JP2022038980A - Hair growing agent - Google Patents
Hair growing agent Download PDFInfo
- Publication number
- JP2022038980A JP2022038980A JP2020143735A JP2020143735A JP2022038980A JP 2022038980 A JP2022038980 A JP 2022038980A JP 2020143735 A JP2020143735 A JP 2020143735A JP 2020143735 A JP2020143735 A JP 2020143735A JP 2022038980 A JP2022038980 A JP 2022038980A
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- JP
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- Prior art keywords
- hair
- compound
- enmain
- cells
- growth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
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Abstract
Description
特許法第３０条第２項適用申請有り 刊行物１：Ｃｏｎｇｒｅｓｓ Ｃｈａｉｒｐｅｒｓｏｎｓ：Ｓｈｕｊｉ Ａｋａｉ，Ｙａｓｕｙｕｋｉ Ｋｉｔａ ２７ｔｈ ＩＳＨＣ Ｓｅｃｒｅｔａｒｉａｔ：Ｌａｂｏｒａｔｏｒｙ ｏｆ Ｓｙｎｔｈｅｔｉｃ Ｍｅｄｉｃｉｎａｌ Ｃｈｅｍｉｓｔｒｙ Ｇｒａｄｕａｔｅ Ｓｃｈｏｏｌ ｏｆ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅｓ，Ｏｓａｋａ Ｕｎｉｖｅｒｓｉｔｙ Ｂｏｏｋ ｏｆ Ａｂｓｔｒａｃｔｓ，２７▲ｔｈ▼ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｓｏｃｉｅｔｙ ｏｆ Ｈｅｔｅｒｏｃｙｃｌｉｃ Ｃｈｅｍｉｓｔｒｙ Ｃｏｎｇｒｅｓｓ，ｐ．５６３ 令和１年８月２８日 刊行物２：一般社団法人 日本生薬学会、日本本生薬学会第６６回年会 東京２０１９ 講演要旨集 第１８３頁、令和１年８月３１日 刊行物３：公益法人日本薬学会 ｈｔｔｐｓ：／／ｃｏｎｆｉｔ．ａｔｌａｓ．ｊｐ／ｇｕｉｄｅ／ｅｖｅｎｔ／ｐｈａｒｍ１４０／ｓｕｂｊｅｃｔ／２Ｙ１４－２４－０８／ａｄｖａｎｃｅｄ 令和２年３月５日Article 30 paragraph 2 apply application there publications Patent Act 1: Congress Chairpersons: Shuji Akai, Yasuyuki Kita 27th ISHC Secretariat: Laboratory of Synthetic Medicinal Chemistry Graduate School of Pharmaceutical Sciences, Osaka University Book of Abstracts, 27 ▲ th ▼ International Society of Heterocyclic Chemistry Congress, p. 563 Reiwa August 28, 1 Publication 2: General Incorporated Association Japan Biopharmacy Society, Japan Society of Biopharmacy 66th Annual Meeting Tokyo 2019 Lecture Abstracts, page 183, Reiwa August 31, 1 Publication 3: Public Interest Corporation Japan Pharmaceutical Association https: // confit. atlas. jp / guide / event / farm140 / subject / 2Y14-24-08 / advanced Reiwa March 5, 2
本発明は、発毛促進剤に関する。より詳細には、男性ホルモンの活性化に影響を与えることなく、毛乳頭細胞増殖作用、ＶＥＧＦ産生促進作用、β―カテニン産生促進作用により、優れた脱毛予防効果、育毛・養毛効果、かゆみの抑制効果及び髪質改善効果を持つ、化粧品、医薬部外品、医薬品等の発毛促進組成物に関する。 The present invention relates to a hair growth stimulant. More specifically, it has excellent hair loss prevention effect, hair growth / hair growth effect, and itching effect due to hair papilla cell proliferation action, VEGF production promoting action, and β-catenin production promoting action without affecting the activation of male hormones. The present invention relates to a hair growth promoting composition for cosmetics, quasi-drugs, pharmaceuticals, etc., which has an inhibitory effect and a hair quality improving effect.
毛髪の質や量には個人差があり、生まれつき、または若いときから、毛髪の本数が少ない、毛髪が細いなどの理由で外見上髪が薄い場合がある。また、加齢とともに、毛髪が細くなったり、早期に抜け易くなったりして、年齢が上がると髪が薄くなる場合がある。さらに、ストレスや薬剤の副作用などの医学的な原因により、頭髪全体又は一部の髪が抜けたり、細くなったり、発毛が低減したりすることもある。
毛髪は、成長期、退行期および休止期からなる毛周期（ヘアサイクル）に従い、生えかわっている。この毛周期（ヘアサイクル）のうち、成長期において髪は成長する。しかしながら、この成長期が短くなることにより薄毛となる。薄毛を改善するためには、この短くなった成長期をもとに戻し、毛周期（ヘアサイクル）を正常な状態とすることが重要である。
There are individual differences in the quality and quantity of hair, and from the time of birth or when you are young, the number of hairs may be small, or the hair may be thin in appearance. In addition, with aging, the hair may become thinner or easier to come off at an early stage, and the hair may become thinner as the age increases. In addition, medical causes such as stress and side effects of drugs may cause the entire or part of the hair to fall out, become thinner, or reduce hair growth.
Hair grows according to a hair cycle consisting of a growth phase, a catagen phase, and a resting phase. In this hair cycle (hair cycle), hair grows during the anagen phase. However, as this growth period becomes shorter, the hair becomes thinner. In order to improve thinning hair, it is important to restore this shortened growth period and restore the hair cycle to a normal state.
毛周期（ヘアサイクル）の成長期において毛乳頭細胞の増殖及び分化が促進されることが知られている（非特許文献１～４）。さらにこの成長を誘導／延長させる生体内因子として、毛乳頭細胞等から分泌されるサイトカインある。このサイトカインとして、線維芽細胞増殖因子（ＦＧＦ－７）、血管内皮細胞（ＶＥＧＦ）等が知られている（非特許文献５～８）。さらに、毛乳頭細胞の増殖およびサイトカインの分泌におけるメカニズムの一つとして細胞内のβ―カテニンの量の増加がある（非特許文献９～１１）。
また、近年、フェナステリドに代表されるように男性ホルモンの活性化を抑制する発毛成分が利用されているが、副作用があるとともに、その適用疾患は男性型脱毛症に限定され、その適応者も男性となっている（非特許文文献１２）。
特許文献１には、アロエエキスが血管内皮細胞増殖因子の産生を促進することから、育毛効果に優れていることが開示されている。特許文献２には、アスコルビン酸リン酸Ｍｇがβ―カテニンの産生の産生を促進することから、育毛効果に優れていることが開示されている。非特許文献１３には、フェニルプロパノイド成分であるシナピン酸の毛乳頭細胞増殖促進効果およびＶＥＧＦの産生促進効果が報告されている。
また、非特許文献１４には、エンメイソウの主成分のひとつであるエンメインのエンメイソウからの精製方法が報告されており、非特許文献１５にはエンメイソウの化学合成法が報告されている。
It is known that the proliferation and differentiation of dermal papilla cells are promoted during the growth phase of the hair cycle (Non-Patent Documents 1 to 4). Further, as an in vivo factor that induces / prolongs this growth, there is a cytokine secreted from dermal papilla cells and the like. As this cytokine, fibroblast growth factor (FGF-7), vascular endothelial cells (VEGF) and the like are known (Non-Patent
In recent years, hair growth components that suppress the activation of male hormones have been used, as typified by phenasteride, but they have side effects and their applicable diseases are limited to androgenetic alopecia. He is a man (Non-Patent Document 12).
Patent Document 1 discloses that aloe extract is excellent in hair growth effect because it promotes the production of vascular endothelial cell growth factor. Patent Document 2 discloses that Mg ascorbic acid phosphate has an excellent hair-growth effect because it promotes the production of β-catenin. Non-Patent Document 13 reports the effect of promoting the proliferation of sinapinic acid, which is a phenylpropanoid component, and the effect of promoting the production of VEGF.
