JP2009040753A - Skin repair composition and screening method thereof - Google Patents
Skin repair composition and screening method thereof Download PDFInfo
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本発明は、損傷したり老化したりした皮膚を、補修して、治癒したり若返らせたりする皮膚補修組成物、及びそれを見出すスクリーニング方法に関するものである。 The present invention relates to a skin repair composition for repairing, healing, and rejuvenating damaged or aged skin, and a screening method for detecting the same.
生体には、主に皮膚を作る線維芽細胞が存在する。この線維芽細胞を増殖させる線維芽細胞増殖因子（ＦＧＦ）という一群のタンパク質が、今までに２０数種類知られている。 In living bodies, there are fibroblasts that mainly make skin. To date, a dozen types of proteins called fibroblast growth factors (FGFs) for growing fibroblasts have been known.
そのうちの一つのタンパク質である線維芽細胞増殖因子−１は、怪我などで皮膚組織が損傷したとき線維芽細胞を増殖させ傷口を塞いで修復させたり、コラーゲンやヒアルロン酸を産生する線維芽細胞を増殖させたりして、新生細胞を産生させるものである。 One of these proteins, fibroblast growth factor-1, is a fibroblast that proliferates fibroblasts and closes and repairs wounds when skin tissue is damaged due to injury, or produces fibroblasts that produce collagen and hyaluronic acid. It is proliferated to produce new cells.
加齢や紫外線曝露によって皮膚の弾力が低下し弛みや皺が増えたり、乾燥肌となったりするのは、この線維芽細胞増殖因子の活性の低下が一因である。 The decrease in fibroblast growth factor activity is one of the reasons why skin elasticity decreases due to aging and exposure to ultraviolet rays, and sagging and wrinkles increase or dry skin.
特許文献１には、線維芽細胞増殖因子−１を含み線維芽細胞又は表皮細胞の増殖を促進する組成物が開示されている。 Patent Document 1 discloses a composition containing fibroblast growth factor-1 and promoting the growth of fibroblasts or epidermal cells.
線維芽細胞増殖因子−１は、生体に内在する稀少なタンパク質であるため簡便に工業的規模で大量生産できるものでなく、多工程を要する面倒な遺伝子工学によって僅かな量が調製されるに過ぎないものである。そのため内服薬やサプリメントのような内用剤や、化粧品のような外用剤に、線維芽細胞増殖因子−１をふんだんに用いることはできない。 Fibroblast growth factor-1 is a rare protein inherent in the living body, so it cannot be easily mass-produced on an industrial scale, and only a small amount is prepared by troublesome genetic engineering that requires multiple steps. There is nothing. Therefore, fibroblast growth factor-1 cannot be used abundantly for internal preparations such as internal medicines and supplements and external preparations such as cosmetics.
本発明は前記の課題を解決するためになされたもので、簡易な構成で、大量に製造でき、線維芽細胞増殖因子−１様の活性物質や線維芽細胞増殖因子−１の活性促進物質により、タンパク質である線維芽細胞増殖因子−１自体を用いなくとも、それの作用を発現したり増強したりでき、それによって損傷した皮膚を補修することができる皮膚補修組成物を提供することを目的とする。また、数多の候補物質の中から皮膚補修物質を効率よく峻別するためのスクリーニング方法を提供することを目的とする。 The present invention has been made to solve the above-described problems, and can be produced in a large amount with a simple configuration, and can be produced by using an active substance like fibroblast growth factor-1 or an activity promoting substance for fibroblast growth factor-1. An object of the present invention is to provide a skin repair composition that can express or enhance its action without using fibroblast growth factor-1 itself, which is a protein, and thereby repair damaged skin. And It is another object of the present invention to provide a screening method for efficiently distinguishing skin repair substances from among a large number of candidate substances.
前記の目的を達成するためになされた特許請求の範囲の請求項１に記載の皮膚補修組成物は、線維芽細胞増殖因子−１様の活性物質、及び線維芽細胞増殖因子−１の活性促進物質の少なくとも何れかの皮膚補修物質を、含有することを特徴とする。 The skin repair composition according to claim 1, which has been made to achieve the above object, comprises an active substance such as fibroblast growth factor-1 and promotion of fibroblast growth factor-1 activity. It contains at least any skin repair substance of the substance.
請求項２に記載の皮膚補修組成物は、請求項１に記載されたもので、前記皮膚補修物質が、植物の組織を、水、有機溶媒、水−有機溶媒混合液、水溶液、水溶液−有機溶媒混合液で抽出した抽出液、その抽出液の濃縮物、前記抽出液の精製物、又は前記濃縮物の精製物であることを特徴とする。精製物は、例えば分離精製画分である。
The skin repair composition according to
請求項３に記載の皮膚補修組成物は、請求項１に記載されたもので、前記植物が、ユリ科、イラクサ科、マメ科、バラ科、リンドウ科、シソ科、ゴマノハグサ科、ドクダミ科、サトイモ科、ウコギ科、ウリ科、アサ科、ユキノシタ科、イネ科、ヤシ科、パイナップル科、トウダイグサ科、カンナ科、ツユクサ科、クスノキ科、ショウガ科、クワ科、キク科、ノウゼンカズラ科、ウコギ科、チャセンシダ科、リュウゼンラン科、ラン科、ソテツ科、コショウ科、クロウメモドキ科、サトイモ科、ミズキ科の少なくとも何れかの植物であることを特徴とする。 The skin repair composition according to claim 3 is the skin repair composition according to claim 1, wherein the plant is a lily family, a net family, a legume family, a rose family, a gentian family, a perilla family, a scorpion family, a scorpion family, Araceae, Araliaceae, Cucurbitaceae, Asaaceae, Yukinoshita, Gramineae, Palmidae, Pineappleaceae, Euphorbiaceae, Cannaaceae, Citrusaceae, Camphoraceae, Gingeraceae, Mulberryaceae, Chrysanthemum, Lepidoptera, Urchinaceae The plant is characterized in that it is at least one of the plants of the family Chacensidae, Ryuzenran, Orchidaceae, Cycadaceae, Pepperaceae, Chrysanthaceae, Araceae, and Viburnum.
