JP2013035800A - Antimicrobial agent and external preparation for skin - Google Patents
Antimicrobial agent and external preparation for skin Download PDFInfo
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Abstract
Description
本発明は、抗菌剤及び皮膚外用剤に関する。 The present invention relates to an antibacterial agent and a skin external preparation.
グラム陽性菌の１種であるアクネ菌（Propionibacterium acnes）やブドウ球菌（Staphylococcus属細菌）は、皮膚の常在菌として知られる一方で、皮膚の炎症にも深く関与している。
アクネ菌は、皮脂をグリセリンと脂肪酸に分解して皮膚を弱酸性に保つことで病原菌の侵入や繁殖を抑える役割を担っている。皮脂が毛穴等に詰まった状態が続くと、皮脂を栄養源としてアクネ菌が増殖し、これが原因となってニキビ等の炎症が引き起こされる。
一方、ブドウ球菌は、ヒトの皮膚や腸等に存在し、通常は無害の細菌である。しかし、黄色ブドウ球菌（Staphylococcus aureus）のように毒性の強いものは、傷口の化膿やとびひ等の原因菌としても知られる。黄色ブドウ球菌が炎症を起こした傷口から体内に侵入して髄膜炎、肺炎、腹膜炎、敗血症等を引き起こすこともある。
Acne ( Propionibacterium acnes ) and Staphylococcus (Bacteria belonging to the genus Staphylococcus ), which are one of Gram-positive bacteria, are known as skin resident bacteria, and are also deeply involved in skin inflammation.
Acne bacteria play a role in suppressing the invasion and propagation of pathogenic bacteria by degrading sebum into glycerin and fatty acids to keep the skin weakly acidic. If sebum is clogged in pores, acne bacteria grow using sebum as a nutrient source, causing inflammation such as acne.
On the other hand, staphylococci are present in human skin, intestines and the like, and are usually harmless bacteria. However, a highly toxic substance such as Staphylococcus aureus is also known as a causative bacterium such as suppuration or jumping of the wound. Staphylococcus aureus may enter the body through inflamed wounds and cause meningitis, pneumonia, peritonitis, sepsis and the like.
皮膚の炎症の予防・改善には、炎症の原因菌の増殖を効果的に制御することが重要であり、皮膚の健康の維持・改善に有用な新たな抗菌剤及び皮膚外用剤の開発が望まれている。例えば、特許文献１及び非特許文献１には、抗アクネ菌活性や抗黄色ブドウ球菌活性を有する化合物を含有する皮膚外用剤が記載されている。 In order to prevent and improve skin inflammation, it is important to effectively control the growth of pathogenic bacteria, and the development of new antibacterial agents and external skin preparations useful for maintaining and improving skin health is hoped for. It is rare. For example, Patent Document 1 and Non-Patent Document 1 describe a skin external preparation containing a compound having anti-acne bacteria activity or anti-Staphylococcus aureus activity.
本発明は、皮膚の炎症の原因菌に対して抗菌活性を示す抗菌剤及び皮膚外用剤の提供を課題とする。 An object of the present invention is to provide an antibacterial agent and an external preparation for skin that exhibit antibacterial activity against bacteria causing inflammation of the skin.
本発明者等は上記課題に鑑み鋭意検討を行った。その結果、皮膚の炎症の原因菌に対して抗菌活性を有する化合物を新たに見い出した。本発明はこの知見に基づき完成させるに至ったものである。 The present inventors have intensively studied in view of the above problems. As a result, a compound having antibacterial activity against the causative bacteria of skin inflammation was newly found. The present invention has been completed based on this finding.
本発明は、下記式（１）〜（４）のいずれかで表される化合物の少なくとも１種を有効成分とする抗菌剤に関する。 The present invention relates to an antibacterial agent containing as an active ingredient at least one compound represented by any one of the following formulas (1) to (4).
また、本発明は、前記式（１）〜（４）のいずれかで表される化合物の少なくとも１種を有効成分とする皮膚外用剤に関する。 Moreover, this invention relates to the skin external preparation which uses as an active ingredient at least 1 sort (s) of the compound represented by either of said Formula (1)-(4).
また、本発明は、前記式（１）で表される化合物に関する。
さらに、本発明は、前記式（２）で表される化合物に関する。
The present invention also relates to a compound represented by the formula (1).
