JP2019019108A - 5α-REDUCTASE INHIBITOR, AND COMPOSITION FOR PREVENTIVE TREATMENT OF ALOPECIA OR HAIR GROWTH PROMOTION, AND METHOD FOR INHIBITING 5α-REDUCTASE IN SCALP AND HAIR GROWTH PROMOTION METHOD - Google Patents
5α-REDUCTASE INHIBITOR, AND COMPOSITION FOR PREVENTIVE TREATMENT OF ALOPECIA OR HAIR GROWTH PROMOTION, AND METHOD FOR INHIBITING 5α-REDUCTASE IN SCALP AND HAIR GROWTH PROMOTION METHOD Download PDFInfo
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Abstract
Description
本発明は、新規の５α−レダクターゼ阻害剤、及びその応用技術に関する。 The present invention relates to a novel 5α-reductase inhibitor and its application technology.
男性においては、男性ホルモンが関与する男性型脱毛症がよく知られている。また、女性の脱毛症においても男性ホルモンが関与する女性男性型脱毛症が知られている。女性は加齢とともに女性ホルモンが低下し、更年期に女性ホルモンの分泌が減少し、相対的に男性ホルモンが過剰になるために発症しやすくなると考えられている。 In men, male pattern baldness involving male hormones is well known. In addition, female androgenetic alopecia involving male hormones is also known in female alopecia. It is believed that females are more likely to develop because female hormones decrease with age, female hormone secretion decreases during menopause, and male hormones are relatively excessive.
５α−レダクターゼは、テストステロンから活性型のジヒドロテストロンへと変換する酵素である。男性ホルモンであるテストステロンは、毛包、皮脂腺等に存在する５α−レダクターゼによりジヒドロテストステロン（ＤＨＴ）に転換され、生成したジヒドロテストステロンによって、毛母細胞の分化を阻害して脱毛を促進させる。そのため、５α−レダクターゼの活性を阻害することにより、脱毛症の緩和等の効能効果が期待できる。 5α-reductase is an enzyme that converts testosterone into active dihydrotestrone. Testosterone, a male hormone, is converted to dihydrotestosterone (DHT) by 5α-reductase present in hair follicles, sebaceous glands and the like, and the generated dihydrotestosterone inhibits the differentiation of hair matrix cells and promotes hair loss. Therefore, by inhibiting the activity of 5α-reductase, efficacy effects such as alopecia alleviation can be expected.
これまでに、様々な５α−レダクターゼ阻害剤が報告されており、その有効成分として、雪霊芝抽出物（特許文献１）、ポリウムオシド（特許文献２）、キツネノマゴ科クロサンドラ属植物抽出物（特許文献３）、コメバツガザクラ抽出物（特許文献４）、非発酵型ルイボス茶由来アスパラチン（特許文献５）等が知られている。 So far, various 5α-reductase inhibitors have been reported, and as their active ingredients, snow ganoderma extract (Patent Document 1), polyumoside (Patent Document 2), foxglove Crosandra plant extract (Patent) Document 3), rice bran cherry extract (patent document 4), non-fermented rooibos tea-derived asparatin (patent document 5) and the like are known.
上述した従来例の有効成分の中には、５α−レダクターゼ阻害作用が十分でないこともあり、さらなる開発が行われていた。
かかる状況下、本発明の目的は、新規の５α−レダクターゼ阻害剤及びその応用用途を提供することにある。
Among the active ingredients of the conventional examples described above, 5α-reductase inhibitory action is not sufficient, and further development has been performed.
Under such circumstances, an object of the present invention is to provide a novel 5α-reductase inhibitor and its application.
本発明者は、上記課題を解決すべく鋭意研究を重ねた結果、フルボ酸にテストステロン５α−レダクターゼ阻害作用を見出し、本発明に至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that fulvic acid has a testosterone 5α-reductase inhibitory action, leading to the present invention.
すなわち、本発明は、以下の発明に係るものである。
＜１＞ フルボ酸を有効成分として含有する５α−レダクターゼ阻害剤。
＜２＞ 前記フルボ酸が、木材分解物由来のフルボ酸である＜１＞に記載の５α−レダクターゼ阻害剤。
＜３＞ 前記フルボ酸が、精製フルボ酸である＜１＞又は＜２＞に記載の５α−レダクターゼ阻害剤。
＜４＞ フルボ酸を含有し、５α−レダクターゼ阻害作用に基づく脱毛症の予防治療用又は育毛促進用組成物。
＜５＞ フルボ酸を含有する組成物を、頭皮投与させることにより、頭皮中の５α−レダクターゼを阻害させる方法（但し、ヒトに対する医療行為を除く。）。
＜６＞ フルボ酸を含有する組成物を、頭皮投与させる育毛促進方法（但し、ヒトに対する医療行為を除く。）。
That is, the present invention relates to the following inventions.
<1> A 5α-reductase inhibitor containing fulvic acid as an active ingredient.
<2> The 5α-reductase inhibitor according to <1>, wherein the fulvic acid is a fulvic acid derived from a wood degradation product.
<3> The 5α-reductase inhibitor according to <1> or <2>, wherein the fulvic acid is purified fulvic acid.
<4> A composition for preventing or treating alopecia or promoting hair growth, comprising fulvic acid and based on a 5α-reductase inhibitory action.
<5> A method of inhibiting 5α-reductase in the scalp by administering a composition containing fulvic acid to the scalp (excluding medical practice for humans).
<6> A method for promoting hair growth in which a composition containing fulvic acid is administered to the scalp (excluding medical treatment for humans).
本発明によれば、フルボ酸を有効成分とすることによって、５α−レダクターゼの活性を阻害することができる５α−レダクターゼ阻害剤が提供される。
また、フルボ酸を有効成分とすることによって、５α−レダクターゼ阻害作用に基づいて、脱毛症の予防治療作用及び育毛促進作用に優れる組成物を提供することができる。
According to the present invention, there is provided a 5α-reductase inhibitor capable of inhibiting the activity of 5α-reductase by using fulvic acid as an active ingredient.
Further, by using fulvic acid as an active ingredient, it is possible to provide a composition that is excellent in the effect of preventing and treating alopecia and the effect of promoting hair growth based on the 5α-reductase inhibitory action.
以下、本発明について例示物等を示して詳細に説明するが、本発明は以下の例示物等に限定されるものではなく、本発明の要旨を逸脱しない範囲において任意に変更して実施できる。なお、本明細書において、「〜」とはその前後の数値又は物理量を含む表現として用いるものとする。 Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited to the following examples and the like, and can be arbitrarily modified and implemented without departing from the gist of the present invention. In the present specification, “to” is used as an expression including numerical values or physical quantities before and after.
＜１．５α−レダクターゼ阻害剤＞
本発明の５α−レダクターゼ阻害剤は、フルボ酸を有効成分とし、５α−レダクターゼの活性を阻害することができる。
<1.5α-reductase inhibitor>
The 5α-reductase inhibitor of the present invention contains fulvic acid as an active ingredient and can inhibit the activity of 5α-reductase.
