JP2010012076A - Method of measuring dermal moisture quantity - Google Patents
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Abstract
Description
本発明は、皮膚水分量の測定方法、およびそれを用いた皮膚内部の水分分布測定方法に関する。 The present invention relates to a method for measuring skin moisture content, and a method for measuring moisture distribution in the skin using the same.
洗顔、入浴、化粧水塗布等の日々の行動により、皮膚内部の水分は大きく変動する。また皮膚の健康状態や、温度、湿度、風量等の生活環境によっても、皮膚内部の水分は変動する。そのため、皮膚内部の水分量を測定し、水の動態を把握することは、より優れた医薬品・化粧品・身体洗浄剤・入浴剤・食器洗浄剤・各種洗浄剤・空調設備機器・機能性衣料・機能性寝具・エステティック機器・エステティック手法、スキンケア技術等の開発を行う上で非常に有用である。 Due to daily actions such as face washing, bathing, and applying lotion, moisture inside the skin varies greatly. The moisture inside the skin also varies depending on the health condition of the skin and the living environment such as temperature, humidity, and air volume. Therefore, measuring the amount of water in the skin and grasping the dynamics of water is better than pharmaceuticals, cosmetics, body cleaners, bathing agents, dishwashing agents, various cleaning agents, air conditioning equipment, functional clothing, It is very useful for developing functional bedding, esthetic equipment, esthetic techniques, skin care technology, etc.
皮膚内部の水分量測定方法として、例えば非特許文献１には、ラマン分光法による皮膚内部の水分量測定方法が記載されている。しかし、従来の方法では、各種処理により皮膚内部に浸透した水分と、もともと皮膚内部に存在していた水分とを区別できない。そのため、皮膚内部での水の動態の解析には限界があった。 As a method for measuring the amount of moisture in the skin, for example, Non-Patent Document 1 describes a method for measuring the amount of moisture in the skin by Raman spectroscopy. However, the conventional method cannot distinguish between moisture that has penetrated into the skin by various treatments and moisture that originally existed in the skin. Therefore, there was a limit to the analysis of water dynamics inside the skin.
本発明は、ラマン分光測定法により、皮膚内部の水の動態を簡便にかつ正確に解析する方法を提供することを目的とする。
さらに、本発明は、皮膚内部の水分分布測定方法を提供することを目的とする。
An object of the present invention is to provide a method for simply and accurately analyzing the dynamics of water inside the skin by Raman spectroscopy.
Furthermore, an object of the present invention is to provide a method for measuring the moisture distribution inside the skin.
本発明者等は上記課題を解決するため、鋭意検討を行った。その結果、重水を皮膚に接触、浸透させることで、外部から浸入した水（重水）と、もともと皮膚内部に存在した水（軽水）を別々に定量でき、皮膚内部での水の動態の解析することができることを見出した。本発明はこの知見に基づきなされるに至ったものである。 In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. As a result, by contacting and penetrating heavy water into the skin, water that has entered from the outside (heavy water) and water originally present in the skin (light water) can be separately quantified, and the dynamics of water inside the skin are analyzed. I found that I can do it. The present invention has been made based on this finding.
本発明は、皮膚内部の水分量の測定方法であって、重水を浸透させた皮膚のラマンスペクトルを測定し、得られたラマンスペクトル中の重水の信号強度より測定する皮膚水分量の測定方法に関する。 The present invention relates to a method for measuring the amount of moisture in the skin, and relates to a method for measuring the amount of moisture in the skin by measuring the Raman spectrum of skin infiltrated with heavy water and measuring the signal intensity of heavy water in the obtained Raman spectrum. .
また、本発明は、前記水分量の測定方法により、皮膚のラマンスペクトルを表面からの深さ方向及び／または水平方向に変えて測定し、皮膚内部の水分分布を測定する方法に関する。 In addition, the present invention relates to a method for measuring the moisture distribution inside the skin by measuring the Raman spectrum of the skin in the depth direction and / or the horizontal direction from the surface by the method for measuring water content.
本発明によれば、ラマン分光測定法により、皮膚内部の水の動態を簡便にかつ正確に解析する方法を提供することができる。
さらに、本発明によれば、皮膚内部の水分分布測定方法を提供することができる。
ADVANTAGE OF THE INVENTION According to this invention, the method of analyzing the dynamics of the water inside skin easily and correctly can be provided by the Raman spectroscopic measurement method.
Furthermore, according to the present invention, it is possible to provide a method for measuring the moisture distribution inside the skin.