請求項４に記載の皮膚補修組成物は、請求項１に記載されたもので、前記植物が、アロエ、イラクサ、ウスベニアオイ、ハリモクシュク、アンズ、クジン、ゲンチアナ、サルビア、ジオウ、ドクダミ、ショウブ、セイヨウキズタ、ナギイカダ、ヘチマ、セイヨウカラハナソウ、ヤグルマソウ、クロゴメ、トウツルモドキ、パイナップル、ダンドク、クロトンノキ、ムラサキオモト、シバニッケイ、ゲットウ、ヒメイタビ、シロバナセンダングサ、クワ、ニンニクカズラ、ホンコンカポック、イヌビワ、オオタニワタリ、ドラセナ、シラン、アコウ、ソウシジュ、クワノハイチゴ、ジュズダマ、カクレミノ、ヒマ、ショウジョウボク、ソテツ、フウトウカズラ、クワズイモ、リュウキュウクロウメモドキ、及びリュウキュウハナイカダの少なくとも何れかであることを特徴とする。 The skin repair composition according to claim 4 is the skin repair composition according to claim 1, wherein the plant is aloe, nettle, usbeneer, harimokushu, apricot, kudin, gentian, salvia, diou, dokudami, shobu, edible kizuta , Nagii kada, loofah, caladium, cornflower, black widow, red clover, pineapple, dangdok, blackwood, purple motomoto, shivanikakei, ghetto, kingfisher, white-bellied duckweed, mulberry, garlic quail, honkon quasi Lesser Sowju, Kwanoha Strawberry, Juzudama, Kakuremino, Castor, Drosophila, Cycad, Pepper, Kuwazuimo, Ryukyu Umebodo, and Ryukyu Hanaikada Characterized in that is also one.
請求項５に記載の皮膚補修剤は、請求項１に記載の皮膚補修組成物を、含んでいることを特徴とする。 The skin repair agent according to claim 5 includes the skin repair composition according to claim 1.
請求項６に記載の皮膚補修剤は、請求項５に記載されたもので、内用剤、又は外用剤であることを特徴とする。 The skin repair agent according to claim 6 is the skin repair agent according to claim 5, and is an internal preparation or an external preparation.
請求項７に記載の皮膚補修剤は、請求項６に記載されたもので、前記内用剤が、医薬品、医薬部外品、食品添加物、又はサプリメントであり、前記外用剤が、医薬品、医薬部外品、又は化粧品であることを特徴とする。 The skin repair agent according to claim 7 is the skin repair agent according to claim 6, wherein the internal preparation is a pharmaceutical, a quasi-drug, a food additive, or a supplement, and the external preparation is a pharmaceutical, It is a quasi-drug or cosmetic.
請求項８に記載の皮膚補修物質のスクリーニング方法は、線維芽細胞増殖因子−１レセプターを持つ細胞を培養し、その培地又は培養液に被験物質を添加して培養し続け、該細胞の生存数の増減を測定し、その増殖を指標として前記被験物質から皮膚補修物質を峻別することを特徴とする。 The method for screening a skin repair substance according to claim 8, wherein cells having fibroblast growth factor-1 receptor are cultured, the test substance is added to the medium or culture solution, and the culture is continued. The skin repair substance is discriminated from the test substance using the increase and decrease as an index.
請求項９に記載の皮膚補修物質のスクリーニング方法は、皮膚線維芽細胞を培養し、その培地又は培養液に被験物質を添加して培養し続け、該細胞の生存数の増減を測定し、その増殖を指標として前記被験物質から皮膚補修物質を峻別することを特徴とする。 The method for screening a skin repair substance according to claim 9 cultivates dermal fibroblasts, continuously adds the test substance to the medium or culture medium, measures the increase or decrease in the number of surviving cells, A skin repair substance is distinguished from the test substance using proliferation as an index.
請求項１０に記載の皮膚補修物質のスクリーニング方法は、非ヒト哺乳動物の除毛した皮膚に、粘着テープを貼り付けてからそれを剥離する操作を繰り返して、角質を取り除いて前記皮膚を損傷させた後、そこへの被験物質の投与群と非投与群とで、各々の皮膚の経皮水分蒸散量を測定し、前記非投与群よりも前記投与群での前記経皮水分蒸散量の低下を指標として前記被験物質から皮膚補修物質を峻別することを特徴とする。
The method for screening for a skin repair substance according to
請求項１１に記載の皮膚補修物質のスクリーニング方法は、請求項８〜１０の何れかに記載のもので、峻別した前記皮膚補修物質が、線維芽細胞増殖因子−１様の活性物質、及び線維芽細胞増殖因子−１の活性促進物質の少なくとも何れかであることを特徴とする。 The screening method for a skin repair material according to claim 11 is the method according to any one of claims 8 to 10, wherein the skin repair material is a fibroblast growth factor-1-like active substance and a fiber. It is at least one of blast growth factor-1 activity promoters.
