JP4268329B2 - Compositions for prevention or treatment of hair nourishing agents, hair regression phase transition inhibitors, etc. - Google Patents

Description

【００３４】
(2)シアロ糖タンパク質フェチュインの影響確認試験
シアロ糖タンパク質フェチュイン（分子量48,400）は、哺乳動物胎児血清や、種々の疾患（特に外傷時）の成体血中などに存在する物質である。またフェチュインは、TGF-βのレセプターに類似したアミノ酸配列と二次構造を持ち、TGF-βのアンタゴニストとして働くことから（Demetriou M et al： J Biol Chem 271： 12755-12761, 1996）、フェチュインの毛髪退行期移行抑制効果を検証した。
【００３５】
フェチュイン（最終濃度１，１０，５０μＭ）またはコントロールのウシ血清アルブミン（最終濃度５０μｍ）を基礎培地に添加した培地に交換し、さらに７日間毛包の伸長と形態変化を観察しながら培養を続けた。コントロールのウシ血清アルブミン添加に比べて、フェチュイン添加で毛伸長は有意に促進された。これらTGF-β中和抗体とフェチュインの結果から、TGF-β2作用抑制による毛髪退行期移行抑制効果が実証された。
【００３６】
[PAI-1の測定によりTGF-βの抑制効果が測定できることの確認]
ペニシリン、ストレプトマイシン及びファンギゾン（Fungizone）の３種類の抗生物質を加えたＤＭＥＭ培地に、１０％ウシ胎児血清を添加した培地を用い、９６穴プレート（１ウエル当たりの細胞数約7,500）でヒト毛乳頭細胞の培養を行った。まず、PAI-1の発現上昇のTGF-β2濃度依存性を調べた。適当な細胞密度になったところで、種々の濃度のTGF-β2を含む培地に交換し、さらに２４時間培養を行った。培地１０μlを取り、市販のPAI-1測定キット（TintElize PAI-1：biopool社）により定量した。その結果では、TGF-β2の濃度依存的にPAI-1の上昇が見られた。
【００３７】
また、TGF-β2と同時に２０μｇ/ｍｌのTGF-β中和抗体を加えておくと、TGF-β2によるPAI-1の上昇が完全に抑制された。この結果は、TGF-βとその作用を打ち消す物質を共存させると、PAI-1の上昇が抑制される例であり、本評価方法がTGF-βの作用抑制物質のスクリーニングに適していることを示すものである。
【００３８】
【実施例】
以下に本発明の実施例を示すが、本発明はこれら実施例により限定されるものではなく、特許請求の範囲により特定されるものであることはいうまでもないことである。
【００３９】
[アマチャエキスに含有される成分についての確認試験]
本発明者は、アマチャエキスの５０％エタノール抽出液を作成し、その含有成分を分析したところ、フィロズルシン、ヒドランゲノール、クロロゲン酸、ウンベリフェロン等の多くの物質が存在することを確認した。本発明者は、前記のとおり確認作業を行ったが、それら物質がアマチャエキス中に存在することは図１に示すように既知の事実である。
【００４０】
ついで、本発明者は、そのうちの１つで、イソクマリン類化合物であるフィロズルシンについて、TGF-β2によるPAI-1の発現上昇を抑制する作用があるかどうかを以下のとおり調査した。フィロズルシンをＤＭＳＯに０．０５〜５０μｇ/ｍｌの濃度で溶解させて用いた。１ng/mlのTGF-β2を含む培地で２４時間培養後、培地１０μｌを用いてPAI-1量を測定した。また、培養終了時にアラマーブルー（Alamar Blue）法により細胞の生残率を測定した。
【００４１】
それは以下のとおり行った。すなわち、新しい培地で細胞を２回洗浄した後、1/10量のアラマーブルー（アラマーバイオサイエンス社）を含む培地に交換、３７℃（５％ＣＯ₂）で４時間インキュベートした。インキュベート後、５９５nm及び５７０nmの吸光度をマイクロプレートリーダーで測定して各ウエルのアラマーブルー還元率を算出し、これをフィロズルシン試料不添加のネガティブコントロールのアラマーブルー還元率で割ることにより、細胞の生残率を算出した。
【００４２】
前者の結果は、図２に図示してあり、それによれば、フィロズルシンはPAI-1の発現上昇を抑制する。すなわち、この図の縦軸はPAI-1の量で、フィロズルシンを添加した場合には１８であり、不添加の場合の１００より大幅に低く、優れたPAI-1の上昇抑制効果を有することが理解できる。
【００４３】
また、図３は、後者のフィロズルシンの細胞毒性を図示しており、それによれば、フィロズルシンには細胞毒性は殆ど認められなかった。すなわち、図３において縦軸は、アラマーブルー還元率（％）を示し、この値が１００％に近いほど細胞毒性は低く、フィロズルシンを添加した場合の還元率は、９５％で、不添加の場合の１００％に近接した値であるから、細胞毒性が低いことがわかる。
【００４４】
(2)毛包伸長効果の確認試験
フィロズルシンの器官培養法における毛伸長促進効果の検証を以下のとおり行った。フィロズルシン（和光純薬：生薬検定用標準品）をＤＭＳＯ５０ｍｇ/ｍｌの濃度で溶解させた。使用した方法は、ヒト毛包器官培養法に従った。培養２日目に、基礎培地に最終濃度０．０６μｇ/ｍｌにフィロズルシン溶液を添加した培地に交換し、さらに７日間毛包の伸長と形態変化を観察しながら培養を続けた。
【００４５】
その結果、図４に図示してあり、それによれば培養８日目においてフィロズルシン０．０６μｇ/ｍｌの添加群で、溶媒コントロールに比べて有意に毛伸長が促進された。このことから、TGF-βの作用抑制物質として選択されたフィロズルシンには、毛髪退行期抑制効果があることは明らかであり、本発明の有効性が確認できる。
【００４６】
(3)毛髪の毛伸長効果の確認試験