Further, Non-Patent Document 14 reports a method for purifying Enmain, which is one of the main components of Enmeisou, from Enmeisou, and Non-Patent Document 15 reports a method for chemically synthesizing Enmeisou.
近年、フェナステリドに代表されるように男性ホルモンの活性化を抑制する発毛成分が利用されているが、副作用のおそれがあるとともに、非特許文文献１２に示されるように、その適用疾患は男性型脱毛症に限定され、その適応者も男性となっている。
そこで、男性ホルモンの活性化に作用しないことにより副作用の懸念も少なく、男女問わず使用でき、より優れた育毛効果を示す発毛促進剤を得るために、毛乳頭細胞増殖促進効果、血管内皮増殖因子（ＶＥＧＦ）産生促進効果、β―カテニン産生促進効果の高い成分が求められている。
In recent years, hair growth components that suppress the activation of male hormones, such as phenasteride, have been used, but there is a risk of side effects, and as shown in Non-Patent Document 12, the applicable disease is male. It is limited to androgenetic alopecia, and its indication is also male.
Therefore, there is less concern about side effects because it does not act on the activation of male hormones, and it can be used by both men and women. A component having a high factor (VEGF) production promoting effect and β-catenin production promoting effect is required.
本発明は、男性ホルモンの活性化に影響を与えることなく、優れた毛乳頭細胞増殖促進効果、血管内皮増殖因子（ＶＥＧＦ）産生促進効果、β―カテニン産生促進効果を有する発毛促進剤を提供することを目的とする。 The present invention provides a hair growth promoting agent having an excellent hair papilla cell proliferation promoting effect, vascular endothelial growth factor (VEGF) production promoting effect, and β-catenin production promoting effect without affecting the activation of androgen. The purpose is to do.
本発明者らは、上記課題を解決すべく鋭意検討を行った結果、以下の本発明を完成させた。
１．下記の化学式１で表される化合物を有効成分とする発毛促進剤。
２．化学式１で表される化合物からなる毛乳頭細胞増殖促進剤。
３．化学式１で表される化合物からなるＶＥＧＦ産生促進剤。
４．化学式１で表される化合物からなるβ―カテニン産生促進剤。
The present inventors have completed the following invention as a result of diligent studies to solve the above problems.
1. 1. A hair growth stimulant containing a compound represented by the following chemical formula 1 as an active ingredient.
2. 2. A hair papilla cell proliferation promoter comprising a compound represented by Chemical Formula 1.
3. 3. A VEGF production promoter comprising a compound represented by Chemical Formula 1.
4. A β-catenin production promoter composed of a compound represented by Chemical Formula 1.
本発明により、副作用の懸念も少なく、男女問わず使用でき、男性ホルモンの活性化に影響を与えることなく、優れた毛乳頭細胞増殖促進効果、血管内皮増殖因子（ＶＥＧＦ）産生促進効果、β―カテニン産生促進効果を有する発毛促進剤の提供ができる。 According to the present invention, there is little concern about side effects, it can be used by both men and women, and it does not affect the activation of male hormones, and has an excellent hair papilla cell proliferation promoting effect, vascular endothelial growth factor (VEGF) production promoting effect, β-. It is possible to provide a hair growth promoting agent having a catenin production promoting effect.
以下、本発明の実施の形態について説明する。
化学式１で示される化合物には、エンメイソウ（延命草）等から抽出される成分である、エンメイン、イソドカルピン、ノドシン、シェリンＣなどが挙げられる。
本明細書において「発毛」の用語は、毛包内で毛の新たな発生を促す、あるいは毛の発育・成長を促すように有利にはたらく効果を包含する。なお、ここで毛とは、ヒトを含む動物における身体に生える任意の毛を意味し、例えば、毛髪（頭髪）、眉毛、睫毛、口髭、顎鬚、胸毛、背部毛などあらゆる体毛を意味し、主には、毛髪、眉毛、又は睫毛を意味する。
Hereinafter, embodiments of the present invention will be described.
Examples of the compound represented by the chemical formula 1 include enmain, isodocarpine, nodocin, and sherin C, which are components extracted from emmeiso (extended life grass) and the like.
As used herein, the term "hair growth" includes an effect that favorably promotes the development of new hair in the hair follicle or promotes the growth and growth of hair. Here, hair means any hair that grows on the body in animals including humans, and means, for example, all body hair such as hair (head hair), eyelashes, eyelashes, mustache, jaw beard, chest hair, and back hair. Mainly means hair, eyelashes, or eyelashes.
エンメイソウ（延命草）としては、シソ科のヒキオコシ Ｉｓｏｄｏｎ ｊａｐｏｎｉｃｕｓ（Ｐｌｅｃｔｒａｎｔｈｕｓ ｊａｐｏｎｉｃｕｓ，Ｒａｂｄｏｓｉａ ｊａｐｏｎｉｃａ）、クロバナヒキオコシＩｓｏｄｏｎ ｔｒｉｃｈｏｃａｒｐｕｓ（Ｐｌｅｃｔｒａｎｔｈｕｓ ｔｒｉｃｈｏｃａｒｐｕｓ，Ｒａｂｄｏｓｉａ，ｔｒｉｃｈｏｃａｒｐａ）およびカメバヒキオコシ Ｉｓｏｄｏｎ ｕｍｂｒｏｓｕｓ（ｌｅｕｃａｎｔｈｕｓ
ｋａｍｅｂａ，Ｉｓｏｄｏｎ ｋａｍｅｂａ，Ｐｌｅｃｔｒａｎｔｈｕｓ ｋａｍｅｂａ）が挙げられ、これらの地上部（葉、茎等）を主に利用する。これらのなかでもクロバナヒキオコシが好ましく、使用する部位としては葉部が好ましい。また、本発明で用いるエンメイソウ抽出物とは、かかる地上部等を乾燥し又は乾燥することなく粉砕あるいは裁断した後、常温又は加温下に、溶剤により抽出するか又はソックスレー抽出器等の抽出器具を用いて抽出することにより得られる各種溶媒抽出液、その希釈液、その濃縮液、あるいはその乾燥粉末である。
ここで、エンメイソウの抽出に使用される溶媒は特に限定されず、例えば、水、メチルアルコール、エチルアルコール等の低級アルコール、プロピレングリコール、１，３－ブチレングリコール等の液状多価アルコール、酢酸エチル等の低級アルキルエステル、ヘプタン、ヘキサン等の炭化水素、ジエチルエーテル、アセトン等の公知の溶媒が挙げられ、これらの溶媒は、１種ないし２種以上を組み合わせて用いることができる。これらの中でも水およびエタノール水溶液が好ましく、７０％エタノール溶液が特に好ましい。
なお、エンメイソウの抽出は常法で行い、得られたエンメイソウ抽出液はそのまま用いてもよいが、さらに必要により精製、濃縮、濾過等の処理したものを用いることができる。
The Enmeisou (emmeisou), Labiatae of cause Isodon japonicus (Plectranthus japonicus, Rabdosia japonica), Kurobanahikiokoshi Isodon trichocarpus (Plectranthus trichocarpus, Rabdosia, trichocarpa) and Kamebahikiokoshi Isodon umbrosus (leucanthus
Kameba, Isodon kameba, Spurflowers kameba), and these above-ground parts (leaves, stems, etc.) are mainly used. Among these, Isodon trichocarpus is preferable, and the leaf part is preferable as the site to be used. Further, the Enmeisou extract used in the present invention is an extraction device such as a Soxhlet extractor or the like, which is obtained by crushing or cutting the above-ground part or the like without drying or drying, and then extracting with a solvent at room temperature or under heating. Various solvent extracts, diluted solutions thereof, concentrated solutions thereof, or dry powders thereof obtained by extraction using the above.
Here, the solvent used for extracting Enmeisou is not particularly limited, and for example, water, lower alcohols such as methyl alcohol and ethyl alcohol, liquid polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, ethyl acetate and the like, etc. Examples thereof include lower alkyl esters of the above, hydrocarbons such as heptane and hexane, known solvents such as diethyl ether and acetone, and these solvents can be used alone or in combination of two or more. Of these, water and an aqueous ethanol solution are preferable, and a 70% ethanol solution is particularly preferable.