本発明に係るフルボ酸（fulvic acid）は、いわゆる腐植物質に含まれる物質であり、酸によって沈殿しない無定形高分子有機酸を意味する。本明細書において「腐植物質」とは、植物の葉や茎などの有機物が多種多様な微生物によって分解し、二次的に生成された有機成分で、糖、タンパク質、脂質などに分類されない有機物の総称をいう。
フルボ酸は、化学構造がただ一つ決まった分子ではなく、その分子内にカルボキシル基、フェノール性水酸基を多く含んだ多価有機酸である。なお、腐植物質には、フルボ酸と共にフミン酸（humic acid）が含まれる。日本腐植学会によると「腐植物質の定義はあくまで疑念的定義」であるが、土壌や堆積物からの腐植物質は、一般には酸及び塩基に対する溶解性に基づいて、フミン酸は一般に塩基性水溶液に可溶であり、フルボ酸は一般に酸性及び塩基性水溶液に可溶であると定義されている。
本発明においては、「フルボ酸」を、「腐植物質（木材分解物を含む）から分離精製することができる物質であって、酸性及び塩基性水溶液に可溶な無定形高分子有機酸」と定義するものとする。
The fulvic acid according to the present invention is a substance contained in so-called humic substances, and means an amorphous high molecular organic acid that is not precipitated by the acid. In this specification, the term “humic substance” refers to organic components that are secondarily generated and decomposed by a wide variety of microorganisms, such as plant leaves and stems, and are not classified as sugar, protein, lipid, etc. A general term.
Fulvic acid is not a molecule with a single chemical structure, but a polyvalent organic acid containing many carboxyl groups and phenolic hydroxyl groups in the molecule. In addition, humic acid is contained in humic substance with fulvic acid. According to the Japan Humus Society, the definition of humic substances is a suspicious definition, but humic substances from soil and sediment are generally based on solubility in acids and bases, and humic acids are generally dissolved in basic aqueous solutions. Soluble and fulvic acid is generally defined as soluble in acidic and basic aqueous solutions.
In the present invention, “fulvic acid” is “a substance that can be separated and purified from humic substances (including wood degradation products) and is soluble in acidic and basic aqueous solutions” Shall be defined.
本発明の５α−レダクターゼ阻害剤が含有するフルボ酸は、ヒト、特にヒトの頭皮に適用が許容されるものであれば特に限定されず、例えば、地上の土壌、海底の土壌等に由来するフルボ酸；河川、湖沼等の水系に由来するもの等の天然由来のフルボ酸、有機廃棄物由来のフルボ酸等の人工的に製造されたフルボ酸のいずれも使用することができる。これらのフルボ酸は１種又は２種以上を使用することもできる。 The fulvic acid contained in the 5α-reductase inhibitor of the present invention is not particularly limited as long as it can be applied to humans, particularly human scalp. For example, fulvic acid derived from ground soil, seabed soil, etc. Acids: Naturally derived fulvic acids such as those derived from water systems such as rivers and lakes, and artificially produced fulvic acids such as fulvic acids derived from organic wastes can be used. These fulvic acids may be used alone or in combination of two or more.
本発明に係るフルボ酸として、木材分解物由来のフルボ酸が好適に用いられる。本発明において、「木材分解物由来のフルボ酸」とは、実質的に原材料に木材を使用し、木材を微生物や酸液等によって分解して得られる木材分解物から分離抽出されたフルボ酸を意味する。抽出源となる木材分解物は、天然由来のものも使用できるが、通常、人工的に木材を微生物や酸液等によって分解して得られる木材分解物が用いられる。 As the fulvic acid according to the present invention, a fulvic acid derived from a wood decomposition product is preferably used. In the present invention, “a fulvic acid derived from a wood decomposition product” means a fulvic acid separated and extracted from a wood decomposition product obtained by substantially using wood as a raw material and decomposing the wood with a microorganism or an acid solution. means. Naturally derived wood degradation products can be used as an extraction source, but usually wood degradation products obtained by artificially degrading wood with microorganisms, acid solutions, or the like are used.
原料木材としては、広葉樹が好適である。但し、原料木材には本発明の目的を損なわない範囲で広葉樹以外の原料（針葉樹、非木材系リグニン等）が含まれていてもよい。
木材分解物由来のフルボ酸として、後述する実施例の方法で得られるクヌギを原料木材として、特定の白色腐朽菌による分解作用により得られる木材分解物から分離精製したフルボ酸が挙げられる。また、市販の木材分解物由来のフルボ酸も好適に使用できる。
A hardwood is suitable as the raw material wood. However, the raw material wood may contain raw materials other than hardwoods (softwood, non-wood lignin, etc.) as long as the object of the present invention is not impaired.
Examples of the fulvic acid derived from the wood decomposition product include fulvic acid obtained by separating and purifying from the wood decomposition product obtained by the decomposition action by a specific white rot fungus, using kunugi obtained by the method of Examples described later as a raw material wood. In addition, fulvic acid derived from commercially available wood degradation products can also be used suitably.
本発明に係るフルボ酸は、５α−レダクターゼ阻害作用を有している限り、フルボ酸のみならず、フルボ酸の塩、エステル又は誘導体を包含するものとする。本発明に係るフルボ酸として、フルボ酸（特には精製フルボ酸）、フルボ酸の塩が好適に使用できる。 The fulvic acid according to the present invention includes not only fulvic acid but also a salt, ester or derivative of fulvic acid as long as it has a 5α-reductase inhibitory action. As the fulvic acid according to the present invention, fulvic acid (particularly purified fulvic acid) and a salt of fulvic acid can be preferably used.
また、本発明に係るフルボ酸として、５α−レダクターゼ阻害作用を有している限り、フルボ酸以外にもフミン酸、アミノ酸、ビタミン、酵素、ミネラル、その他微量元素を含んでいるものも使用できるが、精製フルボ酸を使用することもできる。
本発明において「精製フルボ酸」とは、フルボ酸を含む混合物からフルボ酸以外の成分を除去し、分離精製して得られるフルボ酸を意味する。分離精製処理は目的とするフルボ酸が得られる限り任意であり、フルボ酸及び他の成分を含む溶液をイオン交換樹脂に通すことなどにより行うことができる。特にＩＨＳＳ法(国際腐植学会指定の操作方法及び確認方法)に準じる方法が好ましい。なお、当該分離精製方法の具体例は実施例にて説明する。
Moreover, as long as it has a 5 (alpha) -reductase inhibitory effect as a fulvic acid which concerns on this invention, what contains humic acid, an amino acid, a vitamin, an enzyme, a mineral, and other trace elements besides fulvic acid can also be used. Purified fulvic acid can also be used.
In the present invention, “purified fulvic acid” means fulvic acid obtained by removing components other than fulvic acid from a mixture containing fulvic acid and separating and purifying it. Separation and purification treatment is optional as long as the desired fulvic acid is obtained, and can be performed by passing a solution containing fulvic acid and other components through an ion exchange resin. In particular, a method according to the IHSS method (operation method and confirmation method specified by the International Humus Society) is preferable. In addition, the specific example of the said isolation | separation purification method is demonstrated in an Example.
