JP2019038766A - Sebum production promoter - Google Patents

Sebum production promoter Download PDF

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JP2019038766A
JP2019038766A JP2017161114A JP2017161114A JP2019038766A JP 2019038766 A JP2019038766 A JP 2019038766A JP 2017161114 A JP2017161114 A JP 2017161114A JP 2017161114 A JP2017161114 A JP 2017161114A JP 2019038766 A JP2019038766 A JP 2019038766A
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mobile phase
bamboo
extract
sebum production
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JP7041913B2 (en
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佐藤　隆
Takashi Sato
隆 佐藤
晃治 水野
Koji Nizuno
晃治 水野
弘明 坂上
Hiroaki Sakagami
弘明 坂上
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Ebc&m LLC
Take Plas LLC
Take-Plas LLC
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Take Plas LLC
Take-Plas LLC
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Abstract

To obtain a sebum production promoter promoting sebum producing ability of sebocytes from bamboo extract.SOLUTION: A sebum production promoter contains an ingredient with a retention time of 32 minutes or more to 42 minutes or less when a bamboo extract by normal pressure excessively heated vapor is developed by high performance liquid chromatography in the following analytical conditions. Analytical conditions for high performance liquid chromatography: Waters SunFire C18 Column (carrier particle diameter 5 μm, column inner diameter 4.6 mm, column length 150 mm), column temperature: room temperature, mobile phase A: 0.1% TFA/Water, mobile phase B: 0.1% TFA/CHCN, separation conditions: 0 to 5 min: retaining mobile phase A 98%, mobile phase B 2%, 5 to 40 min: linearly changing from mobile phase A 98%, mobile phase B 2% to mobile phase A 60%, mobile phase B 40%,40 to 50 min: retaining mobile phase A 2%, mobile phase B 98%, mobile phase flow rate: 1 mL/min, measurement wavelength: 214 nm.SELECTED DRAWING: Figure 2

Description

本発明は、竹の常圧過熱水蒸気による抽出物を主成分とする皮脂産生促進剤に関する。   TECHNICAL FIELD The present invention relates to a sebum production promoter mainly composed of an extract of bamboo with normal pressure superheated steam.

外来植物である孟宗竹は、１９５０年代頃までは竹材や筍を得るために管理された竹林で栽培されていた。しかしながら、近年は竹林業が経済的に成立しなくなり、各地で竹林が放置されるようになった。孟宗竹は繁殖力が異常に強いため、放置された竹林では、竹の繁殖域が無秩序に周囲に広がり、既存の植生を破壊するいわゆる竹害が問題となっている。   The exotic plant, Munetake Bamboo, was cultivated in bamboo forests managed to obtain bamboo and bamboo until the 1950s. However, in recent years, bamboo forestry has not been established economically, and bamboo forests have been neglected in various places. Since the reproductive power of Somune bamboo is unusually strong, in the abandoned bamboo forests, the so-called bamboo damage that destroys the existing vegetation is a problem because the bamboo breeding area spreads out randomly.

一方、竹害の問題に対し、竹を有効利用するための研究が進められている。例えば、竹を常圧過熱水蒸気で処理して発生する蒸気を凝縮して得られる竹の過加熱水蒸気抽出物（以下、竹ＳＨＳ(Super-Heated Stream)抽出物という）が、食中毒原因菌選択制抗菌剤として提案されている（特許文献１）。   On the other hand, research on the effective use of bamboo is underway for the problem of bamboo damage. For example, bamboo superheated steam extract (hereinafter referred to as bamboo SHS (Super-Heated Stream) extract) obtained by condensing steam generated by treating bamboo with normal-pressure superheated steam is a food poisoning-causing bacteria selection system. It has been proposed as an antibacterial agent (Patent Document 1).

特開２０１６−１３５７４９号公報Japanese Patent Laid-Open No. 2006-135749

特許文献１に記載の竹ＳＨＳ抽出物も含めて、竹抽出物に抗菌作用があることや低分子量の有機酸（ギ酸、酢酸、コハク酸、リンゴ酸など）が含まれることが知られているが、有機酸の物性から、竹抽出物を外用剤として皮膚疾患治療や美容の目的に利用する場合、皮膚刺激が懸念される。   It is known that the bamboo extract including the bamboo SHS extract described in Patent Document 1 has an antibacterial action and contains low molecular weight organic acids (formic acid, acetic acid, succinic acid, malic acid, etc.). However, due to the physical properties of organic acids, there is a concern about skin irritation when bamboo extract is used as an external preparation for the treatment of skin diseases and cosmetics.

これに対し、本発明は、竹抽出物から新たな有用成分とその薬剤としての用途を見出し、竹の有効利用を図ることを課題とする。   On the other hand, this invention makes it a subject to discover a new useful ingredient and its use as a chemical | medical agent from a bamboo extract, and to aim at the effective utilization of bamboo.

