JP4194669B2 - Hair treatment agent - Google Patents

Description

【００２９】
保持時間18.3分の画分のアミノ酸配列分析結果：
【００３０】
【化２】

【００３１】
（但し、Ｘは未同定のアミノ酸である。）
【００３２】
製造例３、４及び参考例３、４（ヒト毛小皮からの毛小皮由来抽出物、毛小皮由来抽出物誘導体、毛小皮特異蛋白質及び毛小皮特異蛋白質誘導体の製造）
ヒト毛小皮100 mgを用いる以外は、製造例１、２及び参考例１、２と同様にして、ヒト毛小皮からの毛小皮由来抽出物、毛小皮由来抽出物誘導体、毛小皮特異蛋白質及び毛小皮特異蛋白質誘導体を製造した（製造例３、４及び参考例３、４）。
【００３３】
得られた画分を減圧乾燥後、アプライドバイオシステムズ社製471A気相プロテインシークエンサーでアミノ酸配列を分析し、下記の結果を得た。
【００３４】
保持時間14.2分の画分のアミノ酸配列分析結果：
【００３５】
【化３】

【００３６】
保持時間18.3分の画分のアミノ酸配列分析結果：
【００３７】
【化４】

【００３８】
製造例５（変性剤及び還元剤含有緩衝液による毛小皮由来抽出物の製造）
ヒト毛小皮800 mgを、8M尿素及び 0.2M 2-メルカプトエタノールを含む0.2Mトリス（pH 9.5）3ml に加え、50℃１時間インキュベートした後、ガラスホモジナイザーを用いて擦り潰した。更に50℃１時間インキュベートした後、遠心分離（10,000 g）して上清を収集した。残渣をガラスホモジナイザーを用いて擦り潰し、50℃１時間インキュベートした後、遠心分離（10,000 g）し上清を収集する操作を、計６回繰り返し、得られた上清を集めて毛小皮由来抽出物（蛋白質量として21mg）を得た。
【００３９】
製造例６（界面活性剤及び還元剤含有緩衝液による毛小皮由来抽出物の製造）
ヒト毛小皮を抽出する溶液として、2%ドデシル硫酸ナトリウム及び 10mM ジチオスレイトールを含む0.2Mトリス（pH 9.0）3ml を用いる以外は製造例５と同様にして、毛小皮由来抽出物（蛋白質量として15mg）を得た。
【００４０】
製造例７（ヒト毛小皮からの還元剤含有緩衝液による毛小皮由来抽出物の製造）
ヒト毛小皮を抽出する溶液として、0.2M 2-メルカプトエタノールを含む0.2Mトリス（pH 9.5）3ml を用いる以外は製造例５と同様にして、毛小皮由来抽出物（蛋白質量として8.9 mg）を得た。
【００４１】
参考例５（全毛髪のパーマ液による毛小皮特異蛋白質画分の製造）
ヒト毛髪2.5gをパーマ第１液（12％チオグリコール酸アンモニウム、１％エタノールアミン、0.05％エチレンジアミン四酢酸ナトリウム、0.42％アンモニア水溶液）50mlに室温15分間浸した。５回パーマ第１液を交換して溶出液を集め、限外濾過膜を用いて濃縮し毛小皮特異蛋白質画分（蛋白質量として0.13mg）を得た。
【００４２】
製造例８、９及び参考例６、７（羊毛小皮からの毛小皮由来抽出物、毛小皮由来抽出物誘導体、毛小皮特異蛋白質及び毛小皮特異蛋白質誘導体の製造）
羊毛小皮 200mgを用い、製造例１、２及び参考例１、２の（１）と同様の方法により、毛小皮由来抽出物（蛋白質量として5.2 mg；製造例８）を得た。
【００４３】
また、この毛小皮由来抽出物（製造例８）の半量にモノヨード酢酸を25mg加える他は、製造例１、２及び参考例１、２の（２）と同様にして、毛小皮由来抽出物（カルボキシメチル化）誘導体（製造例９）を得た。
【００４４】
毛小皮特異蛋白質ならびにその毛小皮特異蛋白質（カルボキシメチル化）誘導体はSDS-ポリアクリルアミド電気泳動により分離した。上記によって得られた製造例８又は９の抽出物を、6M尿素を含む15％ゲルを用いて電気泳動した後、得られた毛小皮抽出物の分子量約7,000 のバンドならびにその毛小皮抽出物誘導体の分子量約15,000のバンドを切り取り、蛋白質溶出装置（ISCO社製、1750）を用いて抽出し、毛小皮特異蛋白質（参考例６）及び毛小皮特異蛋白質（カルボキシメチル化）誘導体（参考例７）を得た。
【００４５】
得られた画分を6M塩酸で加水分解後、日立製アミノ酸分析機でアミノ酸を分析した、下記の結果を得た。なお、システイン含量は参考例６では過ギ酸酸化後加水分解して得たシステイン酸、参考例７ではS-カルボキシメチルシステインとしての定量値である。
【００４６】
【表１】