ヒトの単離毛髪にフィロズルシンを添加した際の毛髪の伸長を調査した。結果は図５の写真に示すとおりであり、この写真よりフィロズルシンを添加した場合には、コントロールの場合に比較して毛髪が伸長していることが明確に確認できる。
【００４７】
そして、TGF-β2が肝硬変や腎炎の病因の一つであることは既知であり、フィロズルシンには、前記したとおりTGF-β2に対する作用抑制能（拮抗作用）があるので、肝硬変治療薬あるいは腎炎治療剤としての性能を有することも明らかである。また、フィロズルシン以外のイソクマリン類化合物もフィロズルシンと同様にTGF-β2に対する作用抑制能（拮抗作用）を有しており、養毛料、毛髪退行期移行抑制剤、肝硬変治療薬あるいは腎炎治療剤として有効である。
【００４８】
本発明は、前記したとおりイソクマリン類、中でもヒドロイソクマリン類、特にフィロズルシンを有効成分として含有する養毛料、毛髪退行期移行抑制剤、肝硬変治療薬あるいは腎炎治療剤を提供するものであり、そのうちの養毛料については、医薬品、医薬部外品あるいは化粧料の形態として提供することができる。
【００４９】
その剤型については、本発明の効果を発揮できるものであれば、特に制限はなく、任意に選択できる。例えば、液状、乳状、軟膏、クリーム、ゲル、エアゾール、ムース等が例示できる。具体的には、トニック、ローション、コンディショナー、スカルプトリートメント、シャンプー、リンス等が挙げられる。
【００５０】
そして、これの製剤中には、本発明にかかるフィロズルシンを含むイソクマリン類化合物のほかに、通常医薬品や化粧料に配合可能な成分を、本発明の効果が損なわれない範囲で配合することができる。例えば、薬効成分としては、血行促進作用を有する薬剤として、センブリエキス、ビタミンＥ及びその誘導体、ニコチン酸ベンジルエステル等のニコチン酸エステル類などが挙げられる。
【００５１】
また、局所刺激作用により血液循環を促進する薬剤としては、トウガラシチンキ、カンタリスチンキ、カンフル、ノニル酸ワニリルアミド等が挙げられる。毛包賦活作用を有する薬剤としては、ヒノキチオール、プラセンタエキス、感光素、パントテン酸及びその誘導体等が挙げられる。抗男性ホルモン作用を有する薬剤としては、エストラジオール、エチニルエストラジオール、エストロン等のホルモン剤などが挙げられる。抗脂漏作用を有する薬剤としてはイオウ、チオキソロン、ビタミンＢ₆等が挙げれる。
【００５２】
その他、フケの発生を防止するために角質溶解作用、殺菌作用を有するサリチル酸、レゾルシン等が挙げられ、頭皮の炎症を防止するためにグリチルリチン酸及びその誘導体、メントール等が、さらには毛包への栄養補給、酵素活性の賦活のためにセリン、メチオニン、アルギニン等のアミノ酸類、ビオチン等のビタミン類、生薬エキス等が挙げられる。
【００５３】
また、アルテア、ヨクイニン、ペパーミント、ヨウテイ、トウガラシ、アロエ、クコ、ヨモギ、イネ、マンケイシ、マンネンロウ、コッサイホ、エニシダ、リンドウ、タンジン、ヘチマ、キキョウ、マツ、クジン、トウキ、ベニバナ、メギ、ビンロウジ、ユーカリ、カゴソウ、モクツウ、ゴシツ、サイコ、チャ、カンゾウ、ホップ、キク、セネガ、ゴマ、センキュウ、カシュウ、カッコン、マイカイカ、サフラン、ローズマリー、ジオウ、ゼニアオイ等の植物抽出物を配合することもできる。
【００５４】
また、アルコキシカルボニルピリジンＮ−オキシド、塩化カルプロニウム、アセチルコリン誘導体等の血管拡張剤；セファランチン等の皮膚機能亢進剤；ヘキサクロロフェン、ベンザルコニウムクロリド、セチルピリジウムクロリド、ウンデシレン酸、トリクロロカルバニド、ビチオノール、フェノール、イソプロピルメチルフェノール等の抗菌剤；亜鉛及びその誘導体；乳酸又はそのアルキルエステル；クエン酸；コハク酸；リンゴ酸等の有機酸類、トラネキサム酸等のプロテアーゼ阻害剤等を配合することもできる。その他、サイクロスポリン類、オキセンドロン、ジアゾキシド、ミノキシジル等の薬剤も配合可能である。
【００５５】
また、エタノール、イソプロピルアルコール等のアルコール類；グリセリン、プロピレングリコール、ポリエチレングリコール等の多価アルコール類；高級脂肪酸、高級アルコール、炭化水素油、天然油脂、エステル油、シリコーン油等の油分；界面活性剤；香料；キレート剤；1,3−ブチレングリコール、ヒアルロン酸及びその誘導体、マルチトール、アテロコラーゲン、乳酸ナトリウム等の保湿剤；マルメロ粘質物、カルボキシビニルポリマー、キサンタンガム等の増粘剤；高分子化合物；酸化防止剤；紫外線吸収剤；色素；水；安定化剤等、通常医薬品や化粧料に配合される成分を配合することができる。
【００５６】
本発明の毛髪等の予防あるいは治療組成物の有効成分であるイソクマリン類は、医薬品、医薬部外品、化粧料等における皮膚外用剤に配合することができ、特に、ニキビ等の皮膚障害を改善する皮膚障害治療剤として有用である。その皮膚外用剤の形態は、例えば、軟膏、ローション、乳液、クリーム、パック、ジェル、ファンデーション、口紅、フェイスカラー、皮膚洗浄料、浴用剤等、通常皮膚外用剤として適用される剤形であれば特に限定されない。また、皮膚外用剤には、カウレン類の他、通常皮膚外用剤に配合可能な成分を問題の生じない範囲で配合することができる。
【００５７】
また、本発明のイソクマリン類を含有する組成物は、上記以外の医薬用組成物、化粧用組成物としても使用可能である。医薬組成物としては、経口用、非経口用剤型のいずれでもよく、その剤型も任意で、例えば、液状、シロップ状、錠剤、散剤、顆粒剤、カプセル剤、座剤あるいは注射剤等の剤型とすることができる。
【００５８】
その剤型化の際には、必要に応じて希釈剤、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤、安定化剤、可溶化剤、ｐＨ調製剤等、通常使用される添加剤を用いて、常法により製造すればよい。例えば、液状製剤とするには、生理食塩液、エタノール、１，３−ブチレングリコールなどを希釈剤または担体として使用することができる。
