JP4827032B2 - Skin release component measurement method - Google Patents
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- JP4827032B2 JP4827032B2 JP2005170471A JP2005170471A JP4827032B2 JP 4827032 B2 JP4827032 B2 JP 4827032B2 JP 2005170471 A JP2005170471 A JP 2005170471A JP 2005170471 A JP2005170471 A JP 2005170471A JP 4827032 B2 JP4827032 B2 JP 4827032B2
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- Measuring And Recording Apparatus For Diagnosis (AREA)
Description
本発明は、皮膚から放出した皮膚放出成分を測定する方法であって、極微量の皮膚放出成分であっても、短時間のサンプリング及び分析で、簡単に測定することのできる皮膚放出成分測定方法に関する。 The present invention is a method for measuring a skin release component released from the skin, and can easily measure even a very small amount of skin release component by short-time sampling and analysis. About.
従来、芳香成分を摂取することにより、皮膚表面より該成分を放出させ、体臭を改善する目的で、「食べる香水」としてローズオットーの精油を含んだカプセル等が販売されている。
しかし、これらを摂取して、上記精油由来成分が皮膚から放出されているかについて確認されたことがなかった。というのも、該成分は、極微量のため捕集及び分析が困難で、同定及び定量が出来なかったからである。従って、上記精油由来成分を皮膚表面より放出させようとすると、多量に摂取しなければならず、肝臓や腎臓に悪影響を及ぼすおそれがあった。
Conventionally, capsules containing Rose Otto essential oil have been marketed as “eating perfumes” for the purpose of improving the body odor by ingesting an aromatic component to release the component from the skin surface.
However, when these were ingested, it was never confirmed whether the essential oil-derived component was released from the skin. This is because the component is extremely small and difficult to collect and analyze, and cannot be identified and quantified. Therefore, if the essential oil-derived component is to be released from the skin surface, it must be ingested in a large amount, which may adversely affect the liver and kidney.
そこで、皮膚表面より放出した皮膚ガスを測定する方法として、下記のような技術が知られている。
まず、ヒト皮膚より放出される皮膚ガスとして、アセトン、水素、アンモニア、メタン、エタン、エチレンが発見され、報告されている（例えば、非特許文献１参照。）。これによると、上記皮膚ガスを捕集する方法として、手表面を容器で覆い、容器内に放出された蒸気ガスをガスクロマトグラフィーで分析するというものである。
また、上記文献の発表者は、皮膚表面に１０秒間、５％エタノール水溶液を噴霧し、回収したエタノール水溶液を液体クロマトグラフィーで分析する特許（例えば、特許文献１参照。）や、皮膚に密着したカプセル内で、皮膚透過分泌物を除湿空気と拡散混合し、アルコール濃度測定機に注入することで、皮膚透過分泌物中のアルコール濃度を測定する特許（例えば、特許文献２参照）を提案している。
Therefore, the following techniques are known as methods for measuring skin gas released from the skin surface.
First, acetone, hydrogen, ammonia, methane, ethane, and ethylene have been discovered and reported as skin gases released from human skin (see, for example, Non-Patent Document 1). According to this, as a method of collecting the skin gas, the surface of the hand is covered with a container, and the vapor gas released into the container is analyzed by gas chromatography.
In addition, the presenter of the above-mentioned document sprays a 5% ethanol aqueous solution on the skin surface for 10 seconds, and analyzes the collected ethanol aqueous solution by liquid chromatography (see, for example, Patent Document 1), or adheres closely to the skin. Proposing a patent (for example, see Patent Document 2) that measures the alcohol concentration in the skin permeation secretion by diffusing and mixing the skin permeation secretion with dehumidified air in the capsule and injecting it into an alcohol concentration measuring device. Yes.
しかしながら、非特許文献１及び特許文献２記載の測定方法では、揮発性の高い化合物の同定及び定量を開示したものであり、また、特許文献１の測定方法では、ほとんど揮発しないグルコースの同定及び定量を開示しているに過ぎず、ローズオットーの精油に含まれるリナロール、シトロネロール、ゲラニオール等といった、揮発性の低い化合物では、その濃度が低すぎて検出が難しい。
また、上記測定方法では、皮膚表面から放出した皮膚ガスのみを測定しているか、皮膚表
面のみに存在しているグルコースを測定しているかのどちらかであるので、両方を測定するには、２度のサンプリングが必要になり、煩雑で時間を要した。
However, the measurement methods described in
In the above measurement method, either only the skin gas released from the skin surface is measured or glucose existing only on the skin surface is measured. Sampling was necessary, complicated and time consuming.
本発明は、このような事情に鑑みなされたものであって、その目的とするところは、極微量の皮膚放出成分であっても、短時間のサンプリング及び分析で、簡単に測定することのできる皮膚放出成分測定方法を提供するにある。 The present invention has been made in view of such circumstances, and the object of the present invention is to measure even a very small amount of a skin-released component with a short sampling and analysis. It is in providing the measuring method of a skin release component.
上記目的は、皮膚から放出した皮膚放出成分を測定する方法であって、下記（１）〜（４）を順次行うことを特徴とする皮膚放出成分測定方法により達成される。
（１）皮膚から放出した皮膚放出成分を、捕集容器で捕集する工程
（２）上記捕集容器内及び皮膚表面を、親水性溶媒で洗浄する工程
（３）上記（２）で得られた洗浄液を、固相抽出する工程
（４）上記（３）で得られた固相を、ガスクロマトグラフ質量分析する工程
The above object is achieved by a method for measuring a skin release component released from the skin, wherein the following (1) to (4) are sequentially performed.
(1) Step of collecting the skin release component released from the skin with a collection container (2) Step of washing the inside of the collection container and the skin surface with a hydrophilic solvent (3) Obtained by (2) above (4) Step of gas chromatograph mass spectrometry of the solid phase obtained in (3) above
また、上記目的は、皮膚から放出した皮膚放出成分を測定する方法であって、下記（１）〜（４）を順次行うことを特徴とする皮膚放出成分測定方法により達成される。
（１）皮膚から放出した皮膚放出成分を、捕集容器で捕集する工程
（２）上記捕集容器内及び皮膚表面を、親水性溶媒で洗浄する工程
（３）上記（２）で得られた洗浄液を、減圧濃縮する工程
（４）上記（３）で得られた濃縮液を、ガスクロマトグラフ質量分析する工程
Further, the above object is achieved by a method for measuring a skin release component, which is a method for measuring a skin release component released from the skin, wherein the following (1) to (4) are sequentially performed.