Furthermore, this invention relates to the compound represented by said Formula (2).
本発明の抗菌剤及び皮膚外用剤は、皮膚の炎症の原因菌に対して抗菌活性を示す。 The antibacterial agent and the external preparation for skin of the present invention exhibit antibacterial activity against bacteria that cause skin inflammation.
本発明の抗菌剤及び皮膚外用剤は、下記式（１）〜（４）のいずれかで表される化合物の少なくとも１種を有効成分とする。 The antibacterial agent and external preparation for skin of the present invention contain at least one compound represented by any one of the following formulas (1) to (4) as an active ingredient.
式（１）〜（４）のいずれかで表される化合物のうち塩の形態をとり得るものについては、前記化合物を塩の形態で本発明の抗菌剤及び皮膚外用剤に含有させてもよい。このような塩としては特に限定されず、例えば、ナトリウム，カリウム，カルシウム等のアルカリ金属又はアルカリ土類金属塩、アルミニウム、亜鉛等の両性金属塩、アミノ酸塩、アミン塩等が挙げられる。 Among the compounds represented by any one of formulas (1) to (4), those that can take the form of a salt may be contained in the antibacterial agent and external skin preparation of the present invention in the form of a salt. . Such salts are not particularly limited, and examples thereof include alkali metal or alkaline earth metal salts such as sodium, potassium and calcium, amphoteric metal salts such as aluminum and zinc, amino acid salts and amine salts.
本発明の抗菌剤及び皮膚外用剤において、前記式（１）〜（４）のいずれかで表される化合物の１種を単独で有効成分としてもよいし、２種以上を組合わせて有効成分としてもよい。 In the antibacterial agent and skin external preparation of the present invention, one of the compounds represented by any one of the formulas (1) to (4) may be used alone as an active ingredient, or an active ingredient may be used in combination of two or more. It is good.
本発明に用いる前記化合物は、通常の方法により化学合成してもよいし、特定の植物から抽出してもよい。
本発明に用いる前記化合物を抽出することができる植物として、アキノキリンソウ（Solidago virgaurea）が挙げられる。例えば、アキノキリンソウの任意の部位（例えば、根、茎、全草、葉、花等）を原料として特定の溶媒を用いて抽出操作を行い、アクネ菌やブドウ球菌等に対する抗菌活性を有する抽出物を得、該抽出物を液液分配やカラムクロマトグラフィー等の通常の分画手法を組合わせて順次分画していき、得られた画分の抗菌活性を指標に比活性の高い画分を得ていくことで、本発明に用いる有効成分を得ることができる。
The said compound used for this invention may be chemically synthesized by a normal method, and may be extracted from a specific plant.
An example of a plant from which the compound used in the present invention can be extracted is Solidago virgaurea . For example, an extraction operation is performed using a specific solvent using any part of the red ginseng (eg, root, stem, whole grass, leaf, flower, etc.) as a raw material, and an extract having antibacterial activity against acne or staphylococci The extract is fractionated sequentially by combining ordinary fractionation techniques such as liquid-liquid distribution and column chromatography, and a fraction with high specific activity is obtained using the antibacterial activity of the obtained fraction as an index. By doing so, the active ingredient used in the present invention can be obtained.
アキノキリンソウ抽出物の調製には、アキノキリンソウの任意の部位をそのまま、又は乾燥粉砕して用いることができ、これらの水蒸気蒸留物又は圧搾物を用いてもよい。また、これらを精油等により精製したものを用いてもよい。本発明に用いることができるアキノキリンソウ抽出物を得るには、任意の部位の１種のみから抽出してもよいし、２種以上の部位から抽出してもよい。本発明に用いることができるアキノキリンソウ抽出物を得るには、アキノキリンソウの茎部を抽出することが好ましい。また、アキノキリンソウ抽出物として、市販品を用いてもよい。 For the preparation of an Achillinum ginseng extract, any part of the Achillea ginseng can be used as it is or after dry pulverization, and these steam distillates or pressed products may be used. Moreover, you may use what refine | purified these with essential oil etc. In order to obtain the Achillinus extract that can be used in the present invention, it may be extracted from only one kind of any part, or may be extracted from two or more parts. In order to obtain an extract of Achillea ginseng that can be used in the present invention, it is preferable to extract a stem portion of Achillea ginseng. Moreover, you may use a commercial item as a ginkgo biloba extract.