【００４７】
比較製造例１（変性剤及び還元剤含有緩衝液による毛皮質由来抽出物の製造）
原料として、毛小皮を除いた毛髪、すなわち毛皮質 800mgを用いる以外は製造例１と同様にして、可溶性抽出物（蛋白質量として 160mg）を得た。
【００４８】
試験例１（蛋白質の分析）
毛小皮由来抽出物及び毛小皮特異蛋白質画分（製造例１，２，６，７，８，参考例５）ならびに毛皮質由来抽出物（比較製造例１）の蛋白質の分析結果を図１に示した。尚、毛小皮由来抽出物ならびに毛皮質由来抽出物中の蛋白質の分析は、毛小特異蛋白質（参考例１）ならびに毛小皮特異蛋白質（カルボキシメチル化）誘導体（参考例２）の分離に用いたSDS-ポリアクリルアミド電気泳動により行った。
【００４９】
製造例１，２，６，７，８ではいずれも低分子の蛋白質バンドが観察されるのに対し、比較製造例１では観察されなかった。
【００５０】
試験例２（蛋白質の分析）
試験例１と同様にして、製造例５，６，７，比較製造例１の蛋白質の分析を行い、結果を図２に示した。
【００５１】
製造例５，６，７ではいずれも低分子の蛋白質バンドが観察されるのに対し、比較製造例１では観察されなかった。
【００５２】
試験例３（毛小皮特異蛋白質の分析）
毛小皮由来抽出物中の毛小皮特異蛋白質の分析は、毛小皮特異蛋白質に対する特異抗体を用いて、ウエスタンブロッティング法により行った。毛小皮特異蛋白質に対する特異抗体は、アミノ酸配列に基づき合成して得られるペプチド抗原を免疫して得た抗血清を用いた。
【００５３】
ここでは抗原として、毛小皮特異蛋白質の部分アミノ酸配列に基いて合成した下記の構造を持つ多抗原性ペプチドを使用した。
【００５４】
【化５】

【００５５】
多抗原性ペプチド 250μｇを、３週間毎４回、FCA(初回免疫時) 又は、FIA(追加免疫時) と共にウサギに免疫し、初回免疫から10週後に全採血して得た抗血清を、以下の試験に用いた。
【００５６】
製造例１，２，６，７，８，参考例５で得た毛小皮由来抽出物及び毛小皮特異蛋白質画分、並びに比較製造例１で得た毛皮質由来抽出物を試験例１の方法で電気泳動した後、ＰＶＤＦ膜に転写した後、0.8%の塩化ナトリウム及び0.1%のTween 20を含む20mMトリス塩酸緩衝液で1,000倍に希釈した抗毛小皮特異蛋白質血清に浸し、１時間室温で反応させた。結合した抗体をプロテインブロッティングキット（RPN23,Amersham社製）で検出した結果を図３に示した。
【００５７】
製造例１，２，６，７，８ではいずれも低分子の蛋白質バンドへの抗毛小皮特異蛋白質特異抗体の結合が観察されるのに対し、比較製造例１では観察されなかった。従って、製造例１，２，６，７，８の毛小皮由来抽出物がいずれも毛小皮特異蛋白質を多量に含有することは明らかである。
【００５８】
試験例４（毛小皮特異蛋白質の分析）
試験例３と同様にして、製造例５〜７の毛小皮特異蛋白質の分析を行なった結果を、図４に示す。
【００５９】
製造例５〜７ではいずれも低分子の蛋白質バンドへの抗毛小皮特異蛋白質特異抗体の結合が観察されるのに対し、比較製造例１では観察されなかった。従って、製造例５〜７の毛小皮由来抽出物がいずれも毛小皮特異蛋白質を多量に含有することは明らかである。
【００６０】
実施例１〜５（毛小皮由来抽出物含有毛髪処理剤）
製造例１，２，６，７，８で得た毛小皮由来抽出物及び毛小皮由来抽出物誘導体10mgを20mM2-メルカプトエタノールを含む生理リン酸緩衝液１mlに溶解し、毛小皮由来抽出物含有の毛髪損傷修復用毛髪処理剤（実施例１〜５）を調製した。
【００６１】
試験例５（損傷毛髪に対する修復効果）
予め、パーマ処理を３回施した毛髪７束を、実施例１〜５の毛小皮由来抽出物含有毛髪損傷修復剤に各々室温で30分間浸した後、水洗した。無処理毛髪（健常毛），修復剤処理前及び処理後のパーマ処理毛髪の３種の毛髪に、キューティクルの捲れが観察し易い様にそれぞれ結び目を作り、走査型電子顕微鏡観察を行った。パーマ処理毛髪に観察されるキューティクルの捲れが、いずれの処理剤で処理することによっても健常毛程度に修復した様子が観察された。
【００６２】
実施例６〜１１（毛小皮由来抽出物含有毛髪トリートメント剤）
下記に示す組成の、製造例１，２，６，７，８，９で得た毛小皮由来抽出物及び毛小皮由来抽出物誘導体を含有した毛髪トリートメント剤（実施例６〜１１）を調製した。
【００６３】
【表２】