(1) Step of collecting the skin release component released from the skin with a collection container (2) Step of washing the inside of the collection container and the skin surface with a hydrophilic solvent (3) Obtained by (2) above (4) Step of gas chromatograph mass spectrometry of the concentrated solution obtained in (3) above
好ましくは、皮膚放出成分が、極性化合物及びテルペン類の少なくとも一方である。
また、固相抽出工程を有する皮膚放出成分測定方法においては、ガスクロマトグラフが熱脱着式であることが好ましい。
Preferably, the skin release component is at least one of a polar compound and terpenes.
Moreover, in the skin release component measuring method which has a solid-phase extraction process, it is preferable that a gas chromatograph is a thermal desorption type.
すなわち、まず、本発明者らは、市販されているローズオットーの精油を含む飲食品に関し、該精油に含まれる芳香成分が皮膚より放出されているかどうか、またその放出量を検出する方法について、その濃度に着目し鋭意検討した結果、捕集容器を用いて皮膚から放出した成分を捕集し、該捕集容器内及び皮膚表面を親水性溶媒で洗浄して洗浄液回収した後、該洗浄液をそのまま分析するのではなく固相抽出または減圧濃縮により濃縮してからガスクロマトグラフ質量分析することで、ナノグラムレベルの極微量の成分でも、短時間で簡単且つ確実に同定及び定量可能なことを見出した。
更に、上記ローズオットーの精油に含まれる成分のみならず、一般食品に使用される食品香料で多用される成分等の皮膚からの放出有無についても本発明の方法を用いると、短時間で簡単且つ確実に測定できることを確認し、本発明を完成した。
That is, first, the present inventors, regarding food and drink containing the essential oil of Rose Otto that is commercially available, whether or not the fragrance component contained in the essential oil is released from the skin, and a method for detecting the release amount, As a result of diligent investigation focusing on the concentration, the components released from the skin were collected using a collection container, and the washing liquid was collected by washing the collection container and the skin surface with a hydrophilic solvent. It was found that by analyzing by gas chromatograph mass spectrometry after solid phase extraction or vacuum concentration instead of analyzing as it is, it is possible to easily and reliably identify and quantify even a very small amount of components at the nanogram level. .
Furthermore, using the method of the present invention not only for the ingredients contained in the essential oil of Rose Otto, but also for the presence or absence of release from the skin of ingredients frequently used in food fragrances used in general food, It was confirmed that measurement was possible with certainty, and the present invention was completed.
本発明の皮膚放出成分測定方法を用いると、ナノグラムレベルの極微量の皮膚放出成分の同定及び定量が確実に実施できる。
また、極微量の皮膚放出成分にもかかわらず、短時間でサンプリングが可能である。よって、皮膚放出成分を繰返し捕集・測定でき、該成分がどのように放出されているか、時間の経過と共に確認することを容易に行うことができる。
更には、本発明によれば、１回の皮膚放出成分の捕集で、複数成分を同定及び定量できるとともに、洗浄液の処理方法の選択により測定可能な皮膚放出成分の範囲が広がるので、固相抽出と減圧濃縮を選択さえすれば、殆どの皮膚放出成分を測定することができる。
By using the method for measuring a skin release component of the present invention, identification and quantification of a minute amount of a skin release component at a nanogram level can be reliably performed.
In addition, sampling is possible in a short time despite the extremely small amount of skin release components. Therefore, the skin release component can be repeatedly collected and measured, and it can be easily confirmed over time how the component is released.
Furthermore, according to the present invention, a single collection of skin release components can identify and quantify a plurality of components, and the range of skin release components that can be measured by selecting a treatment method for the cleaning liquid is expanded. As long as extraction and vacuum concentration are selected, most skin release components can be measured.
以下に、本発明を詳しく説明する。 The present invention is described in detail below.
まず、本発明における皮膚放出成分とは、皮膚を介して放出した成分であれば、特に限定するものではないが、本発明では、特に従来測定が困難であるとされていた極性化合物やテルペン類等の揮発性の低い化合物であっても好適に測定し得る。
なお、放出とは、皮膚を介して体外に表出していればよく、表出後に皮膚表面上に付着していたり、皮膚から離れて空気中に飛散している等を問わない。これらの例としては、経口及び／又は経皮摂取物由来の皮膚ガスや体内からの分泌物、これらの酸化物、他の皮膚付着物との反応物等が挙げられる。
上記極性化合物、テルペン類の具体的成分としては、例えば芳香族アルデヒド類、テルペン系アルコール類、ラクトン類、アルデヒド類、ケトン類、リモネン等が挙げられる。中でも芳香族アルデヒド類やテルペン系アルコール類が、効率よく測定できる点で好ましい。更に好ましくは、ゲラニオール、リナロール、シトロネロール、バニリン、ｌ−メントールであることがより効率的に測定できる点で好ましい。これらは、単独でも、複数含有されていてもよい。
First, the skin release component in the present invention is not particularly limited as long as it is a component released through the skin, but in the present invention, polar compounds and terpenes that have been particularly difficult to measure conventionally. Even a low volatility compound such as can be suitably measured.
The release is not limited as long as it is exposed outside the body through the skin, and may be attached to the skin surface after being exposed or scattered in the air away from the skin. Examples of these include skin gases derived from oral and / or transdermal ingestions, secretions from the body, oxides thereof, reactants with other skin deposits, and the like.
Specific examples of the polar compounds and terpenes include aromatic aldehydes, terpene alcohols, lactones, aldehydes, ketones, and limonene. Of these, aromatic aldehydes and terpene alcohols are preferred because they can be measured efficiently. More preferably, geraniol, linalool, citronellol, vanillin, and l -menthol are preferable because they can be measured more efficiently. These may be used alone or in combination.