また、本発明に係るフルボ酸として、５α−レダクターゼ阻害作用を有している限り、各種市販されているものをそのまま又は適宜濃縮、希釈して使用することができる。このような市販品としては、株式会社Ｔ＆Ｇから販売されている腐植前駆物質溶液「リードアップ」、株式会社ミヤモンテＪＡＰＡＮから販売されている「キレートバランス」及び「美しさの源気麗−留」、株式会社日本フルボ酸総合研究所から販売されている「濃密生成フルボ酸製品」（フルボ酸植物活性化剤「みどりの神様」）、コヨウ株式会社製のフルボ酸等が挙げられる。
このような市販品には、精製の都合上、不純物としてフミン酸等を含有している場合があるが、その場合は分離精製して使用することもできる。
また、日本腐食物質学会の頒布する愛知県段戸森林土壌由来のフルボ酸（標準フルボ酸）も使用することができる。
Moreover, as long as it has a 5 (alpha) -reductase inhibitory effect as a fulvic acid based on this invention, what is marketed can be used as it is or it concentrates and dilutes suitably. As such commercial products, the humus precursor solution “Lead-up” sold by T & G Co., Ltd., “Chelate balance” and “Beauty source of beauty-tome” sold by Miyamonte Japan Co., Ltd., “Densely-produced fulvic acid products” (manufactured by the fulvic acid plant activator “Midori no Kami”) sold by Japan Research Institute for Fulvic Acid Co., Ltd., fulvic acid manufactured by Koyo Co., Ltd., and the like.
Such a commercial product may contain humic acid or the like as an impurity for the sake of purification. In that case, it can be used after separation and purification.
In addition, fulvic acid (standard fulvic acid) derived from Aichi Prefecture Dando forest soil distributed by the Japanese Society of Corrosive Substances can also be used.
本発明の５α−レダクターゼ阻害剤において、フルボ酸の含有量は、有意な５α−レダクターゼ阻害作用が得られる範囲で適宜決定すればよい。例えば、フルボ酸の量として、０．０００１〜１００質量％である。 In the 5α-reductase inhibitor of the present invention, the content of fulvic acid may be appropriately determined within a range in which a significant 5α-reductase inhibitory action is obtained. For example, the amount of fulvic acid is 0.0001 to 100% by mass.
上述のようなフルボ酸の５α−レダクターゼ阻害作用から、医薬部外品としての様々な用途が期待される。特に、男性型脱毛症、女性男性型脱毛症、育毛、薄毛及び脱毛の予防、毛生促進、発毛促毛、養毛等の効果が期待できる。 Various uses as a quasi-drug are expected from the above-described inhibitory action of fulvic acid on 5α-reductase. In particular, effects such as androgenetic alopecia, female androgenetic alopecia, hair growth, thinning and prevention of hair loss, hair growth promotion, hair growth promotion and hair restoration can be expected.
本発明の５α−レダクターゼ阻害剤は、５α−レダクターゼの活性を阻害できるため、テストステロン５α−レダクターゼの活性過多、テストステロン分泌過多に起因する各種症状の予防又は治療にも有用である。このような症状としては、例えば、脂漏症、座瘡、前立腺肥大症等、が挙げられる。 Since the 5α-reductase inhibitor of the present invention can inhibit the activity of 5α-reductase, it is useful for the prevention or treatment of various symptoms caused by excessive testosterone 5α-reductase activity and excessive testosterone secretion. Examples of such symptoms include seborrhea, acne, prostatic hypertrophy and the like.
本発明の５α−レダクターゼ阻害剤には、フルボ酸の他、食品、化粧品及び医薬品業界で通常使用される配合剤、例えば、賦形剤、防湿剤、防腐剤、強化剤、増粘剤、乳化剤、酸化防止剤、甘味料、酸味料、調味料、着色料、香料、美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、アルコール類、粉末成分、色剤、水性成分、水、各種皮膚栄養剤等を目的に応じて適宜配合することができる。これら配合剤としては、市販品を好適に使用することができる。 The 5α-reductase inhibitor of the present invention includes, in addition to fulvic acid, compounding agents commonly used in the food, cosmetic and pharmaceutical industries, such as excipients, moisture-proofing agents, preservatives, reinforcing agents, thickeners, emulsifiers. , Antioxidant, sweetener, acidulant, seasoning, colorant, fragrance, whitening agent, moisturizer, oily component, ultraviolet absorber, surfactant, alcohol, powder component, colorant, aqueous component, water, Various skin nutrients and the like can be appropriately blended depending on the purpose. As these compounding agents, commercially available products can be suitably used.
＜２．脱毛症の予防治療用又は育毛促進用組成物＞
本発明の脱毛症の予防治療用又は育毛促進用組成物（以下、「本発明の組成物」と称す。）は、フルボ酸を有効成分とし、５α−レダクターゼ阻害作用に基づいて、脱毛症の予防又は治療作用、及び/又は育毛促進作用を奏することを特徴とする。
なお、本発明の組成物に用いるフルボ酸は、本発明の５α−レダクターゼ阻害剤において説明したものと同一であるため、説明を省略する。
<2. Composition for preventing or treating alopecia or promoting hair growth>
The composition for preventing or treating alopecia or promoting hair growth of the present invention (hereinafter referred to as “the composition of the present invention”) comprises fulvic acid as an active ingredient, and is based on 5α-reductase inhibitory action. It has a preventive or therapeutic action and / or a hair growth promoting action.
In addition, since the fulvic acid used for the composition of this invention is the same as what was demonstrated in the 5 (alpha) -reductase inhibitor of this invention, description is abbreviate | omitted.
本発明の組成物は、脱毛症の予防、脱毛症の治療及び育毛促進の少なくともいずれかの作用を有し、特にすべての作用を有することが好ましい。
本明細書において、「脱毛症の予防」には、脱毛症の抑制及び遅延が含まれる。また、「脱毛症の治療」には、脱毛症の改善及び寛解、並びに脱毛症の進展の抑制が含まれる。本発明の対象となる脱毛症としては、５α−レダクターゼの酵素触媒作用が機序となる脱毛症であれば制限はなく、典型的には男性型脱毛症、女性男性型脱毛症が挙げられる。また、本明細書において、「育毛促進」には、毛髪の成長速度の向上及び発毛の促進が含まれる。
The composition of the present invention has at least one action of preventing alopecia, treating alopecia and promoting hair growth, and particularly preferably has all the actions.
As used herein, “prevention of alopecia” includes suppression and delay of alopecia. “Treatment of alopecia” also includes improvement and remission of alopecia and suppression of the progression of alopecia. The alopecia that is the subject of the present invention is not limited as long as the enzyme catalysis of 5α-reductase is the mechanism, and typically includes male pattern alopecia and female male pattern alopecia. In the present specification, “promotion of hair growth” includes improvement of the growth rate of hair and promotion of hair growth.