【００６４】
試験例６（毛小皮由来抽出物含有毛髪トリートメント剤の官能試験）
ヒト毛束（黒髪5g）を実施例６〜１１の毛小皮由来抽出物含有毛髪トリートメント剤に１時間浸漬後、流水で10分間水洗して風乾した。処理後毛束のくし通り、しなやかさ、風合について処理前の毛束とどちらがよいか官能評価を行った結果、いずれも毛小皮由来抽出物含有毛髪トリートメント剤で処理した毛束の方がくし通り、しなやかさ、風合が良好であった。
【００６５】
応用例１（毛小皮由来抽出物を利用した毛髪診断キット）
以下に記載した４種の溶液よりなる毛髪診断キットを作製し、ヒト毛髪で損傷度を診断した。
【００６６】
Ａ液：製造例７で得た毛小皮由来抽出物１mgを、３週間毎４回、FCA(初回免疫時) 又は、FIA(追加免疫時) と共にウサギに免疫し、初回免疫から10週後に全採血して得た抗血清を、0.05%Tween 20 を含むPBS で1,000 倍希釈して作製した。
【００６７】
Ｂ液：ペルオキシダーゼ標識ヤギ抗ウサギIgG 抗体（カッペル社製）を、0.05%Tween20を含むPBS で7,000 倍希釈して作製した。
【００６８】
Ｃ液：オルトフェニレンジアミン20mg及び過酸化水素10μｌを含む0.2Mリン酸二ナトリウム−0.1Mクエン酸緩衝液, pH 5.0（ペルオキシダーゼの基質溶液）
【００６９】
Ｄ液：生理食塩水
【００７０】
髪の傷んだ人と健常な人の毛髪10mgにＡ液を加え37℃、１時間インキュベートした。Ｄ液で６回洗浄した後、Ｂ液と室温、30分間インキュベートした。Ｄ液で６回洗浄した後、Ｃ液を加えると、傷んだ人の毛髪を入れた試験管が発色した。
【００７１】
以上の結果から、毛小皮由来抽出物を抗原として得た抗体で、毛髪の損傷状態あるいは健康状態を知ることができることは明らかである。
【００７２】
尚、Ａ液を加え37℃、１時間インキュベートした時点で、傷んだ毛髪の方は、ハリが戻る等の損傷修復効果も見られた。このことから、本発明の毛小皮由来抽出物を抗原として得た抗体は、毛髪の損傷の修復・予防にも有用であることが明らかとなった。
【００７３】
【発明の効果】
以上の結果から、本発明により、毛髪の損傷状態あるいは健康状態を知るマーカーとして，あるいは損傷毛の修復および予防用素材として有用な、毛小皮由来抽出物、毛小皮由来抽出物誘導体、及び該抽出物を配合した毛髪処理剤を提供できることは明らかである。
【図面の簡単な説明】
【図１】 毛小皮由来抽出物、毛小皮由来抽出物誘導体及び毛皮質由来抽出物を、SDS-ポリアクリルアミド電気泳動（6M尿素存在下、15％ゲル）により分離し、蛋白質をクマシーブリリアントブルー（CBBG）で染色した図面代用写真である。
矢印は、毛小皮特異蛋白質のバンドを示す。
レーン１；製造例１の蛋白質画分
レーン２；製造例２の蛋白質画分
レーン３；製造例６の蛋白質画分
レーン４；製造例７の蛋白質画分
レーン５；参考例５の蛋白質画分
レーン６；製造例８の蛋白質画分
レーン７；比較製造例１の蛋白質画分
【図２】 毛小皮由来抽出物及び毛皮質由来抽出物を、SDS-ポリアクリルアミド電気泳動（6M尿素存在下、15％ゲル）により分離し、蛋白質をクマシーブリリアントブルー（CBBG）で染色した図面代用写真である。
矢印は、毛小皮特異蛋白質のバンドを示す。
レーン１；製造例５の蛋白質画分
レーン２；製造例６の蛋白質画分
レーン３；製造例７の蛋白質画分
レーン４；比較製造例１の蛋白質画分
【図３】 毛小皮由来抽出物、毛小皮由来抽出物誘導体及び毛皮質由来抽出物の毛小皮特異蛋白質を分析した図面代用写真である。試験例１の条件で電気泳動後、PVDF膜に転写し、毛小皮特異蛋白質の特異抗体で検出した。
矢印は、毛小皮特異蛋白質のバンドを示す。
レーン１；製造例１の蛋白質画分
レーン２；製造例２の蛋白質画分
レーン３；製造例６の蛋白質画分
レーン４；製造例７の蛋白質画分
レーン５；参考例５の蛋白質画分
レーン６；製造例８の蛋白質画分
レーン７；比較製造例１の蛋白質画分
【図４】 毛小皮由来抽出物及び毛皮質由来抽出物の毛小皮特異蛋白質を分析した図面代用写真である。試験例２の条件で電気泳動後、PVDF膜に転写し、毛小皮特異蛋白質の特異抗体で検出した。
矢印は、毛小皮特異蛋白質のバンドを示す。
レーン１；製造例５の蛋白質画分
レーン２；製造例６の蛋白質画分
レーン３；製造例７の蛋白質画分
レーン４；比較製造例１の蛋白質画分[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a hair treatment agent useful for preventing or repairing hair damage.
[0002]
[Prior art]
It is known that hair is damaged by permanent treatment or the like, and cuticles curl or decrease in the number of sheets. At this time, there is a report that the protein is eluted from the hair, but its origin is unknown.
[0003]
In addition, attempts have been made to supplement hair coating agents such as silicon, hair keratin, hydrolysates thereof or derivatives thereof as repair agents for damaged hair. However, since hair keratin is an insoluble protein and has completely different physical properties from the protein eluted by perm treatment, it is difficult to obtain a sufficient repair effect on damaged hair.
[0004]
[Problems to be solved by the invention]
The present inventors have result of intensive investigations in view of such circumstances, obtained by performing an extraction operation from the cuticle, specific proteins constituting the surface layer of the hair of the components of the protein eluted by damage of the hair The present invention is completed by finding that the protein can be efficiently produced by extracting the hair with a buffer containing a reducing agent, and that the protein is useful as a hair treatment agent containing the protein. be those leading to, its object is useful in hair damage prevention or damage hair repair, to provide a hair treatment agent.
[0005]