本発明の皮膚補修組成物を含有する皮膚補修剤は、簡易な構成で、大量に入手可能な植物のような素材から簡便に得られる線維芽細胞増殖因子−１様の活性物質や線維芽細胞増殖因子−１の活性促進物質により、それの作用を発現したり増強したりして、損傷した皮膚を補修することができる。この組成物は、大量に製造し難い線維芽細胞増殖因子−１のタンパク質を用いる必要がなく、生産性に優れ高品質のものを安定供給できるものであるから、内用剤や外用剤として、医療や美容のために、汎用できる。さらに、元来、生体内に存在する線維芽細胞増殖因子−１の活性を促進したり、それと同様な薬効活性を示したりするものであるから、安全性が高い。 The skin repair agent containing the skin repair composition of the present invention has a simple structure, and can be easily obtained from a material such as a plant that can be obtained in large quantities. The growth promoter-1 activity promoting substance can repair or damage damaged skin by expressing or enhancing its action. This composition does not require the use of a fibroblast growth factor-1 protein that is difficult to produce in large quantities, and is capable of stably supplying a high-quality product with excellent productivity. Can be used for medical and beauty purposes. Furthermore, since it originally promotes the activity of fibroblast growth factor-1 present in the living body or exhibits the same medicinal activity, it is highly safe.
この皮膚補修物質のスクリーニング方法によれば、数多の候補物質、例えば植物抽出液から皮膚補修物質を効率よく簡便に短時間で見つけ出すことができる。 According to this screening method for skin repair substances, skin repair substances can be found efficiently and simply in a short time from many candidate substances, for example, plant extracts.
以下、本発明の実施のための好ましい形態を詳細に説明するが、本発明の範囲はこれらの形態に限定されるものではない。 Hereinafter, although the preferable form for implementation of this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.
本発明の皮膚補修組成物は、線維芽細胞増殖因子−１様の活性物質、及び線維芽細胞増殖因子−１の活性促進物質の少なくとも何れかを有する植物抽出物からなる物質を、含有するものである。 The skin repair composition of the present invention contains a substance comprising a plant extract having at least one of a fibroblast growth factor-1-like active substance and a fibroblast growth factor-1 activity promoting substance. It is.
この組成物中、植物抽出物をその乾燥重量換算で、０．０００１〜０．３ｗ/ｖ（重量／体積）％含んでいることが好ましい。 In this composition, it is preferable that the plant extract contains 0.0001 to 0.3 w / v (weight / volume)% in terms of its dry weight.
植物抽出物は、一般的には植物の全体、又は植物の特定の組織例えば葉、茎、幹、樹皮、花、実、根、根茎、塊茎等の組織を、生のまま、又は乾燥し、必要に応じ粉砕し、抽出溶媒で抽出したものである。抽出溶媒は、抽出性に優れ、沸点が比較的低い溶媒が好ましく、複数種混合して用いてもよい。より具体的には、水；エタノール等のアルコール、ヘキサン等の炭化水素、酢酸エチル等のエステル、アセトン等のケトンのような有機溶媒；水とこれら有機溶媒との混合液；酸、アルカリ、又は塩の水溶液、例えば生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水；これら水溶液と有機溶媒との混合液が挙げられる。植物抽出物は、用時調製したものであってもよく、調製後保存したものであってもよく、市販品であってもよい。 A plant extract is generally a whole plant or a specific tissue of a plant, such as leaves, stems, stems, bark, flowers, fruits, roots, rhizomes, tubers, etc., raw or dried, If necessary, it is crushed and extracted with an extraction solvent. The extraction solvent is preferably a solvent having excellent extractability and a relatively low boiling point, and a plurality of types may be used as a mixture. More specifically, water; an organic solvent such as an alcohol such as ethanol, a hydrocarbon such as hexane, an ester such as ethyl acetate, a ketone such as acetone; a mixed solution of water and these organic solvents; an acid, an alkali, or An aqueous solution of a salt, for example, physiological saline, phosphate buffer, phosphate buffered saline; a mixed solution of these aqueous solution and an organic solvent can be mentioned. The plant extract may be prepared at the time of use, stored after preparation, or commercially available.
植物の乾燥１重量部に対し、抽出溶媒１０〜１２０重量部で抽出することが好ましい。 It is preferable to extract with 10 to 120 parts by weight of the extraction solvent with respect to 1 part by weight of the dried plant.
抽出は、常温下で行ってもよく、加熱して行ってもよい。加圧下で行ってもよい。抽出時間は、植物の種類や形状や形態、抽出溶媒の種類、抽出温度によって適宜調整されるが、１分間〜１４日間程度である。抽出にはソックスレー抽出器を用いてもよい。 Extraction may be performed at room temperature or by heating. You may carry out under pressure. Although extraction time is suitably adjusted with the kind, shape, and form of a plant, the kind of extraction solvent, and extraction temperature, it is about 1 minute-14 days. A Soxhlet extractor may be used for extraction.