本発明の組成物は、特に男性型脱毛症、女性男性型脱毛症、育毛促進、薄毛及び脱毛の予防、毛生促進、発毛促毛、養毛等の効果が期待できる。 The composition of the present invention can be expected to have effects such as male pattern alopecia, female male pattern alopecia, hair growth promotion, prevention of thinning and hair loss, hair growth promotion, hair growth promotion, hair restoration and the like.
本発明の組成物において、上記フルボ酸の配合量は、使用目的、性別、症状等を考慮して適宜決定すればよく、例えば、フルボ酸の量として、０．０００１〜１０質量％である。 In the composition of the present invention, the blending amount of the fulvic acid may be appropriately determined in consideration of the purpose of use, sex, symptoms, and the like. For example, the amount of fulvic acid is 0.0001 to 10% by mass.
本発明の組成物は、フルボ酸と、必要に応じて適宜選択した各種配合剤とを混合することにより調製できる。フルボ酸の他、食品、化粧品及び医薬品業界で通常使用される配合剤、例えば、賦形剤、防湿剤、防腐剤、強化剤、増粘剤、乳化剤、酸化防止剤、甘味料、酸味料、調味料、着色料、香料、美白剤、保湿剤、油性成分、紫外線吸収剤、界面活性剤、アルコール類、粉末成分、色剤、水性成分、水、各種皮膚栄養剤等を目的に応じて適宜配合することができる。これら配合剤としては、市販品を好適に使用することができる。 The composition of this invention can be prepared by mixing a fulvic acid and the various compounding agents selected suitably as needed. In addition to fulvic acid, compounding agents commonly used in the food, cosmetic and pharmaceutical industries, such as excipients, moisture-proofing agents, preservatives, reinforcing agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, Seasoning, coloring, flavoring, whitening agent, moisturizer, oily component, UV absorber, surfactant, alcohols, powder component, colorant, aqueous component, water, various skin nutrients, etc. Can be blended. As these compounding agents, commercially available products can be suitably used.
本発明の組成物は、溶液、分散液、乳液、軟膏、クリーム、ゲル、エアゾール、パック等の皮膚外用剤の形態で使用されることが好ましく、頭皮外用剤の形態で使用されることが特に好ましい。 The composition of the present invention is preferably used in the form of a skin external preparation such as a solution, dispersion, emulsion, ointment, cream, gel, aerosol, pack, etc., and particularly preferably used in the form of a scalp external preparation. preferable.
本発明の組成物の形態は任意であり、溶液、分散液、乳液、軟膏、クリーム、ゲル、エアゾール、パック等の皮膚外用組成物、特には頭皮外用組成物の形態で使用されることが好ましい。具体的には、ヘアトニック、ヘアクリーム、ヘアローション、ヘアシャンプー、ヘアリンス、ヘアコンディショナー、ヘアスプレー、ヘアエアゾール、ポマード、粉末、ジェル等が挙げられるが、これらに制限されるものではない。 The form of the composition of the present invention is arbitrary, and it is preferably used in the form of an external composition for skin, such as a solution, dispersion, emulsion, ointment, cream, gel, aerosol, pack, etc., particularly an external composition for scalp. . Specific examples include, but are not limited to, hair tonics, hair creams, hair lotions, hair shampoos, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, powders and gels.
本発明の組成物は、本発明の目的を損なわない範囲で、他の毛髪用成分を含んでいてもよい。このような成分として、毛根及び毛包細胞活性成分、毛包細胞血流増強成分、殺菌成分、ふけ防止剤、角質軟化剤、清凉剤、保湿剤などが挙げられる。 The composition of the present invention may contain other hair ingredients as long as the object of the present invention is not impaired. Examples of such components include hair follicle and hair follicle cell active components, hair follicle cell blood flow enhancing components, bactericidal components, anti-dandruff agents, keratin softeners, cleansing agents, moisturizing agents, and the like.
本発明の組成物は、頭皮投与させることにより、頭皮中の５α−レダクターゼを阻害させる方法に用いることができる。また、本発明の組成物は、頭皮投与させる、脱毛症の予防治療方法や育毛促進方法に用いることができる。経皮投与の方法としては噴霧、塗布、湿布等の方法が挙げられ、特に制限されない。当該方法における本発明の組成物の使用量は、含有されるフルボ酸の量、使用目的、性別、症状等を考慮して適宜決定すればよい。 The composition of the present invention can be used in a method of inhibiting 5α-reductase in the scalp by administering it to the scalp. In addition, the composition of the present invention can be used in a method for preventing and treating alopecia and a method for promoting hair growth that are administered to the scalp. Examples of the transdermal administration method include spraying, coating, and poultry, and are not particularly limited. What is necessary is just to determine the usage-amount of the composition of this invention in the said method suitably in consideration of the quantity of the fulvic acid to contain, the intended purpose, sex, a symptom, etc.
なお、本発明の組成物を頭皮投与させることによる頭皮中の５α−レダクターゼを阻害させる方法、及び本発明の組成物を頭皮投与させることによる育毛促進方法については、ヒトの医療行為以外に行うことができる。このようなヒトの医療行為に該当しない行為として、例えば、床屋、美容室、ヘアケアサロン等で行われる本発明の組成物のヒトの頭皮への使用が挙げられる。 The method for inhibiting 5α-reductase in the scalp by administering the composition of the present invention to the scalp and the method for promoting hair growth by administering the composition of the present invention to the scalp are performed in addition to human medical practice. Can do. As an act not corresponding to such a human medical practice, for example, use of the composition of the present invention performed on a human scalp performed in a barber shop, a beauty salon, a hair care salon or the like can be mentioned.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
実施例においては、以下の２種類のフルボ酸を使用して、５α−レダクターゼの阻害活性の評価を行った。
フルボ酸Ａ：クヌギ分解物由来のフルボ酸（製造方法は後述）
フルボ酸Ｂ：株式会社日本フルボ酸総合研究所製 フルボ酸
In Examples, the inhibitory activity of 5α-reductase was evaluated using the following two types of fulvic acid.
Fulvic acid A: Fulvic acid derived from a decomposition product of Kunugi (manufacturing method will be described later)
Fulvic acid B: Fulvic acid manufactured by Japan Fulvic Acid Research Institute
１．フルボ酸Ａの製造及びその同定
以下、フルボ酸Ａの製造方法、及びその同定方法を説明する。
フルボ酸Ａの製造方法に使用した原料木材、白色腐朽菌は以下の通りである。
（１）原料木材
原料木材として、クヌギチップを使用した。大分県の森林樹木伐採現場にて発生したものを使用しており、解体などで発生する建築廃材及び産業廃棄物由来の木材を含まない。また、伐採後一切の化学処理を施してはいない。
（２）白色腐朽菌
供試菌として、独立行政法人製品評価技術基盤機構 特許微生物寄託センターの受託番号NITE P-02428（識別のための表示BMC-110012）で特定される白色腐朽菌（以下、「白色腐朽菌（NITE P-02428）」と記載する。）を使用した。
1. Production of fulvic acid A and identification thereof The production method of fulvic acid A and the identification method thereof will be described below.