[Means for Solving the Problems]
Therefore, the above-mentioned object is to obtain an extract derived from a hair dermis extracted from a hair dermis of a mammal , or a hair dermis derived extract obtained by modifying all or part of the SH group of a protein. It is achieved by a hair treatment agent characterized by compounding.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the configuration of the present invention will be described in detail.
[0007]
As a method obtaining the cuticle (cuticle) used in the present invention include a method that may be separated from the known hair. For example, the hair can be physically peeled off by shaking in water, stirring or ultrasonication (see Journal of the Society of Cosmetic Chemists, 25, 13, 1974). Exfoliated cuticle, by filtration through a gauze or mesh, after separation from the hair, can be collected by centrifugation or filtration. The hair used in the present invention includes human hair, wool, pig hair and the like.
[0008]
As a method for producing cuticle from extracts of the invention, the cuticle obtained by separation from the example collected hair hair, buffer containing only (1) a reducing agent, (2) a reducing agent and soluble Examples include a method of extracting with an extract such as a buffer containing a solubilizer. In particular, when performing the extraction with a buffer containing only the reducing agent (1), it is possible to obtain with less inclusions cuticle derived extract preferably.
[0009]
Extraction operation, performing solution of extraction buffer hair small skin by alkaline. It is preferable that the temperature is 25 to 100 ° C.
[0010]
Also, either when manufacturing the cuticle derived extract from cuticle is stirred a cuticle in extract and the cuticle crush rubs in the extract by using a homogenizer or the like, efficiently present invention the preferred hair cuticle from the extract obtained in.
[0011]
Examples of the reducing agent used in the present invention include mercaptoethanol, dithiothreitol, sodium sulfite, ammonium thioglycolate and the like.
[0012]
The concentration of the reducing agent used in the present invention alone is preferably 1 to 3% by weight of the total amount of the buffer solution.
[0013]
As the solubilizer used in the extract, well-known and publicly known solubilizers, solubilizers such as surfactants can be used. Examples of the denaturants include urea, guanidine hydrochloride, and the like. Is sodium dodecyl sulfate.
[0014]
As hair methods for making a small skin extract derived from derivatives of the present invention, a protein SH groups contained in the cuticle from the extract of the present invention for example, include conventional methods for modifying with monoiodoacetic acid and ethylene imine It is done.
[0015]
Cuticle extract derived from derivatives of the present invention can be applied to a wide range because even in the absence of a reducing agent and a solubilizer are dissolved in water.
[0016]
Cuticle derived extract of the present invention, and since cuticle derived extract derivative itself is a protein that contains a large amount of S-S bridge or SH group, directly shampoos, rinses, hair treatment agents such as hair treatments It can also be used as a protective component for hair by blending.
[0017]