以下、本発明を詳細に説明する。
本発明の皮膚水分量の測定方法では、皮膚のラマンスペクトルを測定する。ラマンスペクトルの測定について、皮膚を採取してラマンスペクトルを測定する方法（侵襲法）や、皮膚のラマンスペクトルを直接測定する方法（非侵襲法）等が挙げられるが、直接肌の状態を測定できる非侵襲法が好ましい。ラマンスペクトルの測定においては、一般的な方法を使用することができ、特に、顕微ラマン法による測定が好ましい。
ラマンスペクトル測定装置についても特に制限はなく、通常の装置を用いることができる。水分量の測定箇所が非常に狭い場合、測定部位が非常に小さな場合でもラマンスペクトルを得ることができる共焦点光学系を有する測定装置を用いることが好ましい。
Hereinafter, the present invention will be described in detail.
In the skin moisture content measuring method of the present invention, the Raman spectrum of the skin is measured. As for the measurement of Raman spectrum, there are a method of collecting the Raman spectrum and measuring the Raman spectrum (invasive method) and a method of directly measuring the Raman spectrum of the skin (non-invasive method). Non-invasive methods are preferred. In the measurement of the Raman spectrum, a general method can be used, and measurement by the micro Raman method is particularly preferable.
There is no particular limitation on the Raman spectrum measuring apparatus, and a normal apparatus can be used. When the measurement location of the moisture content is very narrow, it is preferable to use a measurement apparatus having a confocal optical system that can obtain a Raman spectrum even when the measurement site is very small.
また、ラマンスペクトルの測定に用いられる光源について、波長および入射角は特に限定はない。例えば、被測定物がヒトの皮膚の場合、波長は５００〜１１００ｎｍが好ましく、６００〜９００ｎｍがより好ましい。皮膚表面に対する光源の入射角は０°〜６０°が好ましく、０°〜３０°がより好ましい。なおここでの入射角は、皮膚表面に対して垂直な法線からの角度とする。
本発明において、レーザー光入射部位の深さ方向については特に制限はない。例えば、ヒトの皮膚について測定する場合、ヒトの皮膚の表面から真皮組織までの水分量を測定するのが好ましく、レーザー光入射部位を表面から１０００μｍまでの範囲とするのが好ましく、表面から２００μｍまでの範囲とするのがより好ましい。
使用する対物レンズについて、倍率、開口数（Ｎ．Ａ．）は特に限定はない。例えば、被測定物がヒト皮膚の場合、より高空間分解能での測定を行う場合には、倍率は４０倍〜１００倍、開口数は０．９〜１．５が好ましい。水平方向および深さ方向により広範囲の測定を行う場合は、倍率は１０倍〜４０倍、開口数は０．３〜０．９が好ましい。
Further, the wavelength and incident angle of the light source used for the measurement of Raman spectrum are not particularly limited. For example, when the object to be measured is human skin, the wavelength is preferably 500 to 1100 nm, and more preferably 600 to 900 nm. The incident angle of the light source with respect to the skin surface is preferably 0 ° to 60 °, more preferably 0 ° to 30 °. The incident angle here is an angle from a normal line perpendicular to the skin surface.
In the present invention, there is no particular limitation on the depth direction of the laser light incident site. For example, when measuring on human skin, it is preferable to measure the amount of water from the surface of human skin to the dermis tissue, and the laser light incident site is preferably in the range from the surface to 1000 μm, from the surface to 200 μm It is more preferable to set the range.
There are no particular limitations on the magnification and numerical aperture (NA) of the objective lens used. For example, when the object to be measured is human skin, the magnification is preferably 40 to 100 times and the numerical aperture is 0.9 to 1.5 when performing measurement with higher spatial resolution. When a wide range of measurement is performed in the horizontal direction and the depth direction, the magnification is preferably 10 to 40 times and the numerical aperture is preferably 0.3 to 0.9.
本発明の測定方法における測定対象としては特に限定はなく、ヒトの皮膚の他、マウス、モルモット、ブタ、イヌ、等の動物の皮膚を対象とすることができる。また、測定部位についても特に制限はない。 The measurement target in the measurement method of the present invention is not particularly limited, and the skin of animals such as mice, guinea pigs, pigs, dogs, etc. can be used in addition to human skin. Moreover, there is no restriction | limiting in particular also about a measurement site | part.