The raw wood and white rot fungi used in the method for producing fulvic acid A are as follows.
(1) Raw material wood Kunugi chip was used as raw material wood. It uses what was generated at the Oita Prefecture forest tree cutting site, and does not include building waste and wood from industrial waste generated by demolition. In addition, no chemical treatment has been performed after logging.
(2) White rot fungus As a test bacterium, white rot fungus (hereinafter referred to as BMC-110012) designated by NITE P-02428 (identification label for identification) of the National Institute of Technology and Evaluation, Japan "Described as white rot fungus (NITE P-02428)").
＜工程（１）＞
クヌギ木材（幹の部分）を１０ｍｍ程度のチップに粉砕し、得られたチップを更に解繊機(西邦機工株式会社、製品名：ラブマシーン)で処理し、粉末状（１００μｍ程度）に解繊した。
得られた粉末状クヌギと脱脂米糠（添加栄養物）と水とを、５：０．８：４（重量比）で撹拌・混合した後に混合物（クヌギ木粉培地）を、菌床袋（容積３．６Ｌ）へ１．７ｋｇずつ詰め、金枠に入れて形を整えた。詰め込み時の含水率は５７．９重量％であった。
次いで、常法に従いオートクレーブ（１２１℃、６０分）で殺菌処理、放冷後、常法に従い接種菌（白色腐朽菌（NITE P-02428））を接種した。なお、接種菌である白色腐朽菌（NITE P-02428）は、３００ｍＬ三角フラスコにクヌギ木粉培地を適宜入れ前培養したものを使用した。菌床袋のクヌギ木粉培地への接種菌の接種は、１５ｍｍφの鉄棒で菌床袋の培地をプレスし、二箇所の穴を開け接種孔を形成して行い、接種量は、粉末状クヌギ１ｋｇに対して、１０ｇとした。
<Step (1)>
Kunugi wood (trunk part) is crushed into chips of about 10 mm, and the obtained chips are further processed by a defibrating machine (Seiho Kiko Co., Ltd., product name: Love Machine), and defibrated into powder (about 100 μm). did.
The obtained powdered cucumber, defatted rice bran (additive nutrient), and water were stirred and mixed at 5: 0.8: 4 (weight ratio), and then the mixture (Knugi wood flour medium) 3.6L) was packed in 1.7kg each and placed in a metal frame to adjust the shape. The water content at the time of packing was 57.9% by weight.
Then, in accordance with an ordinary method, sterilization was performed in an autoclave (121 ° C., 60 minutes), allowed to cool, and then inoculated with an inoculum (white rot fungus (NITE P-02428)) according to an ordinary method. In addition, the white rot fungus (NITE P-02428), which is an inoculum, was obtained by appropriately pre-culturing a Kunugi wood flour medium in a 300 mL Erlenmeyer flask. Inoculation of the fungus bed bag into the Kunugi wood flour medium is performed by pressing the medium of the fungus bed bag with a 15 mmφ iron bar, making two holes and forming an inoculation hole. It was 10 g for 1 kg.
工程（１）で得られた木材分解物を使用して、以下の手順でフルボ酸を分離精製した。 Using the wood decomposition product obtained in step (1), fulvic acid was separated and purified by the following procedure.
＜工程（２）＞
工程（１）で得られた木材分解物（３０日分解）１ｋｇと水２Ｌとを容器に入れミキサーで、所定の時間（３時間以上）撹拌して木材分解物に含まれる成分を水に抽出し、木材分解物を含有する液状組成物を得た。
<Step (2)>
Put 1kg of wood degradation product (30-day degradation) obtained in step (1) and 2L of water into a container and stir with a mixer for a predetermined time (3 hours or more) to extract the components contained in the wood degradation product into water. Thus, a liquid composition containing a wood decomposition product was obtained.
＜工程（３）＞
工程（２）で得られた木材分解物を含有する液状組成物を、遠心分離機にて固液分離を行い、液体部分をデカンテーションして得た液体を減圧乾燥器にて減圧乾燥し、腐植酸物質を含有する乾燥粉末を得た。収率は約５％（原料１ｋｇから約５０ｇ生成)であった。
<Step (3)>
The liquid composition containing the wood decomposition product obtained in step (2) is subjected to solid-liquid separation with a centrifuge, and the liquid obtained by decanting the liquid part is dried under reduced pressure with a vacuum dryer, A dry powder containing humic acid material was obtained. The yield was about 5% (generated about 50 g from 1 kg of raw material).
＜工程（４）＞
工程（３）で得られた乾燥粉末を、以下の工程によって分離精製し、フルボ酸を得た。なお、この分離精製方法は、ＩＨＳＳ法(国際腐植学会指定の操作方法及び確認方法)に相当する。
まず、工程（３）で得られた乾燥粉末を、０．１Ｍ-ＮａＯＨ水溶液に入れ、１日放置後に沈殿物を生成させのちに、沈殿物と水溶液を分離した。次いで、水溶液に濃塩酸を加えてｐＨ１になるように調整した。再び沈殿物が生成するので、更に沈殿物と水溶液に分離した。
得られた水溶液はＸＲＤ樹脂を約１５０ｍＬ充填したカラムに通し、ＸＲＤ樹脂にフルボ酸を吸着させた。フルボ酸を吸着させた後のカラムに１Ｍ塩酸３００ｍＬ、続いて０．１Ｍ塩酸３００ｍＬを通過させて洗浄し、不純物を除去した。
次いで、カラムに０．１Ｍ-ＮａＯＨ水溶液、約４５０ｍＬを通過させて、ＸＲＤ樹脂に吸着していたフルボ酸を遊離させて、フルボ酸を含む遊離液を回収した。なお、０．１Ｍ-ＮａＯＨ水溶液の通液は、カラムからでる通過液の色が薄くなるまで行った。
<Process (4)>
The dry powder obtained in step (3) was separated and purified by the following steps to obtain fulvic acid. This separation and purification method corresponds to the IHSS method (operation method and confirmation method designated by the International Humic Society).
First, the dry powder obtained in the step (3) was put into a 0.1 M NaOH aqueous solution, left to stand for 1 day to generate a precipitate, and then the precipitate and the aqueous solution were separated. Next, concentrated hydrochloric acid was added to the aqueous solution to adjust to pH 1. Since a precipitate formed again, it was further separated into a precipitate and an aqueous solution.
The obtained aqueous solution was passed through a column packed with about 150 mL of XRD resin, and fulvic acid was adsorbed on the XRD resin. The column after adsorbing fulvic acid was washed by passing 300 mL of 1M hydrochloric acid and then 300 mL of 0.1M hydrochloric acid to remove impurities.