抽出物は、これら溶媒で抽出した後、遠心分離、ろ過、デカンテートしたものであってもよい。抽出物は、そのままでもよいが、さらにそれを濃縮した濃縮物であってもよく、乾固させた後、粉砕した乾燥粉末であってもよく、それらを希釈した希釈液であってもよい。 The extract may be extracted with these solvents, centrifuged, filtered, and decanted. The extract may be used as it is, or may be a concentrate obtained by further concentrating the extract, or may be a dry powder obtained by pulverizing after drying, or a diluted solution obtained by diluting them.
皮膚補修組成物は、線維芽細胞増殖因子−１様の活性物質や線維芽細胞増殖因子−１の活性促進物質を含んでいる抽出物を、含有するものである。 The skin repair composition contains an extract containing a fibroblast growth factor-1-like active substance or a fibroblast growth factor-1 activity promoting substance.
この皮膚補修組成物を含む皮膚補修剤は、液剤、エリキシル剤、カプセル剤、顆粒剤、丸剤、懸濁剤、乳剤、散剤、錠剤、シロップ剤にして、医薬品、医薬部外品、食品添加物、サプリメントのような内用剤として用いてもよい。この皮膚補修剤は、液剤、乳剤、軟膏剤、硬膏剤、坐剤、貼付剤、湿布剤、ゲル、クリーム、フォーム、ミスト、ジェルのような剤型で、皮膚外用剤のような医薬品、医薬部外品、化粧品として用いてもよい。 Skin repair agents containing this skin repair composition are liquids, elixirs, capsules, granules, pills, suspensions, emulsions, powders, tablets, syrups, pharmaceuticals, quasi drugs, food additives It may be used as an internal preparation such as food or supplement. This skin repair agent is in the form of liquids, emulsions, ointments, plasters, suppositories, patches, poultices, gels, creams, foams, mists, gels, pharmaceuticals such as topical skin preparations, pharmaceuticals It may be used as an quasi-drug or cosmetic.
皮膚外用剤である医薬品、医薬部外品、化粧品のような外用剤として、より具体的には、ヘアトニック、ヘアジェル、ヘアクリーム、ヘアトリートメントローション、ヘアフォーム、ヘアミスト、ヘアシャンプー、ヘアリンス、育毛剤；化粧水、乳液、ローション、リップクリームのような顔用化粧品；石鹸、ボディローション、ボディソープ、ボディクリーム；軟膏、硬膏、湿布が挙げられる。 As external preparations such as pharmaceuticals, quasi-drugs and cosmetics that are external preparations for skin, more specifically, hair tonics, hair gels, hair creams, hair treatment lotions, hair foams, hair mists, hair shampoos, hair rinses, hair restorers Facial cosmetics such as lotions, emulsions, lotions, lip balms; soaps, body lotions, body soaps, body creams; ointments, plasters, poultices.
外用剤には、ヒアルロン酸やセラミドのような保湿剤；脂肪、ロウ、パラフィン、オリーブ油、グリセリンのような油性成分；α−トコフェノールやＬ−アスコルビン酸のような抗酸化剤；界面活性剤；紫外線吸収剤；毛包賦活剤；香料；色素；防菌防黴剤；ｐＨ調整剤；増粘剤；賦形剤・担持剤；消炎剤、抗炎症剤、血行促進剤、鎮痛剤を含んでいてもよい。 External preparations include humectants such as hyaluronic acid and ceramide; oily components such as fat, wax, paraffin, olive oil and glycerin; antioxidants such as α-tocophenol and L-ascorbic acid; surfactants; UV absorbers; hair follicle activators; fragrances; pigments; antibacterial and antifungal agents; pH adjusters; thickeners; excipients / carriers; including anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, analgesics May be.
皮膚補修組成物が含有している線維芽細胞増殖因子−１様の活性物質や線維芽細胞増殖因子−１の活性促進物質の物質は、線維芽細胞増殖因子レセプターを持つ細胞例えば線維芽細胞増殖因子レセプター−Ｂａ／Ｆ３細胞に添加して培養したり、皮膚線維芽細胞例えば正常ヒト皮膚線維芽細胞に添加して培養したりしたときのその細胞の生存数の増加によって、数多の植物抽出物から峻別される。またこれらの物質は、非ヒト哺乳動物例えばマウスの損傷皮膚からの経皮水分蒸散量の経時的な低下によって、数多の植物抽出物から峻別される。 Fibroblast growth factor-1-like active substances and fibroblast growth factor-1 activity-promoting substances contained in the skin repair composition are cells having fibroblast growth factor receptors such as fibroblast proliferation. Numerous plant extracts by increasing the number of surviving cells when added to factor receptor-Ba / F3 cells and cultured, or when cultured with dermal fibroblasts such as normal human skin fibroblasts Distinguished from things. In addition, these substances are distinguished from many plant extracts by a decrease in the amount of transdermal water transpiration from damaged skin of non-human mammals such as mice.
本発明を適用する実施例について、詳細に説明する。 Embodiments to which the present invention is applied will be described in detail.
