JPH09202716A - Hair tonic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hair tonic having effects on hair growth, hair restoration and prevention of hair removal, by using a new peptide having a promoting action on proliferation of hair papilla cell prepared from a pericarp or a sarcocarp of a citrus fruit. SOLUTION: A pericarp of a citrus fruit such as Amanatu (modified Citrus natsudaidai), Citrus iyo, Citrus hassaku, mandarin or lemon is extracted with an alcohol such as methanol and the alcohol is distilled away from a filtrate to give a dried substance. The dried substance is made into an aqueous solution. In the case of a sarcocarp, a crude fruit juice is pressed, the crude fruit juice is filtered to give a filtrate, which is made into a solution of fruit juice. Then the aqueous solution or the solution of fruit juice is adsorbed on an ion exchange resin, eluted with an eluting solvent such as diluted ammonia water and the eluted solution is concentrated under reduced pressure to give a crude substance. The crude substance is purified and used. The peptide thus obtained is dissolved in water or a hydrous alcohol and into pharmaceutically manufactured into a hair tonic, shampoo, hair cream, etc., and used. The hair tonic is preferably mixed with a germicidal component such as an aloe essence. The peptide contains aspartic acid and oxylysine.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】この発明は、柑橘類由来の毛
乳頭細胞増殖促進作用を有するペプチドを含有し、発
毛、育毛促進および脱毛防止効果のある育毛剤に関す
る。TECHNICAL FIELD The present invention relates to a hair-growing agent containing a peptide having a citrus-derived hair papilla cell proliferation-promoting action and having hair growth, hair-growth promotion and hair loss prevention effects.

【０００２】[0002]

【従来の技術】脱毛の原因として、男性ホルモン関与に
よる毛包機能の低下、毛包・毛球部の新陳代謝機能の低
下、頭皮生理機能の低下、頭皮緊張による局所血流障
害、栄養不良、ストレス、薬物による副作用、遺伝等の
要因があげられるが、まだ脱毛の原因は完全には解明さ
れていない。このため、市販の育毛剤に含まれる有効成
分は、頭皮の血行促進、毛根の刺激や毛包賦活等の作用
により、二次的に毛関連細胞を活性化させるものであ
る。この出願人は、先に各種柑橘類の果皮およびアロエ
の日本酒による抽出液の混合液が育毛効果のあることを
見い出した（特開平３−２２７９１１）が、薬効の本体
までは解明されていなかった。2. Description of the Related Art As a cause of hair loss, a decrease in hair follicle function due to male hormone involvement, a decrease in metabolic function of hair follicles / bulbs, a decrease in physiological function of the scalp, a local blood flow disorder due to scalp tension, malnutrition, stress Factors such as drug side effects and heredity can be mentioned, but the cause of hair loss has not been completely clarified. Therefore, the active ingredient contained in the commercially available hair restorer secondaryly activates hair-related cells by the action of promoting blood circulation in the scalp, stimulating hair roots and activating hair follicles. The applicant previously found that a mixture of various citrus peels and aloe extract of Japanese sake has a hair-growing effect (Japanese Patent Laid-Open No. 3-227911), but the medicinal effect has not been clarified.

【０００３】[0003]

【発明が解決しようとする課題】発毛や脱毛メカニズム
の基礎的な研究に基づいて直接毛関連細胞を活性化する
育毛剤が要望されている。この発明の目的は、上記の観
点から、発毛のメカニズムを担っている毛乳頭細胞に直
接作用して、該細胞の増殖を促進するペプチドを含有す
る育毛剤を提供するものである。There is a need for a hair-growing agent that directly activates hair-related cells based on basic research on hair growth and hair loss mechanisms. From the above viewpoints, an object of the present invention is to provide a hair-growing agent containing a peptide which directly acts on hair papilla cells which are responsible for the mechanism of hair growth and promotes the growth of the cells.