本発明者は、竹ＳＨＳ抽出物に含まれる成分を高速液体クロマトグラフィーにより分離し、個々の成分を細胞に作用させることで、比較的疎水性度の高い成分が皮脂産生促進作用を有することを見出し、本発明を想到した。   The present inventor has confirmed that the components contained in the bamboo SHS extract are separated by high performance liquid chromatography and the individual components act on the cells, so that the components having a relatively high hydrophobicity have a sebum production promoting action. The inventor came up with the present invention.

即ち、本発明は、竹の常圧過加熱水蒸気による抽出物であって、高速液体クロマトグラフィーにて次の分析条件で展開した場合にリテンションタイムが３２分以上４２以下の成分を含有してなる皮脂産生促進剤を提供する。
高速液体クロマトグラフィーの分析条件
・カラム Ｗａｔｅｒｓ社製ＳｕｎＦｉｒｅ Ｃ１８（担体粒子径５μｍ、カラム内径４．６ｍｍ、カラム長１５０ｍｍ）
・カラム温度 室温
・移動相Ａ：０．１％ＴＦＡ／Ｗａｔｅｒ、移動相Ｂ：０．１％ＴＦＡ／ＣＨ3ＣＮ
・分離条件
０〜５ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％に保持、
５〜４０ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％から移動相Ａ６０％、移動相Ｂ４０％へ直線的に変化、
４０〜５０ｍｉｎ：移動相Ａ２％、移動相Ｂ９８％に保持、
・移動相流速 １ｍＬ／ｍｉｎ
・測定波長 ２１４ｎｍ That is, the present invention is an extract of bamboo with normal-pressure superheated steam, and contains a component having a retention time of 32 minutes or more and 42 or less when developed under the following analysis conditions by high performance liquid chromatography. A sebum production promoter is provided.
Analysis conditions for high performance liquid chromatography-Column SunFire C18 manufactured by Waters (carrier particle diameter 5 μm, column inner diameter 4.6 mm, column length 150 mm)
Column temperature Room temperature Mobile phase A: 0.1% TFA / Water, mobile phase B: 0.1% TFA / CH 3 CN
Separation conditions 0 to 5 min: held at mobile phase A 98%, mobile phase B 2%,
5-40 min: linear change from mobile phase A 98%, mobile phase B 2% to mobile phase A 60%, mobile phase B 40%,
40 to 50 min: held at mobile phase A 2%, mobile phase B 98%,
・ Mobile phase flow rate 1mL / min
・ Measurement wavelength: 214 nm

本発明の皮脂産生促進剤によれば、脂腺細胞における皮脂産生能力を促進させることができる。そのため、本発明の皮脂産生促進剤は皮脂の減少に伴う皮膚疾患の治療ないし予防に有用となる。また、本発明の皮脂産生促進剤を皮膚に適用すると、表皮に潤いや柔軟性を付与することができ、美容上の効果も得ることができる。   According to the sebum production promoter of the present invention, sebum production ability in sebaceous cells can be promoted. Therefore, the sebum production promoter of the present invention is useful for the treatment or prevention of skin diseases associated with a decrease in sebum. Moreover, when the sebum production promoter of the present invention is applied to the skin, moisture and flexibility can be imparted to the epidermis, and a cosmetic effect can be obtained.

さらに、本発明の皮脂産生促進剤は、竹を原材料とし、常圧過熱水蒸気で竹を処理し、発生した蒸気を凝集するという簡便な方法により製造できるため、安価に製造することができる。   Furthermore, since the sebum production promoter of the present invention can be manufactured by a simple method of using bamboo as a raw material, treating bamboo with atmospheric superheated steam, and aggregating the generated steam, it can be manufactured at low cost.