The extract of Enmeisou is carried out by a conventional method, and the obtained Enmeisou extract may be used as it is, but if necessary, it may be further purified, concentrated, filtered or the like.
本発明の発毛促進剤は、化学式１で示される化合物を有効成分とするものであり、優れた発毛促進効果を有し、かつ安全性も高いので、剤型としては錠剤、カプセル剤、散剤、内服液、細粒剤、顆粒剤等の経口投与剤の形態となすことができ、また、適当な基材、薬剤などと混合した皮膚外用剤や頭髪化粧料等の外用形態とすることができる。具体的には、ローション、乳液、軟膏、クリーム、ジェル、オイル、パック、シャンプー、リンス、トリートメント、ヘアートニック、ヘアーリキッド等の形態をとることができる。さらに上記のような外用剤の他にも、例えば、石けん、入浴剤といったものに配合してもよい。また、本発明においてエンメイソウ抽出物を処理したものとしては、例えば、エンメイソウの地上部からの抽出物を酢酸エチルと水にて液―液分配し、得られた酢酸エチル可溶部を活性炭に吸着させ、その後、活性炭を適当な溶媒にて再度抽出した活性炭吸着エキス等が挙げられる。 The hair growth-promoting agent of the present invention contains the compound represented by the chemical formula 1 as an active ingredient, has an excellent hair growth-promoting effect, and is highly safe. It can be in the form of an oral dosage form such as a powder, an internal solution, a fine granule, or a granule, and it can be in the form of an external skin preparation or hair cosmetic mixed with an appropriate base material or drug. Can be done. Specifically, it can take the form of lotion, milky lotion, ointment, cream, gel, oil, facial mask, shampoo, conditioner, treatment, hair tonic, hair liquid and the like. Further, in addition to the above-mentioned external preparations, for example, soaps and bath salts may be added. Further, as a treatment of the Enmeisou extract in the present invention, for example, the extract from the above-ground part of Enmeisou is liquid-liquid partitioned with ethyl acetate and water, and the obtained ethyl acetate-soluble part is adsorbed on the activated carbon. Then, the activated charcoal adsorption extract or the like in which the activated charcoal is extracted again with an appropriate solvent can be mentioned.
さらに本発明の発毛促進剤は、下記一般式（１）、
上記一般式（１）の化合物には、エンメイン、イソドカルピン、ノドシン、シェリンＣなどが挙げられ、植物からの抽出、単離、または既知の方法による合成により得ることができる。特に、前記の方法によりエンメイソウの地上部からエンメイソウ抽出物を得た後、精製処理をすることにより、好適に得ることができる。
精製処理は、例えば、クロマトグラフ法、イオン交換樹脂、活性炭を使用する溶離法、溶媒による分配抽出、再結晶法等を単独、又は組み合わせて採用することができる。クロマトグラフ法としては、順相クロマトグラフィー、逆相クロマトグラフィー、薄層クロマトグラフィー、遠心液体クロマトグラフィー、高速液体クロマトグラフィー等を挙げることができ、これらのいずれか、又はそれらを組み合わせで行う方法が挙げられる。この際の担体、溶出溶媒等の精製条件は、各種クロマトグラフィーに対応して適宣選択することができる。
Examples of the compound of the general formula (1) include enmain, isodocarpine, nodocin, sherin C and the like, which can be obtained by extraction from a plant, isolation, or synthesis by a known method. In particular, it can be preferably obtained by obtaining an Enmeisou extract from the above-ground part of Enmeisou by the above method and then purifying it.
As the purification treatment, for example, a chromatographic method, an ion exchange resin, an elution method using activated carbon, a partition extraction with a solvent, a recrystallization method and the like can be adopted alone or in combination. Examples of the chromatographic method include normal phase chromatography, reverse phase chromatography, thin layer chromatography, centrifugal liquid chromatography, high performance liquid chromatography and the like, and any of these or a combination thereof can be used. Can be mentioned. The purification conditions such as the carrier and the elution solvent at this time can be appropriately selected according to various chromatographies.
また、本発明においては、上記一般式中のＲ１が水酸基、Ｒ２が水素、Ｒ３が水素、Ｒ４が水酸基である下記式で表されるエンメイン（ｅｎｍｅｉｎ）であることが好ましい。
エンメインは、エンメイソウ抽出物から定法により単離することも可能である。また、非特許文献１３の方法等により精製することもできるし、非特許文献１４の方法等により合成することもできる。 Enmain can also be isolated from the Enmeisou extract by a conventional method. Further, it can be purified by the method of Non-Patent Document 13 or the like, or can be synthesized by the method of Non-Patent Document 14 or the like.
本発明において、エンメイソウ抽出液の配合量は、添加形態および投与形態によっても異なるが、外用剤の場合には、全組成物中０．０００１～９０質量％配合することが好ましく、０．００１～５０質量％配合することがより好ましく、０．０００１～１０質量％配合することがさらにより好ましい。また、経口投与剤の場合には、成人一日あたり、０．００１から１００ｇになるようにするのが好ましい。 In the present invention, the blending amount of the Enmeisou extract varies depending on the addition form and the administration form, but in the case of an external preparation, it is preferable to blend 0.0001 to 90% by mass in the total composition, and 0.001 to 0.001 to 90% by mass. It is more preferable to add 50% by mass, and even more preferably 0.0001 to 10% by mass. In the case of an orally administered agent, the amount is preferably 0.001 to 100 g per adult day.
また、本発明の発毛促進剤は、男性ホルモンの活性化に影響を与えることなく毛乳頭細胞増殖促進効果、ＶＥＧＦ遺伝子発現促進効果、ＶＥＧＦタンパク産生促進効果およびβ―カテニンタンパク発現促進効果を併せ持つ。 Further, the hair growth promoting agent of the present invention has a hair papillary cell proliferation promoting effect, a VEGF gene expression promoting effect, a VEGF protein production promoting effect and a β-catenin protein expression promoting effect without affecting the activation of male hormones. ..
また、本発明の発毛促進剤には、さらに必要に応じて、本発明の効果を損なわない範囲で、化粧品や医薬品等に一般的に用いられている保湿剤、油性成分、界面活性剤、ビタミン類、タンパク分解酵素、増粘剤、防腐剤、紛体、酸化防止剤、紫外線吸収剤、乳化剤、アルコール類、色素、水溶性成分、脂肪酸類、香料、キレート剤、ｐＨ調整剤、精製水等の添加成分を配合することができる。 Further, the hair growth stimulant of the present invention includes, if necessary, a moisturizer, an oily component, a surfactant, which are generally used in cosmetics, pharmaceuticals, etc., as long as the effects of the present invention are not impaired. Vitamin, proteolytic enzymes, thickeners, preservatives, powders, antioxidants, UV absorbers, emulsifiers, alcohols, pigments, water-soluble ingredients, fatty acids, fragrances, chelating agents, pH adjusters, purified water, etc. Can be blended with the additive components of.
保湿剤としては、例えば、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、１，３－ブチレングリコール等の多価アルコール、ソルビット、マンニット等の糖アルコール、ヒアルロン酸ナトリウム、コンドロイチン硫酸、冬虫夏草エキス、延命草エキス、オオムギエキス、ブドウエキス、チューベロースポリサッカライド、桔梗エキス、ヨクイニンエキス、オトギリソウエキス、オーツ麦エキス等が挙げられる。 Examples of the moisturizing agent include polyhydric alcohols such as glycerin, propylene glycol, polyethylene glycol and 1,3-butylene glycol, sugar alcohols such as sorbit and mannit, sodium hyaluronate, chondroitin sulfate, winter worm summer grass extract and life-prolonging grass extract. Examples thereof include corn extract, grape extract, tuberose polysaccharide, kikyo extract, yokuinin extract, otogirisou extract, oat wheat extract and the like.