Cuticle derived extract of the present invention, treatment is as dosage form for hair treatment comprising a cuticle derived extract derivative, rinse, shampoo, hair cream, hair lotion, hair pack, permanent dosage etc., to the hair And various types used.
[0018]
The content of cuticle from the extract and cuticle extract derived from derivatives of the present invention may comprise from 0.01% to 5% preferably 0.1% to 1% by weight based on the total amount of the composition are particularly preferred.
[0019]
In addition, cuticle derived extract of the present invention, and cuticle extract derived from derivatives are useful, for example, as an antigen when producing an antibody that specifically recognizes the cuticle.
[0020]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
[0021]
Production Examples 1, 2 and Reference Examples 1 and 2 (preparation of cuticle derivative extracts cuticle derived extract derivative, cuticle specific proteins and cuticle specific protein derivatives from human cuticle)
(1) Human cuticle 800 mg, was added to 0.2M Tris (pH 9.5) 3 ml containing 8M urea and 0.2M 2-mercaptoethanol and incubated 50 ° C. 1 hour, crushed rubbed using a glass homogenizer. After further incubation 50 ° C. 1 h, the supernatant collected by centrifugation (10,000 g), cuticle derived extract (21 mg as protein mass; Production Example 1) was obtained.
[0022]
(2) In addition the monoiodoacetic acid 75mg to half of the cuticle from extract (Production Example 1), and pH 9.0 with Tris, and 1.5 hr stirring in the dark at room temperature. 10mM Tris - give the hydrochloride buffer cuticle derived extract was dialyzed against (pH 8.0) (carboxymethylated) derivative (preparation 2).
[0023]
(3) cuticle specific proteins and their cuticle specific protein (carboxymethylated) derivatives were separated by SDS- polyacrylamide electrophoresis. The resulting Production Example 1 or 2 of extract by above was electrophoresed by using a 15% gel containing 6M urea, and transferred to a polyvinylidene difluoride (PVDF) membrane, resulting cuticle extract cut a molecular weight of about 7,000 bands and molecular weight of about 15,000 band of the cuticle extract derivative of a product, acetic acid / acetonitrile / water and extracted with a mixed solution of (7: 2: 1), cuticle specific proteins (reference example 1) and was obtained cuticle specific protein (carboxymethylated) derivative (reference example 2).
[0024]
After cuticle specific protein on the PVDF membrane (carboxymethylated) derivative was treated with 0.5% polyvinyl pyrrolidone and reacted at 24 hours 37 ° C. was added trypsin (Promega). The resulting degradation product was subjected to reverse phase liquid chromatography under the following conditions, and fractions having retention times of 14.2 minutes and 18.3 minutes were collected.
[0025]
Column: A-312 ODS (6.O x 150 mm, YMC)
Elution: Water-acetonitrile-trifluoroacetic acid (90: 10: 0.1) to water-acetonitrile-trifluoroacetic acid (30: 70: 0.1) linear concentration gradient (concentration gradient over 60 minutes)
Flow rate: 2.O ml / min
Detection: Absorbance at 220 nm
The obtained fraction was dried under reduced pressure, and then the amino acid sequence was analyzed with a 471A gas phase protein sequencer manufactured by Applied Biosystems. The following results were obtained.
[0027]
Results of amino acid sequence analysis of a fraction with a retention time of 14.2 minutes:
[0028]
[Chemical 1]