先ず様々な植物の抽出物を被験物質として、線維芽細胞増殖因子（ＦＧＦ）−１の活性促進物質を探索した。 First, fibroblast growth factor (FGF) -1 activity promoting substances were searched for using various plant extracts as test substances.
（植物の抽出物の調製）
先ず、植物の抽出物を得た。例えば、クロゴメの場合、乾燥重量２ｇに対し、７０％エタノール水溶液２０ミリリットルの割合で、室温にて７日間浸漬して、可溶性成分を抽出した。抽出液と抽出残渣との分離は、遠心分離またはろ過によって、行った。得られた上清を被験物質とした。
(Preparation of plant extracts)
First, a plant extract was obtained. For example, in the case of black widow, the soluble component was extracted by dipping for 7 days at room temperature in a ratio of 20 ml of 70% aqueous ethanol to 2 g of dry weight. Separation of the extract from the extraction residue was performed by centrifugation or filtration. The obtained supernatant was used as a test substance.
他の植物の全体又は特定した葉や茎等の組織からも同様にして抽出物が得られる。抽出物は、抽出溶媒が水の場合、１００℃で２０分間抽出し、抽出液と抽出残渣とを遠心分離又はろ過したものであってもよい。抽出物は、市販の抽出物であってもよい。 Extracts can be obtained in the same manner from the whole of other plants or tissues such as specified leaves and stems. When the extraction solvent is water, the extract may be extracted at 100 ° C. for 20 minutes, and the extract and the extraction residue may be centrifuged or filtered. The extract may be a commercially available extract.
（一次スクリーニング方法及びその評価）
本発明を適用するスクリーニング方法のうち一次スクリーニングとして、遺伝子組み換えマウス細胞を用い、線維芽細胞増殖因子−１に特異的に細胞増殖率が変化する培養条件を検討し、３４１種の植物の抽出物を被験物質として、細胞増殖率を指標にしてスクリーニングを行った。その詳細を以下に示す。
(Primary screening method and its evaluation)
As a primary screening method among the screening methods to which the present invention is applied, genetically modified mouse cells are used, and the culture conditions in which the cell growth rate changes specifically to fibroblast growth factor-1 are examined, and 341 plant extracts are extracted. Was screened using the cell growth rate as an index. Details are shown below.
用いた細胞は、ＦＲ−Ｂａ／Ｆ３細胞である。その親細胞株である「Ｂａ／Ｆ３細胞」（マウスＩＬ−３依存性ｐｒｏ−Ｂ細胞株）は、独立行政法人理化学研究所バイオリソースセンターの理研セルバンクから入手可能したものであるが、それ以外の様々なところから自由に入手可能なものである。「ＦＲ−Ｂａ／Ｆ３」のＦＲとは、線維芽細胞増殖因子（ＦＧＦ）レセプターの略であり、線維芽細胞増殖因子−１（ＦＧＦ−１）を意味しており、具体的には「ＦＧＦレセプター １ｃ」を指している。ＦＲ−Ｂａ／Ｆ３細胞は、「Ｂａ／Ｆ３」へ発現カセット付の「ＦＧＦレセプター １ｃ」のｃＤＮＡを遺伝子導入することにより、安定に「ＦＧＦレセプター １ｃ」を発現させたものである。この細胞は、親株である「Ｂａ／Ｆ３」と同様に、ＩＬ−３依存性、即ちＩＬ−３非存在下では生存・増殖できないという性質を、有している。さらにＦＲを発現させたことによりＦＧＦ−１、さらにＦＧＦ−２，５などに対する応答性を獲得し、ＩＬ−３非存在下であっても、ＦＧＦ−１及びヘパリンが存在すれば、生存・増殖可能である。なお、ＦＧＦがＦＲに結合しシグナルを入力する際にはヘパリンの結合が必須である。このスクリーニング方法では、自家培養して対数増殖期にある細胞を使用した。 The cells used are FR-Ba / F3 cells. The parent cell line “Ba / F3 cell” (mouse IL-3-dependent pro-B cell line) was obtained from RIKEN Cell Bank of RIKEN BioResource Center. It can be freely obtained from various places. FR of “FR-Ba / F3” is an abbreviation for fibroblast growth factor (FGF) receptor, which means fibroblast growth factor-1 (FGF-1). Receptor 1c ". FR-Ba / F3 cells are those in which “FGF receptor 1c” is stably expressed by introducing a cDNA of “FGF receptor 1c” with an expression cassette into “Ba / F3”. Similar to the parent strain “Ba / F3”, these cells have the property of being IL-3-dependent, that is, not viable and proliferating in the absence of IL-3. Furthermore, by expressing FR, responsiveness to FGF-1, further FGF-2, 5, etc. is acquired, and even in the absence of IL-3, if FGF-1 and heparin are present, survival / proliferation Is possible. When FGF binds to FR and inputs a signal, heparin binding is essential. In this screening method, cells in autologous culture and in the logarithmic growth phase were used.