Subsequently, about 450 mL of 0.1 M NaOH aqueous solution was passed through the column to release the fulvic acid adsorbed on the XRD resin, and the free liquid containing fulvic acid was recovered. The 0.1 M NaOH aqueous solution was passed until the color of the passing solution coming out of the column became light.
得られた遊離液は、ＩＲＣ樹脂を約３００ｍＬ充填したカラムに通し、ＮａＯＨ水溶液を除去した。また、カラムを残存したフルボ酸を回収するために、カラムを蒸留水３００ｍＬで洗浄し、洗浄液と遊離したフルボ酸溶液と合わせた。
得られたフルボ酸溶液は減圧乾燥で水を留去し、目的とする乾燥粉末（フルボ酸Ａ）を得た。収率は約０．６重量％（１ｋｇから約６ｇ生成)であった。
The obtained free liquid was passed through a column packed with about 300 mL of IRC resin, and the aqueous NaOH solution was removed. Moreover, in order to collect | recover the fulvic acid which remained the column, the column was wash | cleaned with 300 mL of distilled water, and it combined with the washing | cleaning liquid and the liberated fulvic acid solution.
The obtained fulvic acid solution was dried under reduced pressure to distill off water, thereby obtaining the intended dry powder (fulvic acid A). The yield was about 0.6% by weight (from 1 kg to about 6 g produced).
分離精製後の乾燥粉末（以下、「実施例の試料」と称す）をとして、ＦＴ−ＩＲ、ＵＶ、1Ｈ−ＮＭＲ、元素分析（Ｃ・Ｈ・Ｎコーダ）により解析し、市販品のフルボ酸（コヨウ株式会社製）と比較検討した。
使用した装置は以下の通りである。
ＩＲ：日本分光(株)、FT/IR-5000型
ＵＶ：日本分光(株)、Ubest V-560型
ＮＭＲ：ブルカーバイオスピン(株)、S-NMR 600
元素分析：(株)パーキンエルマージャパン、CHNS/O アナライザー 2400II
The dried powder after separation and purification (hereinafter referred to as “sample of the example”) was analyzed by FT-IR, UV, 1 H-NMR, elemental analysis (C / H / N coder), Comparison was made with acid (manufactured by Koyo Co., Ltd.).
The equipment used is as follows.
IR: JASCO Corporation, FT / IR-5000 type UV: JASCO Corporation, Ubest V-560 type NMR: Bruker Biospin Corporation, S-
Elemental analysis: PerkinElmer Japan, CHNS / O analyzer 2400II
図１に実施例と市販品のフルボ酸のＩＲスペクトルを示す。３４００ｃｍ-1に水酸基（ＯＨ）、２９８０ｃｍ-1にメチレン基（-ＣＨ2-）及び１７００ｃｍ-1にカルボキシル基（ＣＯＯＨ）の波長に特徴的なピークが見られた。文献（環境中の腐植物質-その特徴と研究法-、ISBN-10: 4782705778）からも同様なピークが確認されており、フルボ酸特有の水酸基やカルボキシル基を多く有する構造であると考えられる。
図２に実施例と市販品のＵＶスペクトルを示す。どちらも同様な吸収曲線を示したが、実施例の方が２７０ｎｍ付近の吸収が大きかった。この付近の吸収帯は芳香環に由来するものと考えられ、実施例の試料は芳香環を市販のものより比較的多く含む構造であると考えられる。
FIG. 1 shows IR spectra of fulvic acid of Examples and commercial products. Hydroxyl (OH) in 3400 cm -1, methylene group 2980cm -1 (-CH 2 -) characteristic peaks at wavelengths of and a carboxyl group in the 1700 cm -1 (COOH) was observed. A similar peak has been confirmed in the literature (humic substances in the environment-its characteristics and research methods-ISBN-10: 4782705778), and it is thought that the structure has many hydroxyl groups and carboxyl groups peculiar to fulvic acid.
FIG. 2 shows UV spectra of the examples and commercial products. Both showed similar absorption curves, but the absorption in the vicinity of 270 nm was larger in the example. The absorption band in the vicinity is considered to be derived from the aromatic ring, and the samples of the examples are considered to have a structure containing relatively more aromatic rings than those commercially available.
図３に実施例の試料の1Ｈ−ＮＭＲを測定した結果を示した。０．８ｐｐｍに弱いシグナルの末端メチル基、１．１〜１．３ｐｐｍにβ位のメチル基及びメチレンのシグナルにそれぞれ帰属され、３．５〜４．０ｐｐｍにメトキシル基、アルコール性水酸基を含む置換脂肪族水素のシグナルに帰属された。
図４にKononovaにより提唱されているＥ４/Ｅ６比の結果を示した。Ｅ４/Ｅ６比は４６５ｎｍと６６５ｎｍの吸光度の比率であり、フルボ酸の特性付けに用いられている。文献（環境中の腐植物質-その特徴と研究法-、ISBN-10: 4782705778）によるとフルボ酸のＥ４/Ｅ６比は６．０〜８．５の間で変動すると定義されている。実施例の試料のＥ４/Ｅ６比は７．８となった。
また、元素分析を行ったところ、各試料のＣ，Ｈ，Ｎの含有量は市販品（Ｃ:29.16％、Ｈ：4.71％、Ｎ：4.99％）、実施例（Ｃ：38.65％、Ｈ：4.63％、Ｎ：2.93％）となった。文献（環境中の腐植物質-その特徴と研究法-、ISBN-10: 4782705778）と比較したところ、窒素含量が多いことが確認された。また、平均分子量は約１０００００であった。これらの結果から、実施例の試料（分離精製後の乾燥粉末）がフルボ酸であることが確認された。
FIG. 3 shows the result of measuring 1 H-NMR of the sample of the example. Substituent methyl group with weak signal at 0.8 ppm, methyl group at β-position at 1.1-1.3 ppm and methylene signal, substitution with methoxyl group and alcoholic hydroxyl group at 3.5-4.0 ppm Assigned to aliphatic hydrogen signal.
FIG. 4 shows the result of the E4 / E6 ratio proposed by Koononova. The E4 / E6 ratio is the ratio of absorbance at 465 nm and 665 nm and is used to characterize fulvic acid. According to the literature (humic substances in the environment-its characteristics and research methods-ISBN-10: 4782705778), the E4 / E6 ratio of fulvic acid is defined to vary between 6.0 and 8.5. The E4 / E6 ratio of the sample of the example was 7.8.
When elemental analysis was performed, the C, H, and N contents of each sample were commercially available (C: 29.16%, H: 4.71%, N: 4.99%), and Examples (C: 38.65%, H: 4.63%, N: 2.93%). Compared with literature (environmental humic substances-characteristics and research methods-ISBN-10: 4782705778), it was confirmed that the nitrogen content was high. The average molecular weight was about 100,000. From these results, it was confirmed that the sample of the example (dry powder after separation and purification) was fulvic acid.