油性成分としては、例えばユーカリ油、サフラワー油、月見草油、ホホバ油等の植物油、不飽和脂肪酸アルキルエステル（オレイン酸エチル、リノール酸イソプロピル等）、リノレイン酸エステル、ミリスチン酸メチル、ミリスチン酸イソプロピル等のエステル油、多価アルコール脂肪酸エステル（トリ－２エチルヘキサン酸グリセリン、トリイソステアリン酸トリメチロールプロパン酸等の多価アルコール脂肪酸エステル等のエステル類、精油類、シリコーン油類などが挙げられる。 Examples of the oily component include vegetable oils such as eucalyptus oil, safflower oil, evening primrose oil, and jojoba oil, unsaturated fatty acid alkyl esters (ethyl oleate, isopropyl linoleate, etc.), linoleic acid esters, methyl myristate, isopropyl myristate, and the like. Examples thereof include esters such as polyhydric alcohol fatty acid esters such as glycerin tri-2ethylhexanoic acid and trimethylolpropanoic acid triisostearate, essential oils, and silicone oils.
界面活性剤としては、例えば、ソルビタン脂肪酸エステル類（ソルビタンモノラウレート、ソルビタンモノオレート等）、ポリオキシエチレン硬化ヒマシ油、ポリオキシエチレン硬化ヒマシ油モノ又はイソステアレート、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル（モノミリスチン酸デカグリセリン、モノミリスチン酸ペンタグリセリン等の非イオン性界面活性剤、ラウリル硫酸ナトリウム、ラウリル硫酸カリウム等の高級アルキル硫酸エステル塩、ＰＯＥ－ラウリル硫酸トリエタノールアミン、ＰＯＥ－ラウリル硫酸ナトリウム等のアルキルエーテル硫酸エステル塩、ラウロイルサルコシンナトリウム等のＮ－アシルサルコシン酸、Ｎ－ミリストイル－Ｎ－メチルタウリンナトリウム、ヤシ油脂肪酸メチルタウリッドナトリウム、ラウリルメチルタウリッドナトリウム等の高級脂肪酸アミドスルホン酸塩、ＰＯＥオレイルエーテルリン酸ナトリウム、ＰＯＥステアリルエーテルリン酸等のリン酸エステル塩、ジ－２－エチルヘキシルスルホコハク酸ナトリウム、モノラウロイルモノエタノールアミドポリオキシエチレンスルホコハク酸ナトリウム、ラウリルポリプロピレングリコールスルホコハク酸ナトリウム等のスルホコハク酸塩、リニアドデシルベンゼンスルホン酸ナトリウム、リニアドデシルベンゼンスルホン酸トリエタノールアミン、ドデシルベンゼンスルホン酸等のアルキルベンゼンスルホン酸塩、Ｎ－ラウロイルグルタミン酸モノナトリウム、Ｎ－ステアロイルグルタミン酸ジナトリウム、Ｎ－ミリストイル－Ｌ－グルタミン酸モノナトリウム等のＮ－アシルグルタミン酸塩、
硬化ヤシ油脂肪酸グリセリン硫酸ナトリウム等の高級脂肪酸エステル硫酸エステル塩、ＰＯＥ－アルキルエーテルカルボン酸、ＰＯＥ－アルキルアリルエーテルカルボン酸塩、α－オレフィンスルホン酸塩、高級脂肪酸エステルスルホン酸塩、二級アルコール硫酸エステル塩、高級脂肪酸アルキロールアミド硫酸エステル塩、ラウロイルモノエタノールアミドコハク酸ナトリウム、Ｎ－パルミトイルアスパラギン酸ジトリエタノールアミン、カゼインナトリウム等のアニオン界面活性剤、第四級アンモニウム塩型カチオン性界面活性剤、塩化ポリ（Ｎ，Ｎ’－ジメチル－３，５－メチレンピペリジニウム）、アルキルイソキノリニウム塩、ジアルキルモリホニウム塩、ＰＯＥ－アルキルアミン、アルキルアミン塩、ポリアミン脂肪酸誘導体、アミルアルコール脂肪酸誘導体、塩化ベンザルコニウム、塩化ベンゼトニウム等が挙げられる。第四級アンモニウム塩型カチオン性界面活性剤としては、例えば、塩化ステアリルトリメチルアンモニウム、塩化セチルトリメチルアンモニウム、塩化ベヘニルトリメチルアンモニウム、臭化ステアリルトリメチルアンモニウム、塩化ジステアリルジメチルアンモニウム、塩化ラウリルベンジルジメチルアンモニウム等のカチオン界面活性剤、第四級アンモニウム塩型カチオン性界面活性剤、塩化ポリ（Ｎ，Ｎ’－ジメチル－３，５－メチレンピペリジニウム）、アルキルイソキノリニウム塩、ジアルキルモリホニウム塩、ＰＯＥ－アルキルアミン、アルキルアミン塩、ポリアミン脂肪酸誘導体、アミルアルコール脂肪酸誘導体、塩化ベンザルコニウム、塩化ベンゼトニウム等が挙げられる。第四級アンモニウム塩型カチオン性界面活性剤としては、例えば、塩化ステアリルトリメチルアンモニウム、塩化セチルトリメチルアンモニウム、塩化ベヘニルトリメチルアンモニウム、臭化ステアリルトリメチルアンモニウム、塩化ジステアリルジメチルアンモニウム、塩化ラウリルベンジルジメチルアンモニウム等のカチオン界面活性剤、２－ウンデシル－Ｎ，Ｎ，Ｎ－（ヒドロキシエチルカルボキシメチル）－２－イミダゾリンナトリウム等のイミダゾリン系両性界面活性剤、２－ヘプタデシル－Ｎ－カルボキシメチル－Ｎ－ヒドロキシエチルイミダゾリニウムベタイン、ラウリルジメチルアミノ酢酸ベタイン、アルキルベタイン、アミドベタイン、スルホベタイン等のベタイン系界面活性剤等の両性界面活性剤が挙げられる。
Examples of the surface active agent include sorbitan fatty acid esters (sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene cured castor oil, polyoxyethylene cured castor oil mono or isostearate, glycylline fatty acid ester, and polyglycerin fatty acid. Esters (nonionic surfactants such as decaglycylline monomyristylate and pentaglyceryl monomyristylate, higher alkyl sulfates such as sodium lauryl sulfate and potassium lauryl sulfate, POE-triethanolamine lauryl sulfate, and sodium POE-sodium lauryl sulfate Alkyl ether sulfate ester salts such as, N-acyl sarcosic acid such as sodium lauroyl sulcosin, sodium N-myristyl-N-methyl taurin, sodium coconut oil fatty acid methyl taurid, sodium lauryl methyl taurid, and the higher fatty acid amide sulfonates. , POE oleyl ether phosphate sodium, POE stearyl ether phosphate and other phosphate ester salts, di-2-ethylhexyl sulfosuccinate sodium, monolauryl monoethanolamide polyoxyethylene sulfosuccinate sodium, lauryl polypropylene glycol sulfosuccinate sodium and the like. Alkylbenzene sulphate, sodium dodecylbenzene sulphate, triethanolamine lineard decylbenzene sulphonate, sodium dodecylbenzene sulnate, monosodium N-lauroyl glutamate, dissodium N-stearoyl sulphate, N-myristoyl-L- N-acylglutamate, such as monosodium glutamate,
Higher fatty acid ester sulfate ester salts such as hardened coconut oil fatty acid glycerin sulfate sodium, POE-alkyl ether carboxylic acid, POE-alkylallyl ether carboxylate, α-olefin sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate Anionic surfactants such as ester salts, higher fatty acid alquilolamide sulfate ester salts, sodium lauroylmonoethanolamide succinate, ditriethanolamine N-palmitoyle aspartate, sodium caseinate, quaternary ammonium salt-type cationic surfactants, Polychloride (N, N'-dimethyl-3,5-methylenepiperidinium), alkylisoquinolinium salt, dialkylmoriphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative , Benzalconium chloride, benzethonium chloride and the like. Examples of the quaternary ammonium salt-type cationic surfactant include stearyltrimethylammonium chloride, cetyltrimethylammonium chloride, behenyltrimethylammonium chloride, stearyltrimethylammonium bromide, disstearyldimethylammonium chloride, and laurylbenzyldimethylammonium chloride. Cationic surfactant, quaternary ammonium salt type cationic surfactant, polychloride (N, N'-dimethyl-3,5-methylenepiperidinium), alkylisoquinolinium salt, dialkylmoriphonium salt, Examples thereof include POE-alkylamines, alkylamine salts, polyamine fatty acid derivatives, ammonium alcohol fatty acid derivatives, benzalconium chloride, and benzethonium chloride. Examples of the quaternary ammonium salt-type cationic surfactant include stearyltrimethylammonium chloride, cetyltrimethylammonium chloride, behenyltrimethylammonium chloride, stearyltrimethylammonium bromide, distearyldimethylammonium chloride, and laurylbenzyldimethylammonium chloride. Cationic surfactants, imidazoline-based amphoteric surfactants such as 2-undecyl-N, N, N- (hydroxyethylcarboxymethyl) -2-imidazoline sodium, 2-heptadecyl-N-carboxymethyl-N-hydroxyethyl imidazole Examples thereof include amphoteric surfactants such as betaine-based surfactants such as nium betaine, lauryldimethylaminoacetic acid betaine, alkyl betaine, amide betaine, and sulfobetaine.