本発明において、ラマンスペクトルを測定する際に、皮膚に重水を浸透させる。皮膚に浸透させる重水の量は、重水のラマンスペクトルを測定できる量であれば特に制限はない。重水の浸透方法についても特に制限はなく、重水を満たした容器を用いて皮膚に重水を接触させ浸透させる方法、重水に浸したパッチなどを皮膚に貼る方法、重水を還流させながら皮膚に接触させる方法などが挙げられる。また温度や流速の制御装置を有する重水の浸透装置を用いると、より厳密な水分量の測定を行うことが可能となるため、好ましい。 In the present invention, when measuring the Raman spectrum, heavy water is permeated into the skin. The amount of heavy water that permeates into the skin is not particularly limited as long as the Raman spectrum of heavy water can be measured. There is no particular limitation on the method of penetrating heavy water, and a method of bringing heavy water into contact with the skin using a container filled with heavy water, a method of sticking a patch soaked in heavy water, etc., contacting the skin while refluxing heavy water The method etc. are mentioned. In addition, it is preferable to use a heavy water permeation device having a temperature and flow rate control device because it enables more accurate measurement of water content.
本発明において、重水を浸透させた皮膚のラマンスペクトルを測定し、ラマンスペクトル中の重水の信号強度より、皮膚内部に存在する水分量を測定する。重水と軽水は分子量、密度などが異なる。しかし、通常、重水と軽水は皮膚内部で同様の挙動を示すものである。そのため、本発明において、重水を水分量測定の指標として用いる。 In the present invention, the Raman spectrum of the skin infiltrated with heavy water is measured, and the amount of water present in the skin is measured from the signal intensity of heavy water in the Raman spectrum. Heavy water and light water differ in molecular weight and density. However, normally, heavy water and light water exhibit the same behavior inside the skin. Therefore, in the present invention, heavy water is used as an index for measuring the amount of water.
本発明の方法において、重水を皮膚外部から皮膚内部に浸透させる。従って、本発明において、ラマンスペクトル中の重水由来の信号強度を測定することにより、皮膚外部から皮膚内部に浸透した水分量（浸透量）を測定することができる。
また、本発明において、ラマンスペクトル中には前記重水由来の信号の他に、軽水由来の信号も観測される。この軽水は、皮膚内部にもともと存在していたものである。従って、本発明において、ラマンスペクトル中の軽水の信号強度を測定することで、皮膚外部から浸透した水分量の他に、皮膚内部にもともと存在していた水分量も測定することができる。
In the method of the present invention, heavy water is permeated into the skin from the outside of the skin. Therefore, in the present invention, by measuring the signal intensity derived from heavy water in the Raman spectrum, it is possible to measure the amount of water permeated from the outside of the skin into the skin (the amount of penetration).
In the present invention, in addition to the signal derived from heavy water, a signal derived from light water is also observed in the Raman spectrum. This light water was originally present inside the skin. Therefore, in the present invention, by measuring the signal intensity of light water in the Raman spectrum, it is possible to measure the amount of moisture originally present inside the skin in addition to the amount of moisture permeated from outside the skin.
測定したラマンスペクトルから皮膚水分量を測定する方法について説明するが、本発明はこれに限定されるものではない。
皮膚の角層の主要成分は水（軽水）と蛋白質である。したがって、角層を水と蛋白質の二成分系で近似した場合、角層蛋白質に由来（主にＣＨ伸縮振動に由来）する２９００ｃｍ-1付近のラマン信号強度Ｉprotein、水のＯＨ伸縮振動および蛋白質のＮＨ伸縮振動に由来する３４００ｃｍ-1付近のラマン信号強度ＩOH、および角層中の軽水と蛋白質の質量比ＲH2O/proteinの間には以下の関係が与えられる。
A method for measuring the skin moisture content from the measured Raman spectrum will be described, but the present invention is not limited thereto.
The main components of the stratum corneum of the skin are water (light water) and protein. Therefore, when the stratum corneum is approximated by a binary system of water and protein, the Raman signal intensity I protein near 2900 cm −1 derived from the stratum corneum protein (mainly derived from CH stretching vibration), the OH stretching vibration of water and the protein The following relationship is given between the Raman signal intensity I OH in the vicinity of 3400 cm −1, which is derived from the NH stretching vibration, and the light water to protein mass ratio R H2O / protein in the stratum corneum.
ここで、Ａ及びＢは、水分量既知の蛋白質のラマン測定より実験的に導出される定数である。
式（１）において、ＩOHおよびＩproteinは以下の式で表すことができる。
Here, A and B are constants derived experimentally from Raman measurement of a protein with a known water content.
In formula (1), I OH and I protein can be represented by the following formulas.