【０００４】[0004]

【課題を解決するための手段】この発明者は、天然産物
中に毛乳頭細胞増殖促進作用を示す生理活性物質を広範
囲に探索した結果、柑橘類の果皮、果肉エキスから得ら
れた新規ペプチドが該活性を有することを見い出した。Means for Solving the Problems The present inventor has extensively searched for a physiologically active substance having a dermal papilla cell proliferation promoting action in natural products, and as a result, a novel peptide obtained from citrus peels and pulp extracts has been identified. It was found to have activity.

【０００５】この発明は、柑橘類由来の次の物理化学的
性質を有するペプチドを含有する育毛剤である。 （１）分子量：２，０００〜４，０００（ゲルろ過法に
よる） （２）赤外線吸収スペクトル（ＫＢｒ法）：図１に示す
とおりであり、３，３５６、３，０８０、１，６７６、
１，６４７、１，６１４、１，５００、１，４２５、
１，４０２、１，３５８、１，３３６、１，２５０、
１，１５１、１，０７２、９８５cm^-1に吸収帯を有す
る。紫外線吸収スペクトル（水中）は、２００nmまで特
徴的な吸収極大を示さない。 （３）溶解性：水に可溶。エーテル、ベンゼン、クロロ
ホルムに難溶。 （４）呈色反応：ニンヒドリン反応およびニトロプルシ
ドナトリウム反応は陽性；アニスアルデヒド反応、フェ
ノール硫酸反応およびドラーゲンドルフ反応は陰性。 （５）塩基性、中性、酸性の区別：中性 （６）性状：白色の結晶または結晶性粉末であって、わ
ずかに特有の臭いを有する。 （７）構成アミノ酸：ホスホセリン、アスパラギン酸、
セリン、グルタミン酸、グリシン、アラニン、バリン、
シスチン、メチオニン、オキシリシン、オルニチン、リ
シンおよびヒスチジンを含み、そのモル比はリシンを１
とした場合、それぞれ約８：９，１７４：８４：４：
６：４：４：１７：２：１３，６０６：２：１：２であ
り、オキシリシンおよびアスパラギン酸を多く含む。他
にタウリン、ホスホエタノールアミン、尿素を含む。The present invention is a hair restorer containing a peptide derived from citrus fruits having the following physicochemical properties. (1) Molecular weight: 2,000 to 4,000 (by gel filtration method) (2) Infrared absorption spectrum (KBr method): As shown in FIG. 1, 3,356, 3,080, 1,676,
1,647, 1,614, 1,500, 1,425,
1,402, 1,358, 1,336, 1,250,
It has absorption bands at 1,151,1,072,985 cm ^-1 . The UV absorption spectrum (in water) does not show a characteristic absorption maximum up to 200 nm. (3) Solubility: Soluble in water. Hardly soluble in ether, benzene and chloroform. (4) Color reaction: ninhydrin reaction and sodium nitroprusside reaction are positive; anisaldehyde reaction, phenol sulfuric acid reaction and Dragendorff reaction are negative. (5) Distinguishing between basic, neutral, and acidic: neutral (6) Property: white crystals or crystalline powder with a slight unique odor. (7) Constituent amino acids: phosphoserine, aspartic acid,
Serine, glutamic acid, glycine, alanine, valine,
Contains cystine, methionine, oxylysine, ornithine, lysine and histidine, the molar ratio of which is 1 lysine
, Respectively, about 8: 9, 174: 84: 4:
It is 6: 4: 4: 17: 2: 13, 606: 2: 1: 2 and is rich in oxylysine and aspartic acid. Others include taurine, phosphoethanolamine, and urea.

【０００６】この発明の育毛剤の有効成分であるペプチ
ドは、次のようにして得ることができる。甘夏カン、イ
ヨカン、ハッサク、ミカン、レモン系の柑橘類の果皮を
メタノール、エタノールのようなアルコールで抽出し、
抽出液をろ過して得たろ液からアルコールを留去し、乾
燥物を得る。この乾燥物を精製水に溶解して水溶液とす
る。果肉の場合は、粗果汁液として絞り、粗果汁液をろ
過して得たろ液を果汁溶液とする。The peptide which is the active ingredient of the hair-growing agent of the present invention can be obtained as follows. Amanatsukan, Iyokan, Hassaku, Mandarin, Lemon-based citrus peels are extracted with alcohol such as methanol and ethanol.
Alcohol is distilled off from the filtrate obtained by filtering the extract to obtain a dried product. The dried product is dissolved in purified water to give an aqueous solution. In the case of pulp, the crude juice is squeezed and the crude juice is filtered to obtain a filtrate.