ＦＲ−Ｂａ／Ｆ３細胞を１．０×１０６個／ｍＬの濃度に調整し、９６穴マイクロプレートで、１０％ＦＢＳ（ウシ胎児血清）存在下、培養した。培養液（ＳＩＧＭＡ社製；商品名ＲＰＭＩ−１６４０）に、ＦＧＦ−１（ＰＥＰＲＯ ＴＥＣＨ ＥＣ社製；商品名１００−１７Ａ）、へパリン（ＳＩＧＭＡ社製；商品名Ｈ３１４９）、及び適当に希釈した評価試料である被験物質（植物の抽出物）を添加して培養した。ＦＧＦ−１の添加量は最終濃度１０〜５０ｎｇ／ｍＬを基本とし、ヘパリンの添加量は最終濃度５μｇ／ｍＬとし、被験物質は希釈倍率を最低でも４点設定した。各ウェルに、「ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８」（株式会社同仁化学研究所製；商品番号３４１−０８００１）を添加し、３時間培養した後、細胞の増殖の程度を測定した。マイクロプレートリーダーで吸光度を測定し、生存細胞数を計算した。上記培養細胞の生存率が高いものは、ＦＧＦ−１様活性又はＦＧＦ−１様活性を促進する効果があると考えられる。 FR-Ba / F3 cells were adjusted to a concentration of 1.0 × 10 6 cells / mL and cultured in a 96-well microplate in the presence of 10% FBS (fetal bovine serum). Culture solution (manufactured by SIGMA; trade name RPMI-1640), FGF-1 (manufactured by PEPRO TECH EC; trade name 100-17A), heparin (manufactured by SIGMA; trade name H3149), and appropriately diluted evaluation A test substance (plant extract) as a sample was added and cultured. The addition amount of FGF-1 was based on a final concentration of 10 to 50 ng / mL, the addition amount of heparin was set to a final concentration of 5 μg / mL, and the dilution ratio of the test substance was set to at least 4 points. To each well, “cell counting kit-8” (manufactured by Dojindo Laboratories Co., Ltd .; product number 341-080001) was added and cultured for 3 hours, and then the degree of cell proliferation was measured. Absorbance was measured with a microplate reader and the number of viable cells was calculated. Those having a high survival rate of the cultured cells are considered to have an effect of promoting FGF-1-like activity or FGF-1-like activity.
この一次スクリーニングの結果、表１〜３に挙げる４６種類の被験物質が、ＦＧＦ−１様活性物質、又はＦＧＦ−１様活性活性促進物質として、峻別された。その結果を、まとめて図１〜６に示す。
As a result of this primary screening, 46 kinds of test substances listed in Tables 1 to 3 were distinguished as FGF-1-like active substances or FGF-1-like activity promoting substances. The results are collectively shown in FIGS.
（二次スクリーニング方法及びその評価）
本発明を適用するスクリーニング方法のうち二次スクリーニングとして、前記の一次スクリーニングで峻別された被験物質のうち、ヒトの皮膚細胞の増殖を促進するものを更に峻別するスクリーニングを行った。二次スクリーニングは、凍結正常ヒト皮膚線維芽細胞（ＮＨＤＦ：Normal Human Dermal Fibroblasts）を用い、その細胞増殖率を指標にしてスクリーニングを行った。その詳細を以下に示す。
(Secondary screening method and its evaluation)
As a secondary screening among the screening methods to which the present invention is applied, a screening for further distinguishing those that promote the proliferation of human skin cells among the test substances distinguished by the primary screening was performed. In the secondary screening, frozen normal human dermal fibroblasts (NHDF) were used, and screening was performed using the cell growth rate as an index. Details are shown below.
用いたＮＨＤＦ細胞は、市販の凍結正常ヒト皮膚線維芽細胞（新生児）（ＣａｍＢｒｅｘ社製；商品名ＣＣ−２５０９）である。このスクリーニング方法では、その細胞を購入後、自家培養して対数増殖期にある細胞、又は解凍後初回培養を行った細胞を使用した。 The NHDF cells used are commercially available frozen normal human dermal fibroblasts (newborn) (manufactured by CamBrex; trade name CC-2509). In this screening method, after the cells were purchased, they were self-cultured and were in a logarithmic growth phase, or cells that were first cultured after thawing.