前記式（１）中の第２項（切片）に相当する−Ｂは、軽水のＯＨ伸縮振動由来のピークに重畳している蛋白質のＮＨ伸縮振動由来のピークの寄与を排除するためのものである。軽水と蛋白質由来の信号強度比は第一項の傾きＡに相当する。重水のＯＤ伸縮振動由来のピークが出現する領域には、重畳する蛋白質由来の信号は存在しないため、第一項のみを考慮すればよい。
蛋白質のＣＨ伸縮振動に由来する信号と重水のＯＤ伸縮振動由来の信号の強度比を直接求めることは容易ではない。したがって、軽水のＯＨ伸縮振動由来のピークと重水のＯＤ伸縮振動由来のピークの強度比より、間接的に蛋白質のＣＨと重水のＯＤの信号強度比を求めることが好ましい。ここで、重水と軽水の密度比は１．１１である。また、下記参考例で示すように、重水のＯＤ伸縮振動と軽水のＯＨ伸縮振動にぞれぞれ由来するピークの強度比は実質的には一定値Ｃを示す。これらの値を用いると、角層中の重水と蛋白質の質量比ＲD2O/proteinは以下のように与えられる。
-B corresponding to the second term (intercept) in the formula (1) is for eliminating the contribution of the peak derived from the NH stretching vibration of the protein superimposed on the peak derived from the OH stretching vibration of light water. is there. The signal intensity ratio derived from light water and protein corresponds to the slope A of the first term. In the region where the peak derived from the OD stretching vibration of heavy water appears, there is no superimposed protein-derived signal, so only the first term needs to be considered.
It is not easy to directly determine the intensity ratio between the signal derived from the CH stretching vibration of protein and the signal derived from the OD stretching vibration of heavy water. Therefore, it is preferable to indirectly determine the signal intensity ratio between the CH of protein and the OD of heavy water from the intensity ratio of the peak derived from the OH stretching vibration of light water and the peak derived from the OD stretching vibration of heavy water. Here, the density ratio of heavy water and light water is 1.11. Moreover, as shown in the following reference example, the intensity ratio of the peaks derived from the OD stretching vibration of heavy water and the OH stretching vibration of light water substantially shows a constant value C. Using these values, the mass ratio R D2O / protein of heavy water and protein in the stratum corneum is given as follows.
ここで、角層の成分を蛋白質・軽水・重水の３成分で近似した場合、軽水分量ＣH2O(ｍａｓｓ％）および重水分量ＣD2O(ｍａｓｓ％）は以下のように与えられる。 Here, when the stratum corneum components are approximated by three components of protein, light water, and heavy water, the light water content C H2O (mass%) and the heavy water content C D2O (mass%) are given as follows.
皮膚における軽水と重水は基本的な振る舞いはほとんど同じであるが、上述のように重水ではＨ（水素）がＤ（重水素）になり分子量が増加した分だけ、密度が上昇している。１．０００ｇの軽水と１．１１０ｇの重水は同じ体積を示すので、これらを同等に取り扱った方が、皮膚における水の動態を解析する上で好ましい。そこで本明細書では、重水を軽水に換算した場合の濃度ＣD2O’を以下のように定義した。本明細書での重水分量および軽水分量は、式（８）および式（９）より得られる換算量を用いる。 The basic behavior of light water and heavy water in the skin is almost the same, but as described above, in heavy water, the density increases as H (hydrogen) becomes D (deuterium) and the molecular weight increases. Since 1.000 g of light water and 1.110 g of heavy water have the same volume, it is preferable to treat them equally in analyzing the dynamics of water in the skin. Therefore, in this specification, the concentration C D2O ′ when heavy water is converted to light water is defined as follows. For the heavy water content and the light water content in the present specification, the converted amounts obtained from the equations (8) and (9) are used.
なお重水の添加により、蛋白質中のＮＨの一部で重水のＤとＨＤ交換が起こり、ＮＤに変化することが考えられる。しかしＮＤとＯＤの伸縮振動は、ほぼ同じ振動モードを持ち、かつ同じ位置に出現する。そのため、両者のラマン散乱断面積も近似的には同じと取り扱うことができる。従って、ＨＤ交換によってＮＤが生じたとしても、ＮＤの信号をＯＤの信号として読みかえてＯＤの浸透挙動、即ち重水の浸透挙動とみなして問題ないと考えられる。 By adding heavy water, it is conceivable that D and HD exchange of heavy water occurs in a part of NH in the protein and changes to ND. However, the stretching vibration of ND and OD has substantially the same vibration mode and appears at the same position. Therefore, both Raman scattering cross sections can be treated as approximately the same. Therefore, even if ND occurs due to the HD exchange, it is considered that there is no problem when the ND signal is read as the OD signal and regarded as the OD permeation behavior, that is, the permeation behavior of heavy water.