【００５９】
本発明の組成物中におけるイソクマリン類化合物の配合濃度については、組成物の形態または使用目的等に応じて適宜決定されるが、通常組成物中に０．００１〜２重量％配合される。０．００１重量％未満では、本発明効果を十分に発揮することが難しく、他方２重量％を超えると製剤上好ましくない。
また、その投与量についても、組成物の形態または使用目的等に応じて適宜決定されるが、養毛料又は外用剤の場合には、通常０．００１〜１００ｍｇを１日１回または数回に分けて頭皮または皮膚に塗布または散布することが好ましい。
【００６０】
【発明の効果】
本発明は、腫瘍増殖因子（TGF-β2)が毛髪成長サイクルにおける成長期から退行期への移行を促進し、TGF-β2の拮抗作用物質（アンタゴニスト）により退行期への移行を抑制することにより育毛効果が得られることを本発明者が見出したことに基づいて開発した新規なスクリーニング方法により、フィロズルシンを含むイソクマリン類に、養毛料、毛髪退行期移行抑制剤、肝硬変治療薬あるいは腎炎治療薬の有効成分としての特性を持つことを見出したことにより開発されたものである。
【００６１】
本発明は、これら組成物中に、フィロズルシンを含むイソクマリン類を含有することにより、養毛料、毛髪退行期移行抑制剤、肝硬変治療薬あるいは腎炎治療薬として有効に効能を発現することができるものである。また、イソクマリン類は、細胞毒性もほとんどなく、養毛料あるいは毛髪退行期移行抑制剤等として適正な性能を発現することができるものである。さらに、その化合物は飲料用として利用されているアマチャ中の成分でもあり、この点からしても、安心して使用できる有効成分である。
【図面の簡単な説明】
【図１】アマチャエキス中の既知成分の化学構造式一覧。
【図２】 TGF-β2によるPAI-1の発現上昇を抑制する作用に関し、フィロズルシンについて調査した結果を示す。
【図３】フィロズルシンの細胞毒性について、アラマーブルー還元率を測定した結果を示す。
【図４】フィロズルシンについて実施した毛包伸長促進効果に関する試験結果を示す。
【図５】ヒトの単離毛髪にフィロズルシンを添加した際の毛髪の伸長調査結果を示す写真。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a composition for preventing or treating hair or the like containing isocoumarins, particularly phyllozulcin as an active ingredient. More specifically, the present invention relates to a hair nourishing agent, a hair regression phase inhibitor, a liver cirrhosis therapeutic agent, or a nephritis therapeutic agent containing isocoumarins, particularly phyllozulcin as an active ingredient. More preferably, the present invention relates to a hair nourishing agent or a hair regression phase inhibitor containing isocoumarins that promote hair elongation, especially dihydroisocoumarins, especially phyllozulcin as an active ingredient.
[0002]
[Prior art]
The hair cosmetics called hair restorers or hair restorers are expected to promote hair elongation, which penetrates the hair root, dilates blood vessels, promotes blood circulation, and further enhances the hair papilla. It contains drugs that help stimulate and generate hair and promote hair growth. When the drugs contained in the hair nourishing agent are shown by action, components having actions such as blood circulation promotion, local stimulation, hair follicle activation, anti-androgen, anti-seborrheic, keratolysis, sterilization or anti-inflammation are blended.
[0003]
Ingredients that have been blended in the past in anticipation of their action include concrete extract, vitamin E, chili pepper, vitamin B ₆ Hinokitiol, estradiol, sulfur, salicylic acid, menthol, glycyrrhizin or pantothenic acid.