図１は、竹ＳＨＳ抽出物を逆相クロマトグラフィーで展開したクロマトグラムである。FIG. 1 is a chromatogram of a bamboo SHS extract developed by reverse phase chromatography. 図２は、図１に対し、竹ＳＨＳ抽出物の注入量および分離条件を変更して逆相クロマトグラフィーで展開した場合のクロマトグラムである。FIG. 2 is a chromatogram in the case where the injection amount and separation conditions of the bamboo SHS extract are changed with respect to FIG. 1 and developed by reverse phase chromatography. 図３Ａは、竹ＳＨＳ抽出物又はそのＨＰＬＣ画分の脂腺細胞に対する作用を示す写真である。FIG. 3A is a photograph showing the action of bamboo SHS extract or its HPLC fraction on sebaceous cells. 図３Ｂは、竹ＳＨＳ抽出物のＨＰＬＣ画分の脂腺細胞に対する作用を示す写真である。FIG. 3B is a photograph showing the effect of bamboo SHS extract on the sebocytes of the HPLC fraction. 図３Ｃは、竹ＳＨＳ抽出物のＨＰＬＣ画分の脂腺細胞に対する作用を示す写真である。FIG. 3C is a photograph showing the action of the bamboo SHS extract on the sebocytes of the HPLC fraction. 図３Ｄは、竹ＳＨＳ抽出物のＨＰＬＣ画分の脂腺細胞に対する作用を示す写真である。FIG. 3D is a photograph showing the effect of bamboo SHS extract on the sebocytes of the HPLC fraction. 図４は、脂腺細胞のＤＮＡ当たりの皮脂（ＴＧ）の産生量に及ぼす竹ＳＨＳ抽出物の影響を示す図である。FIG. 4 is a diagram showing the influence of bamboo SHS extract on the production amount of sebum (TG) per sebaceous cell DNA.

以下、本発明の皮脂産生促進剤について詳細に説明する。
本発明の皮脂産生促進剤は、竹を常圧過熱水蒸気で処理することにより発生する蒸気を凝縮して得られる竹ＳＨＳ抽出物であって、後述する逆相クロマトグラフィーで展開した場合に、リテンションタイムが特定の範囲となる成分を含有する。 Hereinafter, the sebum production promoter of the present invention will be described in detail.
The sebum production promoter of the present invention is a bamboo SHS extract obtained by condensing steam generated by treating bamboo with normal-pressure superheated steam, which is retained when developed by reverse phase chromatography described later. Contains ingredients with a specific time range.

ここで、竹はイネ目イネ科タケ亜科のうち、茎が木質化する種の総称であり、竹の代表的な種類としては、モウソウチク(孟宗竹)、マダケ、ハチク等が挙げられる。本発明が抽出対象とする竹には、孟宗竹をはじめとする上述の代表的な竹の他に、アズマザサ、ヤダケ、アズマネザサ、スズタケ、クマザサやチシマザサなどのイネ科タケ亜科に属するササ類を含めることができる。   Here, bamboo is a general term for species of stems that turn into woody among the rice family Gramaceae, and typical examples of bamboo include Mosouchiku, Madake, Hachiku and the like. Bamboo to be extracted by the present invention includes not only the above-mentioned representative bamboos such as Miso bamboo, but also Sasa belonging to the Gramineae subfamily, such as Azumazasa, Yadatake, Azumaneza, Suzutake, Kumazasa and Chishimasa. be able to.

抽出する竹の部位としては、稈、枝、葉又は根をあげることができる。   Bamboo parts to be extracted can include bamboo shoots, branches, leaves or roots.

竹から竹ＳＨＳ抽出物を得る方法は、特許文献１に記載されている方法によればよい。即ち、竹を入れた容器に、好ましくは１８０〜２５０℃、より好ましくは２００〜２３０℃の常圧過加熱水蒸気を、好ましくは０．２〜１．０ｋｇ／竹１ｋｇの流量で導入し、それにより発生する加水分解生成物の蒸気（即ち、竹の分解により生じた揮発竹酢成分）を冷却凝縮させる。こうして得られた竹ＳＨＳ抽出物は、タール分を含まず、発がん性を有するベンゾピレンの濃度が低く、好ましくは０．７ｐｐｂ以下である。   A method for obtaining a bamboo SHS extract from bamboo may be the method described in Patent Document 1. That is, normal pressure superheated steam of preferably 180 to 250 ° C., more preferably 200 to 230 ° C. is introduced into a container containing bamboo, preferably at a flow rate of 0.2 to 1.0 kg / bamboo 1 kg. The steam of the hydrolyzed product generated by (i.e., the volatile bamboo vinegar component produced by the decomposition of bamboo) is cooled and condensed. The bamboo SHS extract thus obtained does not contain tar and has a low carcinogenic benzopyrene concentration, preferably 0.7 ppb or less.

また、こうして得られた竹ＳＨＳ抽出物は種々の成分の混合物となっているが、本発明の皮膚産生促進剤には、室温（例えば２５℃）で実施例に記載の逆相クロマトグラフィーで展開した場合にリテンションタイムが３２分以上４２分以下の成分を含有させる。一方、この逆相クロマトグラフィーでリテンションタイム１０分以下の成分は、ギ酸、酢酸、コハク酸、リンゴ酸などの低分子量の有機酸であり、細胞毒性があるので、皮脂産生促進剤には含有させないことが好ましい。   The bamboo SHS extract thus obtained is a mixture of various components. The skin production promoter of the present invention is developed by reverse phase chromatography described in Examples at room temperature (for example, 25 ° C.). In this case, a component having a retention time of 32 minutes or more and 42 minutes or less is contained. On the other hand, components with a retention time of 10 minutes or less in this reverse phase chromatography are low molecular weight organic acids such as formic acid, acetic acid, succinic acid, malic acid, and are cytotoxic, so they are not included in the sebum production promoter. It is preferable.