では、次に、本発明の測定方法を説明する。
まず、皮膚から放出した皮膚放出成分を、捕集容器で捕集する。すなわち、皮膚放出成分には、皮膚から表出後に皮膚表面上に付着する成分や、空気中に飛散する成分が存在することは上述の通りであり、当該工程では、両方の成分を同時に捕集する。
上記捕集容器とは、皮膚放出成分を採取する皮膚の一部を覆うことができ、皮膚から放出した皮膚放出成分を捕集できるものであれば、特に限定するものではないが、捕集した皮膚放出成分を確実に捕集容器内に留めておく点で略密閉容器であることが好ましい。更に好ましくは、後述する親水性溶媒による洗浄の点から、親水性溶媒を注入できる開閉自在のコックが設けられていることが望ましい。
その材質は、親水性溶媒により捕集容器の構成成分が溶出したり、捕集した皮膚放出成分が該構成成分に吸着したりする等の問題が生じなければ、特に限定するものではない。好適には、略密閉状態に維持する観点から、柔軟性を有する材質であることが好ましい。例えば、フッ素系樹脂等が挙げられる。具体的製品例としては、ポリフッ化ビニル製のデュポン社製のテドラーバッグ等が挙げられる。
Next, the measurement method of the present invention will be described.
First, the skin release component released from the skin is collected in a collection container. That is, in the skin release component, there are components that adhere to the skin surface after being exposed from the skin and components that are scattered in the air as described above. In this process, both components are collected simultaneously. To do.
The collection container is not particularly limited as long as it can cover a part of the skin from which the skin release component is collected and can collect the skin release component released from the skin. It is preferable that the container is a substantially hermetically sealed container in that the skin release component is surely kept in the collecting container. More preferably, an openable / closable cock capable of injecting the hydrophilic solvent is provided from the viewpoint of washing with the hydrophilic solvent described later.
The material is not particularly limited as long as it does not cause problems such as elution of the components of the collection container by the hydrophilic solvent or adsorption of the collected skin release components to the components. Preferably, the material is flexible from the viewpoint of maintaining a substantially sealed state. For example, a fluorine-type resin etc. are mentioned. Specific examples of the product include a Tedlar bag made by DuPont made of polyvinyl fluoride.
上記捕集容器を用いて、皮膚放出成分を捕集する方法は、特に限定されるものではないが、例えば、捕集容器で皮膚の一部を覆い、約３０分間略密閉状態を維持して、皮膚から飛散する皮膚放出成分を、捕集容器内に留める等が挙げられる。皮膚から表出後に皮膚表面上に付着している成分の捕集は、例えば、捕集容器を覆っている部位を振動させる等で皮膚表面から捕集容器に脱落させればよい。なお、振動等でも皮膚表面から脱落しない皮膚放出成分は、後の洗浄工程で直接洗浄すればよい。この時、より高い密閉性を得るために、捕集容器の開口縁部をパラフィルム等で密閉させる等、捕集容器の開口部における気相の出入りを完全に閉鎖した状態にすることが好適である。 The method of collecting the skin release component using the collection container is not particularly limited. For example, a part of the skin is covered with the collection container and kept in a substantially sealed state for about 30 minutes. For example, the skin release component scattered from the skin is retained in the collection container. The component adhering to the skin surface after being exposed from the skin may be removed from the skin surface to the collecting container by, for example, vibrating a portion covering the collecting container. In addition, what is necessary is just to wash | clean the skin discharge | release component which does not fall out from the skin surface by vibration etc. directly at a later washing process. At this time, in order to obtain higher sealing performance, it is preferable to completely close the entrance and exit of the gas phase at the opening of the collection container, such as sealing the opening edge of the collection container with parafilm or the like. It is.
皮膚放出成分を捕集する皮膚の部位は、限定されるものではないが、好ましくは手首から指の末端までの手のひら部分、足のうら、わき等の汗腺の集中している部分、更に好ましくは手のひら部分は、効率良く皮膚放出成分を捕集し得る点で望ましい。 The part of the skin that collects the skin release component is not limited, but preferably the palm part from the wrist to the end of the finger, the part where the sweat glands such as the back of the foot and the arm are concentrated, more preferably The palm portion is desirable because it can efficiently collect the skin-released component.
次に、上記のようにして皮膚放出成分を捕集した捕集容器内と、皮膚放出成分を捕集した部分の皮膚表面を、親水性溶媒で洗浄する。
上記親水性溶媒とは、例えば、水、エタノール、イソプロパノール、アセトン、ブタノール、酢酸エチル、アセトニトリル、ジエチルエーテル等が挙げられる。この中でも、エタノールは、皮膚放出成分を確実に溶解する一方、体に悪影響を与えない点で好ましい。また、上記親水性溶媒は、単独でも、複数組合せて用いてもよい。
また、上記親水性溶媒を組合せて水溶液として用いる場合の濃度は、溶媒の種類や、測定する皮膚放出成分の特性にあわせて調整すればよい。例えば、エタノール水溶液を用い、後の工程で固相抽出を実施する場合は、１〜５０容量％エタノール水溶液を用いることが、効率よく固相抽出を行えるので、皮膚放出成分の回収を高められる点で好適である。また、エタノール水溶液を用い、後の工程で減圧濃縮を実施する場合は、８０〜１００容量％エタノール水溶液を用いることが、短時間で減圧濃縮を実施できる点及び皮膚放出成分の留去が抑えられる点で好適である。
Next, the inside of the collection container in which the skin release component is collected as described above and the skin surface of the part in which the skin release component is collected are washed with a hydrophilic solvent.
Examples of the hydrophilic solvent include water, ethanol, isopropanol, acetone, butanol, ethyl acetate, acetonitrile, diethyl ether and the like. Among these, ethanol is preferable in that it dissolves the skin-releasing component reliably and does not adversely affect the body. Moreover, the said hydrophilic solvent may be used individually or in combination with multiple.