[0004]
As methods for evaluating the hair nourishing agent, there are existing methods such as a method using an animal such as a mouse, rabbit or monkey, a method using a human, a method using tissue culture, and the like. Among them, the method using humans is a direct method in which a test substance is applied to the human head and the like, and the growth of hair is observed, and the method using tissue culture is to test hair follicles separated from the skin, for example. There is a method of culturing in the presence of a substance and measuring the degree of elongation of the cultured hair.
[0005]
[Problems to be solved by the invention]
Regarding these conventional methods, the former method using humans takes a long time to obtain results, and the latter method requires a microscope to obtain results, is complicated in operation, and a large number of samples are once collected. There are problems such as being difficult to test. Therefore, a screening method that can be carried out in a short time and with a simple operation and that can test a large number of samples at once has been desired.
[0006]
Therefore, the present inventors have developed a novel screening method that can be applied to screening of hair nourishing agents and that can test a large number of samples at once in a short time and with a simple operation. A patent application has already been filed (Japanese Patent Application No. 11-86406).
[0007]
In this new screening method, tumor growth factor (Transforming Growth Factor-β2; TGF-β2) promotes the transition from the growth phase to the regression phase in the hair growth cycle, and is regressed by an antagonist of TGF-β2. It was developed on the basis of the inventor's finding that the hair growth effect can be obtained by suppressing the transition to the stage.
[0008]
The present inventors further show that plasminogen-activator inhibitor produced in the absence of an antagonist when human papillary cells are cultured in the presence or absence of TGF-β2 and an antagonist to TGF-β2. It was confirmed that the production amount of PAI-1 was decreased in the presence of the TGF-β2 antagonist as compared with the amount of 1 (PAI-1). The present invention was developed based on the discovery of this confirmed new matter.
[0009]
In the process of confirming the effectiveness of this screening method, various herbal medicines are screened, and it is confirmed that many herbal medicines have an antagonistic action against TGF-β2. A patent application was filed with the invention. In addition, it was also confirmed that amacha extract has the strongest antagonism against TGF-β2.
[0010]
Further research on the amacha extract, which was found to have the most powerful antagonism (TGF-β inhibitory action using PAI-1 as an indicator, human hair follicle organ culture), Could be identified. That is, it was confirmed that isocoumarins, particularly phyllozulcin, which is one of dihydroisocoumarins, is the main body of its action.
The present invention has been made on the basis of the above research results and novel findings.
[0011]
[Means for Solving the Problems]
The present invention relates to a hair nourishing agent, a hair regression phase inhibitor, a cirrhosis therapeutic agent or a nephritis therapeutic agent based on the research results and novel findings as described above, and the hair nourishing agent includes isocoumarins ( Compound), especially phyllozulcin as an active ingredient. In addition, other hair regression phase inhibitors, liver cirrhosis therapeutic agents, or nephritis therapeutic agents all contain isocoumarins (compounds), particularly phyllozulcin as an active ingredient.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described. First, the novel screening method that has produced the present invention will be specifically described.
The human hair growth cycle begins after 5 to 6 years of growth, 2-3 weeks of regression, and 2-3 months of rest, and eventually new hair is generated and the growth begins. Is done.
[0013]
The present inventors first experimentally proved that TGF-β2 induces and initiates the regression phase, that is, the growth phase is shortened. That is, when the growing follicles obtained from human scalp are organ-cultured in the presence and absence of TGF-β2 and cultured in the presence of TGF-β2, the natural follicle enters the regression phase It was confirmed that a morphological change similar to that of the transition occurred. In addition, the distribution of TGF-β2 in human hair follicles in the growth phase and in human hair follicles in the regression phase was observed by immunohistochemical staining using an anti-TGF-β2 antibody. It was found that TGF-β2 was expressed and distributed remarkably in the hair follicles.
[0014]
In addition, human hair follicles were organ-cultured in the presence and absence of anti-TGF-β-antibodies and fetuins known to be antagonists of TGF-β2, and hair elongation was measured. . As a result, in the presence of anti-TGF-β2-antibody or fetuin, hair elongation is larger than in the absence of them, and TGF-β2 is neutralized by the antagonist of TGF-β2 and the regression phase It was confirmed that the transition to is suppressed.
[0015]
Next, in order to find a simple means of producing an antagonistic effect on TGF-β2, culturing hair papilla cells in medium containing TGF-β2 at various concentrations and measuring PAI-1 in the culture Thus, it was confirmed that TGF-β2 promotes the production of PAI-1 in dermal papilla cells. Furthermore, when dermal papilla cells are cultured in the presence of TGF-β2, an increase in PAI-1 production (production) is suppressed by adding an anti-TGF-β2-antibody that is an antagonist of TGF-β2. I found out that From these experiments, it was confirmed that the presence, amount, activity, etc. of antagonists to TGF-β2 can be easily measured by measuring PAI-1 in the dermal papilla cell culture according to a conventional method. .
[0016]
Based on the above new findings, the present inventors have also developed a screening method and have already filed a patent application as described above. That is, dermal papilla cells are cultured in the presence of TGF-β2 and various test substances, and the amount of PAI-1 in the culture is measured. When the elongation by organ culture was measured, it was confirmed that the test substance that decreases the measured value of PAI-1 promotes hair elongation, and the effectiveness of this screening method was confirmed.