本発明の皮脂産生促進剤の剤型は、固形状、半固形状、液状等のいずれであってもよく、製品の形態に応じて適宜決定される。   The dosage form of the sebum production promoter of the present invention may be any of solid, semi-solid, liquid, etc., and is appropriately determined according to the form of the product.

また、本発明の皮脂産生促進剤は、本発明の効果を損なわない範囲で、ｐＨ調整剤、増粘剤、酸化防止剤、紫外線吸収剤、防腐剤、香料、色素などの各種添加剤や賦形剤を含有することができる。   The sebum production promoter of the present invention is not limited to the effects of the present invention, and various additives and additives such as pH adjusters, thickeners, antioxidants, ultraviolet absorbers, preservatives, fragrances, and pigments. A dosage form can be included.

本発明の皮脂産生促進剤の投与形態は、経口、経腸、径粘膜、注射等とすることができる。また、医薬品、化粧品、飲食品、ペットフード等に配合して使用することができる。   The administration form of the sebum production promoter of the present invention can be oral, enteral, radial mucosa, injection or the like. Moreover, it can mix | blend and use for a pharmaceutical, cosmetics, food-drinks, pet food, etc.

（１）竹ＳＨＳ抽出物の調製
特許文献１の実施例の「選択性抗菌剤の製造実施例１」に記載されている方法で、孟宗竹の稈部分から竹ＳＨＳ抽出物を得た。 (1) Preparation of Bamboo SHS Extract Bamboo SHS extract was obtained from the cocoon part of Sosetsu bamboo by the method described in “Example 1 of production of selective antibacterial agent” in Examples of Patent Document 1.

（２）ＨＰＬＣによる竹ＳＨＳ抽出物の解析
（１）で得た竹ＳＨＳ抽出物を試料とし、高速液体クロマトグラフ（Ｃｈｒｏｍａｓｔｅｒ（Ｏｒｇａｎｉｚｅｒ，Ｉｎｔｅｒｆａｃｅ Ｂｏｘ，５４１０ ＵＶ Ｄｅｔｅｃｔｏｒ，５１１０ Ｐｕｍｐ）、株式会社日立ハイテクサイエンス)を用いて次の逆相クロマトグラフィーの分離条件で展開し、図１のクロマトグラムを得た。図１から疎水性度の異なる複数の化合物が竹ＳＨＳ抽出物に含まれることがわかる。 (2) Analysis of bamboo SHS extract by HPLC Using bamboo SHS extract obtained in (1) as a sample, high performance liquid chromatograph (Chromamaster (Organizer, Interface Box, 5410 UV Detector, 5110 Pump), Hitachi High-Tech Science Co., Ltd. 1) was developed under the following reverse phase chromatography separation conditions to obtain the chromatogram of FIG. It can be seen from FIG. 1 that a plurality of compounds having different hydrophobicities are contained in the bamboo SHS extract.

ＨＰＬＣ分析条件
・カラム Ｗａｔｅｒｓ社製ＳｕｎＦｉｒｅ Ｃ１８（担体粒子径５μｍ、カラム内径４．６ｍｍ、カラム長１５０ｍｍ）
・カラム温度 室温
・移動相Ａ：０．１％ＴＦＡ／Ｗａｔｅｒ、移動相Ｂ：０．１％ＴＦＡ／ＣＨ3ＣＮ
・分離条件
０〜１７０ｍｉｎ：移動相Ａ１００％、移動相Ｂ０％から移動相Ａ６０％、移動相Ｂ４０％へ直線的に変化
・移動相流速 １ｍＬ／ｍｉｎ
・測定波長 ２１４ｎｍ
・試料注入量 １００μＬ HPLC analysis conditions / column SunFire C18 manufactured by Waters (carrier particle diameter 5 μm, column inner diameter 4.6 mm, column length 150 mm)
Column temperature Room temperature Mobile phase A: 0.1% TFA / Water, mobile phase B: 0.1% TFA / CH 3 CN
Separation conditions 0 to 170 min: linear change from mobile phase A 100%, mobile phase B 0% to mobile phase A 60%, mobile phase B 40% mobile phase flow rate 1 mL / min
・ Measurement wavelength: 214 nm
・ Sample injection volume 100μL

（３）ＨＰＬＣによる竹ＳＨＳ抽出物分画試料の調製
次に、竹ＳＨＳ抽出物に含まれる個々の成分を分離し、分離した成分ごとに細胞に対する作用を調べるため、逆相クロマトグラフィーの分離条件及び試料注入量を次のように変更し、細胞培養に用いるために十分な量の分画成分を得た。この場合のクロマトグラムを図２に示す。 (3) Preparation of bamboo SHS extract fraction sample by HPLC Next, separation of individual components contained in the bamboo SHS extract and the separation conditions of reversed-phase chromatography for examining the action on cells for each separated component And the sample injection amount was changed as follows to obtain a sufficient amount of fraction components for use in cell culture. The chromatogram in this case is shown in FIG.