以下に実施例を説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Examples will be described below, but the present invention is not limited to these examples.
（実施例１）
実施例１は、エンメイソウから抽出物を抽出した実施例である。
１）エンメイソウの地上部の刻み３ｋｇ（日本産、三国（株）社製）を７０％エタノール溶液による抽出を３回繰り返し行い、７０％エタノール溶液抽出液を得た。得られたメタノール抽出液を減圧下溶媒留去して７０％エタノール溶液抽出エキスを得た。
２）７０％エタノール溶液抽出エキスについて、酢酸エチル／水にて分液を行い、酢酸エチル層を得た。
３）酢酸エチル層について、溶媒を留去し、さらに順相および逆相カラムクロマトグラフィー、次いでＨＰＬＣにて精製を行い、化合物１～２０（不図示）を得た。
(Example 1)
Example 1 is an example in which an extract is extracted from Enmeisou.
1) Extraction of 3 kg of the above-ground part of Enmeisou (made in Japan, manufactured by Mikuni Co., Ltd.) with a 70% ethanol solution was repeated 3 times to obtain a 70% ethanol solution extract. The obtained methanol extract was distilled off under reduced pressure to obtain a 70% ethanol solution extract.
2) The 70% ethanol solution extract was separated with ethyl acetate / water to obtain an ethyl acetate layer.
3) The solvent was distilled off from the ethyl acetate layer, and further purification was performed by normal phase and reverse phase column chromatography and then HPLC to obtain compounds 1 to 20 (not shown).
（実施例２）
実施例２は、実施例１で得た７０％エタノール溶液抽出エキス、酢酸エチル層および単離した４種類のエントカウラン型ジテルペノイド（化合物１～４）について、毛乳頭細胞増殖促進作用試験を行った実施例である。
まず、正常ヒト毛乳頭細胞（タカラバイオ（株）社製）をウシ胎児結成（ＦＢＳ）（１０％）、ペニシリンＧ（１００ｕｎｉｔｓ/ｍＬ）およびストレプトマイシン（１００μｇ/ｍＬ）を含むＤｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ（ＤＭＥＭ）培地を用い、３７℃、５％ＣＯ２下で培養した。
このように培養された細胞を、トリプシン／ＥＤＴＡ溶液（０.０５％濃度）によりトリプシン処理した。処理後の細胞を、ウシ胎児血清（ＦＢＳ）不含ＤＭＥＭ培地を用いて
、１．０×１０５細胞／ｍＬの濃度に希釈した。希釈された細胞を、９６ウエルのマイクロプレートに、１００μＬ／ウエルで播種し、１日間培養した。
(Example 2)
In Example 2, the 70% ethanol solution extract obtained in Example 1, the ethyl acetate layer, and the isolated four types of entocaulan-type diterpenoids (Compounds 1 to 4) were subjected to a hair papilla cell proliferation promoting action test. This is an example.
First, normal human hair papilla cells (manufactured by Takara Bio Inc.) containing fetal bovine serum (FBS) (10%), penicillin G (100 units / mL) and streptomycin (100 μg / mL) in Dulvecco's modified Eagle'. Using s Medium (DMEM) medium, the cells were cultured at 37 ° C. under 5% CO 2 .
The cells thus cultured were treated with trypsin / EDTA solution (0.05% concentration). The treated cells were diluted with fetal bovine serum (FBS) -free DMEM medium to a concentration of 1.0 × 105 cells / mL. Diluted cells were seeded in 96-well microplates at 100 μL / well and cultured for 1 day.
被験試料としての化合物１～４を、所定の濃度でＦＢＳ不含ＤＭＥＭに溶解した。濃度は、５μｍｏｌ／Ｌ、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌ、４０μｍｏｌ／Ｌのそれぞれとした。
溶解された各被験試料を、上記の細胞を播種した９６ウエルマイクロプレートの各ウエルに１００μＬ／ウエルの量で添加し、さらに４日間培養した。毛乳頭細胞増殖効果は、ＷＳＴ-８アッセイを用いて測定した。即ち、培養後、各ウエルにＷＳＴ-８溶液（株式会社同仁化学研究所社製）を加え、３７℃、５％ＣＯ２下で１時間培養を行い、細胞増殖の指標としてＯ.Ｄ.値（４５０－６５５ｎｍ）の測定を行った。毛乳頭細胞増殖促進率（％）は、以下の式（１）により算出した。
毛乳頭細胞増殖率（％） ＝ Ａ１ ／ Ｂ１ × １００ ・・・式（１）
Ａ１：被験試料添加時のＯ.Ｄ．４５０－６５０値
Ｂ１：被験試料無添加時（コントロール）のＯ.Ｄ．４５０－６５０値
Compounds 1 to 4 as test samples were dissolved in FBS-free DMEM at a predetermined concentration. The concentrations were 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L, respectively.
Each lysed test sample was added to each well of a 96-well microplate seeded with the above cells in an amount of 100 μL / well and cultured for an additional 4 days. The dermal papilla cell proliferation effect was measured using the WST-8 assay. That is, after culturing, WST-8 solution (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.) was added to each well, and the cells were cultured at 37 ° C. under 5% CO 2 for 1 hour, and the O.D. value was used as an index of cell proliferation. Measurements were made at (450-655 nm). The hair papilla cell proliferation promotion rate (%) was calculated by the following formula (1).
Hair papilla cell proliferation rate (%) = A1 / B1 × 100 ・ ・ ・ Equation (1)
A1: O.D. at the time of adding the test sample. 450-650 value B1: O.D. of the test sample without addition (control). 450-650 value
メタノール抽出エキス、酢酸エチル層および化合物１～４のそれぞれについて算出された毛乳頭細胞増殖促進率を、以下の表１に示す。毛乳頭細胞増殖率は、化合物１～４の各濃度についてそれぞれ、３回測定した。その３回の測定値の平均値および標準誤差を、「平均値±標準誤差」として示す。なお、各測定値は、コントロールを１００％とした相対値である。陽性対照として、現在、育毛成分として使用されている硫酸ミノキシジルおよび毛乳頭細胞増殖促進効果が明らかとなっているシナピン酸（非特許文献１５）を用いた。
上記表１に示されるように、７０％エタノール溶液、酢酸エチル層、メタノール層、エンメイン（化合物１）、ノドシン（化合物３）のそれぞれにおける１０μｍｏｌ／Ｌにおいて、また、イソドカルピン（化合物２）の５μｍｏｌ／Ｌおいて、さらに、シェリンＣ（化合物４）の１μｍｏｌ／Ｌおいて、陽性対照である硫酸ミノキシジルおよびシナピン酸を上回る活性強度を有していた。
実施例２の結果から、エンメイン（化合物１）、イソドカルピン（化合物２）、ノドシン（化合物３）、セリシンＣ（化合物４）は、毛乳頭細胞増殖促進剤の有効成分となり得ることが確認された。これらの化合物１～４は、低濃度で硫酸ミノキシジルおよびシナピン酸を超える毛乳頭細胞増殖促進効果を示している。したがって、エンメイン（化合物１）、イソドカルピン（化合物２）、ノドシン（化合物３）、セリシンＣ（化合物（化合物４）は、硫酸ミノキシジルおよびシナピン酸より毛乳頭細胞増殖促進効果が優れていることがわかる。
As shown in Table 1 above, at 10 μmol / L in each of 70% ethanol solution, ethyl acetate layer, methanol layer, enmain (Compound 1), nodocin (Compound 3), and 5 μmol of isodocarpine (Compound 2). At / L, and further at 1 μmol / L of Sherin C (Compound 4), it had an activity intensity higher than that of the positive controls minoxidil sulfate and sinapic acid.