[0017]
According to this method, cells capable of producing PAI-1 (hair papilla cells, fibroblasts) are cultured in the presence of TGF-β2 and a test substance, and the amount of PAI-1 in the culture is measured. That's fine. In that case, when the measured value of PAI-1 is significantly lower than the control in which the test substance was not added, the test substance is determined to be TGF-β2 (antagonist) and can be used as a hair nourishing candidate. .
[0018]
The hair papilla cells used in this method are isolated from human scalp according to the method of Messenger (Messenger AG: BR J Dermatol 110, 685, 1984). In addition, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) is used for the culture. In addition, as the hair papilla cells to be used, immortalized cultured hair papilla cells can also be used. Immortalized hair papilla cells can be obtained in large quantities at any time by culturing them, and have the advantage that a large number of test substances can be screened at once or over several orders.
[0019]
Immortalized hair papilla cells are infected with SV40-T antigen in normal hair papilla cells, and after several weeks of cultivation, a large number of cells are cloned from a growing colony, and have excellent proliferative ability and normal morphology and properties. An immortalized cell line can be established by continuing the culture of clones close to cells. For example, it can be established according to the method proposed by Handa et al. (Japanese Patent Application No. 9-271927).
[0020]
Papilla cells can be cultured in a conventional medium for culturing animal cells. The concentration of TGF-β2 in the screening medium is about 0.01 ng / ml to 100 ng / ml. ₂ Done in the presence of The culture time until PAI-1 is measured is approximately 8 to 72 hours.
Fibroblasts can also be isolated and cultured by conventional methods, and immortalized cells can be established. PAI-1 is measured by a conventional method, for example, by sandwich ELISA (enzyme immunoassay) using an anti-PAI-1 antibody.
[0021]
The present invention, which was developed by the present inventors and confirmed the effectiveness of the hair restoration or hair regression phase inhibitor of the present invention by using the screening method that confirmed its effectiveness, Hereinafter, the effectiveness of the screening method will be further described with a specific confirmation test.
[0022]
[Confirmation that TGF-β2 promotes onset of regression phase]
The confirmation was performed by the following test.
(1) Confirmation study that TGF-β2 promotes the onset of the regression phase
A medium supplemented with three antibiotics, penicillin, streptomycin and fungizone, and 10 ng / ml hydrocortisone, 10 μg / ml insulin, sodium pentaselenate and 10 μg / ml transferrin, on Williams E medium (Gibco). An insulin-containing medium and a medium without addition were used as a basal medium for human hair follicle organ culture.
[0023]
Growing follicles were isolated from the human scalp using a micro-sword under a stereomicroscope. The isolated hair follicles are washed with basal medium, measured for length, and submerged in an insulin-containing medium (using a 24-well microplate: 1 ml per well) at 37 ° C., 5% CO ₂ Incubated overnight under gas phase conditions with air. After measuring the length again, hair follicles having elongation of 0.25 mm or more and maintaining the growth phase were selected and divided into groups of 10 to 12 so that the elongation was uniform.
[0024]
Each group was replaced with a medium containing a test substance, and then cultured under the above gas phase conditions. The elongation of the hair follicle was measured over time by inserting a micrometer into the eyepiece part of an inverted microscope. Pictures of the entire hair follicle and hair bulb were taken with a camera connected to an inverted microscope. On the second day of culture, the medium was replaced with a medium in which TGF-β2 was added to the basic medium at a final concentration of 50 μg / ml, and the culture was continued for another 5 days while observing hair follicle elongation and morphological changes.
[0025]
On the 6th day of culture, the hair follicles added with TGF-β2 promoted a regression-like morphological change compared to the control without addition. In other growth factors and cytokines, the above-mentioned phenomenon that promotes regression-like morphological changes was not observed. Therefore, it was considered that TGF-β2 has a regression effect.
We also observed changes in hair follicle morphology during the transition from the growth phase to the regression phase in natural hair. From the comparison of the observation results, it is clear that TGF-β2 promotes the transition to the regression phase.
[0026]
(2) Confirmation of localization of TGF-β2 in human hair follicles
Human scalp tissue or organ-cultured hair follicles are washed with PBS, fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours, dehydrated with ethanol series, embedded in paraffin, and 5 μm thick. Tissue sections were prepared.
To clarify the role of TGF-β2 in human hair follicles, we investigated the localization of TGF-β2 in human hair follicles. Observation of TGF-β2 localization in human hair follicles was performed by anti-TGF-β2 antibody immunostaining of human scalp tissue sections.
[0027]
Using a human scalp tissue section that has been deparaffinized and hydrophilically treated with the ethanol series, anti-TGF-β2 antibody (Santa Cruz) as the primary antibody, and peroxidase-labeled rabbit IgG as the secondary antibody, avidin-biotin-peroxidase complex The presence of TGF-β2 in human hair follicles was immunohistochemically stained by body method. As a result, TGF-β2 (its immunostaining property) was found in the outermost layer of the outer root sheath in the hair follicles in the late growth phase. On the other hand, in the hair follicle in the late regression phase, TGF-β2 (its immunostaining property) was observed in the outermost root sheath cells and the regressing epithelial cells in the upper part of the hair papilla. This suggests that TGF-β2 is active in the late regression phase.
[0028]
(3) Confirmation of localization of TGF-β2 in the early regression phase of human hair follicles
In the case of hair follicle organ culture, when growing hair follicle fragments are cultured in an insulin-containing medium, the growth phase is maintained for about 2 weeks, whereas when cultured in a basal medium not containing insulin. The hair follicle changes to a form resembling that of a regressing hair follicle.