ＨＰＬＣ分析条件
・カラム Ｗａｔｅｒｓ社製ＳｕｎＦｉｒｅ Ｃ１８（担体粒子径５μｍ、カラム内径４．６ｍｍ、カラム長１５０ｍｍ）
・カラム温度 室温
・移動相Ａ：０．１％ＴＦＡ／Ｗａｔｅｒ、移動相Ｂ：０．１％ＴＦＡ／ＣＨ3ＣＮ
・分離条件
０〜５ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％に保持、
５〜４０ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％から移動相Ａ６０％、移動相Ｂ４０％へ直線的に変化、
４０〜５０ｍｉｎ：移動相Ａ２％、移動相Ｂ９８％に保持、
・移動相流速 １ｍＬ／ｍｉｎ
・測定波長 ２１４ｎｍ
・試料注入量 １０００μＬ HPLC analysis conditions / column SunFire C18 manufactured by Waters (carrier particle diameter 5 μm, column inner diameter 4.6 mm, column length 150 mm)
Column temperature Room temperature Mobile phase A: 0.1% TFA / Water, mobile phase B: 0.1% TFA / CH 3 CN
Separation conditions 0 to 5 min: held at mobile phase A 98%, mobile phase B 2%,
5-40 min: linear change from mobile phase A 98%, mobile phase B 2% to mobile phase A 60%, mobile phase B 40%,
40 to 50 min: held at mobile phase A 2%, mobile phase B 98%,
・ Mobile phase flow rate 1mL / min
・ Measurement wavelength: 214 nm
・ Sample injection volume 1000μL

図２が示すように、竹ＳＨＳ抽出物は１１の成分に分画した。このようにして得られた１１の画分を凍結乾燥した。   As shown in FIG. 2, the bamboo SHS extract was fractionated into 11 components. The 11 fractions thus obtained were lyophilized.

このうち、画分１は、ＤＭＳＯに溶解した成分（Ｆ１Ｄ）と、ＤＭＳＯに不溶性のものを超純水にて溶解したもの（Ｆ１Ｗ）の２つに分けた。画分２から画分９は全てＤＭＳＯに溶解した（Ｆ２−Ｆ９）。なお、リテンションタイム１０分以下の成分には、特許文献１に記載されている、ギ酸、酢酸、コハク酸、リンゴ酸などの低分子量の有機酸が含まれていると考えられる。   Of these, fraction 1 was divided into two components: a component dissolved in DMSO (F1D) and a component insoluble in DMSO dissolved in ultrapure water (F1W). Fractions 2 to 9 were all dissolved in DMSO (F2-F9). The component having a retention time of 10 minutes or less is considered to contain low molecular weight organic acids such as formic acid, acetic acid, succinic acid and malic acid described in Patent Document 1.

（４）竹ＳＨＳ抽出物の皮脂産生促進作用
(4-1)ハムスター脂腺細胞の単離
ゴールデンハムスター（雄、５週齢）の左右耳介部を切除し、ｐｅｎｉｃｉｌｌｉｎ Ｇ（１００ｕｎｉｔｓ／ｍＬ）および硫酸ｓｔｒｅｐｔｏｍｙｃｉｎ（１００μｇ／ｍＬ）を添加したＤＭＥＭに浸し、１時間以上４℃にて放置した後にリン酸緩衝食塩液（ＰＢＳ(−)）で洗浄した。耳介部周囲の毛を刈り、５×５ｍｍ程度に細切し、２．４ｕｎｉｔｓ／ｍＬ ｄｉｓｐａｓｅ溶液中で４℃、１３．５時間静置した。酵素処理後、ピンセットにて耳介部より表皮を剥離し、皮脂腺を含む真皮を脂腺細胞培養液（ＳＧＭ）［ＤＭＥＭ/Ｆ１２／６％(v/v)ＦＢＳ／２％(v/v)ｈｕｍａｎ ｓｅｒｕｍ／０．６８ｍＭ Ｌ−ｇｌｕｔａｍｉｎｅ／ｐｅｎｉｃｉｌｌｉｎ（１００ｕｎｉｔｓ／ｍＬ）／硫酸ｓｔｒｅｐｔｏｍｙｃｉｎ（１００μｇ／ｍＬ）］に浸し、実体顕微鏡下で皮脂腺を単離した。 (4) Sebum production promoting action of bamboo SHS extract
(4-1) Isolation of hamster sebaceous gland cells The left and right auricles of a golden hamster (male, 5 weeks old) were excised and immersed in DMEM supplemented with penicillin G (100 units / mL) and streptomycin (100 μg / mL). After standing at 4 ° C. for 1 hour or longer, it was washed with a phosphate buffered saline (PBS (−)). The hair around the auricle was cut, chopped to about 5 × 5 mm, and left in a 2.4 units / mL displace solution at 4 ° C. for 13.5 hours. After the enzyme treatment, the epidermis is peeled off from the auricle with tweezers, and the dermis including the sebaceous glands is sebaceous cell culture solution (SGM) [DMEM / F12 / 6% (v / v) FBS / 2% (v / v) human serum / 0.68 mM L-glutamine / penicillin (100 units / mL) / sulfate streptomycin (100 μg / mL)], and sebaceous glands were isolated under a stereomicroscope.