From the results of Example 2, it was confirmed that enmain (Compound 1), isodocarpine (Compound 2), nodocin (Compound 3), and sericin C (Compound 4) can be active ingredients of the dermal papilla cell proliferation promoter. .. These compounds 1 to 4 show a hair papillary cell proliferation promoting effect over minoxidil sulfate and sinapic acid at low concentrations. Therefore, it can be seen that enmain (Compound 1), isodocarpine (Compound 2), nodocin (Compound 3), and sericin C (Compound (Compound 4) are more effective in promoting hair papilla cells than minoxidil sulfate and sinapic acid. ..
（実施例３）
実施例３は、実施例１で単離したエンメイン（化合物１）について、ヘアサイクルに関与する増殖因子ＶＥＧＦ遺伝子に対するｍＲＮＡ産生促進試験を行ったものである。産生促進試験は、ｍＲＮＡの発現を評価するｍＲＮＡ発現促進作用試験（リアルタイムＰＣＲ法）により行った。
実施例２で用いたものと同様に、３７℃、５％ＣＯ２下で培養したで培養されたヒト正常毛乳頭細胞を、トリプシン／ＥＤＴＡ溶液（０．０５％濃度）によりトリプシン処理した。
処理後の細胞を、ウシ胎児血清（ＦＢＳ）含有ＤＭＥＭ培地を用いて、４．０×１０４細胞／ｍＬの濃度に希釈した。希釈された細胞を、６ウエルのマイクロプレートに、１ｍＬ／ウエルで播種し、４日間培養した。培養後、培地を吸引除去し、ＰＢＳ（－）を用いて洗浄後、ウシ胎児血清（ＦＢＳ）不含ＤＭＥＭ培地（１ｍＬ）を加え、１日間培養した。
その後、被験試料としてのエンメイン（化合物１）を、２．５μｍｏｌ／Ｌ、５μｍｏｌ／Ｌ、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌのそれぞれの濃度でＦＢＳ不含ＤＭＥＭに溶解させた試験液を１ｍＬ／ウエルで加え、さらに１日間培養した。
(Example 3)
In Example 3, the mRNA production promoting test for the growth factor VEGF gene involved in the hair cycle was performed on the enmain (Compound 1) isolated in Example 1. The production promotion test was performed by an mRNA expression promoting action test (real-time PCR method) for evaluating the expression of mRNA.
Human normal dermal papilla cells cultured at 37 ° C. under 5% CO 2 were trypsinized with a trypsin / EDTA solution (0.05% concentration) in the same manner as used in Example 2.
The treated cells were diluted with Fetal Bovine Serum (FBS) -containing DMEM medium to a concentration of 4.0 × 104 cells / mL. Diluted cells were seeded in 6-well microplates at 1 mL / well and cultured for 4 days. After culturing, the medium was removed by suction, washed with PBS (−), DMEM medium (1 mL) free of fetal bovine serum (FBS) was added, and the cells were cultured for 1 day.
Then, 1 mL / well of the test solution in which Enmain (Compound 1) as a test sample was dissolved in FBS-free DMEM at the respective concentrations of 2.5 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L. In addition, the cells were cultured for another day.
培養後、一般的な方法によりｔｏｔａｌＲＮＡを調製した。被験試料無添加で培養した細胞についても、同様にｔｏｔａｌＲＮＡを調製した。それぞれのＲＮＡ量を分光光度計で測定し、０．２μｇ/μＬになるようにｔｏｔａｌＲＮＡを調製した。このｔｏｔａｌＲＮＡを鋳型とし、市販のキットにて、ｃＤＮＡを調製した。このｃＤＮＡを鋳型とし、ヘアサイクルに関与する各種増殖因子であるＶＥＧＦ及び内部標準であるＲＰＬＰ０のｍＲＮＡ発現量を測定した。検出は、リアルタイムＰＣＲ装置（ＣＦＸ Ｃｏｎｎｅｃｔ Ｒｅａｌ－Ｔｉｍｅ ＰＣＲ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｂｉｏ－Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ社製））を用いて、ＳｓｏＡｄｖａｎｃｅｄｔｍ Ｕｎｉｖｅｒｓａｌ ＳＹＢＲ（登録商標）Ｇｒｅｅｎ Ｓｕｐｅｒｍｉｘ（Ｂｉｏ－Ｒａｄ
Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ社製）によるリアルタイムＰＣＲ反応により行った。
増殖因子ＶＥＧＦのｍＲＮＡ発現量は、ＲＰＬＰ０のｍＲＮＡの発現量で補正した。
なお、増殖因子ＶＥＧＦのｍＲＮＡの発現値は以下の式（２）により算出した。
増殖因子ＶＥＧＦのｍＲＮＡの発現値＝Ａ２／Ｂ２ 式（２）
Ａ２：被験試料添加時の補正値
Ｂ２：被験試料無添加時（コントロール）の補正値
After culturing, total RNA was prepared by a general method. TotalRNA was prepared in the same manner for the cells cultured without the addition of the test sample. The amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 0.2 μg / μL. Using this total RNA as a template, cDNA was prepared using a commercially available kit. Using this cDNA as a template, the mRNA expression levels of VEGF, which is a growth factor involved in the hair cycle, and RPLP0, which is an internal standard, were measured. Detection was performed using a real-time PCR device (CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.)), SsoAdvancedtm Universal SYBR (registered trademark) GreenSuper.
It was carried out by a real-time PCR reaction by (Laboratories, Inc.).
The mRNA expression level of the growth factor VEGF was corrected by the mRNA expression level of RPLP0.
The expression value of mRNA of growth factor VEGF was calculated by the following formula (2).
Expression value of growth factor VEGF mRNA = A2 / B2 formula (2)
A2: Correction value when the test sample is added B2: Correction value when the test sample is not added (control)
エンメイン（化合物１）についての増殖因子ＶＥＧＦのｍＲＮＡ発現値を、以下の表２に示す。表２に示すｍＲＮＡ発現値は、それぞれ１点について、コントロールを１とした相対値である。
上記の表２に示されるように、エンメイン（化合物１）には、被験試料無添加の２倍程度の増殖因子ＶＥＧＦのｍＲＮＡ産生促進効果が認められた。
このように、実施例３の結果から、エンメイン（化合物１）は、増殖因子ＶＥＧＦのｍＲＮＡ産生促進剤の有効成分となり得ることが確認された。
As shown in Table 2 above, Enmain (Compound 1) was found to have about twice the effect of promoting mRNA production of the growth factor VEGF as that without the addition of the test sample.
As described above, from the results of Example 3, it was confirmed that Enmain (Compound 1) can be an active ingredient of the mRNA production promoter of the growth factor VEGF.
（実施例４）
実施例４は、実施例２で単離したエンメイン（化合物１）について、ヘアサイクルに関与する増殖因子ＶＥＧＦタンパクに対する産生促進試験を行ったものである。産生促進試験は、ヒト毛乳頭細胞から培地へ分泌されたＶＥＧＦタンパク量をエライザ法にて定量し、評価する方法により行った。
実施例２で用いたものと同様に、３７℃、５％ＣＯ２下で培養されたヒト正常毛乳頭細胞を、トリプシン／ＥＤＴＡ溶液（０．０５％濃度）によりトリプシン処理した。処理後
の細胞を、ウシ胎児血清（ＦＢＳ）含有ＤＭＥＭ培地を用いて、４．０×１０４細胞／ｍＬの濃度に希釈した。希釈された細胞を、６ウエルのマイクロプレートに、５００μＬ／ウエルで播種し、４日間培養した。培養後、培地を吸引除去し、ＰＢＳ（－）を用いて洗浄後、ウシ胎児血清（ＦＢＳ）不含ＤＭＥＭ培地（２００μＬ）を加え、１日間培養した。
その後、被験試料としてのエンメイン（化合物１）を、５μｍｏｌ／Ｌ、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌのそれぞれの濃度でＦＢＳ不含ＤＭＥＭに溶解させた試験液を２００μＬ／ウエルで加え、さらに１日間培養した。
(Example 4)
In Example 4, the enmain (Compound 1) isolated in Example 2 was subjected to a production promotion test for the growth factor VEGF protein involved in the hair cycle. The production promotion test was carried out by a method of quantifying and evaluating the amount of VEGF protein secreted from human dermal papilla cells into the medium by the Eliza method.