[0029]
Results of amino acid sequence analysis of the fraction with a retention time of 18.3 minutes:
[0030]
[Chemical 2]

[0031]
(However, X is an unidentified amino acid.)
[0032]
Production Examples 3 and 4 and Reference Examples 3 and 4 (manufacture of cuticle derivative extracts cuticle derived extract derivative, cuticle specific proteins and cuticle specific protein derivatives from human cuticle)
But using human cuticle 100 mg, in the same manner as in Preparation Example 1, 2 and Reference Examples 1 and 2, cuticle from extracts from human cuticle, cuticle extract derived from derivatives, hair Small It was produced skin-specific protein and cuticle specific protein derivatives (preparation 3, 4 and reference examples 3 and 4).
[0033]
The obtained fraction was dried under reduced pressure, and then the amino acid sequence was analyzed with a 471A gas phase protein sequencer manufactured by Applied Biosystems. The following results were obtained.
[0034]
Results of amino acid sequence analysis of a fraction with a retention time of 14.2 minutes:
[0035]
[Chemical 3]

[0036]
Results of amino acid sequence analysis of the fraction with a retention time of 18.3 minutes:
[0037]
[Formula 4]

[0038]
Production Example 5 (Production of cuticle from the extract by denaturing and reducing agents containing buffer)
The 800 mg human cuticle, in addition to 0.2M Tris (pH 9.5) 3 ml containing 8M urea and 0.2M 2-mercaptoethanol and incubated 50 ° C. 1 hour, crushed rubbed using a glass homogenizer. After further incubation at 50 ° C. for 1 hour, the supernatant was collected by centrifugation (10,000 g). The residue is triturated with a glass homogenizer, after incubation 50 ° C. 1 h, the operation of collecting the centrifuged (10,000 g) and the supernatant, repeated six times, from cuticle collecting resulting supernatant An extract (21 mg as protein mass) was obtained.
[0039]
Production Example 6 (Production of cuticle from the extract with a surfactant and a reducing agent-containing buffer solution)
As a solution for extracting the human cuticle, 0.2 M Tris (pH 9.0) containing 2% sodium dodecyl sulfate and 10mM dithiothreitol except using 3ml in the same manner as in Production Example 5, cuticle derived extract (protein 15 mg) was obtained.
[0040]
Production Example 7 (Production of cuticle from the extract with a reducing agent-containing buffer from human cuticle)
As a solution for extracting the human cuticle, 0.2 M Tris (pH 9.5) containing 0.2 M 2-mercaptoethanol except using 3ml in the same manner as in Production Example 5, cuticle derived extract (8.9 mg as protein content )
[0041]
Reference Example 5 (Production of hair cuticle-specific protein fractions by the perm solution of all hair)
2.5 g of human hair was immersed in Perm 1st liquid (12% ammonium thioglycolate, 1% ethanolamine, 0.05% sodium ethylenediaminetetraacetate, 0.42% aqueous ammonia) for 15 minutes at room temperature. 5 times to replace the permanent first liquid collected eluant to afford an ultrafiltration membrane and concentrated using a cuticle-specific protein fraction (0.13 mg as protein mass).
[0042]
Production Examples 8, 9 and Reference Examples 6 and 7 (cuticle from extracts from wool cuticle, production of cuticle from extract derivative, cuticle specific proteins and cuticle specific protein derivatives)
Using wool cuticle 200mg, in the same manner as in Production Example 1, 2 and Reference Examples 1 and 2 (1), cuticle derived extract (5.2 mg as protein mass; Production Example 8) was obtained.
[0043]
Another added 25mg is a monoiodoacetic acid to half of the cuticle from extract (Preparation Example 8), in the same manner as in Production Example 1, 2 and Reference Examples 1 and 2 (2), cuticle extract derived from A product (carboxymethylated) derivative ( Production Example 9 ) was obtained.
[0044]
Cuticle specific proteins and their cuticle specific protein (carboxymethylated) derivatives were separated by SDS- polyacrylamide electrophoresis. The resulting preparation 8 or 9 extracts the above, was electrophoresed by using a 15% gel containing 6M urea, resulting hair molecular weight of about 7,000 bands and their cuticle Extraction of cuticle extract cut a molecular weight of about 15,000 band of articles derivatives, protein dissolution apparatus (ISCO, Inc., 1750) was extracted using a cuticle-specific proteins (reference example 6) and cuticle specific protein (carboxymethylated) derivative ( Reference Example 7 ) was obtained.
[0045]
The obtained fraction was hydrolyzed with 6M hydrochloric acid, and amino acids were analyzed with an amino acid analyzer manufactured by Hitachi. The following results were obtained. The cysteine content is a quantitative value as cysteic acid obtained by hydrolysis after formic acid oxidation in Reference Example 6 , and as S-carboxymethylcysteine in Reference Example 7 .
[0046]
[Table 1]