本発明の方法によれば、皮膚の局所における水分量を測定することができる。ここで、「局所」とは、レーザー光が照射されたときの焦点部分となる皮膚内部の一部分をいう。
また、本発明の水分量の測定方法を用いて、皮膚のラマンスペクトルを表面からの深さ方向及び／または水平方向に変えて測定することで、皮膚内部の水分分布を測定することができる。
According to the method of the present invention, the amount of water in the local area of the skin can be measured. Here, “local” refers to a part of the skin that becomes a focal portion when laser light is irradiated.
Moreover, the moisture distribution inside the skin can be measured by changing the Raman spectrum of the skin in the depth direction and / or the horizontal direction from the surface using the method for measuring the amount of moisture of the present invention.
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.
（１） 軽水と重水のラマン散乱強度比の測定
軽水（イオン交換水）と重水（Ｍｅｒｃｋ製、ＮＭＲ用、９９．９６％）を軽水：重水＝１：０（軽水のみ）、３：１、２：１、１：１、１：２、１：３および０：１（重水のみ）の体積比で混合し、ガラスボトムディッシュ（マツナミ製）に入れ、ラマンスペクトルを測定した。その結果を図１に示す。なお、図１中の各スペクトルはスペクトルの最大値および最小値を用いて規格化して表示した（オフセット表示）。
ラマンスペクトル測定装置としては、共焦点ラマン分光器 ナノファインダー３０（商品名、東京インスツルメンツ製）を用いた。また、測定条件は以下のとおりである。
＜測定条件＞
対物レンズ：４０×、ＮＡ０．９
レーザー：６３３ｎｍ、８ｍＷ（試料上）
積算時間：６０ｓ
ピンホール径：８０μｍ
回折格子：１５０ｇｒ／ｍｍ
(1) Measurement of Raman scattering intensity ratio of light water and heavy water Light water (ion exchange water) and heavy water (Merck, for NMR, 99.96%) light water: heavy water = 1: 0 (light water only), 3: 1 The mixture was mixed at a volume ratio of 2: 1, 1: 1, 1: 2, 1: 3 and 0: 1 (heavy water only), placed in a glass bottom dish (manufactured by Matsunami), and a Raman spectrum was measured. The result is shown in FIG. Each spectrum in FIG. 1 was normalized and displayed using the maximum and minimum values of the spectrum (offset display).
As a Raman spectrum measuring apparatus, a confocal Raman spectroscope Nano Finder 30 (trade name, manufactured by Tokyo Instruments) was used. The measurement conditions are as follows.
<Measurement conditions>
Objective lens: 40 ×, NA 0.9
Laser: 633 nm, 8 mW (on sample)
Total time: 60s
Pinhole diameter: 80 μm
Diffraction grating: 150 gr / mm
図１で示すように、軽水（軽水：重水＝１：０）のラマンスペクトルにおいては、１６４０ｃｍ-1付近にＨＯＨの変角振動由来のピーク（δ（ＨＯＨ））、３２５０ｃｍ-1付近にＯＨ対称伸縮振動由来のピーク（νsy（ＯＨ））、３４１０ｃｍ-1付近にＯＨ逆対称伸縮振動由来のピーク（νasy（ＯＨ））を示すラマンスペクトルが得られた。一方、重水（軽水：重水＝１：０）のラマンスペクトルにおいては、１２００ｃｍ-1付近にＤＯＤの変角振動由来のピーク（δ（ＤＯＤ））、２４００ｃｍ-1付近にＯＤ対称伸縮振動由来のピーク（νsy（ＯＤ））、２５００ｃｍ-1付近にＯＤ逆対称伸縮振動由来のピーク（νasy（ＯＨ））を示すラマンスペクトルが得られた。
軽水に少量の重水を添加（例えば、軽水：重水＝３：１の体積比で軽水に重水を添加）すると、前記の重水由来のピークに加え、１４５０ｃｍ-1付近に新たなピークが出現した。１４５０ｃｍ-1のラマン線はＨＯＤの変角振動由来のピークと判断できる。また２本のＯＨ伸縮振動由来のピーク（対称、逆対称）の強度比に関しては、３２５０ｃｍ-1付近のＯＨ対称伸縮振動由来のピークの相対強度が低下することも観測された。ＯＨ伸縮振動由来のピークに関しては、ＯＨ変角振動由来のピークの波数の二倍に相当するため、フェルミ共鳴による信号強度の増大現象が生じる事が知られている。軽水に重水を加えることによりフェルミ共鳴が抑制され、ＯＨ伸縮振動由来のピークの強度が低下したと考えられる。
重水に少量の軽水を添加（例えば、軽水：重水＝１：３の体積比で重水に軽水を添加）した場合でも同様に、ＯＤ対称伸縮振動由来のピークの強度低下が観測された。
As shown in Figure 1, light water (light water: heavy water = 1: 0) in the Raman spectrum of a peak derived from bending vibration of HOH around 1640 cm -1 ([delta] (HOH)), OH symmetrically around 3250cm -1 A Raman spectrum showing a peak derived from stretching vibration (ν sy (OH)) and a peak derived from OH reverse symmetrical stretching vibration (ν asy (OH)) in the vicinity of 3410 cm −1 was obtained. On the other hand, in the Raman spectrum of heavy water (light water: heavy water = 1: 0), a peak derived from DOD bending vibration (δ (DOD)) near 1200 cm −1 and a peak derived from OD symmetrical stretching vibration near 2400 cm −1. (Ν sy (OD)) A Raman spectrum showing a peak (ν asy (OH)) derived from OD inversely symmetric stretching vibration in the vicinity of 2500 cm −1 was obtained.