Moreover, what is necessary is just to adjust the density | concentration at the time of using it as an aqueous solution combining the said hydrophilic solvent according to the kind of solvent, and the characteristic of the skin release component to measure. For example, when solid-phase extraction is carried out in a later step using an aqueous ethanol solution, the use of a 1 to 50% by volume ethanol aqueous solution can efficiently perform the solid-phase extraction, thereby improving the recovery of the skin-released components. It is suitable. In addition, when an ethanol aqueous solution is used and vacuum concentration is performed in a later step, the use of an 80 to 100% by volume ethanol aqueous solution can suppress the vacuum concentration in a short time and the evaporation of the skin release component can be suppressed. This is preferable in terms of points.
上記親水性溶媒の総使用量は、後の濃縮工程において支障がなく、洗浄時に皮膚放出成
分を１ｎｇ以上溶解し得る量とすることが好適である。具体的には、３〜８ｍｌ程度が好ましい。なお、上記総使用量とは、捕集容器と皮膚表面の洗浄を別々に行った際に両者で用いた溶媒の合計量を指し、それぞれの洗浄に使用する量は問わない。
It is preferable that the total amount of the hydrophilic solvent used is an amount that does not hinder the subsequent concentration step and can dissolve 1 ng or more of the skin release component during washing. Specifically, about 3 to 8 ml is preferable. In addition, the said total usage-amount refers to the total amount of the solvent used by both when the collection container and the skin surface were wash | cleaned separately, and the quantity used for each washing | cleaning is not ask | required.
上記親水性溶媒で洗浄する方法は、特に限定されるものではないが、例えば、次のように行えばよい。
すなわち、まず、捕集終了後、捕集容器のコックを開いて親水性溶媒を注入し、コックを閉じた後、該容器をまんべんなく振る等することにより、容器内で飛散していたり、容器内壁に付着している皮膚放出成分を洗浄する。
次に、捕集容器を皮膚表面から外し、該捕集容器が覆っていた皮膚表面に親水性溶媒を適当量噴霧し、該皮膚表面に付着している皮膚放出成分を洗浄する。
その後、それぞれで得られた洗浄液を回収し、合わせる。
なお、上記のように、２段階に分けずに、捕集容器内で容器と皮膚表面の両者の洗浄を行ってもよい。
The method of washing with the hydrophilic solvent is not particularly limited, and may be performed as follows, for example.
That is, first, after the collection is completed, the cock of the collection container is opened, a hydrophilic solvent is injected, the cock is closed, and then the container is shaken evenly, etc. Wash away skin-release components adhering to.
Next, the collection container is removed from the skin surface, and an appropriate amount of a hydrophilic solvent is sprayed on the skin surface covered by the collection container to wash the skin-released component adhering to the skin surface.
Thereafter, the cleaning liquid obtained in each is collected and combined.
As described above, both the container and the skin surface may be washed in the collection container without dividing into two stages.
次に、上記のようにして得られた洗浄液を、固相抽出あるいは減圧濃縮で濃縮する。
上記濃縮方法のうち、固相抽出と減圧濃縮のどちらを用いるかは、測定する皮膚放出成分の性質に合わせて選択すればよい。すなわち、固相抽出で固相に吸着し難い皮膚放出成分の場合は、減圧濃縮を用いる等適宜選択すればよい。例えば、ゲラニオール、リナロール、シトロネロール、ｌ−メントール等は固相抽出が適しており、バニリン等の比較的水溶性の高い化合物は減圧濃縮が適している。
なお、必要であれば、洗浄液を分割し、固相抽出と減圧濃縮のそれぞれを行ってもよい。
Next, the washing liquid obtained as described above is concentrated by solid phase extraction or vacuum concentration.
Of the above-mentioned concentration methods, whether to use solid phase extraction or vacuum concentration may be selected according to the properties of the skin release component to be measured. That is, in the case of a skin release component that is difficult to be adsorbed to the solid phase by solid phase extraction, it may be appropriately selected such as using reduced pressure concentration. For example, geraniol, linalool, citronellol, l -menthol and the like are suitable for solid phase extraction, and relatively highly water-soluble compounds such as vanillin are suitable for concentration under reduced pressure.
If necessary, the washing solution may be divided and subjected to solid phase extraction and vacuum concentration.
上記固相抽出とは、化学結合型シリカゲル・ポーラスポリマー・アルミナ・活性炭等の固定相（固相）を用いながら、複雑な組成を示す試料中から特定の目的成分のみを選択的に抽出し、分離・精製を行う手法のことである。
本発明で使用する固相は特に限定しないが、後工程のガスクロマトグラフ質量分析で、熱脱着式ガスクロマトグラフを用いる場合には、特にポリジメチルシロキサン等が好ましい。製品例としては、例えばGerstel社製の固相抽出用攪拌子Twister等が挙げられる。
The above solid-phase extraction is to selectively extract only a specific target component from a sample having a complicated composition while using a stationary phase (solid phase) such as chemically bonded silica gel, porous polymer, alumina, activated carbon, It is a technique for separation and purification.
The solid phase used in the present invention is not particularly limited, but polydimethylsiloxane and the like are particularly preferable when a thermal desorption gas chromatograph is used in the gas chromatograph mass analysis in the subsequent step. Examples of products include a solid phase extraction stirrer Twister manufactured by Gerstel.
上記固相抽出の条件は、上記洗浄液に蒸留水と塩化ナトリウムを加え適当量にした後、固相抽出用攪拌子で、約１時間攪拌して、攪拌子表面に洗浄液中に含まれる皮膚放出成分を吸着させればよい。また、固相抽出の回収率は２０％以上であることが、定量性の点で望ましい。 The conditions for the solid phase extraction are as follows. After adding distilled water and sodium chloride to the washing solution to make an appropriate amount, stir with a solid phase extraction stirrer for about 1 hour, and release the skin contained in the washing solution on the stirrer surface. What is necessary is just to adsorb | suck a component. Further, the recovery rate of solid phase extraction is preferably 20% or more from the viewpoint of quantitativeness.
