＡに逆転写した後、ＡＢＩ７３００(Applied Biosystems社製)により、下記プライマーセットを用いてリアルタイムＰＣＲ（９５℃：１５秒間、６０℃：３０秒間、４０ｃｙｃｌｅｓ）を実施し、ＣＸＣＬ１２の発現を確認した。その他の操作は定められた方法に従って実施した。 The CXCL12 gene expression analysis was performed as follows. Hair follicle stem cells after addition of the extract were washed twice with PBS (-), and then RNA was extracted from the cells by Trizol Reagent (manufactured by Invitrogen). After reverse transcribing the extracted RNA to cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95 ° C: 15 seconds) using the following primer set using ABI7300 (manufactured by Applied Biosystems). , 60 ° C.: 30 seconds, 40 cycles), and the expression of CXCL12 was confirmed. Other operations were performed according to the prescribed method.
ＣＸＣＬ１２用プライマーセット：
５’－ＣＡＴＧＣＣＧＡＴＴＣＴＴＣＧＡＡＡＧＣ－３’（配列番号１）
５’－ＣＧＡＧＴＧＧＧＴＣＴＡＧＣＧＧＡＡＡＧ－３’（配列番号２）
１８ｓｒＲＮＡ（内部標準）用プライマーセット：
５’－ＣＣＧＡＧＣＣＧＣＣＴＧＧＡＴＡＣ－３’（配列番号３）
５’－ＣＡＧＴＴＣＣＧＡＡＡＡＣＣＡＡＣＡＡＡＡＴＡＧＡ－３’(配列番号４）
Primer set for CXCL12:
5'-CATGCCGATTCTTCGAAAGC-3'(SEQ ID NO: 1)
5'-CGAGTGGGTCTAGCGGAAAG-3'(SEQ ID NO: 2)
Primer set for 18srRNA (internal standard):
5'-CCGAGCCGCCTGGATAC-3'(SEQ ID NO: 3)
5'-CAGTTCGAAAACCAAAAAATAGA-3'(SEQ ID NO: 4)
ＣＸＣＬ１２の発現促進効果については、抽出物を添加せずに培養した毛包幹細胞（コントロール）におけるＣＸＣＬ１２のｍＲＮＡの発現量を内部標準である１８ｓ ｒｉｂｏｓｏｍａｌ ＲＮＡ（１８ｓｒＲＮＡ）の発現量に対する割合として算出したＣＸＣＬ１２遺伝子相対発現量（ＣＸＣＬ１２遺伝子発現量／１８ｓｒＲＮＡ遺伝子発現量）の値を１とし、これに対し、抽出物を添加して培養した毛包幹細胞におけるＣＸＣＬ１２の遺伝子相対発現量の値を算出し、評価した。その結果を下記の表１に示す。 Regarding the expression-promoting effect of CXCL12, the expression level of CXCL12 mRNA in hair follicles stem cells (controls) cultured without adding an extract was calculated as the ratio of the expression level of 18s ribosomal RNA (18srRNA), which is an internal standard, to the expression level of CXCL12. The value of the gene relative expression level (CXCL12 gene expression level / 18srRNA gene expression level) was set to 1, and the value of the gene relative expression level of CXCL12 in the hair follicle stem cells cultured by adding the extract was calculated and evaluated. did. The results are shown in Table 1 below.
表１に示すように、トリュフの抽出物（製造例１）、蒸気加熱処理後、加熱乾燥処理を行った(修治)オタネニンジンの抽出物（製造例２～５）を添加した培地で培養した毛包幹細胞では、未修治オタネニンジン（白参）の抽出物（比較製造例１）に比べ、ＣＸＣＬ１２遺伝子の発現量が亢進し、ＣＸＣＬ１２遺伝子の発現促進効果を有することが示された。また、蒸気加熱処理前に乾燥させたオタネニンジンの抽出物（製造例３～５）は、生のオタネニンジン（紅参）の抽出物（製造例２）よりも当該効果が高かった。さらに、トリュフの抽出物（製造例１）とオタネニンジンの抽出物（製造例２～５）を併用すると、格別に効果が高まることが確認できた。よって、トリュフの抽出物、修治オタネニンジンの抽出物、及びこれらの混合物は、ＣＸＣＬ１２遺伝子の発現促進作用に基づき、毛包内バルジ領域における色素幹細胞の異所性分化を抑制して未分化な状態を維持するとともに、メラノサイトの分化関連遺伝子の発現を抑制することができるので、白髪化を根本的に抑制及び阻止できるものといえる。 As shown in Table 1, hair cultured in a medium supplemented with an extract of truffle (Production Example 1) and an extract of Otaneninjin (Production Examples 2 to 5) that had been heat-dried after steam heat treatment (Shuji). It was shown that the expression level of the CXCL12 gene was enhanced in the envelope stem cells as compared with the unrepaired Otaneninjin (white ginseng) extract (Comparative Production Example 1), and the expression promoting effect of the CXCL12 gene was exhibited. In addition, the ginseng extract (Production Examples 3 to 5) dried before the steam heat treatment had a higher effect than the raw ginseng (red ginseng) extract (Production Example 2). Furthermore, it was confirmed that the combined use of the truffle extract (Production Example 1) and the Panax ginseng extract (Production Examples 2 to 5) significantly enhances the effect. Therefore, the truffle extract, the Shuji Otaneninjin extract, and a mixture thereof suppress the ectopic differentiation of pigment stem cells in the bulge region in the hair follicle based on the expression promoting action of the CXCL12 gene, resulting in an undifferentiated state. Since it can be maintained and the expression of melanosite differentiation-related genes can be suppressed, it can be said that the whitening of hair can be fundamentally suppressed and prevented.