アキノキリンソウ抽出物を得る方法は特に限定されず、通常の抽出方法を採用することができる。
抽出に用いる溶媒に特に制限はなく、植物成分の抽出に通常用いられる溶媒を用いることができる。このような溶媒として、例えば、水；メタノール、エタノール、プロパノール、ブタノール等のアルコール類；プロピレングリコール、ブチレングリコール等の多価アルコール類；アセトン、メチルエチルケトン等のケトン類；酢酸メチル、酢酸エチル等のエステル類；テトラヒドロフラン、ジエチルエーテル等の鎖状及び環状エーテル類；ジクロロメタン、クロロホルム、ジクロロエタン等のハロゲン化炭化水素類；スクワラン、ヘキサン、シクロヘキサン、石油エーテル等の炭化水素類；トルエン等の芳香族炭化水素類；ポリエチレングリコール等のポリエーテル類；及びピリジン類等が挙げられ、これら２種以上混合溶媒を用いることができる。本発明において、水性アルコールを含有する溶媒を用いることが好ましく、エタノールを含有する溶媒を用いることがより好ましい。エタノールを含有する溶媒はエタノール水溶液であることが好ましく、エタノールの含有量は２０〜８０質量％が好ましく、３０〜７０質量％がより好ましい。
There is no particular limitation on the method for obtaining the red ginseng extract, and a normal extraction method can be employed.
There is no restriction | limiting in particular in the solvent used for extraction, The solvent normally used for extraction of a plant component can be used. Examples of such solvents include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate. Chain and cyclic ethers such as tetrahydrofuran and diethyl ether; Halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane; Hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; Aromatic hydrocarbons such as toluene Polyethers such as polyethylene glycol; and pyridines, and the like. Two or more mixed solvents can be used. In the present invention, a solvent containing an aqueous alcohol is preferably used, and a solvent containing ethanol is more preferably used. The ethanol-containing solvent is preferably an ethanol aqueous solution, and the ethanol content is preferably 20 to 80% by mass, more preferably 30 to 70% by mass.
アキノキリンソウ抽出物を得るための抽出条件も通常の条件を適用できる。例えば、抽出原料となるアキノキリンソウの任意の部位を、好ましくは０〜１００℃、より好ましくは５〜６０℃、さらに好ましくは１０〜３０℃の温度下で、好ましくは１時間〜４週間、より好ましくは５時間〜１週間、上記溶媒中に浸漬又は加熱還流することでアキノキリンソウ抽出物を得ることができる。 Ordinary conditions can also be applied as the extraction conditions for obtaining the ginkgo biloba extract. For example, an arbitrary part of the ginkgo biloba as an extraction raw material is preferably 0 to 100 ° C., more preferably 5 to 60 ° C., and still more preferably 10 to 30 ° C., preferably 1 hour to 4 weeks, more preferably Can be obtained by immersing or heating to reflux for 5 hours to 1 week in the above solvent.
本明細書において「抗菌」とは、菌の増殖を抑制すること、菌を死滅させること、の両概念が包含される。本発明に用いる有効成分は、アクネ菌やブドウ球菌等のグラム陽性菌に対して優れた抗菌活性を示す。このうち、後述する実施例でも示すように、前記式（１）〜（４）のいずれかで表される化合物は、アクネ菌や黄色ブドウ球菌に対する抗菌活性に特に優れる。 In the present specification, the term “antibacterial” includes both the concepts of suppressing the growth of bacteria and killing the bacteria. The active ingredient used in the present invention exhibits excellent antibacterial activity against gram-positive bacteria such as acne and staphylococci. Among these, as shown also in the Example mentioned later, the compound represented by either of said Formula (1)-(4) is especially excellent in the antibacterial activity with respect to acne bacteria and Staphylococcus aureus.