When a small amount of heavy water was added to light water (for example, heavy water was added to light water at a volume ratio of light water: heavy water = 3: 1), a new peak appeared in the vicinity of 1450 cm −1 in addition to the above peak derived from heavy water. The Raman line at 1450 cm −1 can be judged to be a peak derived from the HOD bending vibration. It was also observed that the relative intensity of the peak derived from the OH symmetric stretching vibration in the vicinity of 3250 cm −1 was lowered with respect to the intensity ratio of the two OH stretching vibration derived peaks (symmetrical and inversely symmetric). Since the peak derived from OH stretching vibration corresponds to twice the wave number of the peak derived from OH bending vibration, it is known that a signal intensity increase phenomenon due to Fermi resonance occurs. It is considered that Fermi resonance was suppressed by adding heavy water to light water, and the intensity of the peak derived from OH stretching vibration was reduced.
Even when a small amount of light water was added to heavy water (for example, light water was added to heavy water at a volume ratio of light water: heavy water = 1: 3), a decrease in the intensity of the peak derived from the OD symmetric stretching vibration was also observed.
また、図１に示す結果に基づき、重水と軽水の混合比と、ＯＤ伸縮振動とＯＨ伸縮振動にそれぞれ由来する信号の強度比との関係を以下のとおりに算出した。
重水と軽水の各混合比での、重水と軽水の信号強度比ＲOD/OHを下記式で算出した。
Further, based on the results shown in FIG. 1, the relationship between the mixing ratio of heavy water and light water and the intensity ratio of signals derived from OD stretching vibration and OH stretching vibration was calculated as follows.
The signal intensity ratio R OD / OH of heavy water and light water at each mixing ratio of heavy water and light water was calculated by the following formula.
重水と軽水の混合体積比と、ラマンスペクトルにおける重水と軽水の信号強度比との関係を図２に示す。
図２で示すように、混合体積比と信号強度比の関係は直線性を示し、その傾きは約１．２０となった。前述のように、軽水と重水の混合過程では、ＨＯＤ分子の出現やフェルミ共鳴の発生等があり、混合体積比と信号強度比の間には直線性が原理的に保証されているわけではない。しかし、図２より、軽水と重水の存在比を重水及び軽水の伸縮振動由来のピークの面積比から見積もれば、実用上は十分である判断できる。従って、単位体積当たりの重水と軽水の分子数を同じと近似する（２５℃での重水と軽水の密度比１．１１０は、重水と軽水の分子量比１．１１２とほぼ一致する）と、モル当たりのラマン散乱強度の比較においては、重水のＯＤ伸縮振動由来のピークの強度（面積）は、軽水のＯＨ伸縮振動由来のピークの強度（面積）の１．２０倍と見積もることができる。したがって式（４）のＣの実験値として、１．２０を得ることができた。
FIG. 2 shows the relationship between the mixing volume ratio of heavy water and light water and the signal intensity ratio of heavy water and light water in the Raman spectrum.