[0029]
Therefore, if culturing hair follicles in a basal medium containing no insulin and examining the localization of TGF-β2 in hair follicles with slight morphological changes, is TGF-β2 working in the regression phase? Can be estimated. Therefore, after organ culture for only one day, hair follicles that retain the growth phase morphology and hair follicles that showed a morphological change slightly similar to the regression hair follicle were immunostained with anti-TGF-β2.
[0030]
As a result, hair follicles that completely maintained the growth phase showed almost no TGF-β2 (immunostaining) in the hair matrix or hair papilla.
On the other hand, in hair follicles that showed a morphological change slightly similar to that in the regressing stage, strong TGF-β2 immunostaining was observed at the boundary between the hair matrix and the hair papilla. From this, it became clear that TGF-β2 also works in vitro to induce the regression phase.
[0031]
[Confirmation of inhibitory effect of TGF-β2 on transition to hair regression stage]
From the above results, it was shown that in the human hair cycle, TGF-β2 works to induce the regression phase. That is, if the action of TGF-β2 is suppressed in the hair follicle during the growth phase, it is considered that the transition to the regression phase can be prevented or delayed. In other words, the effect of extending the hair growth period by suppressing TGF-β2 can be expected. Specifically, when a substance that suppresses the action of TGF-β2 in human hair follicle organ culture is added, hair growth is promoted or the regression phase-like morphological change is suppressed. The migration inhibition effect was verified.
[0032]
(1) Effect confirmation test of TGF-β neutralizing antibody
The effect of TGF-β neutralizing antibody (which is known to have a neutralizing action on human TGF-β1 and TGF-β2) on the transition to the hair regression phase was examined. The method followed the human hair follicle organ culture method. On the second day of culture, an anti-TGF-β antibody having a neutralizing effect on TGF-β (Genzyme) or control mouse IgG was added to the basal medium to a final concentration of 20 μg / ml or 100 μg / ml. The culture medium was replaced, and the culture was continued while observing hair follicle elongation and morphological changes for an additional 4-7 days.
According to it, the tendency for hair elongation to be promoted by addition of the TGF-β neutralizing antibody was observed. In addition, as shown in Table 1, the ratio of hair follicles in which the hair bulb portion was retained was increased by the addition of the TGF-β neutralizing antibody.
[0033]
[Table 1]

[0034]
(2) Effect confirmation test of sialoglycoprotein fetuin
Sialoglycoprotein fetuin (molecular weight 48,400) is a substance present in mammalian fetal serum and adult blood of various diseases (particularly during trauma). Fetuin has an amino acid sequence and secondary structure similar to the receptor for TGF-β and acts as an antagonist of TGF-β (Demetriou M et al: J Biol Chem 271: 12755-12761, 1996). The effect of suppressing the transition to the hair regression phase was verified.
[0035]
The medium was replaced with a medium supplemented with fetuin (final concentration 1,10,50 μM) or control bovine serum albumin (final concentration 50 μm), and further cultured for 7 days while observing hair follicle elongation and morphological changes. . Compared with the control bovine serum albumin, the hair elongation was significantly promoted by the addition of fetuin. From the results of these TGF-β neutralizing antibodies and fetuin, the effect of inhibiting the transition to the hair regression phase by inhibiting the action of TGF-β2 was demonstrated.
[0036]
[Confirmation that the inhibitory effect of TGF-β can be measured by measuring PAI-1]
Human hair papilla in a 96-well plate (approximately 7,500 cells per well) using a medium containing 10% fetal bovine serum in DMEM medium containing 3 types of antibiotics, penicillin, streptomycin and fungizone Cells were cultured. First, the TGF-β2 concentration dependency of the increase in PAI-1 expression was examined. When the cell density reached an appropriate level, the medium was replaced with a medium containing various concentrations of TGF-β2, and further cultured for 24 hours. 10 μl of the medium was taken and quantified with a commercially available PAI-1 measurement kit (TintElize PAI-1: biopool). As a result, an increase in PAI-1 was observed depending on the concentration of TGF-β2.
[0037]
Further, when 20 μg / ml neutralizing antibody of TGF-β was added simultaneously with TGF-β2, the increase in PAI-1 by TGF-β2 was completely suppressed. This result is an example in which the increase of PAI-1 is suppressed when TGF-β and a substance that cancels its action coexist, and it is shown that this evaluation method is suitable for screening for a substance that suppresses the action of TGF-β. It is shown.
[0038]
【Example】
Examples of the present invention are shown below, but the present invention is not limited by these Examples, and it is needless to say that the present invention is specified by the claims.
[0039]
[Confirmation test for ingredients contained in amateur extract]
The present inventor prepared a 50% ethanol extract of an amateur extract and analyzed the components contained therein. As a result, it was confirmed that many substances such as phyllozulcin, hydrangenol, chlorogenic acid, and umbelliferone exist. The present inventor carried out the confirmation work as described above, and it is a known fact that these substances are present in the amateur extract as shown in FIG.
[0040]
Next, the present inventor investigated, as one of them, whether or not phyllozulcin, which is an isocoumarin compound, has an action of suppressing the increase in the expression of PAI-1 by TGF-β2. Filozurcin was used by dissolving it in DMSO at a concentration of 0.05 to 50 μg / ml. After culturing for 24 hours in a medium containing 1 ng / ml TGF-β2, the amount of PAI-1 was measured using 10 μl of the medium. At the end of the culture, the survival rate of the cells was measured by the Alamar Blue method.