また、上記減圧濃縮とは、減圧下で、皮膚放出成分よりも沸点の低い溶媒を留去することである。
その減圧条件は、目的とする皮膚放出成分及び溶媒の沸点を考慮して決定すればよい。好ましくは、減圧時の試料の品温が常温（１０〜３０℃）程度であるのが、効率性の点からよい。また、濃縮倍率は、好適には３倍〜５倍であることが、定量性の点で望ましい。
Moreover, the said vacuum concentration is distilling off the solvent whose boiling point is lower than a skin release component under reduced pressure.
The decompression condition may be determined in consideration of the target skin release component and the boiling point of the solvent. Preferably, the product temperature of the sample during decompression is about room temperature (10 to 30 ° C.) from the viewpoint of efficiency. The concentration factor is preferably 3 to 5 times from the viewpoint of quantitativeness.
次に、上記のようにして得られた固相又は濃縮液を、ガスクロマトグラフ質量分析計で分析する。
その分析方法は、特に限定するものではないが、例えば固相を使用する場合は、熱脱着式ガスクロマトグラフを用いてもよく、固相を再度適当な溶媒中に戻して濃縮液を得た後、ガスクロマトグラフにかけてもよい。好適には、熱脱着式ガスクロマトグラフを用いることが、検出感度が高くなる点でよい。
Next, the solid phase or concentrated liquid obtained as described above is analyzed with a gas chromatograph mass spectrometer.
The analysis method is not particularly limited. For example, when a solid phase is used, a thermal desorption gas chromatograph may be used. After returning the solid phase to an appropriate solvent again to obtain a concentrated solution. Alternatively, a gas chromatograph may be applied. Preferably, use of a thermal desorption type gas chromatograph is sufficient in terms of high detection sensitivity.
ガスクロマトグラフ質量分析計における測定条件は、測定する皮膚放出成分の科学的性
質及び、装置の特性を考慮し、適宜設定すればよい。
The measurement conditions in the gas chromatograph mass spectrometer may be appropriately set in consideration of the scientific properties of the skin release component to be measured and the characteristics of the apparatus.
以上の測定方法により、皮膚放出成分を測定することができるが、例えば、実際に摂取した成分が皮膚表面から放出しているかの測定を行う場合には、測定する成分を摂取することで皮膚表面から放出させればよい。すなわち、皮膚放出成分を含む剤又は飲食品等を、経口及び／又は経皮摂取等により、腸管、口腔粘膜、皮膚等から体内へ吸収すると、該成分は皮膚上の汗腺または細胞間隙より、汗や水蒸気等と共に表出するというメカニズムを利用し、測定する成分を摂取し、放出させるのである。 The skin release component can be measured by the above measurement method. For example, when measuring whether the actually ingested component is released from the skin surface, the skin surface can be obtained by ingesting the component to be measured. Can be released from That is, when an agent or food / drink containing a skin-releasing component is absorbed into the body from the intestinal tract, oral mucosa, skin, etc. by oral and / or transdermal ingestion, the component is absorbed from the sweat glands or cell gaps on the skin. Ingest and release the components to be measured using the mechanism of expression with water and water vapor.
上記飲食品としては、具体的には、例えば、飲料（スープ、コーヒー、茶類、ジュース、ココア、酒類等）や、冷菓や、菓子類（錠菓、ハードキャンディ、ソフトキャンディ、グミ、ゼリー、チューインガム、チョコレート等）や、ベーカリー食品（パン、クッキー等）や、麺類を始めとする澱粉系食品や、粉末食品や、健康食品等が挙げられる。この中でもチューインガム、ソフトキャンディは、口腔内の滞留時間が長いため、粘膜から、測定する成分が効率よく吸収される点、及び該成分の放出持続性の点で好適である。
なお、チューインガムの場合は、各種成分がガムベースに吸着されやすい傾向にあるので、上記成分を閉じ込めたカプセルを使用することが好ましい。カプセルが使用できない場合には、チューインガム全体重量中ガムベース含量を好ましくは４０重量％以下、更に好ましくは２０重量％以下に設定した外殻層を設けた形態にし、該成分が外殻層より口腔粘膜から吸収されやすくする設計とすることが好ましい。
Specific examples of the food and drink include beverages (soups, coffee, teas, juices, cocoa, alcoholic beverages), frozen desserts, confectionery (tablets, hard candy, soft candy, gummi, jelly, Chewing gum, chocolate, etc.), bakery foods (bread, cookies, etc.), starch-based foods such as noodles, powdered foods, health foods and the like. Among these, chewing gum and soft candy are preferable in terms of efficient absorption of the component to be measured from the mucous membrane and long-term release of the component because the residence time in the oral cavity is long.
In the case of chewing gum, since various components tend to be easily adsorbed to the gum base, it is preferable to use a capsule containing the above components. When capsules cannot be used, an outer shell layer in which the gum base content in the total weight of the chewing gum is preferably set to 40% by weight or less, more preferably 20% by weight or less is provided. It is preferable to design it so that it can be easily absorbed.
なお、本発明における皮膚放出成分の測定には、目的とする成分が確実に放出するよう、強い香料を含む飲食品を摂取するのは控え、更に白湯等を摂取したり、軽い運動を実施する等して、発汗が促進される状態とし、成分が皮膚から放出されやすい状態にするのが好ましい。 In addition, in the measurement of the skin release component in the present invention, in order to surely release the target component, refrain from ingesting foods and drinks containing strong fragrances, further ingest white water, etc., or carry out light exercise. For example, it is preferable that sweating is promoted and components are easily released from the skin.
以下に、本発明の実施例を用いて例示する。 In the following, examples of the present invention are used to illustrate.
まず、極性化合物及びテルペン類を経口摂取した後の皮膚放出について、最適な測定方法を試験した。
≪実施例１〜４及び比較例１≫
＜チューインガムの調製＞
皮膚放出成分検出用に、ローズオットーの精油に含まれるゲラニオール、リナロール、シトロネロール、及び、食品香料で多用されるｌ−メントール、バニリンの合計５成分について、これら単独もしくは複数含有するチューインガムを、表１の組成に従い、原料を加熱混合し、均質化した後、縦２０ｍｍ×横１３．５ｍｍ×厚み1０．５ｍｍ、１粒当り３．１ｇとなるように成型した。
First, the optimal measurement method was tested for skin release after ingesting polar compounds and terpenes.