本発明において、前記有効成分を抗菌剤及び皮膚外用剤としても用いてもよいし、前記有効成分と前記有効成分以外の成分とを本発明の抗菌剤及び皮膚外用剤に含有させてもよい。本発明に用いることができる有効成分以外の成分としては特に制限はなく、例えば、酸化チタン、炭酸カルシウム、精製水、乳糖、デンプン、アルコール、界面活性剤、油性物質、保湿剤、皮膚老化防止剤、美白剤、高分子化合物、防腐剤、増粘剤、乳化剤、薬効成分、粉体、紫外線吸収剤、色素、香料、乳化安定剤、ｐＨ調整剤等が挙げられる。
本発明の抗菌剤及び皮膚外用剤の形態に特に制限はなく、例えば、液体、流動体、粉体、固体、半固形体、乳化物、ゲル、クリーム、軟膏、ローション等が挙げられる。
In this invention, you may use the said active ingredient also as an antibacterial agent and a skin external preparation, and you may make the antibacterial agent and skin external preparation of this invention contain the said active ingredient and components other than the said active ingredient. Ingredients other than the active ingredient that can be used in the present invention are not particularly limited. For example, titanium oxide, calcium carbonate, purified water, lactose, starch, alcohol, surfactant, oily substance, moisturizer, skin antiaging agent , Whitening agents, polymer compounds, preservatives, thickeners, emulsifiers, medicinal ingredients, powders, ultraviolet absorbers, dyes, fragrances, emulsion stabilizers, pH adjusters and the like.
There is no restriction | limiting in particular in the form of the antibacterial agent and skin external preparation of this invention, For example, a liquid, a fluid, a powder, a solid, a semi-solid body, an emulsion, a gel, cream, an ointment, a lotion etc. are mentioned.
本発明の抗菌剤及び皮膚外用剤における前記有効成分の含有量に特に制限はないが、固形分換算で０．０００００１〜１０質量％であることが好ましく、０．０００１〜５質量％であることがより好ましい。 Although there is no restriction | limiting in particular in content of the said active ingredient in the antibacterial agent and skin external preparation of this invention, It is preferable that it is 0.000001-10 mass% in conversion of solid content, and it is 0.0001-5 mass%. Is more preferable.
本発明の抗菌剤は、医薬品、医薬部外品及び化粧料でありうる。本発明の抗菌剤の形態に特に制限はなく、用途に応じて皮膚外用剤、経口剤、注射剤、点鼻剤、坐剤、点滴剤、点眼剤等の形態をとることができる。
皮膚外用剤の形態では、水溶液系、可溶化系、乳化系、粉末系、ゲル系、軟膏系、クリーム、ミスト、エアゾール、水−油２層系、水−油−粉末３層系など、幅広い形態をとりうる。また、皮膚外用剤の形態における製品形態としては、ボディー化粧料、化粧水、乳液、クリーム及びパック等のフェーシャル化粧料、ファンデーション、おしろい、頬紅、口紅、アイシャドー、アイライナー、マスカラ及びサンスクリーン等のメーキャップ化粧料、メーク落とし、洗顔料及びボディーシャンプー等の皮膚洗浄料、ヘアーリンス及びシャンプー等の毛髪化粧料、浴用剤、軟膏、制御放出パッチ剤、貼付剤、あぶら取り紙、芳香化粧料等が挙げられる。
経口剤の形態の例としては、錠剤、顆粒剤、細粒剤、散剤、カプセル剤、チュアブル剤、ペレット剤、シロップ剤、液剤、懸濁剤及び吸入剤などが挙げられる。
Antibacterial agents of the present invention can be pharmaceuticals, quasi drugs and cosmetics. There is no restriction | limiting in particular in the form of the antibacterial agent of this invention, According to a use, it can take forms, such as an external preparation for skin, an oral preparation, an injection, a nasal drop, a suppository, an instillation, an eye drop.
In the form of an external preparation for skin, a wide range such as aqueous solution, solubilization system, emulsification system, powder system, gel system, ointment system, cream, mist, aerosol, water-oil two-layer system, water-oil-powder three-layer system Can take form. In addition, examples of product forms in the form of external preparation for skin include facial cosmetics such as body cosmetics, lotions, emulsions, creams and packs, foundations, funny, blushers, lipsticks, eye shadows, eyeliners, mascaras, sunscreens, etc. Makeup cosmetics, makeup removers, skin cleansers such as facial cleansers and body shampoos, hair cosmetics such as hair rinses and shampoos, bath preparations, ointments, controlled release patches, patches, oil blotting paper, aromatic cosmetics, etc. Is mentioned.