[0047]
Comparative Production Example 1 ( Production of fur- derived extract with a denaturing agent and a reducing agent-containing buffer)
As the raw material, the hair except the hair cuticle, ie except for using cortex 800mg in the same manner as in Preparation Example 1 to obtain a soluble extract (160 mg as a protein amount).
[0048]
Test Example 1 (protein analysis)
Cuticle derived extract and cuticle specific protein fraction (preparation 1,2,6,7,8, Reference Example 5) FIG results of analysis of proteins and cortex-derived extract (Comparative Production Example 1) It was shown in 1. The analysis of the cuticle from the extract and protein cortex-derived extract is, hair small specific protein isolation (Example 1) and cuticle specific protein (carboxymethylated) derivative (Reference Example 2) This was performed by SDS-polyacrylamide electrophoresis.
[0049]
In Production Examples 1 , 2 , 6 , 7 , and 8, low molecular weight protein bands were observed, whereas in Comparative Production Example 1, they were not observed.
[0050]
Test Example 2 (protein analysis)
In the same manner as in Test Example 1, the proteins of Production Examples 5 , 6 , 7 and Comparative Production Example 1 were analyzed, and the results are shown in FIG.
[0051]
In Production Examples 5 , 6 , and 7 , low molecular weight protein bands were observed, whereas in Comparative Production Example 1, they were not observed.
[0052]
Test Example 3 (analysis of the hair cuticle-specific protein)
Analysis of cuticle from hair extract cuticle specific proteins using specific antibodies against cuticle specific proteins was carried out by Western blotting. The specific antibody against cuticle specific proteins using the antiserum obtained by immunizing peptide antigen obtained by, based on the amino acid sequence synthesis.
[0053]
As here antigens, using multiple antigenic peptides with synthesized having the following structure based on the partial amino acid sequence of the cuticle-specific proteins.
[0054]
[Chemical formula 5]

[0055]
Antiserum obtained by immunizing rabbits with 250 μg of multi-antigenic peptide 4 times every 3 weeks with FCA (at the time of the first immunization) or FIA (at the time of the booster immunization) Used in the test.
[0056]
Preparation 1,2,6,7,8, Reference Example cuticle derived extract was obtained at 5 and cuticle specific protein fraction, and Comparative Preparation Example 1 obtained in the cortex extract derived from Test Example 1 after electrophoresis method, after transferred to a PVDF membrane immersed in anti-cuticle specific protein serum diluted 1,000-fold with 20mM tris-HCl buffer containing 0.8% sodium chloride and 0.1% Tween 20, 1 The reaction was performed at room temperature for an hour. The result of detecting the bound antibody with a protein blotting kit (RPN23, manufactured by Amersham) is shown in FIG.
[0057]
While both in the manufacturing example 1,2,6,7,8 binding of anti-cuticle specific protein specific antibodies to protein bands of lower molecular observed, it was not observed in Comparative Production Example 1. Therefore, it is clear cuticle from extracts of Preparation 1,2,6,7,8 of containing a large amount of both cuticle specific proteins.
[0058]
Test Example 4 (analysis of the hair cuticle-specific protein)
In the same manner as in Test Example 3, the result of performing an analysis of cuticle specific proteins of Preparation 5-7, shown in FIG.
[0059]
While both in the manufacturing example 5-7 binding of anti-cuticle specific protein specific antibodies to protein bands of lower molecular observed, it was not observed in Comparative Production Example 1. Therefore, it is clear cuticle from extracts of Preparation 5 to 7 which contains a large amount of both cuticle specific proteins.
[0060]
Examples 1-5 (cuticle derived extract-containing hair treatment)
Dissolved cuticle derived extracts prepared in Preparation Examples 1,2,6,7,8 and cuticle derived extract derivative 10mg physiological phosphate buffer 1ml containing 20mM2- mercaptoethanol, from cuticle An extract- containing hair treatment agent for hair damage repair (Examples 1 to 5 ) was prepared.
[0061]
Test Example 5 (Repair effect on damaged hair)
Previously, the 7 bundles hair subjected 3 times the permanent process, it was immersed for 30 minutes each at room temperature cuticle derived extract-containing hair damage repair agents of Examples 1 to 5 was washed with water. A knot was made on each of the three types of untreated hair (healthy hair), perm treated hair before and after treatment with the restoration agent so that cuticle wrinkles could be easily observed, and observed with a scanning electron microscope. It was observed that the curl of the cuticle observed in the perm-treated hair was restored to the level of healthy hair by treatment with any treatment agent.
[0062]
Example 6-11 (cuticle derived extract-containing hair treatment agent)
Having the following composition, the hair obtained in Production Example 1,2,6,7,8,9 cuticle derived extract and cuticle derived extract derivative hair treatment agent containing (Example 6-11) Prepared.
[0063]
[Table 2]