(4-2)ハムスター脂腺細胞の皮脂産生に対する竹ＳＨＳ抽出物の作用
継代培養したハムスター脂腺細胞（ｐ＝５）を９６ｗｅｌｌ ｐｌａｔｅ （ＩＷＡＫＩ製）に１．０×１０４ｃｅｌｌｓ／ｗｅｌｌの細胞数で播種した。この場合、ハムスター脂腺細胞を未分画の竹ＳＨＳ抽出物（０．０００３％、０．０１％、０．１％及び１％）、及び竹ＳＨＳ抽出物のＨＰＬＣ分画のＦ１Ｄ、Ｆ１Ｗ、Ｆ２、Ｆ３、Ｆ４、Ｆ５、Ｆ６、Ｆ７、Ｆ８及びＦ９（０．０００３％、０．０１％、及び１％）でそれぞれ処理し、９日間培養した。培養後、リン酸緩衝液（ＰＢＳ(−)）にて細胞を洗浄し、４％パラホルムアルデヒドにて固定し、細胞標本を得た。 (4-2) Effect of bamboo SHS extract on sebum production of hamster sebaceous gland cells Subcultured hamster sebaceous gland cells (p = 5) were added to 96 well plate (manufactured by IWAKI) at 1.0 × 10 4 cells / well. Sowing. In this case, hamster sebaceous gland cells were separated from unfractionated bamboo SHS extract (0.0003%, 0.01%, 0.1% and 1%), and F1D, F1W, F2 of HPLC fractions of bamboo SHS extract. , F3, F4, F5, F6, F7, F8 and F9 (0.0003%, 0.01% and 1%), respectively, and cultured for 9 days. After culturing, the cells were washed with a phosphate buffer (PBS (−)) and fixed with 4% paraformaldehyde to obtain a cell specimen.

皮脂産生能に対する竹ＳＨＳ抽出液の影響を評価するため、得られた細胞標本をｏｉｌ ｒｅｄ Ｏ染色法により脂肪滴を染色した。また、対照として、ＤＨＴや竹ＳＨＳ抽出物を添加せずに作製した細胞標本（Ｃｔｌ）、ＤＨＴの存在下(濃度１０μＭ)で竹ＳＨＳ抽出物を添加せずに作製した細胞標本（ＤＨＴ）、又は皮脂産生促進作用が知られているインスリン（Ｉｎｓ）の存在下（濃度１０μＭ）で作製した細胞標本についても同様に染色した。   In order to evaluate the effect of bamboo SHS extract on sebum production ability, the obtained cell specimen was stained with fat red O staining method. In addition, as a control, a cell specimen (Dtl) prepared without adding DHT or bamboo SHS extract, a cell specimen (DHT) prepared without adding bamboo SHS extract in the presence of DHT (concentration 10 μM), Alternatively, cell specimens prepared in the presence of insulin (Ins), which is known to promote sebum production (concentration 10 μM), were also stained in the same manner.

ここで、Ｏｉｌ ｒｅｄ Ｏ染色液は、０．３％(w/v)ｏｉｌ ｒｅｄ Ｏ を含む ｉｓｏｐｒｏｐａｎｏｌ溶液と精製水を３：２(v:v)で混合し、密閉式超音波細胞破砕装置（コスモ・バイオ株式会社製）により１５分間超音波処理を行い、１０分間室温に放置し、ろ過した上清である。   Here, the Oil red O staining solution is prepared by mixing an isopropanol solution containing 0.3% (w / v) oil red O and purified water at a ratio of 3: 2 (v: v), and a sealed ultrasonic cell disruption device ( The supernatant was sonicated for 15 minutes by Cosmo Bio Co., Ltd., left at room temperature for 10 minutes, and filtered.