ＮＨＤＦ細胞を３．２×１０４個／ｍＬの濃度に調整し、培地としてＦＧＭ−２ ＢｕｌｌｅｔＫｉｔ（Ｃａｍｂｒｅｘ社製；商品名ＣＣ−３１３２）を用い、９６穴マイクロプレート上に播種し、４８時間培養した。培養開始４８時間後に、無血清培地としてＤＭＥＭ（Ｇｉｂｃｏ社製；１１８８５）へ培地交換し、２４時間培養した。２４時間培養後、ＤＭＥＭ（Ｇｉｂｃｏ社製；商品名１１８８５）にＦＧＦ−１（ＰＥＰＲＯ ＴＥＣＨ ＥＣ社製；商品名１００−１７Ａ）、ヘパリン（ＳＩＧＭＡ社製；商品名Ｈ３１４９）、及び適当に希釈した評価試料である被験物質（植物の抽出物）を添加して２４時間培養した。このとき、ＦＧＦ−１の添加量は最終濃度０．２〜１μｇ／ｍＬとし、ヘパリンの添加量は最終濃度５μｇ／ｍＬとし、被験物質は希釈倍率を最低でも４点設定した。「ＢｒｄＵ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ ＥＬＩＳＡ Ｃｏｌｏｒｉｍｅｔｒｉｃ Ａｓｓａｙ」（ｅｘａｌｐｈａ社製；商品名Ｘ１３２７Ｋ１）を用いて、細胞を発色させた。マイクロプレートリーダーで吸光度を測定し、生存細胞数を算出した。上記培養細胞の生存率が高いものは、ＦＧＦ−１様活性又はＦＧＦ−１様活性を促進する効果があると考えられる。 NHDF cells were adjusted to a concentration of 3.2 × 10 4 cells / mL, seeded on a 96-well microplate using FGM-2 BulletKit (manufactured by Cambrex; trade name CC-3132) as a medium, and cultured for 48 hours. did. 48 hours after the start of the culture, the medium was changed to DMEM (Gibco; 11885) as a serum-free medium and cultured for 24 hours. After culturing for 24 hours, DMEM (manufactured by Gibco; trade name 11885), FGF-1 (manufactured by PEPRO TECH EC; trade name 100-17A), heparin (manufactured by SIGMA; trade name H3149), and appropriately diluted evaluation A test substance (plant extract) as a sample was added and cultured for 24 hours. At this time, the addition amount of FGF-1 was set to a final concentration of 0.2 to 1 μg / mL, the addition amount of heparin was set to a final concentration of 5 μg / mL, and the dilution ratio of the test substance was set to at least 4 points. Using “BrdU Cell Proliferation ELISA Colorimetric Assay” (manufactured by Exalpha; trade name: X1327K1), the cells were colored. Absorbance was measured with a microplate reader, and the number of viable cells was calculated. Those having a high survival rate of the cultured cells are considered to have an effect of promoting FGF-1-like activity or FGF-1-like activity.
この二次スクリーニングの結果、ＥＣ５０（５０％生存率）が高いＦＧＦ−１様活性物質、又はＦＧＦ−１様活性活性促進物質被験物質として、表４に挙げる３種類の被験物質が、峻別された。その結果を、まとめて図７に示す。 As a result of this secondary screening, three types of test substances listed in Table 4 were distinguished as FGF-1-like active substances with high EC 50 (50% survival rate) or FGF-1-like activity promoting substances test substances. It was. The results are collectively shown in FIG.
表４及び図７から明らかな通り、これら３種の植物の抽出液は、実際のヒト皮膚細胞の増殖に関し有効に作用することが明らかとなった。 As is apparent from Table 4 and FIG. 7, it was revealed that the extracts of these three kinds of plants acted effectively on the actual proliferation of human skin cells.
（三次スクリーニング方法及びその評価）
本発明を適用するスクリーニング方法のうち三次スクリーニングとして、前記の二次スクリーニングで峻別された３種類の被験物質について、非ヒト哺乳動物を用いテープストリッピング法により物理的に皮膚を損傷させたモデルを作成し、体液が体温で蒸発する量、即ち経皮水分蒸散量（ＴＥＷＬ）を指標にして、スクリーニングを行った。その詳細を以下に示す。
(Tertiary screening method and its evaluation)
As a tertiary screening among the screening methods to which the present invention is applied, a model in which the skin is physically damaged by the tape stripping method using a non-human mammal for the three types of test substances distinguished by the secondary screening described above is created. Then, screening was performed using the amount of body fluid evaporated at body temperature, that is, the transdermal water transpiration (TEWL) as an index. Details are shown below.
用いた非ヒト哺乳動物は、５週齢のＣｒｌｊ：ＣＤ−１（ＩＣＲ）マウス（日本チャールス・リバー株式会社から入手）で、雄性 ＳＰＦ，３５例である。 The non-human mammal used was a 5-week-old Crlj: CD-1 (ICR) mouse (obtained from Charles River Japan, Inc.), 35 male SPFs.
テープストリッピング法用の皮膚損傷モデルを、以下のようにして作製した。前記マウス７匹を１群としてそれらの体幹部の背側部の広い範囲の被毛を電気バリカンで除毛した。セロハンテープ（積水化学工業株式会社製）を、一定回数（約５回程度）皮膚に当てて，やや皮膚表面に光沢を認める状態まで角質を取り除いた。処置領域は背側部の正中線からやや左側にずらした領域で，中央部から頭側の部分とした。塗布部位は２×２ｃｍの領域で、セロハンテープは同一品を用い、作製部位の状態を均質に揃えた。表皮各層を剥がす度合いは、経皮水分蒸散量（ＴＥＷＬ）を５００ｇ／ｈ・ｍ２となる程度にして、テープストリッピング法による皮膚損傷モデルとした。 A skin damage model for the tape stripping method was prepared as follows. A group of 7 mice was used to remove hair from a wide range on the back side of their trunks with an electric clipper. Cellophane tape (manufactured by Sekisui Chemical Co., Ltd.) was applied to the skin a certain number of times (about 5 times), and the keratin was removed until the skin surface was slightly glossy. The treatment area was shifted slightly to the left from the midline on the dorsal side, and the area from the center to the head side. The application site was an area of 2 × 2 cm, and the same cellophane tape was used, and the state of the production site was made uniform. The degree of peeling of each epidermis was such that the transdermal moisture transpiration (TEWL) was 500 g / h · m 2, and a skin damage model by the tape stripping method was used.