Human normal dermal papilla cells cultured at 37 ° C. and 5% CO 2 were trypsinized with a trypsin / EDTA solution (0.05% concentration) in the same manner as used in Example 2. The treated cells were diluted with Fetal Bovine Serum (FBS) -containing DMEM medium to a concentration of 4.0 × 104 cells / mL. Diluted cells were seeded in 6-well microplates at 500 μL / well and cultured for 4 days. After culturing, the medium was removed by suction, washed with PBS (−), DMEM medium (200 μL) free of fetal bovine serum (FBS) was added, and the cells were cultured for 1 day.
Then, a test solution prepared by dissolving Enmain (Compound 1) as a test sample in FBS-free DMEM at concentrations of 5 μmol / L, 10 μmol / L, and 20 μmol / L was added at 200 μL / well for another day. It was cultured.
培養後、培地上精２００μＬをマイクロチューブにとり、遠心分離（５０００ｒｐｍ／５ｍｉｎ）を行った後、その上精を市販のエライザキットにて培地中の増殖因子ＶＥＧＦタンパク量を測定した。
なお、増殖因子ＶＥＧＦタンパク量産生促進率（％）は、以下の式（３）により算出した
増殖因子ＶＥＧＦタンパク量産生促進率（％）＝Ａ３／Ｂ３×１００ 式（３）
Ａ３：被験試料添加時の増殖因子ＶＥＧＦタンパク量
Ｂ３：被験試料無添加時（コントロール）の増殖因子ＶＥＧＦタンパク量
After culturing, 200 μL of the supernatant on the medium was placed in a microtube, centrifuged (5000 rpm / 5 min), and then the supernatant was measured for the amount of growth factor VEGF protein in the medium using a commercially available Eliza kit.
The growth factor VEGF protein production promotion rate (%) is calculated by the following formula (3). Growth factor VEGF protein production promotion rate (%) = A3 / B3 × 100 formula (3).
A3: Growth factor VEGF protein amount when test sample is added B3: Growth factor VEGF protein amount when test sample is not added (control)
エンメイン（化合物１）についての増殖因子ＶＥＧＦタンパク量産生促進率（％）を、以下の表３に示す。表３に示す増殖因子ＶＥＧＦタンパク量産生促進率（％）は、それぞれ１点について、コントロールを１００％とした相対値である
上記の表３に示されるように、エンメイン（化合物１）には、被験試料無添加の１．２倍程度の増殖因子ＶＥＧＦタンパク量産生促進効果が認められた。
このように、実施例４の結果から、エンメイン（化合物１）は、ＶＥＧＦタンパク量産生促進剤の有効成分となり得ることが確認された。
As shown in Table 3 above, Enmain (Compound 1) was found to have an effect of promoting the production of the growth factor VEGF protein by about 1.2 times that of the non-added test sample.
As described above, from the results of Example 4, it was confirmed that Enmain (Compound 1) can be an active ingredient of the VEGF protein amount production promoter.
（実施例５）
実施例５は、実施例２で単離したエンメイン（化合物１）について、細胞の分化・増殖に関与するシグナル伝達分子であるβ―カテニンに対する産生促進試験を行った実施例である。産生促進試験は、ヒト毛乳頭細胞内のβ―カテニン量をウェスタンブロット法にて評価する方法により行った。
実施例２で用いたものと同様に、３７℃、５％ＣＯ２下で培養されたヒト正常毛乳頭細胞を、トリプシン／ＥＤＴＡ溶液（０．０５％濃度）によりトリプシン処理した。処理後の細胞を、ウシ胎児血清（ＦＢＳ）含有ＤＭＥＭ培地を用いて、５．０×１０５細胞／ｍＬの濃度に希釈した。希釈された細胞を、７５ｃｍ２培養フラスコに１．０ｍＬ播種し、サブコンフルエントになるまで培養した。培養後、培地を吸引除去、ＰＢＳ（－）を用いて洗浄後、ウシ胎児血清（ＦＢＳ）不含ＤＭＥＭ培地（１０ｍＬ）加え、１日間培養した。
その後、培地を除去し、被験試料としてのエンメイン（化合物１）を、５μｍｏｌ／Ｌ
、１０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌのそれぞれの濃度でＦＢＳ不含ＤＭＥＭに溶解させた試験液を１０ｍＬ／ウエルで加え、さらに1日間培養した。
(Example 5)
Example 5 is an example in which the production promotion test for β-catenin, which is a signal transduction molecule involved in cell differentiation and proliferation, was carried out on the enmain (compound 1) isolated in Example 2. The production promotion test was carried out by a method of evaluating the amount of β-catenin in human dermal papilla cells by Western blotting.
Human normal dermal papilla cells cultured at 37 ° C. and 5% CO 2 were trypsinized with a trypsin / EDTA solution (0.05% concentration) in the same manner as used in Example 2. The treated cells were diluted with Fetal Bovine Serum (FBS) -containing DMEM medium to a concentration of 5.0 × 105 cells / mL. The diluted cells were seeded in 1.0 mL in a 75 cm 2 culture flask and cultured until subconfluent. After culturing, the medium was removed by suction, washed with PBS (-), and then Fetal bovine serum (FBS) -free DMEM medium (10 mL) was added and cultured for 1 day.
Then, the medium was removed, and Enmain (Compound 1) as a test sample was added to 5 μmol / L.
The test solution dissolved in FBS-free DMEM at the respective concentrations of 10 μmol / L and 20 μmol / L was added at 10 mL / well, and the cells were further cultured for 1 day.
培養後、一般的な方法により細胞溶解液を調製した。被験試料無添加で培養した細胞についても、同様に細胞溶解液を調製した。それぞれの細胞溶解液を市販のＢＣＡタンパク定量キットにて総タンパク量で測定し、総タンパク量を１μｇ／μＬになるように細胞溶解液を調製した。この細胞溶解液２５μＬをポリアクリドゲルに充填し、電気泳動を行った。
電気泳動後、ニトロセルロース膜にブロティングした後、抗β―カテニン抗体および抗β―アクチン抗体をそれぞれ反応させＨＲＰが結合された２次抗体を反応させた。その後ケミルミシステムにてタンパクを蛍光検出し、その蛍光強度を数値化し、β―カテニン量を求めた。
β―カテニン量は、β―アクチン量で補正し算出した。β―カテニン産生促進量は以下の式（４）により算出した。
β―カテニン産生促進量（％）＝Ａ４／Ｂ４×１００ 式（４）
Ａ４：被験試料添加時の補正値
Ｂ４：被験試料無添加時（コントロール）の補正時値
After culturing, a cytolytic solution was prepared by a general method. Cytolytic solutions were similarly prepared for cells cultured without the addition of the test sample. Each cytolytic solution was measured by the total protein amount with a commercially available BCA protein quantification kit, and the cytolytic solution was prepared so that the total protein amount was 1 μg / μL. 25 μL of this cytolytic solution was filled in polyacrid gel and electrophoresed.
After electrophoresis, blotting was performed on a nitrocellulose membrane, and then an anti-β-catenin antibody and an anti-β-actin antibody were reacted with each other to react with a secondary antibody to which HRP was bound. After that, the protein was fluorescently detected by the Chemilmi system, the fluorescence intensity was quantified, and the amount of β-catenin was determined.
The amount of β-catenin was corrected and calculated by the amount of β-actin. The amount of β-catenin production promotion was calculated by the following formula (4).