この染色法では、固定した細胞標本にｏｉｌ ｒｅｄ Ｏ 染色液を加えて３７℃で１５分間染色した。   In this staining method, oil red O staining solution was added to the fixed cell specimen and stained at 37 ° C. for 15 minutes.

染色後、細胞をリン酸緩衝液（ＰＢＳ(−)）にて洗浄し、光学顕微鏡（オリンパス株式会社製）により脂肪滴形成を観察した。この観察写真を「竹ＳＨＳ抽出物（画分）」として図３Ａ、図３Ｂ、図３Ｃ及び図３Ｄに示す。   After staining, the cells were washed with a phosphate buffer (PBS (−)), and lipid droplet formation was observed with an optical microscope (Olympus Corporation). This observation photograph is shown in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D as a “bamboo SHS extract (fraction)”.

また、皮脂の主成分であるトリアシルグリセロール（ＴＧ）を定量するため、ＤＨＴや竹ＳＨＳ抽出物を添加しない細胞（Ｃｔｌ）、ＤＨＴの存在下(濃度１０μＭ)で竹ＳＨＳ抽出物を添加せずに処理した細胞（ＤＨＴ）、皮脂産生抑制作用が知られているレチノイン酸を処理した細胞（ＲＡ）（濃度１ｎＭ）、皮脂産生促進作用が知られているインスリンを処理した細胞（Ｉｎｓ）（濃度１０μＭ）、ＤＨＴ存在下でＲＡを処理した細胞（ＤＨＴ＋ＲＡ）、ＤＨＴ存在下で竹ＳＨＳ抽出物を処理した細胞（ＤＨＴ＋竹ＳＨＳ抽出物０．１％又は１％）をそれぞれＰＢＳ(−)で２回洗浄し、０．２５％ (w/v) ｔｒｙｐｓｉｎ/０．０２％ (w/v) ＥＤＴＡ／ＰＢＳ(−)溶液を用いて回収した。得られた細胞懸濁液を氷冷下、密閉式超音波細胞破砕装置(コスモ・バイオ株式会社製)により超音波処理を行い、細胞を破砕した。この細胞内のＴＧ量をアクアオートカイノスＴＧ−II試薬（株式会社カイノス製）を用いて次の方法で測定した。すなわち、上記で調製した細胞懸濁液試料(５０μＬ)に反応試液Ｒ−１(９０μＬ)を添加し、３７℃で１０分間反応させた後、直ちに反応試液Ｒ−２(３０μＬ)を添加し、さらに３７℃で１０分間反応させた。反応終了後、５９５ｎｍにおける吸光度をマイクロプレートリーダーにて測定した。この吸光度からＴＧ量を、同様に実施したトリオレイン標準液(２００ｍｇ／ｄＬ）の吸光度に基づいて算出した。一方、細胞中のＤＮＡ量を３，５−ジアミノ安息香酸二塩酸塩（ＤＡＢＡ）法を用いて測定し、ＤＮＡ１μｇあたりの皮脂量（ＴＧ／ＤＮＡ）を求め、このＴＧ／ＤＮＡのコントロールに対する比を求めた。結果を図４に示す。   In addition, in order to quantify triacylglycerol (TG), which is the main component of sebum, cells without the addition of DHT or bamboo SHS extract (Ctl), without the addition of bamboo SHS extract in the presence of DHT (concentration 10 μM) Treated cells (DHT), cells treated with retinoic acid known to have sebum production inhibitory effect (RA) (concentration 1 nM), cells treated with insulin known to promote sebum production (Ins) (concentration) 10 μM), cells treated with RA in the presence of DHT (DHT + RA), and cells treated with the bamboo SHS extract in the presence of DHT (DHT + bamboo SHS extract 0.1% or 1%) were each 2 with PBS (−). Washed twice and collected using 0.25% (w / v) trypsin / 0.02% (w / v) EDTA / PBS (−) solution. The obtained cell suspension was subjected to sonication with a sealed ultrasonic cell crusher (manufactured by Cosmo Bio Inc.) under ice cooling to crush the cells. The amount of TG in the cells was measured by the following method using Aqua Auto Kinos TG-II reagent (manufactured by Kinos Co., Ltd.). That is, the reaction sample solution R-1 (90 μL) was added to the cell suspension sample (50 μL) prepared above, reacted at 37 ° C. for 10 minutes, and then immediately added with the reaction sample solution R-2 (30 μL). Furthermore, it was made to react at 37 degreeC for 10 minutes. After completion of the reaction, the absorbance at 595 nm was measured with a microplate reader. From this absorbance, the amount of TG was calculated based on the absorbance of the same triolein standard solution (200 mg / dL). On the other hand, the amount of DNA in the cells was measured using the 3,5-diaminobenzoic acid dihydrochloride (DABA) method to determine the amount of sebum per 1 μg of DNA (TG / DNA), and the ratio of this TG / DNA to the control was Asked. The results are shown in FIG.