<< Examples 1-4 and Comparative Example 1 >>
<Preparation of chewing gum>
Table 1 shows chewing gum containing a single or a plurality of chewing gums for a total of five components, geraniol, linalool, citronellol, and l -menthol and vanillin, which are frequently used in food fragrances, for detection of skin release components. According to the composition, the raw materials were heated and mixed and homogenized, and then molded so as to have a length of 20 mm × width of 13.5 mm × thickness of 10.5 mm and 3.1 g per grain.
＜皮膚放出成分の測定＞
上記チューインガムを用いて、下記のように測定を実施した。
<Measurement of skin release components>
Using the chewing gum, the measurement was performed as follows.
≪ブランクの皮膚放出成分の測定≫
＜被験者の条件＞
被験者は、測定前日の入浴後より、香粧品を使用せず、また、測定試験開始前２時間程度は、香料の強い飲食品を喫食しないようにした。
≪Measurement of blank skin release component≫
<Subject's conditions>
The subject did not use cosmetics after bathing on the day before the measurement, and did not eat foods and drinks with strong perfume for about 2 hours before the start of the measurement test.
＜ブランクの皮膚放出成分の捕集及び洗浄液の回収＞
被験者の手を無香料の石鹸で良く洗浄し、良く濯いだのち、清浄な状態で自然乾燥させ、フッ素系樹脂製のテドラーバッグを左の手首より先全体に装着し、テドラーバッグの開口縁部を手首部分でパラフィルムで密閉し、３０分間そのままで保持した。
３０分後、２５容量％エタノールをテドラーバッグのコックから注入し、テドラーバッグ内及び左の手首より先を洗浄した。次に、テドラーバッグから左手を取出し、左の手首より先の全体に２５容量％エタノールを噴霧し、洗浄して、先の洗浄液と合わせた。結果、洗浄液を約２．５ｍｌ回収した。
<Collection of blank skin release components and recovery of cleaning solution>
Wash the subject's hand thoroughly with unscented soap, rinse well, let it dry naturally in a clean state, wear a fluororesin-made Tedlar bag over the left wrist, and cover the opening edge of the Tedlar bag The wrist was sealed with parafilm and held for 30 minutes.
After 30 minutes, 25 vol% ethanol was injected from the cock of the Tedlar bag, and the inside of the Tedlar bag and the tip of the left wrist were washed. Next, the left hand was taken out from the Tedlar bag, and 25 vol% ethanol was sprayed over the entire area from the left wrist, washed, and combined with the previous washing solution. As a result, about 2.5 ml of washing liquid was collected.
＜固相抽出による熱脱着式ガスクロマトグラフ質量分析＞
次いで、得られた洗浄液を２５容量％エタノール水溶液で、５ｍｌにメスアップした。このうち、２ｍｌを取り、蒸留水８ｍｌ、塩化ナトリウム２．０ｇを、バイアル瓶に入れ、固相としてポリジメチルシロキサンを用いた固相抽出用攪拌子（Gerstel社製Twister）を入れ、１時間攪拌した。この時のゲラニオール回収率は５０％であった。
<Thermal desorption gas chromatograph mass spectrometry by solid phase extraction>
Next, the obtained cleaning solution was diluted to 5 ml with a 25 vol% ethanol aqueous solution. Of these, 2 ml was taken, 8 ml of distilled water and 2.0 g of sodium chloride were placed in a vial, and a stirrer for solid phase extraction (Twister manufactured by Gerstel) using polydimethylsiloxane as the solid phase was added and stirred for 1 hour. did. The geraniol recovery rate at this time was 50%.
次いで、TDS-GC/MS（熱脱着式ガスクロマトグラフ質量分析計）測定を行った。測定条件は以下の通りであった。
すなわち、測定装置は、GERSTEL TDS2 AGILENT6890N 5973N、使用したカラムは、DB
-５ｍｓ、０．２５ｍｍ×３０ｍ、オーブン温度は、５０℃（１分）→５℃／分→１８０℃→２０℃／分→３００℃（Hold）、CIS条件は、−120℃→340分（３分）、TDS条件が２０℃→６０℃／分→３００℃（３分）であった。
Subsequently, TDS-GC / MS (thermal desorption type gas chromatograph mass spectrometer) measurement was performed. The measurement conditions were as follows.
That is, the measuring device is GERSTEL TDS2 AGILENT6890N 5973N, and the column used is DB
-5ms, 0.25mm x 30m, oven temperature is 50 ° C (1 minute) → 5 ° C / minute → 180 ° C → 20 ° C / minute → 300 ° C (Hold), CIS condition is -120 ° C → 340 minutes ( 3 minutes) and TDS conditions were 20 ° C. → 60 ° C./min→300° C. (3 minutes).
＜減圧濃縮によるガスクロマトグラフ質量分析＞
上記と同様にしてブランクの皮膚放出成分を捕集してから、洗浄液を回収した。なお、今回の洗浄液は、１００容量％エタノールを用いた。
得られた洗浄液３ｍｌをミニバイアル瓶に移し、品温２５〜３０℃で減圧下で約２時間１ｍｌになるまで濃縮した。
次いで、GC/MS（ガスクロマトグラフ質量分析計）測定を行った。測定条件は以下の通りであった。
すなわち、測定装置はGERSTEL TDS2 AGILENT6890N 5973Nで、使用したカラムはDB-５ｍｓ、０．２５ｍｍ×３０ｍ、オーブン温度は５０℃（１分）→５℃／分→１８０℃→２０℃／分→３００℃（Hold）、GC注入口温度が３４０℃であった。
<Gas chromatography mass spectrometry by concentration under reduced pressure>
The blank skin release component was collected in the same manner as described above, and then the washing liquid was collected. In addition, 100 volume% ethanol was used for the washing | cleaning liquid this time.
3 ml of the obtained washing solution was transferred to a mini vial and concentrated to 1 ml under reduced pressure at a product temperature of 25-30 ° C. for about 2 hours.
Subsequently, GC / MS (gas chromatograph mass spectrometer) measurement was performed. The measurement conditions were as follows.