As shown in FIG. 2, the relationship between the mixing volume ratio and the signal intensity ratio showed linearity, and the slope thereof was about 1.20. As described above, in the mixing process of light water and heavy water, there are appearance of HOD molecules, generation of Fermi resonance, etc., and linearity is not guaranteed in principle between the mixing volume ratio and the signal intensity ratio. . However, it can be judged from FIG. 2 that it is practically sufficient if the abundance ratio of light water and heavy water is estimated from the area ratio of peaks derived from stretching vibration of heavy water and light water. Therefore, when the number of molecules of heavy water and light water per unit volume is approximated to be the same (the density ratio 1.110 of heavy water to light water at 25 ° C. is substantially equal to the molecular weight ratio of 1.112 of heavy water to light water), In comparison of the hit Raman scattering intensity, the peak intensity (area) derived from the OD stretching vibration of heavy water can be estimated as 1.20 times the peak intensity (area) derived from the OH stretching vibration of light water. Therefore, 1.20 was able to be obtained as an experimental value of C in the formula (4).
（２）蛋白質の水分量とラマンスペクトルとの関係の検証
式（１）における定数ＡおよびＢを得るために、含水量の異なる角層のラマンスペクトルの測定を以下のとおりに行った。
健常男性（４０歳代）のかかとより角層片（３．０〜５．５ｍｇ）を剥離し、クロロホルムおよび水に浸漬し、脂質等の油溶性成分と、アミノ酸等の水溶性成分を除去した。本処理後の角層片を、角層蛋白質のモデルとした。各角層を５酸化りんの調湿ボックス中に３日間放置し、調湿ボックス中の角層片に対してレーザーを照射し、ラマンスペクトルを測定した。その後に各角層を調湿ボックスより取り出し、速やかに重量を測定した。このときのラマンスペクトルおよび角層重量を、近似的に含水量０％における各角層片の重量およびラマンスペクトルと定義した。
その後、各角層片を順次調湿環境下に放置した後の、ラマンスペクトルの測定と重量の測定を繰り返した。調湿環境の調製には、乾燥シリカゲル、飽和塩化ナトリウム水溶液、飽和臭化ナトリウム水溶液、飽和硫酸ナトリウム水溶液および飽和リン酸水素二ナトリウム水溶液を用いた。このときのラマンスペクトルの変化を図３に示す。吸湿により、蛋白質のＣＨ伸縮振動に由来する信号（２９２０ｃｍ-1付近）に対する、水のＯＨ伸縮振動に由来する信号強度（３１００ｃｍ-1〜３７００ｃｍ-1付近、蛋白質のＮＨ伸縮振動に由来する信号を含む）が変化していることがわかる。
ここで上記秤量により得た、各調湿環境下での各角層片の含水率Ｒwaterを以下のように定義した。
(2) Verification of relationship between moisture content of protein and Raman spectrum In order to obtain the constants A and B in the formula (1), the Raman spectrum of the stratum corneum having different moisture contents was measured as follows.
The stratum corneum piece (3.0-5.5 mg) was peeled off from the heel of a healthy male (40s) and immersed in chloroform and water to remove oil-soluble components such as lipids and water-soluble components such as amino acids. . The stratum corneum piece after this treatment was used as a model of stratum corneum protein. Each stratum corneum was left in a humidity control box of phosphorus pentoxide for 3 days, a laser was irradiated to the stratum corneum pieces in the humidity control box, and a Raman spectrum was measured. Thereafter, each stratum corneum was taken out from the humidity control box, and the weight was quickly measured. The Raman spectrum and stratum corneum weight at this time were defined as the weight and Raman spectrum of each stratum corneum at a water content of approximately 0%.
Thereafter, the Raman spectrum measurement and the weight measurement were repeated after each of the stratum corneum pieces was sequentially left in a humidity control environment. Dry silica gel, saturated sodium chloride aqueous solution, saturated sodium bromide aqueous solution, saturated sodium sulfate aqueous solution, and saturated disodium hydrogen phosphate aqueous solution were used for the preparation of the humidity control environment. The change of the Raman spectrum at this time is shown in FIG. The moisture absorption for the signal derived from the CH stretching vibrations of protein (2920 cm around -1), the signal intensity derived from the OH stretching vibration of water (3100cm -1 ~3700cm around -1, a signal derived from the NH stretching vibration of the protein (Including) is changing.
Here, the moisture content R water of each corner layer piece obtained under the above-mentioned weighing under each humidity control environment was defined as follows.
各調湿環境下での角層片のラマンスペクトルよりラマン信号強度比（IOH/ICH）を算出し、そのときの含水率Ｒwaterとの関係をプロットした結果を図４に示す。IOH/ICHとＲwaterとの関係より、式（１）のＡの値として０．６４８を、Ｂの値として０．１７３を得た。 FIG. 4 shows the results of calculating the Raman signal intensity ratio (I OH / I CH ) from the Raman spectrum of the stratum corneum under each humidity control environment and plotting the relationship with the water content R water at that time. From the relationship between I OH / I CH and R water , 0.648 was obtained as the value of A in formula (1), and 0.173 was obtained as the value of B.