Examples of oral dosage forms include tablets, granules, fine granules, powders, capsules, chewables, pellets, syrups, solutions, suspensions and inhalants.
本発明の皮膚外用剤は、医薬品、医薬部外品及び化粧料でありうる。本発明の皮膚外用剤の形態に特に制限はなく、水溶液系、可溶化系、乳化系、粉末系、ゲル系、軟膏系、クリーム、ミスト、エアゾール、水−油２層系、水−油−粉末３層系など、幅広い形態をとりうる。また、本発明の皮膚外用剤の製品形態も任意であり、例えば、ボディー化粧料、化粧水、乳液、クリーム及びパック等のフェーシャル化粧料、ファンデーション、おしろい、頬紅、口紅、アイシャドー、アイライナー、マスカラ及びサンスクリーン等のメーキャップ化粧料、メーク落とし、洗顔料及びボディーシャンプー等の皮膚洗浄料、ヘアーリンス及びシャンプー等の毛髪化粧料、浴用剤、軟膏、制御放出パッチ剤、貼付剤、あぶら取り紙、芳香化粧料等が挙げられる。 The external preparation for skin of the present invention can be a pharmaceutical, a quasi-drug, and a cosmetic. There is no restriction | limiting in particular in the form of the skin external preparation of this invention, Aqueous solution type | system | group, solubilization type | system | group, emulsification type | system | group, powder type | system | group, gel type | system | group, ointment type | system | group, cream, mist, aerosol, water-oil two layer system, water-oil- It can take a wide variety of forms, such as a powder three-layer system. In addition, the product form of the external preparation for skin of the present invention is also arbitrary, for example, facial cosmetics such as body cosmetics, lotions, emulsions, creams and packs, foundations, funny, blushers, lipsticks, eye shadows, eyeliners, Makeup cosmetics such as mascara and sunscreen, makeup remover, skin cleansers such as facial cleansers and body shampoos, hair cosmetics such as hair rinses and shampoos, bath preparations, ointments, controlled release patches, patches, oil blotting paper And aromatic cosmetics.
本発明の抗菌剤及び皮膚外用剤の使用量は、前記有効成分の含有量により異なるが、例えば、クリーム状、軟膏状及び液状製剤の場合、皮膚面１ｃｍ2当たり０．００００１〜２０ｍｇ使用するのが好ましい。 The use amount of the antibacterial agent and the external preparation for skin of the present invention varies depending on the content of the active ingredient. For example, in the case of cream, ointment and liquid preparations, 0.00001 to 20 mg per 1 cm 2 of the skin surface is used. Is preferred.
本発明の抗菌剤及び皮膚外用剤は、前記化合物の少なくとも１種を有効成分とし、皮膚炎の炎症原因菌であるアクネ菌、黄色ブドウ球菌等のブドウ球菌等に対する抗菌作用を示す。この抗菌作用により、ニキビや化膿等の皮膚炎の症状を予防することができ、また、これらの皮膚炎の症状を緩和・改善することができる。 The antibacterial agent and the external preparation for skin of the present invention contain at least one of the above compounds as an active ingredient, and exhibit an antibacterial action against staphylococci such as acne and staphylococcus aureus that are inflammatory causes of dermatitis. This antibacterial action can prevent dermatitis symptoms such as acne and suppuration, and can alleviate and improve these dermatitis symptoms.