本発明は、白髪の予防及び／又は改善を目的とした化粧品や医薬部外品の製造分野において利用できる。 The present invention can be used in the field of manufacturing cosmetics and quasi-drugs for the purpose of preventing and / or improving white hair.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001288098A (en) | 2000-04-10 | 2001-10-16 | Maruzen Pharmaceut Co Ltd | Melanogenesis promoter and gray hair ameliorant |
| US20040009143A1 (en) | 2000-10-13 | 2004-01-15 | Karin Golz-Berner | Cosmetic preparation containing plant extracts |
| CN101152432A (en) | 2006-09-27 | 2008-04-02 | 马骥先 | Traditional Chinese medicine for treating alopecia and white hair |
| CN104223080A (en) | 2014-09-26 | 2014-12-24 | 青岛金佳慧食品有限公司 | Health composite food for treating hair loss |
| CN104338009A (en) | 2014-10-25 | 2015-02-11 | 重庆市大足区季家镇卫生院 | Traditional Chinese medicine formula for treating leukotrichia |
| CN104435445A (en) | 2014-12-02 | 2015-03-25 | 成都锦汇科技有限公司 | Liniment for inhibiting growth of canities |
| JP2015124185A (en) | 2013-12-26 | 2015-07-06 | 日本メナード化粧品株式会社 | Sebum synthesis promoter |
| WO2017057891A1 (en) | 2015-09-30 | 2017-04-06 | 주식회사 아모레퍼시픽 | Composition for prevention of gray hair |
| JP2018027920A (en) | 2016-08-19 | 2018-02-22 | 日本メナード化粧品株式会社 | Pigment stem cell differentiation inhibitor, white hair preventing agent, and white hair evaluation method |
| CN108354874A (en) | 2018-05-23 | 2018-08-03 | 佛山云裳化妆品有限公司 | A kind of black hair composition and application |
| JP2019038790A (en) | 2017-08-28 | 2019-03-14 | 共栄化学工業株式会社 | Hair cosmetic |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6038314A (en) * | 1983-08-10 | 1985-02-27 | Osaka Chem Lab | Hair tonic |
| JPS611619A (en) * | 1984-06-14 | 1986-01-07 | Osaka Chem Lab | Hair growth and hair tonic food |
| KR20110078361A (en) * | 2009-12-31 | 2011-07-07 | (주)아모레퍼시픽 | Composition containing ginseng berry extract for preventing gray hair and for treatment of leukoplakia |
| KR102175249B1 (en) * | 2013-09-11 | 2020-11-06 | (주)아모레퍼시픽 | Composition for improving condition of hair and scalp containing Panax ginseng extract and Green tea extract |
| KR101849924B1 (en) * | 2014-04-28 | 2018-04-18 | 고인순 | Composition Having Promoting Effect on Induction of Black Hair and Inhibitory Effect on Induction of White Hair |
-
2018
- 2018-06-05 JP JP2018107531A patent/JP7100351B2/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001288098A (en) | 2000-04-10 | 2001-10-16 | Maruzen Pharmaceut Co Ltd | Melanogenesis promoter and gray hair ameliorant |
| US20040009143A1 (en) | 2000-10-13 | 2004-01-15 | Karin Golz-Berner | Cosmetic preparation containing plant extracts |
| CN101152432A (en) | 2006-09-27 | 2008-04-02 | 马骥先 | Traditional Chinese medicine for treating alopecia and white hair |
| JP2015124185A (en) | 2013-12-26 | 2015-07-06 | 日本メナード化粧品株式会社 | Sebum synthesis promoter |
| CN104223080A (en) | 2014-09-26 | 2014-12-24 | 青岛金佳慧食品有限公司 | Health composite food for treating hair loss |
| CN104338009A (en) | 2014-10-25 | 2015-02-11 | 重庆市大足区季家镇卫生院 | Traditional Chinese medicine formula for treating leukotrichia |
| CN104435445A (en) | 2014-12-02 | 2015-03-25 | 成都锦汇科技有限公司 | Liniment for inhibiting growth of canities |
| WO2017057891A1 (en) | 2015-09-30 | 2017-04-06 | 주식회사 아모레퍼시픽 | Composition for prevention of gray hair |
| JP2018027920A (en) | 2016-08-19 | 2018-02-22 | 日本メナード化粧品株式会社 | Pigment stem cell differentiation inhibitor, white hair preventing agent, and white hair evaluation method |
| JP2019038790A (en) | 2017-08-28 | 2019-03-14 | 共栄化学工業株式会社 | Hair cosmetic |
| CN108354874A (en) | 2018-05-23 | 2018-08-03 | 佛山云裳化妆品有限公司 | A kind of black hair composition and application |
Non-Patent Citations (1)
| Title |
|---|
| Stromal Cell Derived Factor-1 mRNA Expression of Peripheral Blood Stem Cells was Suppressed in a Dose Dependent Manner of Korean Red Ginseng，TRANSFUSION, "Abstract Presentations from the AABB Annual Meeting and CTTXPO, Denver, CO, October 12-15, 2013"，2013年，Vol. 53, Supplement，pp. 66A-67A，SP34 |
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