[0041]
It was done as follows. That is, after washing the cells twice with a new medium, the cells were replaced with a medium containing 1/10 amount of Alamar Blue (Alamar Bioscience), and 37 ° C (5% CO ₂ ) For 4 hours. After incubation, the absorbance at 595 nm and 570 nm was measured with a microplate reader to calculate the alamar blue reduction rate of each well, and this was divided by the alamar blue reduction rate of the negative control without the addition of the phyllozulcin sample. Survival rate was calculated.
[0042]
The former result is illustrated in FIG. 2, according to which phyllozulcin suppresses the increase in PAI-1 expression. That is, the vertical axis of this figure is the amount of PAI-1, which is 18 when phyllozulcin is added, which is significantly lower than 100 when no phyllozulcin is added, and has an excellent PAI-1 increase suppression effect. Understandable.
[0043]
FIG. 3 illustrates the cytotoxicity of the latter phyllozulcin. According to this, little cytotoxicity was observed in phyllozurcin. That is, the vertical axis in FIG. 3 indicates the Alamar Blue reduction rate (%). The closer this value is to 100%, the lower the cytotoxicity. The reduction rate when phyllozurcin is added is 95%, which is not added. Since the value is close to 100% of the case, it can be seen that the cytotoxicity is low.
[0044]
(2) Confirmation test of hair follicle elongation effect
Verification of the hair elongation promoting effect of phyllozulcin in the organ culture method was performed as follows. Filozulcin (Wako Pure Chemical: standard for crude drug assay) was dissolved at a concentration of DMSO 50 mg / ml. The method used followed the human hair follicle organ culture method. On the second day of the culture, the medium was replaced with a basal medium supplemented with a phyllozurcin solution at a final concentration of 0.06 μg / ml, and the culture was continued for another 7 days while observing hair follicle elongation and shape change.
[0045]
As a result, as shown in FIG. 4, hair elongation was significantly promoted in the group to which phyllozulcin 0.06 μg / ml was added on the 8th day of culture as compared with the solvent control. From this, it is clear that phyllozulcin selected as a TGF-β action inhibitory substance has a hair regression phase inhibitory effect, and the effectiveness of the present invention can be confirmed.
[0046]
(3) Confirmation test of hair elongation effect
The elongation of hair when phyllozulcin was added to human isolated hair was investigated. The results are as shown in the photograph of FIG. 5. From this photograph, it can be clearly confirmed that when phyllozulcin is added, the hair is elongated as compared with the control.
[0047]
And it is known that TGF-β2 is one of the pathogenesis of cirrhosis and nephritis, and phyllozulcin has the ability to suppress the action (antagonism) against TGF-β2 as described above. It is also clear that it has performance as an agent. In addition, isocoumarin compounds other than phyllozulcin have the ability to suppress the action (antagonism) on TGF-β2 as well as phyllozurcin, and are effective as hair nourishing agents, hair regression transition inhibitors, cirrhosis treatments, or nephritis treatments. is there.
[0048]
The present invention, as described above, provides a hair nourishing agent, especially a hair restoration containing a hydroisocoumarin, particularly phyllozulcin as an active ingredient, a hair regression transition inhibitor, a cirrhosis therapeutic agent or a nephritis therapeutic agent, of which The hair nourishing agent can be provided in the form of a pharmaceutical, a quasi drug or a cosmetic.
[0049]
About the dosage form, if the effect of this invention can be exhibited, there will be no restriction | limiting in particular, It can select arbitrarily. For example, liquid, milky, ointment, cream, gel, aerosol, mousse and the like can be exemplified. Specific examples include tonics, lotions, conditioners, scalp treatments, shampoos, and rinses.
[0050]
And in these preparations, in addition to the isocoumarin compounds containing phyllozulcin according to the present invention, components that can be usually compounded in pharmaceuticals and cosmetics can be blended within a range that does not impair the effects of the present invention. . For example, as the medicinal component, as a drug having a blood circulation promoting action, assembly extract, vitamin E and its derivatives, nicotinic acid esters such as nicotinic acid benzyl ester and the like can be mentioned.
[0051]
Examples of the drug that promotes blood circulation by local stimulation include capsicum tincture, cantharis tincture, camphor, nonyl acid vanillylamide, and the like. Examples of the agent having a hair follicle activating activity include hinokitiol, placenta extract, photosensitizer, pantothenic acid and derivatives thereof. Examples of the drug having an anti-androgenic action include hormone agents such as estradiol, ethinyl estradiol, and estrone. Anti-seborrheic agents include sulfur, thioxolone, vitamin B ₆ Etc.
[0052]
Other examples include salicylic acid and resorcin, which have keratolytic action and bactericidal action, to prevent the occurrence of dandruff, and glycyrrhizic acid and its derivatives, menthol, etc. to prevent inflammation of the scalp, and further to the hair follicle. Examples include amino acids such as serine, methionine, and arginine, vitamins such as biotin, and herbal extracts for nutritional supplementation and activation of enzyme activity.