２．５α−レダクターゼの阻害活性の評価
以下の方法で、フルボ酸Ａ（上記方法で得たフルボ酸）及びフルボ酸Ｂ（株式会社日本フルボ酸総合研究所製）についての５α−レダクターゼの阻害活性を評価した。なお、フルボ酸Ｂは、広葉樹由来の木材分解物（樹木エキス）から分離精製したフルボ酸である。
また、酵素源（５α−レダクターゼ）としては、ラット肝ホモジネート(S-9)を用いた。
2.5 Evaluation of inhibitory activity of α-reductase Inhibitory activity of 5α-reductase with respect to fulvic acid A (fulvic acid obtained by the above method) and fulvic acid B (manufactured by Nihon Fulvic Acid Research Institute) by the following method. Evaluated. Note that fulvic acid B is fulvic acid that is separated and purified from a wood decomposition product (tree extract) derived from hardwood.
Further, rat liver homogenate (S-9) was used as an enzyme source (5α-reductase).
（１）測定溶液の調整
（標準溶液の調整）
テストステロン、ジヒドロテストステロン（ＤＨＴ）をそれぞれ約１ｍｇ精秤し、エタノールで１ｍＬとなるように溶解し、等量混合したものを標準溶液とした。
(1) Measurement solution adjustment (standard solution adjustment)
About 1 mg each of testosterone and dihydrotestosterone (DHT) was precisely weighed, dissolved to 1 mL with ethanol, and mixed in equal amounts to obtain a standard solution.
（テストステロン溶液の調整）
テストステロン約４．２ｍｇを精秤し、プロピレングリコールで１ｍＬとして、テストステロン溶液を得た。
(Preparation of testosterone solution)
About 4.2 mg of testosterone was precisely weighed and adjusted to 1 mL with propylene glycol to obtain a testosterone solution.
（Ｔｒｉｓ−ＨＣｌ緩衝液の調整＞
2-Amino-2-hydroxymethyl-1,3-propanediol ３０．３ｍｇを精秤し、精製水に溶解した後 １Ｍ ＨＣｌでｐＨ７．１３に調整し、精製水で５０ｍＬにメスアップして、Ｔｒｉｓ−ＨＣｌ緩衝液を得た。
(Preparation of Tris-HCl buffer>
2-Amino-2-hydroxymethyl-1,3-propanediol 30.3mg is precisely weighed, dissolved in purified water, adjusted to pH 7.13 with 1M HCl, made up to 50mL with purified water, Tris-HCl A buffer was obtained.
（ＮＡＤＰＨ溶液の調整）
ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰＨ）約１０ｍｇを精秤し、Ｔｒｉｓ−ＨＣｌ緩衝液緩衝液に溶解し、１０ｍＬにメスアップして、ＮＡＤＰＨ溶液を得た（用時調製）。
(Adjustment of NADPH solution)
About 10 mg of nicotinamide adenine dinucleotide phosphate (NADPH) was precisely weighed, dissolved in Tris-HCl buffer solution, and made up to 10 mL to obtain an NADPH solution (prepared at the time of use).
（被験試料溶液の調整＞
評価対象のフルボ酸（フルボ酸Ａ又はフルボ酸Ｂ）０．５ｍｇ/ｍＬを精秤し、精製水に溶解して、被験試料溶液を得た。
フルボ酸Ａの濃度は０．０５重量％、フルボ酸Ｂの濃度は０．０５重量％である。
(Preparation of test sample solution)
A fulvic acid (fulvic acid A or fulvic acid B) 0.5 mg / mL to be evaluated was precisely weighed and dissolved in purified water to obtain a test sample solution.
The concentration of fulvic acid A is 0.05% by weight, and the concentration of fulvic acid B is 0.05% by weight.
（ラット肝ホモジネート（Ｓ−９）溶液の調整）
−８０℃で凍結したラット肝ホモジネート（Ｓ−９）を氷中にて溶解し、ラット肝ホモジネート溶液（Ｓ−９溶液）を得た。
Ｓ−９の濃度は、（Ｓ９フラクション：プロテイン濃度 １９．８ｍｇ/ｍＬＳ９（５５．４ｍｇ/ｇ liver））である。
(Preparation of rat liver homogenate (S-9) solution)
Rat liver homogenate (S-9) frozen at −80 ° C. was dissolved in ice to obtain a rat liver homogenate solution (S-9 solution).
The concentration of S-9 is (S9 fraction: protein concentration 19.8 mg / mL S9 (55.4 mg / g liver)).
（２）５α−レダクターゼ阻害試験
試験例１：テストステロン+水を同時に反応（Ｓ−９無添加の場合：テストステロン基準）
ふた付きＶ底試験管１５ｍＬにテストステロン溶液２０μＬとＮＡＤＰＨ溶液８２５μＬを加えて混合し、水浴中３７℃で保温した。これに被験試料溶液（０．０５重量％フルボ酸Ａ溶液）８０μＬを加え再び混合し、水浴中３７℃にて６０分間反応させた。
反応終了後、塩化メチレンを正確に１ｍＬ添加し激しく振とうして基質のテストステロンとその反応生成物を抽出し、反応を止めた。
その後、１６００Ｇで１０分間遠心分離して塩化メチレン層を分離して、その内３００μＬを取り出し気化させ、メタノールで溶解した後、ガスクロマトグラフィー質量分析法（ＧＣ-ＭＳ）による測定を行った。ＧＣ−ＭＳは(株)島津製作所 ＧＣＭＳ ＱＰ２０１０ Ｕｌｔｒａを使用した。なお、ＧＣ-ＭＳの測定条件は、以下の表１及び表２の通りである。
(2) 5α-reductase inhibition test test example 1: Testosterone + water are simultaneously reacted (when S-9 is not added: testosterone standard)
A
After completion of the reaction, exactly 1 mL of methylene chloride was added and shaken vigorously to extract the substrate testosterone and its reaction product, and the reaction was stopped.
Thereafter, the methylene chloride layer was separated by centrifuging at 1600 G for 10 minutes, and 300 μL of the methylene chloride layer was taken out, vaporized, dissolved in methanol, and then measured by gas chromatography mass spectrometry (GC-MS). The GC-MS used was Shimadzu Corporation GCMS QP2010 Ultra. The measurement conditions for GC-MS are as shown in Tables 1 and 2 below.
試験例２：テストステロン+水+Ｓ−９を同時に反応
ふた付きＶ底試験管１５ｍＬにテストステロン溶液２０μＬとＮＡＤＰＨ溶液８２５μＬを加えて混合し、水浴中３７℃で保温した。これに被験試料溶液（水）８０μＬとＳ−９溶液７５μＬを加え再び混合し、水浴中３７℃にて６０分間反応させた。
反応終了後、塩化メチレンを正確に１ｍＬ添加し激しく振とうして基質のテストステロンとその反応生成物を抽出し、反応を止めた。
その後、１６００Ｇで１０分間遠心分離して塩化メチレン層を分離して、その内３００μＬを取り出し気化させ、メタノールで溶解した後、ガスクロマトグラフィー質量分析法（ＧＣ-ＭＳ）による測定を行った。
Test Example 2: Testosterone + water + S-9 were reacted at the same time. A testosterone solution (20 μL) and an NADPH solution (825 μL) were added to and mixed with 15 mL of a V-bottom test tube with a lid, and the mixture was kept at 37 ° C. in a water bath. To this, 80 μL of the test sample solution (water) and 75 μL of the S-9 solution were added and mixed again, and reacted at 37 ° C. for 60 minutes in a water bath.