β-catenin production promotion amount (%) = A4 / B4 × 100 formula (4)
A4: Correction value when the test sample is added B4: Correction value when the test sample is not added (control)
エンメイン（化合物１）についてのβ―カテニン産生促進量を、以下の表４に示す。表４に示す産生促進量は、それぞれ１点について、コントロールを１とした相対値である。
上記の表４に示されるように、エンメイン（化合物１）には、被験試料無添加時と比較してβ―カテニン産生促進効果が認められた。
このように、実施例５の結果から、エンメイン（化合物１）は、β―カテニン産生促進剤の有効成分となり得ることが確認された。
As shown in Table 4 above, Enmain (Compound 1) was found to have a β-catenin production promoting effect as compared with the case where no test sample was added.
As described above, from the results of Example 5, it was confirmed that Enmain (Compound 1) can be an active ingredient of the β-catenin production promoter.
（実施例６）
実施例６は、実施例２で単離したエンメイン（化合物１）について、男性ホルモンの活性化酵素である５α―リダクターゼに対する酵素阻害活性試験を行ったものである。酵素阻害活性試験は、下記の方法による評価を行った。
ここで、４８穴マイクロプレートを使用し、緩衝液には４０ｍＭのＫＨ２ＰＯ４－Ｋ２ＨＰＯ４ ｂｕｆｆｅｒ ｐＨ６．５（富士フイルム和光純薬（株）社製）を用いて実験を行った。
０．３５ｎｍｏｌのｔｅｓｔｏｓｔｅｒｏｎｅ（東京化成（株）社製）と１０ｎｍｏｌのＮＡＤＰＨ（富士フイルム和光純薬（株）社製）との混合溶液を各ウエルに４９０μＬずつ添加し、被験物質（ＤＭＳＯで希釈）を５μＬずつ添加した。室温で２０分間インキュベートした後、１０倍希釈したＳ９ Ｒａｔ Ｌｉｖｅｒ Ｆｒａｃｔｉｏｎｓ（オリエンタル酵母工業（株）社製）を１０μＬずつ添加し、３７℃で３０分間加温して反応させた。反応後、沸騰水浴で２分間加熱し、酵素を失活させた。酵素失活後の溶液５００μＬ／ｗｅｌｌを抜き取り、酢酸エチル５００μＬと混ぜ、遠心分離（１００００ｒｐｍ、５ｍｉｎ）した。酢酸エチル層３００μＬを取りだし、酢酸エチルを除去してテストステロンを得た。得られたテストステロンを内部標準物質（ｆｌｕｄｒｏｃｏｒｔｉｓｏｎｅ
ａｃｅｔａｔｅ ２０μｇ／ｍＬ、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）含有のアセトニトリル３０μＬで溶かし、ＨＰＬＣにてテストステロン濃度を定量した。
(Example 6)
In Example 6, the enzyme inhibitory activity test against 5α-reductase, which is an androgen activating enzyme, was performed on the enmain (compound 1) isolated in Example 2. The enzyme inhibitory activity test was evaluated by the following method.
Here, an experiment was conducted using a 48-well microplate and a 40 mM KH 2 PO 4 -K 2 HPO 4 buffer pH 6.5 (manufactured by Wako Pure Chemical Industries, Ltd.) as a buffer solution.
Add 490 μL of a mixed solution of 0.35 nmol testosterone (manufactured by Tokyo Kasei Co., Ltd.) and 10 nmol NADPH (manufactured by Wako Pure Chemical Industries, Ltd.) to each well, and test substance (diluted with DMSO). Was added in 5 μL increments. After incubating at room temperature for 20 minutes, 10 μL of S9 Rat Liver Fractions (manufactured by Oriental Yeast Co., Ltd.) diluted 10-fold was added, and the mixture was heated at 37 ° C. for 30 minutes for reaction. After the reaction, the enzyme was inactivated by heating in a boiling water bath for 2 minutes. 500 μL / well of the solution after enzyme deactivation was withdrawn, mixed with 500 μL of ethyl acetate, and centrifuged (10000 rpm, 5 min). 300 μL of the ethyl acetate layer was taken out and ethyl acetate was removed to obtain testosterone. The obtained testosterone is used as an internal standard substance (fludrocortisone).
Acetate 20 μg / mL, Sigma-Aldrich) -containing acetonitrile was dissolved in 30 μL, and the testosterone concentration was quantified by HPLC.
テストステロンのピーク面積と内部標準物質のピーク面積との比を縦軸に、テストステロンの濃度を横軸にして、検量線を作成した。検量線より求めたテストステロン定量値から、以下の式（５）より阻害率（％）を算出した。
阻害率（％）＝（Ａ５－Ｂ５）／（Ｃ５－Ｂ５）×１００ 式（５）
Ａ５：被験物質添加
Ｂ５：被験物質未添加（コントロール）
Ｃ５：酵素未添加
A calibration curve was prepared with the ratio of the peak area of testosterone to the peak area of the internal standard substance on the vertical axis and the concentration of testosterone on the horizontal axis. The inhibition rate (%) was calculated from the following formula (5) from the testosterone quantitative value obtained from the calibration curve.
Inhibition rate (%) = (A5-B5) / (C5-B5) × 100 formula (5)
A5: Addition of test substance
B5: No test substance added (control)
C5: No enzyme added
上記の表５に示されるように、エンメイン（化合物１）は、有意な５α―リダクターゼ酵素阻害活性を有さなかった。すなわち、エンメイン（化合物１）は、男性ホルモンの活性化に影響を与えないことが確認された。 As shown in Table 5 above, Enmain (Compound 1) did not have significant 5α-reductase enzyme inhibitory activity. That is, it was confirmed that enmain (compound 1) does not affect the activation of male hormones.
（実施例７）
実施例７として、実施例２で単離したエンメインを用いた育毛剤を表６に示す。
As Example 7, the hair growth agent using Enmain isolated in Example 2 is shown in Table 6.
実施例７に示すように、エンメインを有効成分とする毛乳頭細胞増殖促進効果、ＶＥＧＦ産生促進効果、β―カテニン産生促進効果を有する育毛剤が調製できる。 As shown in Example 7, a hair-growth agent containing enmain as an active ingredient and having a hair papilla cell proliferation promoting effect, a VEGF production promoting effect, and a β-catenin production promoting effect can be prepared.
（実施例８）
実施例８として、実施例２で単離したエンメインを用いた育毛剤を以下の表７に示す。
As Example 8, the hair-growth agent using Enmain isolated in Example 2 is shown in Table 7 below.
実施例８に示すように、エンメインを有効成分とする毛乳頭細胞増殖促進効果、ＶＥＧＦ産生促進効果、β―カテニン産生促進効果を有する育毛剤が調製できる。 As shown in Example 8, a hair-growth agent containing enmain as an active ingredient and having a hair papilla cell proliferation promoting effect, a VEGF production promoting effect, and a β-catenin production promoting effect can be prepared.
（実施例９）
実施例９として、実施例２で単離したエンメインを用いた毛髪洗浄剤を以下の表８に示す。
As Example 9, the hair cleansing agent using Enmain isolated in Example 2 is shown in Table 8 below.
実施例９に示すように、エンメインを有効成分とする毛乳頭細胞増殖促進効果、ＶＥＧＦ産生促進効果、β―カテニン産生促進効果を有する毛髪洗浄剤が調製できる。 As shown in Example 9, a hair cleanser having a hair papilla cell proliferation promoting effect, a VEGF production promoting effect, and a β-catenin production promoting effect containing enmain as an active ingredient can be prepared.
本発明によれば、副作用の懸念も少なく、男女問わず使用でき、男性ホルモンの活性化に影響を与えることなく、優れた毛乳頭細胞増殖促進効果、血管内皮増殖因子（ＶＥＧＦ）産生促進効果、β―カテニン産生促進効果を有する発毛促進剤の提供ができる。 According to the present invention, there is little concern about side effects, it can be used by both men and women, and it has an excellent hair papilla cell proliferation promoting effect and vascular endothelial growth factor (VEGF) production promoting effect without affecting the activation of male hormones. It is possible to provide a hair growth promoter having an effect of promoting β-catenin production.
Claims (4)
A β-catenin production promoter composed of a compound represented by Chemical Formula 1.
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