That is, the measuring device is GERSTEL TDS2 AGILENT6890N 5973N, the column used is DB-5ms, 0.25mm × 30m, oven temperature is 50 ℃ (1 minute) → 5 ℃ / minute → 180 ℃ → 20 ℃ / minute → 300 ℃ (Hold), the GC inlet temperature was 340 ° C.
≪チューインガム摂取後の皮膚放出成分の測定≫
上記ブランクを測定後、被験者は、直ちに実施例１〜４及び比較例１のチューインガムを、表１に記載の皮膚放出成分摂取量となるよう喫食した。なお、被験者はサンプリング終了まで白湯しか喫食しなかった。
摂取後１時間目に、ブランクの測定と同様に、皮膚放出成分の測定を行った。各皮膚放出成分における測定方法は、表１に記載の通りである。上記分析結果を、表１にあわせて示す。
≪Measurement of skin release components after chewing gum intake≫
After measuring the above blank, the subject immediately ate the chewing gums of Examples 1 to 4 and Comparative Example 1 so that the amounts of the skin release components in Table 1 were obtained. The subject ate only white water until the end of sampling.
At 1 hour after ingestion, the skin release component was measured in the same manner as the blank measurement. The measurement method for each skin release component is as shown in Table 1. The analysis results are shown in Table 1.
測定の結果、実施例１〜４は、ピークの増加が認められた。この中でも実施例１〜３は、その増加が明らかで、測定方法が適していた。一方実施例４では、実施例３と比較してピークの増加が分かり難くかったものの、ある程度は増加が見られた。すなわち、バニリンの場合は、実施例４の固相抽出よりも実施例３の減圧濃縮による濃縮方法の方が適していた。
これに対し、比較例１では、固相抽出または減圧濃縮を行わなかったため、検出限界外となり、検出できなかった。
As a result of measurement, in Examples 1 to 4, an increase in peak was observed. Among these, Examples 1 to 3 showed an obvious increase, and the measurement method was suitable. On the other hand, in Example 4, although it was difficult to understand the increase in peak as compared with Example 3, an increase was observed to some extent. That is, in the case of vanillin, the concentration method by vacuum concentration in Example 3 was more suitable than the solid phase extraction in Example 4.
On the other hand, in Comparative Example 1, since solid phase extraction or vacuum concentration was not performed, it was outside the detection limit and could not be detected.
以上の結果より、本発明の測定方法が有効であることが確認できたので、より食品として適当な風味・食感に調整したチューインガム及びソフトキャンディを体臭改善食品として調製し、摂取した皮膚放出成分が検出されるかどうか確認した。検出する成分は、表１でピークの増加が認められた皮膚放出成分のうち、食品として良好な風味であるゲラニオールとバニリンで行った。 From the above results, it can be confirmed that the measurement method of the present invention is effective. Thus, chewing gum and soft candy adjusted to a more appropriate flavor and texture as foods are prepared as body odor-improving foods and ingested skin release components To see if is detected. The components to be detected were geraniol and vanillin, which are good flavors as foods, among the skin-released components in which an increase in peak was observed in Table 1.
≪実施例５≫
＜ローズ風味ゲラニオール含有チューインガムの調製＞
表２の組成に従い、ローズ風味のゲラニオール含有チューインガムを調製した。すなわち、原料を加熱混合し、均質化した後、縦２０ｍｍ×横１３．５ｍｍ×厚み１０．５ｍｍ、１粒当り３．１ｇとなるよう成型することによりチューインガムを得た。
Example 5
<Preparation of chewing gum containing rose-flavored geraniol>
According to the composition of Table 2, a rose-flavored geraniol-containing chewing gum was prepared. That is, after the raw materials were heated and mixed and homogenized, the chewing gum was obtained by molding so as to be 20 mm long × 13.5 mm wide × 10.5 mm thick and 3.1 g per grain.
≪実施例６≫
＜ローズ風味バニリン含有ソフトキャンディの調製＞
表２の組成に従い、ローズ風味のバニリン含有ソフトキャンディを調製した。すなわち、各原料を４０℃程度で混合し、エクストルーダーで更に均質化したのち、スタンピング成型して、１粒２．６ｇの球状ソフトキャンディを得た。
Example 6
<Preparation of rose-flavored vanillin-containing soft candy>
According to the composition of Table 2, rose-flavored vanillin-containing soft candy was prepared. That is, each raw material was mixed at about 40 ° C., further homogenized with an extruder, and then stamped to obtain 2.6 g of a spherical soft candy.
＜皮膚放出成分の測定＞
実施例１と同様に被験者のブランクの皮膚放出成分を測定した後、被験者は直ちに表２に記載のゲラニオール摂取量となるように実施例５のチューインガムを摂取した。その後、摂取後１時間ごとに、５時間まで略連続で、実施例１と同様にゲラニオール放出量の測定を行った。その結果を図１に示す。
<Measurement of skin release components>
After measuring the skin release component of the subject's blank as in Example 1, the subject immediately ingested the chewing gum of Example 5 so that the geraniol intake shown in Table 2 was obtained. Thereafter, the amount of geraniol released was measured in the same manner as in Example 1 approximately every 5 hours after ingestion until approximately 5 hours. The result is shown in FIG.
また、実施例６についても、実施例３と同様に被験者のブランクの皮膚放出成分を測定した後、被験者は直ちに表２に記載のバニリン摂取量となるように実施例６のソフトキャンディを摂取した。その後、摂取３時間後でのバニリン放出量を測定した結果を図２に示す。 Moreover, also about Example 6, after measuring the skin discharge | release component of a test subject's blank similarly to Example 3, a test subject ingested the soft candy of Example 6 so that it might become vanillin intake of Table 2 immediately. . Then, the result of having measured the amount of vanillin released 3 hours after ingestion is shown in FIG.