この損傷直後の損傷部位に、５０μＬ／ｈｅａｄずつ、被験物質を塗布した。塗布方法は、２×２ｃｍの面積のプラスチックラップ（基材）を用い，基材と損傷皮膚との間に、被験物質を挟みこみ，５分間圧着した。５分後に基材をはがし，開放状態とした。圧着および投与は、同一者により、同一条件で行った。 The test substance was applied to the damaged site immediately after the injury in an amount of 50 μL / head. As a coating method, a plastic wrap (base material) having an area of 2 × 2 cm was used, and the test substance was sandwiched between the base material and the damaged skin and pressed for 5 minutes. After 5 minutes, the substrate was peeled off and opened. Crimping and administration were performed under the same conditions by the same person.
水分蒸散量測定装置（有限会社アサヒバイオメッド製；商品名 水分蒸散モニターＡＳ−ＣＴ１）を用いて、経皮水分蒸散量（ＴＥＷＬ）を測定した。テープストリッピング前（角層除去前）、テープストリッピング直後（０時間，角層除去直後）、塗布投与終了直後、角層除去してから１時間後，８時間後，２３．５時間後，３０．５時間後，４７．５時間後，５２．５時間後に、経皮水分蒸散量を測定した。なお対照のため被験物質に代えて、陰性対照物質としてリン酸緩衝液（ＰＢＳ）を用いた陰性コントロール群と、陽性対照物質として（ＦＧＦ−１）を用いた陽性コントロール群とについても、同様に、測定した。その結果を図８に示す。 The transdermal water transpiration rate (TEWL) was measured using a water transpiration amount measuring device (manufactured by Asahi Biomed Co., Ltd .; trade name water transpiration monitor AS-CT1). Before tape stripping (before stratum corneum removal), immediately after tape stripping (0 hours, immediately after stratum corneum removal), immediately after application administration, 1 hour, 8 hours, 23.5 hours after stratum corneum removal, 30. After 5 hours, 47.5 hours, and 52.5 hours, the transdermal moisture transpiration was measured. For the control, instead of the test substance, a negative control group using a phosphate buffer (PBS) as a negative control substance and a positive control group using (FGF-1) as a positive control substance are similarly used. ,It was measured. The result is shown in FIG.
図８から明らかな通り、３種類の何れの被験物質を用いた群とも、陰性コントロール群よりも経皮水分蒸散量が低く、皮膚の損傷の回復が早いことが示された。特に、時間の経過とともに、３種類の何れの被験物質を用いた群とも、陽性コントロール群よりも経皮水分蒸散量が低く、ＦＧＦ−１だけを投与するよりも、皮膚の損傷の回復が早いことが示された。 As is clear from FIG. 8, the group using any of the three types of test substances showed a lower transdermal moisture transpiration rate than the negative control group, indicating that recovery of skin damage was quicker. In particular, with the passage of time, the group using any of the three types of test substances has a lower transdermal moisture transpiration rate than the positive control group, and the recovery of skin damage is faster than administration of FGF-1 alone. It was shown that.
従って、これら３種類の被験物質は、皮膚補修組成物として、又、皮膚補修剤として、生体内に存するＦＧＦ−１と同等以上の効果を奏したり、ＦＧＦ−１の活性を促進したりするものであることが示された。 Therefore, these three kinds of test substances have effects equivalent to or higher than those of FGF-1 existing in the living body as skin repair compositions and skin repair agents, and promote the activity of FGF-1. It was shown that.
これにより、線維芽細胞増殖因子様活性物質、及び線維芽細胞増殖因子活性促進物質を含有する皮膚補修組成物は、医薬品、医薬部外品、食品添加物、若しくはサプリメントのような内用剤として、又は医薬品、医薬部外品、若しくは化粧品のような外用剤として、有用な皮膚補修剤となることが立証された。 Accordingly, a skin repair composition containing a fibroblast growth factor-like active substance and a fibroblast growth factor activity promoting substance can be used as an internal preparation such as a pharmaceutical, a quasi-drug, a food additive, or a supplement. Or as a skin repair agent useful as an external preparation such as pharmaceuticals, quasi-drugs, or cosmetics.
本発明の皮膚補修組成物を含む皮膚補修剤は、加齢や紫外線曝露やホルモンバランス異常などにより皮膚が受けた弛み・シミ・小皺・色素沈着・乾燥肌・ニキビなどの損傷を修復して改善するスキンケア用品・美容用品等の化粧品・医薬部外品のような外用剤として、怪我による創傷や火傷や褥瘡を治療するための外用剤や内用剤のような医療用医薬品・医薬部外品として、また、食品添加物やサプリメントのような内用剤として、有用である。 The skin repair agent containing the skin repair composition of the present invention improves and repairs damage such as loosening, spots, small wrinkles, pigmentation, dry skin, acne, etc., caused by aging, ultraviolet exposure, abnormal hormone balance, etc. As an external agent such as cosmetics and quasi-drugs such as skin care products and beauty products, medical drugs and quasi-drugs such as external and internal medicines for treating wounds, burns and pressure ulcers due to injury It is also useful as an internal preparation such as food additives and supplements.
また、本発明の皮膚補修物質のスクリーニング方法は、この皮膚補修物質を効率よく見つけ出すのに有用であり、それを製剤化して内用剤や外用剤等の皮膚補修剤を創製するのに有用である。 The screening method of the skin repair substance of the present invention is useful for efficiently finding out this skin repair substance, and is useful for creating a skin repair agent such as an internal preparation or an external preparation by formulating it. is there.
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