以下、本発明を実施例に基づきさらに詳細に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
製造例 式（１）〜（４）のいずれかで表される化合物の抽出
アキノキリンソウの茎部の乾燥物（American botanicals社より入手）１６０．７９ｇを５０体積％エタノール水溶液８００ｍＬに３日間常温で浸漬した。この浸漬液をろ過して得られたろ液をロータリーエバポレーターを用いて濃縮し、固形分２４．８２ｇを得た。この固形分を液液分配（酢酸エチル／水）に供し、酢酸エチル相を濃縮することで酢酸エチル可溶画分４．６７ｇを得た。これをハイフラッシュカラム（山善社製；６０×１８０ｍｍ）に通し、ヘキサン／酢酸エチル混合溶媒で溶出させることで１５の画分（画分ＥＡ−１〜ＥＡ−１５）を得た。
これらの画分のうち、画分ＥＡ−３（全量：５３２．２ｍｇ）の５２３．８ｍｇをＵＬＴＲＰＡＣＫ Ｂカラム(山善社製；２６×３００ｍｍ）に通し、クロロホルム／メタノール混合溶媒で溶出させて、１２の画分（画分ＥＡ−３−１〜ＥＡ−３−１２）を得た。
これらの画分のうち、画分ＥＡ−３−６（全量：６７．６ｍｇ）の４６．７１ｍｇをＯＤＳ−３カラム(ＧＬサイエンス社製；１０×２５０ｍｍ）に通し、アセトニトリル／水（０．１％ギ酸）混合溶媒で溶出させて、１１の画分（画分ＥＡ−３−６−１〜ＥＡ−３−６−１１）を得た。また、画分ＥＡ−３−８（全量：２９０．２ｍｇ）の４６．７１ｍｇをＯＤＳ−３カラム(ＧＬサイエンス社製；１０×２５０ｍｍ）に通し、アセトニトリル／水（０．１％ギ酸）混合溶媒で溶出させて、１３の画分（画分ＥＡ−３−８−１〜ＥＡ−３−８−１３）を得た。
Production Example Extraction of the compound represented by any one of the formulas (1) to (4) 160.79 g of the dried stem of Achillea ginger (obtained from American botanicals) was immersed in 800 mL of 50% by volume ethanol aqueous solution at room temperature for 3 days. did. The filtrate obtained by filtering this immersion liquid was concentrated using a rotary evaporator to obtain 24.82 g of a solid content. This solid content was subjected to liquid-liquid partition (ethyl acetate / water), and the ethyl acetate phase was concentrated to obtain 4.67 g of an ethyl acetate soluble fraction. This was passed through a high flash column (Yamazen Co., Ltd .; 60 × 180 mm) and eluted with a mixed solvent of hexane / ethyl acetate to obtain 15 fractions (fractions EA-1 to EA-15).
Among these fractions, 523.8 mg of fraction EA-3 (total amount: 532.2 mg) was passed through an ULTRPACK B column (manufactured by Yamazensha; 26 × 300 mm) and eluted with a chloroform / methanol mixed solvent. Fractions (fractions EA-3-1 to EA-3-12) were obtained.
Among these fractions, 46.71 mg of fraction EA-3-6 (total amount: 67.6 mg) was passed through an ODS-3 column (GL Science, 10 × 250 mm), and acetonitrile / water (0.1 Eluting with a mixed solvent of% formic acid, 11 fractions (fraction EA-3-6-1 to EA-3-6-11) were obtained. In addition, 46.71 mg of fraction EA-3-8 (total amount: 290.2 mg) was passed through an ODS-3 column (GL Science, Inc .; 10 × 250 mm) and mixed with acetonitrile / water (0.1% formic acid). To obtain 13 fractions (fraction EA-3-8-1 to EA-3-8-13).
このようにして得られた画分のうち、ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８の各画分に存在する単離成分（以下、それぞれ、単離成分ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８という）に関して、ＮＭＲスペクトルを測定した。
得られたＮＭＲスペクトルの結果、及びこれらのＮＭＲスペクトルから決定した各単離成分の化学構造を表１〜４に示す。
Among the fractions thus obtained, these fractions are present in the fractions EA-3-6-5, EA-3-6-9, EA-3-8-3 and EA-3-8-8. Measure NMR spectra for isolated components (hereinafter referred to as isolated components EA-3-6-5, EA-3-6-9, EA-3-8-3 and EA-3-8-8, respectively) did.
The chemical structure of each isolated component determined from the results of the obtained NMR spectra and these NMR spectra is shown in Tables 1-4.