After completion of the reaction, exactly 1 mL of methylene chloride was added and shaken vigorously to extract the substrate testosterone and its reaction product, and the reaction was stopped.
Thereafter, the methylene chloride layer was separated by centrifuging at 1600 G for 10 minutes, and 300 μL of the methylene chloride layer was taken out, vaporized, dissolved in methanol, and then measured by gas chromatography mass spectrometry (GC-MS).
試験例３：テストステロン+フルボ酸Ａ+Ｓ−９を同時に反応
ふた付きＶ底試験管１５ｍＬにテストステロン溶液２０μＬとＮＡＤＰＨ溶液８２５μＬを加えて混合し、水浴中３７℃で保温した。これに被験試料溶液（０．０５重量％フルボ酸Ａ溶液）８０μＬとＳ−９溶液７５μＬを加え再び混合し、水浴中３７℃にて６０分間反応させた。
反応終了後、塩化メチレンを正確に１ｍＬ添加し激しく振とうして基質のテストステロンとその反応生成物を抽出し、反応を止めた。
その後、１６００Ｇで１０分間遠心分離して塩化メチレン層を分離して、その内３００μＬを取り出し気化させ、メタノールで溶解した後、ガスクロマトグラフィー質量分析法（ＧＣ-ＭＳ）による測定を行った。
Test Example 3: Testosterone + fulvic acid A + S-9 were reacted at the same time. 15 mL of testosterone solution and 825 μL of NADPH solution were added to and mixed with 15 mL of a V-bottom test tube with a lid, and kept at 37 ° C. in a water bath. To this, 80 μL of the test sample solution (0.05 wt% fulvic acid A solution) and 75 μL of the S-9 solution were added and mixed again, and reacted at 37 ° C. for 60 minutes in a water bath.
After completion of the reaction, exactly 1 mL of methylene chloride was added and shaken vigorously to extract the substrate testosterone and its reaction product, and the reaction was stopped.
Thereafter, the methylene chloride layer was separated by centrifuging at 1600 G for 10 minutes, and 300 μL of the methylene chloride layer was taken out, vaporized, dissolved in methanol, and then measured by gas chromatography mass spectrometry (GC-MS).
試験例４：テストステロン+フルボ酸Ｂ+Ｓ−９を同時に反応
ふた付きＶ底試験管１５ｍＬにテストステロン溶液２０μＬとＮＡＤＰＨ溶液８２５μＬを加えて混合し、水浴中３７℃で保温した。これに被験試料溶液（０．０５重量％フルボ酸Ｂ溶液）８０μＬとＳ−９溶液７５μＬを加え再び混合し、水浴中３７℃にて６０分間反応させた。
反応終了後、塩化メチレンを正確に１ｍＬ添加し激しく振とうして基質のテストステロンとその反応生成物を抽出し、反応を止めた。
その後、１６００Ｇで１０分間遠心分離して塩化メチレン層を分離して、その内３００μＬを取り出し気化させ、メタノールで溶解した後、ガスクロマトグラフィー質量分析法（ＧＣ-ＭＳ）による測定を行った。
Test Example 4: Testosterone + fulvic acid B + S-9 were reacted at the same time
After completion of the reaction, exactly 1 mL of methylene chloride was added and shaken vigorously to extract the substrate testosterone and its reaction product, and the reaction was stopped.
Thereafter, the methylene chloride layer was separated by centrifuging at 1600 G for 10 minutes, and 300 μL of the methylene chloride layer was taken out, vaporized, dissolved in methanol, and then measured by gas chromatography mass spectrometry (GC-MS).
５α−レダクターゼ阻害活性（阻害率（％））は、テストステロン量の初期量（初期テストステロン量）（基準量）に対する、５α−レダクターゼによってジヒドロテストステロン又は３アルファ−アンドロスタンジオールに変換又は分解されずに残存したテストステロン量（残存テストステロン量）として以下の式で求められる。なお、５α−レダクターゼ（Ｓ−９)を含まない試験例１のピーク面積は初期テストステロン量、試験例２〜４のピーク面積は、残存テストステロン量に相当する。
阻害率（％）＝（残存テストステロン量）/（初期テストステロン量）×１００
5α-reductase inhibitory activity (inhibition rate (%)) is not converted or decomposed into dihydrotestosterone or 3alpha-androstanediol by 5α-reductase relative to the initial amount of testosterone (initial amount of testosterone) (reference amount). The remaining testosterone amount (residual testosterone amount) is obtained by the following formula. The peak area of Test Example 1 that does not contain 5α-reductase (S-9) corresponds to the initial testosterone amount, and the peak areas of Test Examples 2 to 4 correspond to the residual testosterone amount.
Inhibition rate (%) = (residual testosterone amount) / (initial testosterone amount) × 100
結果を表３に示す。 The results are shown in Table 3.
表３に示したようにテストステロンのＳ−９による酵素阻害率は試験例１を初期としたテストステロンの量（３３４μｇ）を基準値とした。試験例２では被検試料を水として行った場合はＳ−９酵素がテストステロンに対して活性がみられ、そのほとんどがテストステロンからジヒドロテストステロン又は３アルファ−アンドロスタンジオールに変換又は分解したため、初期のテストステロン量は８２．７％減少した。また、試験例３でフルボ酸Ｂを被検試料として添加した場合は６６．３％の阻害率を示し、阻害効果が認められた。一方、試験例４ではフルボ酸Ａを被検試料として添加した場合であり、その阻害率は８５．２％となり、さらに阻害効果が認められた。 As shown in Table 3, the enzyme inhibition rate of testosterone by S-9 was based on the amount of testosterone (334 μg) starting from Test Example 1 as a reference value. In Test Example 2, when the test sample was used as water, the S-9 enzyme showed activity against testosterone, most of which was converted or decomposed from testosterone to dihydrotestosterone or 3alpha-androstanediol. The amount of testosterone decreased by 82.7%. Further, when fulvic acid B was added as a test sample in Test Example 3, an inhibition rate of 66.3% was shown, and an inhibitory effect was observed. On the other hand, in Test Example 4, fulvic acid A was added as a test sample, the inhibition rate was 85.2%, and an inhibitory effect was further observed.
本発明によれば、優れた５α−レダクターゼ阻害を有する５α−レダクターゼ阻害剤及び脱毛症の予防治療用又は育毛促進用組成物が提供されるので、産業的に有望である。 According to the present invention, a 5α-reductase inhibitor having excellent 5α-reductase inhibition and a composition for preventing or treating alopecia or promoting hair growth are industrially promising.
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