【０００７】得られた水溶液または果汁溶液を更にイオ
ン交換樹脂等に吸着させ、希アンモニア水等の溶出溶媒
で溶出し、得られた溶出液を減圧濃縮することにより粗
物質を得る。イオン交換樹脂の担体の具体例としては強
酸性陽イオン交換樹脂のアンバーライトＩＲ−１２０Ｂ
等が挙げられる。The obtained aqueous solution or fruit juice solution is further adsorbed on an ion exchange resin or the like, eluted with an eluting solvent such as dilute aqueous ammonia, and the obtained eluate is concentrated under reduced pressure to obtain a crude substance. Specific examples of the carrier of the ion exchange resin include Amberlite IR-120B which is a strongly acidic cation exchange resin.
And the like.

【０００８】得られた粗物質は、更に水溶性物質の精製
において通常用いられている公知の方法、例えば活性
炭、セルロース、セファデックスＧ−２５、シリカゲ
ル、ゲルろ過等の担体を用いるカラムクロマトグラフィ
ーまたはイオン交換担体を用いる高速液体クロマトグラ
フィーにより精製することができる。このようにして得
られたペプチドを水または含水アルコールに溶解し、要
すれば毛髪用化粧品に常用されている各種成分を配合し
て、ヘアトニック、シャンプー、ヘアクリーム等に製剤
して用いられる。特にアロエエキスのような殺菌成分を
配合したものは好ましい製剤である。The obtained crude substance is further subjected to a known method usually used for purification of water-soluble substances, for example, column chromatography using a carrier such as activated carbon, cellulose, Sephadex G-25, silica gel, gel filtration or the like. It can be purified by high performance liquid chromatography using an ion exchange carrier. The peptide thus obtained is dissolved in water or hydrous alcohol, and if necessary, various components commonly used in hair cosmetics are blended to prepare a hair tonic, shampoo, hair cream or the like for use. In particular, those containing a bactericidal component such as aloe extract are preferable formulations.

【０００９】[0009]