試験例１ アクネ菌に対する抗菌試験
単離成分ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８を各々エタノールに溶解し、濃度１ｍｇ／ｍＬの評価用サンプル溶液を調製し、終濃度０．１ｍｇ／ｍＬでアクネ菌に対する抗菌活性を評価した。抗菌活性は、ルシフェラーゼを利用してＡＴＰ量を定量する市販キット（商品名：BacTiter-GloTM Microbial Cell Viability Assay、Progega社製）を用いて評価した。
変法ＧＡＭ培地（日水製薬社製）を用いて３７℃で３日間嫌気培養したアクネ菌JCM6425株の培養液１０μＬと、変法ＧＡＭ培地８０μＬ及び評価用サンプル溶液１０μＬを９６穴プレートのウェル内で混合し、３７℃で２０時間嫌気培養した。培養後、BacTiter-GloTM 試薬１００μＬをウェルに添加し、1420 Multilabel Counter（Wallac社製）を用いて、５３０ｎｍの蛍光強度（励起光：４８８ｎｍ）を定量した。また、上記単離成分に代えて、エタノールを添加した系を陰性コントロールとして同様の試験を行った。
結果を下記表５に示す。
Test Example 1 Antibacterial test against acne bacteria Isolated components EA-3-6-5, EA-3-6-9, EA-3-8-3 and EA-3-8-8 were dissolved in ethanol, respectively, and the concentration A sample solution for evaluation of 1 mg / mL was prepared, and antibacterial activity against acne was evaluated at a final concentration of 0.1 mg / mL. Antibacterial activity was evaluated using a commercially available kit (trade name: BacTiter-Glo ™ Microbial Cell Viability Assay, manufactured by Progega) that quantifies ATP using luciferase.
In a well of a 96-well plate, 10 μL of a culture solution of Acne JCM6425 strain anaerobically cultured at 37 ° C. for 3 days using a modified GAM medium (manufactured by Nissui Pharmaceutical), 80 μL of the modified GAM medium and 10 μL of the sample solution for evaluation And anaerobically cultured at 37 ° C. for 20 hours. After incubation, 100 μL of BacTiter-Glo ™ reagent was added to the wells, and the fluorescence intensity (excitation light: 488 nm) at 530 nm was quantified using a 1420 Multilabel Counter (manufactured by Wallac). Moreover, it replaced with the said isolation | separation component, and performed the same test by making the system which added ethanol the negative control.
The results are shown in Table 5 below.
表５より、単離成分ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８について蛍光強度の明らかな低下が認められた。したがって、前記式（１）〜（４）のいずれかで表される化合物がアクネ菌に対して優れた抗菌活性を示し、本発明の抗菌剤が抗アクネ菌剤として好適であることが明らかとなった。 From Table 5, the clear fall of the fluorescence intensity was recognized about isolated component EA-3-6-5, EA-3-6-9, EA-3-8-3, and EA-3-8-8. Therefore, it is clear that the compound represented by any one of the formulas (1) to (4) exhibits excellent antibacterial activity against acne bacteria, and the antibacterial agent of the present invention is suitable as an anti-acne fungicide. became.
試験例２ 黄色ブドウ球菌に対する抗菌試験
単離成分ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８を各々エタノールに溶解し、濃度１ｍｇ／ｍＬの評価用サンプル溶液を調製し、終濃度０．１ｍｇ／ｍＬで黄色ブドウ球菌に対する抗菌活性を評価した。抗菌活性は、ルシフェラーゼを利用してＡＴＰ量を定量する市販キット（商品名：BacTiter-GloTM Microbial Cell Viability Assay、Progega社製）を用いて評価した。
ＳＣＤ培地（ダイゴ社製）を用いて３７℃で１日間好気培養した黄色ブドウ球菌IFO13276株の培養液１０μＬと、ＳＣＤ培地８０μＬ及び評価用サンプル溶液１０μＬを９６穴プレートのウェル内で混合し、３７℃で２０時間好気培養した。培養後、BacTiter-GloTM 試薬１００μＬをウェルに添加し、1420 Multilabel Counter（Wallac社製）を用いて、５３０ｎｍの蛍光強度を定量した。また、上記単離成分に代えて、エタノールを添加した系を陰性コントロールとして同様の試験を行った。
結果を下記表６に示す。
Test Example 2 Antibacterial test against Staphylococcus aureus Isolated components EA-3-6-5, EA-3-6-9, EA-3-8-3 and EA-3-8-8 were each dissolved in ethanol, A sample solution for evaluation having a concentration of 1 mg / mL was prepared, and antibacterial activity against S. aureus was evaluated at a final concentration of 0.1 mg / mL. Antibacterial activity was evaluated using a commercially available kit (trade name: BacTiter-Glo ™ Microbial Cell Viability Assay, manufactured by Progega) that quantifies ATP using luciferase.