図１によると、実施例５のチューインガムを摂取すると、摂取後１時間から５時間まで、ゲラニオールが放出された。特に摂取後１〜２時間での放出が顕著に見られた。また、摂取後３〜５時間でも、摂取前（０時間）の放出量より、ピークの増加が見られる傾向にあり、放出が継続的に行われていた。 According to FIG. 1, when the chewing gum of Example 5 was ingested, geraniol was released from 1 to 5 hours after ingestion. In particular, the release was noticeable 1 to 2 hours after ingestion. In addition, even after 3 to 5 hours after ingestion, there was a tendency for a peak to increase from the amount released before ingestion (0 hour), and the release was continuously performed.
図２によると、実施例６のソフトキャンディを摂取すると、摂取３時間後には、ブランク（摂取前）と比較して２倍量以上のバニリンの放出が有意に見られた。 According to FIG. 2, when the soft candy of Example 6 was ingested, the release of vanillin more than twice as much as that of the blank (before ingestion) was significantly observed after 3 hours of ingestion.
以上のように、ゲラニオール及びバニリンの放出は測定できたのであるが、上記実施例５及び６について、実際にゲラニオール、バニリンが放出されていることを官能的に体感できるかについても、併せて確認した。 As described above, the release of geraniol and vanillin could be measured, but it was also confirmed whether or not the geraniol and vanillin were actually released with respect to Examples 5 and 6 above. did.
≪体感試験≫
＜ブランクの体感試験＞
４名の被験者は、測定前日の入浴後より、香粧品を使用せず、測定試験開始前２時間は、香料の強い飲食品を喫食せず、水または白湯のみ摂取した。
次に、被験者らは、無香料の石鹸で、手を良く洗浄し、上水道水で良く濯いだのち、清浄な状態で３０分程度自然乾燥させた。
その後、専門パネラー２名が、手表面の体臭を確認し、ゲラニオール、バニリンが放出していないことを確認した。
≪Experience test≫
<Blank experience test>
Four test subjects did not use cosmetics after taking a bath on the day before the measurement, and took only water or white hot water for 2 hours before the start of the measurement test without eating a fragrant food or drink.
Next, the subjects thoroughly washed their hands with unscented soap, rinsed well with tap water, and then naturally dried in a clean state for about 30 minutes.
After that, two professional panelists confirmed the body odor of the hand surface and confirmed that geraniol and vanillin were not released.
＜体臭改善食品の摂取＞
被験者らは、実施例５についてはゲラニオール摂取量が０．８ｍｇとなるよう、また実施例６についてはバニリン摂取量が５２ｍｇとなるように摂取した。
<Ingestion of body odor improving food>
The subjects ingested the geraniol intake of 0.8 mg for Example 5 and the vanillin intake of 52 mg for Example 6.
＜実施例及びブランクの体感試験＞
摂取１時間後に、被験者らは、手を無香料の石鹸で良く洗浄し、上水道水で良く濯いだのち、清浄な状態で３０分程度自然乾燥させた。
その後、専門パネラー２名が、被験者が何れのチューインガム、ソフトキャンディを食べたか分からない（ブラインド）状態で、各被験者の手のひらの匂いを確認し、＋＋；明らかに香りがする、＋；香りがする、−；ブランクと変化なし、の３点で評価した。
<Example and blank body sensation test>
One hour after ingestion, the subjects thoroughly washed their hands with unscented soap, rinsed well with tap water, and then naturally dried in a clean state for about 30 minutes.
After that, two expert panelists confirmed the smell of the palm of each subject without knowing which chewing gum or soft candy the subject had eaten (blind), and ++; clearly smelled, +; smelled ,-; Evaluated with three points of blank and no change.
この結果、４名の被験者のうち、実施例５では、バラ様の香りが漂っているとして＋＋を選択されたのが２名、特定は出来ないが甘い芳香が漂っているとして＋を選択されたのが１名、−が１名であった。
また、実施例６では、バラ様の香りが漂っているとして＋＋を選択されたのが１名、特定は出来ないが甘い芳香が漂っているとして＋とされたのが２名、−が１名であった。
以上より、実施例５及び６では、皮膚放出成分の放出が測定できる摂取量またはそれ以下の摂取量で、芳香が漂っていると体感できた。従って、本発明の測定方法と体感試験との相関性が認められた。
As a result, among the four test subjects, in Example 5, ++ was selected as having a rose-like scent, and two were selected, and + was selected as having a sweet fragrance that could not be specified. There was one person and-was one person.
Further, in Example 6, one person was selected as ++ because there was a rose-like scent, 2 people were selected as + when there was a sweet fragrance that could not be specified, and-was 1 It was a name.
From the above, in Examples 5 and 6, it was possible to feel that the fragrance was drifting at an intake that can measure the release of the skin release component or an intake that is less than that. Therefore, the correlation between the measurement method of the present invention and the sensory test was recognized.
Claims (6)
（１）皮膚から放出した皮膚放出成分を、捕集容器で捕集する工程
（２）上記捕集容器内及び皮膚表面を、親水性溶媒で洗浄する工程
（３）上記（２）で得られた洗浄液を、固相抽出する工程
（４）上記（３）で得られた固相を、ガスクロマトグラフ質量分析する工程 A method for measuring a skin release component released from the skin, wherein the skin release component is an essential oil component or a food fragrance component, and the following (1) to (4) are sequentially performed: .
(1) Step of collecting the skin release component released from the skin with a collection container (2) Step of washing the inside of the collection container and the skin surface with a hydrophilic solvent (3) Obtained by (2) above (4) Step of gas chromatograph mass spectrometry of the solid phase obtained in (3) above
（１）皮膚から放出した皮膚放出成分を、捕集容器で捕集する工程
（２）上記捕集容器内及び皮膚表面を、親水性溶媒で洗浄する工程
（３）上記（２）で得られた洗浄液を、減圧濃縮する工程
（４）上記（３）で得られた濃縮液を、ガスクロマトグラフ質量分析する工程 A method for measuring a skin release component released from the skin, wherein the skin release component is an essential oil component or a food fragrance component, and the following (1) to (4) are sequentially performed: .
(1) Step of collecting the skin release component released from the skin with a collection container (2) Step of washing the inside of the collection container and the skin surface with a hydrophilic solvent (3) Obtained by (2) above (4) Step of gas chromatograph mass spectrometry of the concentrated solution obtained in (3) above
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