[0053]
Altea, Yokuinin, Peppermint, Peppermint, Pepper, Aloe, Cucumber, Mugwort, Rice, Mankeishi, Mannenrou, Kosaijo, Enishida, Gentian, Tanjin, Loofah, Kyokyo, Pine, Kujijin, Toki, Safflower, Eucalyptus, Eucalyptus, Plant extracts such as Kagoso, Mokutsu, Goshitsu, Psycho, Cha, Licorice, Hops, Chrysanthemum, Senega, Sesame, Senkyu, Kashou, Kakon, Mikaika, Saffron, Rosemary, Giant, Mallow are also included.
[0054]
In addition, vasodilators such as alkoxycarbonylpyridine N-oxide, carpronium chloride, acetylcholine derivatives; skin function enhancers such as cephalanthin; hexachlorophene, benzalkonium chloride, cetylpyridium chloride, undecylenic acid, trichlorocarbanide, bithionol, Antibacterial agents such as phenol and isopropylmethylphenol; zinc and derivatives thereof; lactic acid or alkyl esters thereof; citric acid; succinic acid; organic acids such as malic acid; protease inhibitors such as tranexamic acid and the like can also be added. In addition, drugs such as cyclosporines, oxendron, diazoxide, minoxidil and the like can be blended.
[0055]
Also, alcohols such as ethanol and isopropyl alcohol; polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol; oils such as higher fatty acids, higher alcohols, hydrocarbon oils, natural oils and fats, ester oils, and silicone oils; surfactants Fragrance; chelating agent; 1,3-butylene glycol, hyaluronic acid and its derivatives, moisturizer such as maltitol, atelocollagen, sodium lactate; thickener such as quince mucilage, carboxyvinyl polymer, xanthan gum; polymer compound; Components such as antioxidants, ultraviolet absorbers, dyes, water, stabilizers and the like that are usually blended in pharmaceuticals and cosmetics can be blended.
[0056]
The isocoumarins that are the active ingredients of the composition for preventing or treating hair according to the present invention can be incorporated into external preparations for skin in pharmaceuticals, quasi-drugs, cosmetics, etc., especially for improving skin disorders such as acne. It is useful as a therapeutic agent for skin disorders. The form of the external preparation for skin is, for example, an ointment, lotion, emulsion, cream, pack, gel, foundation, lipstick, face color, skin cleansing agent, bath preparation, etc. There is no particular limitation. In addition to the kaurenes, components that can be blended in normal skin external preparations can be blended in the skin external preparation as long as no problem occurs.
[0057]
Moreover, the composition containing the isocoumarins of the present invention can be used as a pharmaceutical composition and a cosmetic composition other than those described above. The pharmaceutical composition may be either an oral or parenteral dosage form, and the dosage form is also optional, such as liquid, syrup, tablet, powder, granule, capsule, suppository or injection. It can be made into a dosage form.
[0058]
In the formulation, as necessary, diluents, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, stabilizers, solubilizers, pH adjusters, etc. What is necessary is just to manufacture by a conventional method using the additive used normally. For example, in order to obtain a liquid preparation, physiological saline, ethanol, 1,3-butylene glycol and the like can be used as a diluent or carrier.
[0059]
About the compounding density | concentration of the isocoumarin compound in the composition of this invention, although it determines suitably according to the form of a composition or the intended purpose, it is normally mix | blended 0.001 to 2weight% in a composition. If it is less than 0.001% by weight, it is difficult to sufficiently exert the effect of the present invention. On the other hand, if it exceeds 2% by weight, it is not preferable for the preparation.
In addition, the dosage is appropriately determined according to the form or purpose of use of the composition, but in the case of a hair nourishing agent or an external preparation, it is usually 0.001 to 100 mg once or several times a day It is preferable to apply or spray separately on the scalp or skin.
[0060]
【The invention's effect】
In the present invention, tumor growth factor (TGF-β2) promotes the transition from the growth phase to the regression phase in the hair growth cycle, and suppresses the transition to the regression phase by an antagonistic substance (antagonist) of TGF-β2. By a novel screening method developed based on the discovery of the present inventors that hair growth effects can be obtained, isocoumarins containing phyllozulcin can be used as a hair nourishing agent, hair regression phase transition inhibitor, liver cirrhosis therapeutic agent or nephritis therapeutic agent. It was developed by finding out that it has properties as an active ingredient.
[0061]
In the present invention, by containing isocoumarins containing phyllozulcin in these compositions, it is possible to effectively exhibit efficacy as a hair nourishing agent, a hair regression transition inhibitor, a cirrhosis therapeutic agent or a nephritis therapeutic agent. is there. In addition, isocoumarins have little cytotoxicity, and can express appropriate performance as a hair nourishing agent or a hair regression phase inhibitor. Further, the compound is also an ingredient in amateurs used for beverages, and from this point, it is an active ingredient that can be used with confidence.
[Brief description of the drawings]
FIG. 1 is a list of chemical structural formulas of known components in amateur extract.
FIG. 2 shows the results of investigating phyllozulcin for the action of suppressing the increase in PAI-1 expression by TGF-β2.
FIG. 3 shows the results of measurement of Alamar Blue reduction rate for the cytotoxicity of phyllozulcin.
FIG. 4 shows the results of a test on the effect of promoting hair follicle elongation performed on phyllozulcin.
FIG. 5 is a photograph showing the results of an investigation of hair elongation when phyllozulcin is added to isolated human hair.

Claims (2)

Active ingredient and be RuYokeryo the phyllodulcins. Hair catagen transition inhibitor shall be the active ingredient phyllodulcins.

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