10 μL of the culture solution of S. aureus IFO13276 strain aerobically cultured at 37 ° C. for 1 day using SCD medium (manufactured by Daigo), 80 μL of SCD medium and 10 μL of the sample solution for evaluation were mixed in a well of a 96-well plate, The aerobic culture was performed at 37 ° C. for 20 hours. After incubation, 100 μL of BacTiter-Glo ™ reagent was added to the wells, and the fluorescence intensity at 530 nm was quantified using a 1420 Multilabel Counter (manufactured by Wallac). Moreover, it replaced with the said isolation | separation component, and performed the same test by making the system which added ethanol the negative control.
The results are shown in Table 6 below.
表６より、単離成分ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８において明らかな蛍光強度の低下が認められた。したがって、前記式（１）〜（４）のいずれかで表される化合物が黄色ブドウ球菌に対して抗菌活性を示し、本発明の抗菌剤が抗黄色ブドウ球菌剤として好適であることが明らかとなった。特に単離成分ＥＡ−３−８−３及びＥＡ−３−８−８において、黄色ブドウ球菌に対して高い抗菌活性が認められた。 From Table 6, a clear decrease in fluorescence intensity was observed in isolated components EA-3-6-5, EA-3-6-9, EA-3-8-3, and EA-3-8-8. Therefore, it is clear that the compound represented by any one of the formulas (1) to (4) exhibits antibacterial activity against S. aureus, and the antibacterial agent of the present invention is suitable as an anti-S. Aureus agent. became. In particular, in the isolated components EA-3-8-3 and EA-3-8-8, high antibacterial activity was observed against S. aureus.
試験例３ 単離成分を配合したローション（皮膚外用剤及び抗菌剤の一形態）の評価
単離成分（ＥＡ−３−６−５、ＥＡ−３−６−９、ＥＡ−３−８−３及びＥＡ−３−８−８）を用いて、これらを１％（ｗ／ｖ）配合したローション（１）〜（４）を下記表７に示す処方で調製した。
Test Example 3 Evaluation of lotion (form of skin external preparation and antibacterial agent) containing an isolated component Isolated component (EA-3-6-5, EA-3-6-9, EA-3-8-3) And EA-3-8-8), lotions (1) to (4) containing 1% (w / v) of these were prepared according to the formulation shown in Table 7 below.
上記ローション（１）〜（４）について、上記試験例１と同様に市販キットを用いて、培養液中の終濃度１０％（単離成分の終濃度１ｍｇ／ｍＬ）でのアクネ菌に対する抗菌評価を行った。その結果、単離成分を含まないローションを用いたコントロールに比べて、単離成分を含むローション（１）〜（４）では蛍光強度が有意に減少していた。すなわち、前記式（１）〜（４）のいずれかで表される化合物は、皮膚外用剤の形態においても抗菌活性を示すことがわかった。 For the lotions (1) to (4), using a commercially available kit in the same manner as in Test Example 1, antibacterial evaluation against acne bacteria at a final concentration of 10% in the culture solution (final concentration of isolated component 1 mg / mL) Went. As a result, the fluorescence intensity was significantly decreased in lotions (1) to (4) containing an isolated component as compared with a control using a lotion containing no isolated component. That is, it was found that the compound represented by any one of the formulas (1) to (4) exhibits antibacterial activity even in the form of a skin external preparation.
以上のように、式（１）〜（４）のいずれかで表される化合物は、アクネ菌や黄色ブドウ球菌等のグラム陽性菌に対して優れた抗菌活性を示す。したがって、式（１）〜（４）のいずれかで表される化合物の少なくとも１種を有効成分とする抗菌剤及び皮膚外用剤は、皮膚の炎症の原因菌に対する抗菌作用を有する。 As described above, the compound represented by any one of formulas (1) to (4) exhibits excellent antibacterial activity against gram-positive bacteria such as acne bacteria and Staphylococcus aureus. Therefore, the antibacterial agent and the skin external preparation containing at least one of the compounds represented by any one of the formulas (1) to (4) as active ingredients have an antibacterial action against bacteria that cause skin inflammation.
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