【実施例】【Example】

実施例１；甘夏果皮の細片に２〜４倍量のメタノールを
加えてよく撹拌し、果皮抽出エキス１．８L を得た。こ
の抽出エキスをろ過し、沈殿物を除いたろ液１．７L を
減圧下メタノールを留去し、濃縮乾固して粗抽出物を得
た。この粗抽出物を精製水１．７L に溶解して水溶液と
し、活性炭カラムに通塔させ、通過液と水洗画分を一緒
にして、強酸性陽イオン交換樹脂アンバーライトＩＲ−
１２０Ｂ〔Ｈ⁺ 〕のカラムに吸着させ、０．５N のアン
モニア水で活性画分を溶出し、粗粉末７．６６ｇを得
た。この粗粉末をセルロースカラムクロマトグラフィー
にかけ、ブタノール：酢酸：水＝２０：１：１、１０：
１：１および５：１：１の各２L でカラムを洗浄後、ブ
タノール：酢酸：水＝３：１：１の溶出液２L で活性物
質を溶出させ、粗精製物２．１ｇを得た。この粗精製物
をセファデックスＧ−２５を担体とするゲルろ過（展開
溶媒：水）にかけ、粗精製物１．１２ｇを得た。次いで
セルロースカラムクロマトグラフィーにかけ、エタノー
ル：水＝７．５：１および５：１の各０．５L で洗浄
後、エタノール：水＝３：１〜１：１の溶出液１L で活
性物質を溶出させ、粗精製物４６０mgを得た。更に陽イ
オン交換系の高速液体クロマトグラフィー（展開溶媒：
水）により、毛乳頭細胞増殖促進作用を有するペプチド
３００mgを得た。Example 1 2 to 4 times the amount of methanol was added to the strip of sweet summer peel and stirred well to obtain 1.8 L of the peel extract. The extract was filtered, and 1.7 L of the filtrate from which the precipitate had been removed was evaporated under reduced pressure to remove methanol, and concentrated to dryness to obtain a crude extract. This crude extract was dissolved in 1.7 L of purified water to form an aqueous solution, which was passed through an activated carbon column, and the passed liquid and the water-washed fraction were combined to obtain a strongly acidic cation exchange resin Amberlite IR-
It was adsorbed on a 120 B [H ⁺ ] column, and the active fraction was eluted with 0.5 N aqueous ammonia to obtain 7.66 g of a crude powder. This crude powder was subjected to cellulose column chromatography, butanol: acetic acid: water = 20: 1: 1, 10:
After washing the column with 2 L each of 1: 1 and 5: 1: 1, the active substance was eluted with 2 L of an eluent of butanol: acetic acid: water = 3: 1: 1 to obtain 2.1 g of a crude product. The crude product was subjected to gel filtration using Sephadex G-25 as a carrier (developing solvent: water) to obtain 1.12 g of the crude product. Then, it was subjected to cellulose column chromatography, washed with 0.5 L each of ethanol: water = 7.5: 1 and 5: 1, and the active substance was eluted with 1 L of an eluent of ethanol: water = 3: 1 to 1: 1. Thus, 460 mg of a crude product was obtained. Furthermore, cation exchange high performance liquid chromatography (developing solvent:
(Water) to obtain 300 mg of a peptide having a dermal papilla cell growth promoting action.

【００１０】実験例１ ウサギ髭培養毛乳頭細胞に対す
る効果；日本白色系ウサギから分離した髭毛乳頭をコラ
ーゲン（タイプIV）コートしたシャーレ表面に接着し、
勝岡らの方法（Katuoka, K., et al.: Arch. Dermatol.
Res., 279, 20-25 (1986)）を改変した２０％牛胎児血
清（ＦＢＳ）および抗生物質添加Dulbecco変法Eagle 培
地（ＤＭＥＭ）にて培養を行い、髭毛乳頭より遊走した
初代培養細胞を得た。この細胞を更に１０％ＦＢＳ−Ｄ
ＭＥＭにて継代培養し、培養毛乳頭細胞とした。アッセ
イ法は培養毛乳頭細胞を１０％ＦＢＳ−ＤＭＥＭに浮遊
させて９６穴プレートに播き、実施例１で得たペプチド
の所定濃度水溶液を２回添加して、６日間培養した。培
養毛乳頭細胞の増殖の有無はＭＴＴ法で調べた。その結
果を表１に示した。Experimental Example 1 Effect on rabbit beard culture hair papilla cells; Beard papillae isolated from Japanese white rabbits were adhered to a collagen (type IV) coated petri dish surface,
Katsuoka's method (Katuoka, K., et al .: Arch. Dermatol.
Res., 279, 20-25 (1986)) modified 20% fetal bovine serum (FBS) and antibiotic-added Dulbecco's modified Eagle medium (DMEM) were cultured, and primary cells migrated from the papillae of the mustache. Got These cells were further mixed with 10% FBS-D
Subculture was performed with MEM to obtain cultured hair papilla cells. As the assay method, the cultured hair papilla cells were suspended in 10% FBS-DMEM and seeded on a 96-well plate, and the aqueous solution of the peptide having the predetermined concentration obtained in Example 1 was added twice, and the cells were cultured for 6 days. The presence or absence of proliferation of cultured hair papilla cells was examined by the MTT method. The results are shown in Table 1.

【００１１】[0011]

【表１】 [Table 1]

【００１２】この結果から、本発明のペプチドが培養毛
乳頭細胞を１５０％増殖促進する濃度は、１．４μg/ml
と低濃度で有効であることがわかる。From these results, the concentration of the peptide of the present invention that promotes the proliferation of cultured hair papilla cells by 150% is 1.4 μg / ml.
It can be seen that it is effective at low concentrations.