図３Ａ〜図３Ｄ及び図４から、未分画の竹ＳＨＳ抽出物には、その濃度依存的にＤＨＴにより誘導した皮脂産生を抑制する作用が認められ、竹ＳＨＳ抽出物の濃度が１％であると外観観察から細胞死が疑われた。また、竹ＳＨＳ抽出物のＦ１Ｄには皮脂産生に対して強い抑制作用が認められた。しかしながら、竹ＳＨＳ抽出物のＦ２〜Ｆ５には細胞毒性が認められず、Ｆ６〜Ｆ８には皮脂産生の促進作用が認められた。したがって、Ｆ６〜Ｆ８を含む画分、即ち、リテンションタイムが３２分以上４２分以下の画分は、皮脂産生促進剤の有効成分となることがわかる。また、観察された脂肪滴を蓄積する細胞数と各画分の濃度との関係から、この有効成分の適正濃度は、画分濃度として１％未満であることがわかる。   From FIG. 3A to FIG. 3D and FIG. 4, the unfractionated bamboo SHS extract has an effect of suppressing sebum production induced by DHT in a concentration-dependent manner, and the concentration of the bamboo SHS extract is 1%. In some cases, cell death was suspected from appearance observation. In addition, F1D of bamboo SHS extract showed a strong inhibitory effect on sebum production. However, no cytotoxicity was observed in F2 to F5 of the bamboo SHS extract, and a sebum production promoting effect was observed in F6 to F8. Therefore, it can be seen that the fraction containing F6 to F8, that is, the fraction having a retention time of 32 minutes or more and 42 minutes or less is an active ingredient of the sebum production promoter. In addition, from the relationship between the observed number of cells that accumulate lipid droplets and the concentration of each fraction, it is understood that the appropriate concentration of this active ingredient is less than 1% as the fraction concentration.

Claims (3)

竹の常圧過加熱水蒸気による抽出物であって、高速液体クロマトグラフィーにて次の分析条件で展開した場合にリテンションタイムが３２分以上４２分以下の成分を含有してなる皮脂産生促進剤。
高速液体クロマトグラフィーの分析条件
・カラム Ｗａｔｅｒｓ社製ＳｕｎＦｉｒｅ Ｃ１８（担体粒子径５μｍ、カラム内径４．６ｍｍ、カラム長１５０ｍｍ）
・カラム温度 室温
・移動相Ａ：０．１％ＴＦＡ／Ｗａｔｅｒ、移動相Ｂ：０．１％ＴＦＡ／ＣＨ3ＣＮ
・分離条件
０〜５ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％に保持、
５〜４０ｍｉｎ：移動相Ａ９８％、移動相Ｂ２％から移動相Ａ６０％、移動相Ｂ４０％へ直線的に変化、
４０〜５０ｍｉｎ：移動相Ａ２％、移動相Ｂ９８％に保持、
・移動相流速 １ｍＬ／ｍｉｎ
・測定波長 ２１４ｎｍ A sebum production promoter, which is an extract of bamboo under normal pressure superheated steam, and contains a component having a retention time of 32 minutes or more and 42 minutes or less when developed under the following analysis conditions by high performance liquid chromatography.
Analytical conditions for high performance liquid chromatography
Column SunFire C18 manufactured by Waters (carrier particle diameter 5 μm, column inner diameter 4.6 mm, column length 150 mm)
Column temperature Room temperature Mobile phase A: 0.1% TFA / Water, mobile phase B: 0.1% TFA / CH 3 CN
Separation conditions 0 to 5 min: held at mobile phase A 98%, mobile phase B 2%,
5-40 min: linear change from mobile phase A 98%, mobile phase B 2% to mobile phase A 60%, mobile phase B 40%,
40 to 50 min: held at mobile phase A 2%, mobile phase B 98%,
・ Mobile phase flow rate 1mL / min
・ Measurement wavelength: 214 nm 前記高速液体クロマトグラフィーの分析条件において、リテンションタイム１０分以下の成分を含有しない請求項１記載の皮脂産生促進剤。   The sebum production promoter according to claim 1, which does not contain a component having a retention time of 10 minutes or less under the analysis conditions of the high performance liquid chromatography. 竹が孟宗竹である請求項１又は２記載の皮脂産生促進剤。   The sebum production promoter according to claim 1 or 2, wherein the bamboo is Miso bamboo.
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JP2023008338A (en) * 2021-07-05 2023-01-19 伊豆食文化公園株式会社 Oral compositions for improving skin conditions comprising bamboo shoot skin extracts
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