[0064]
Test Example 6 (Sensory Test of cuticle from extract-containing hair treatment agent)
After 1 hour immersion human hair bundle (black hair 5g) in cuticle derived extract-containing hair treatment agent of Example 6 to 11, air-dried and washed with running water for 10 minutes. Combing of processing after the hair bundle, suppleness, comb towards the pre-treatment of the hair bundle and the result of which makes a good or sensory evaluation, the hair bundle both treated with the hair cuticle from the extract-containing hair treatment agent for feeling Street, suppleness and texture were good.
[0065]
Application Example 1 (hair hair diagnostic kit using a small skin-derived extract)
A hair diagnosis kit composed of the four types of solutions described below was prepared, and the degree of damage was diagnosed with human hair.
[0066]
A solution: the cuticle extract derived from 1mg obtained in Production Example 7, 3 weeks every 4 times, FCA (at first immunization) or, immunizing a rabbit with FIA (time booster), 10 weeks after the first immunization Antiserum obtained by whole blood collection was prepared by diluting 1,000 times with PBS containing 0.05% Tween 20.
[0067]
Solution B: Peroxidase-labeled goat anti-rabbit IgG antibody (manufactured by Kappel) was prepared by diluting 7,000 times with PBS containing 0.05% Tween20.
[0068]
Solution C: 0.2 M disodium phosphate-0.1 M citrate buffer solution containing 20 mg orthophenylenediamine and 10 μl hydrogen peroxide, pH 5.0 (peroxidase substrate solution)
[0069]
D liquid: physiological saline [0070]
The solution A was added to 10 mg of hair of a person with damaged hair and a healthy person and incubated at 37 ° C. for 1 hour. After washing 6 times with solution D, it was incubated with solution B for 30 minutes at room temperature. After washing 6 times with solution D, when solution C was added, the test tube containing the damaged person's hair developed color.
[0071]
From the above results, an antibody was obtained cuticle derived extract as an antigen, it is clear that it is possible to know the damage state or health of the hair.
[0072]
In addition, when the solution A was added and incubated at 37 ° C. for 1 hour, the damaged hair also showed an effect of repairing damage such as rejuvenation. Therefore, antibodies to the cuticle from extracts of the present invention was obtained as an antigen, was found to be useful in the repair and prevention of damage to the hair.
[0073]
【The invention's effect】
From the above results, the present invention, as a marker know damage conditions or the health of the hair, or useful as damage hair repair and prophylactic material, cuticle derivative extracts cuticle extract derived from derivatives and, It is clear that a hair treatment agent containing the extract can be provided.
[Brief description of the drawings]
[1] cuticle derived extract, the cuticle extract derived from derivatives and cortex-derived extract, SDS-polyacrylamide electrophoresis (6M urea presence, 15% gel) was separated by Coomassie Brilliant proteins It is a drawing substitute photograph dye | stained with blue (CBBG).
The arrow indicates the band of hair cuticle-specific protein.
Lane 1; protein fraction lane 2 of Production Example 1; protein fraction lane 3 of Production Example 2 ; protein fraction lane 4 of Production Example 6 ; protein fraction lane 5 of Production Example 7 ; protein fraction of Reference Example 5 lane 6; protein fraction lane 7 of preparation 8; the protein fraction of Comparative production example 1 [2] cuticle derived extract and cortex-derived extract, SDS-polyacrylamide electrophoresis (6M urea presence , 15% gel), and is a drawing-substituting photograph in which the protein is stained with Coomassie Brilliant Blue (CBBG).
The arrow indicates the band of hair cuticle-specific protein.
Lane 1; protein fraction Lane 2 of Preparation 5, protein fraction Lane 4 of Production Example 7; protein fraction Lane 3 of Preparation 6 protein fraction of Comparative Production Example 1 [3] cuticle extract derived from things, a drawing-substitute photograph of analyzing the cuticle specific protein cuticle extract derived from derivatives and cortex-derived extract. After electrophoresis under the conditions of Test Example 1, it was transferred to a PVDF membrane and detected with specific antibodies of cuticle specific proteins.
The arrow indicates the band of hair cuticle-specific protein.
Lane 1; protein fraction lane 2 of Production Example 1; protein fraction lane 3 of Production Example 2 ; protein fraction lane 4 of Production Example 6 ; protein fraction lane 5 of Production Example 7 ; protein fraction of Reference Example 5 lane 6; a drawing substitute photograph of analyzing the cuticle specific protein protein fraction [4] cuticle derived extract of Comparative manufacturing example 1 and the cortex-derived extract; protein fraction lane 7 of preparation 8 is there. After electrophoresis under the conditions of Test Example 2, and transferred to a PVDF membrane and detected with specific antibodies of cuticle specific proteins.
The arrow indicates the band of hair cuticle-specific protein.
Lane 1; Protein fraction lane 2 of Production Example 5 ; Protein fraction lane 3 of Production Example 6 ; Protein fraction lane 4 of Production Example 7 ; Protein fraction of Comparative Production Example 1

Claims (4)

A hair treatment agent comprising a hair dermis-derived extract obtained by extraction from mammalian hair dermis under alkaline reducing conditions . The hair according to claim 1, wherein the hair cuticle-derived extract is a hair skin-derived extract derivative obtained by modifying all or part of the SH groups of the protein contained in the extract. Treatment agent . The hair treatment agent according to claim 2, wherein the modification of the SH group is modification with monoiodoacetic acid or ethyleneimine . The hair treatment agent according to any one of claims 1 to 3, wherein the mammal is selected from humans, sheep or pigs .

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