【００１３】実験例２ マウスの体毛の再生試験；実施
例１で得たペプチドの５０％エタノール溶液（４０mg/m
l)を、マウスの背部の毛を剃った皮膚に、６０μl ／マ
ウス／２回／日を毎日塗布した。毛の再生状態を写真撮
影して、この写真から毛の再生部位の面積をデジタイザ
ーで読み取り、塗布部位全体の面積に対する毛の再生面
積％を図２に示す。塗布開始から３２日目には毛の再生
が完了し、一方体重変化および毛の生育には何等悪影響
はなかった。Experiment 2 Mouse hair regeneration test: 50% ethanol solution of the peptide obtained in Example 1 (40 mg / m 2
l) was applied daily to the shaved skin of the back of mice at 60 μl / mouse / 2 times / day. A photograph of the regenerated state of the hair is taken, the area of the regenerated area of the hair is read by a digitizer, and the regenerated area% of the hair with respect to the entire area of the applied area is shown in FIG. Hair regeneration was completed 32 days after the start of application, while there was no adverse effect on weight change and hair growth.

【図面の簡単な説明】[Brief description of drawings]

【図１】本発明のペプチドの赤外線吸収スペクトルを示
す。FIG. 1 shows an infrared absorption spectrum of the peptide of the present invention.

【図２】マウスの体毛の再生試験の結果を示す。FIG. 2 shows the results of a hair regeneration test in mice.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 小宮山 寛機 東京都港区白金５丁目９番１号 社団法人 北里研究所内 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hiroki Komiyama 5-9-1 Shirokane, Minato-ku, Tokyo Inside Kitasato Institute

Claims (3)

【特許請求の範囲】[Claims] 【請求項１】 柑橘類由来の次の物理的化学的性質を有
するペプチドを含有する育毛剤。 （１）分子量 ２，０００〜４，０００（ゲルろ過法による） （２）赤外線吸収スペクトル（ＫＢｒ法）：図１に示す
とおり （３）溶解性 水に可溶。エーテル、ベンゼン、クロロホルムに難溶 （４）呈色反応 ニンヒドリン反応およびニトロプルシドナトリウム反応
は陽性；アニスアルデヒド反応、フェノール硫酸反応お
よびドラーゲンドルフ反応は陰性 （５）塩基性、中性、酸性の区別 中性 （６）性状 無色の結晶または白色の結晶性粉末であって、わずかに
特有の臭いを有する （７）構成アミノ酸 アスパラギン酸およびオキシリシンを含む1. A hair restorer containing a peptide derived from citrus fruits having the following physical and chemical properties. (1) Molecular weight 2,000 to 4,000 (by gel filtration method) (2) Infrared absorption spectrum (KBr method): As shown in FIG. 1 (3) Solubility Soluble in water. Insoluble in ether, benzene and chloroform (4) Color reaction Ninhydrin reaction and sodium nitroprusside reaction are positive; Anisaldehyde reaction, phenolsulfate reaction and Dragendorff reaction are negative (5) Basic, neutral and acidic (6) Properties Colorless crystals or white crystalline powder with a slight unique odor (7) Constituent amino acids Aspartic acid and oxylysine 【請求項２】 構成アミノ酸として、ホスホセリン、ア
スパラギン酸、セリン、グルタミン酸、グリシン、アラ
ニン、バリン、シスチン、メチオニン、オキシリシン、
オルニチン、リシンおよびヒスチジンを含む、請求項１
の育毛剤。2. As constituent amino acids, phosphoserine, aspartic acid, serine, glutamic acid, glycine, alanine, valine, cystine, methionine, oxylysine,
A composition comprising ornithine, lysine and histidine.
Hair restorer. 【請求項３】 アロエエキスを配合した、請求項１また
は２の育毛剤。3. The hair restorer according to claim 1, which contains an aloe extract.