（３）皮膚の水分量及び水分分布の測定
健常男性（４０歳代）の前腕内側を顕微ラマン分光器のガラス窓に接触させ、ガラス窓越しにラマンスペクトル（深さ方向の連続スキャン）を測定した。
その後、上記でラマンスペクトルを測定した部位と同じ部位に重水（４０℃）を満たした円筒状のカップを１０分間接触させた。その後カップを外し、皮膚上に残っている重水を軽くタオルで除いた後、速やかに前腕内側のラマンスペクトルを深さ方向の連続スキャンで測定した。その結果を図５に示す。各スペクトルは最大・最小値を用いて縦軸を規格化後、オフセットをとって表示している。
なお、ラマン測定条件は以下のとおりである。
＜測定条件＞
装置：顕微ラマン分光器 ナノファインダー（商品名、東京インスツルメンツ製）
励起光：６３３ｎｍ、８ｍＷ（試料上）
ピンホール：１５０μｍ
対物レンズ：ＮＡ１．３
スキャン間隔：深さ方向に２μｍ間隔
(3) Measurement of moisture content and distribution of skin Contact the inner side of the forearm of a healthy male (40s) with the glass window of a microscopic Raman spectrometer, and measure the Raman spectrum (continuous scanning in the depth direction) through the glass window. did.
Thereafter, a cylindrical cup filled with heavy water (40 ° C.) was brought into contact with the same site where the Raman spectrum was measured as described above for 10 minutes. Thereafter, the cup was removed, the heavy water remaining on the skin was lightly removed with a towel, and the Raman spectrum inside the forearm was immediately measured by continuous scanning in the depth direction. The result is shown in FIG. Each spectrum is displayed with an offset after normalizing the vertical axis using the maximum and minimum values.
The Raman measurement conditions are as follows.
<Measurement conditions>
Apparatus: Microscopic Raman spectrometer Nano finder (trade name, manufactured by Tokyo Instruments)
Excitation light: 633 nm, 8 mW (on sample)
Pinhole: 150 μm
Objective lens: NA1.3
Scan interval: 2 μm interval in the depth direction
図５において、皮膚外部側で観測されているスペクトルは、皮膚が接触している窓材（ガラス）のスペクトルである。重水由来の信号強度（対ＣＨ強度比）は皮膚内部３μｍで最も高い。また、それより深部では重水由来の信号強度は減少するが、１３μｍまでは重水由来の信号が現れていることがわかる。一方、軽水由来の信号強度（対ＣＨ強度比）は、皮膚内部ほど強くなっていることがわかる。
以上の結果から、皮膚表面に接触させた重水が、皮膚表面から約１３μｍの深さまで浸透していることがわかる。
In FIG. 5, the spectrum observed on the outside of the skin is the spectrum of the window material (glass) in contact with the skin. The signal intensity derived from heavy water (vs. CH intensity ratio) is highest at 3 μm inside the skin. Further, it can be seen that the signal intensity derived from heavy water decreases deeper than that, but the signal derived from heavy water appears up to 13 μm. On the other hand, it can be seen that the signal intensity derived from light water (to the CH intensity ratio) is stronger in the skin.
From the above results, it can be seen that heavy water in contact with the skin surface penetrates to a depth of about 13 μm from the skin surface.
図５に示すスペクトルに基づき、前記式（１）、式（４）、式（７）および式（８）を適用して、皮膚の深さ方向における水分（軽水および重水）量を測定し、得られた値をプロットして、皮膚内部における水分分布を算出した。その結果を図６及び図７に示す。図６には、重水を浸透させる前の水分（軽水）分布も併せて示した。また、図７は、重水処理後の軽水分量と重水分量の合計値をプロットしたものである。なお、図６及び図７に示すデータは、皮膚を蛋白質、軽水、重水の３成分で近似して算出したものであり、重水分量は軽水換算量である。 Based on the spectrum shown in FIG. 5, the amount of water (light water and heavy water) in the depth direction of the skin is measured by applying the above formula (1), formula (4), formula (7) and formula (8), The obtained values were plotted to calculate the moisture distribution inside the skin. The results are shown in FIGS. FIG. 6 also shows water (light water) distribution before permeating heavy water. Moreover, FIG. 7 plots the total value of the light water content and heavy water content after a heavy water process. The data shown in FIGS. 6 and 7 are calculated by approximating the skin with three components of protein, light water, and heavy water, and the heavy water amount is a light water equivalent amount.
図６における重水浸透前の軽水分布は、皮膚表層部の濃度勾配の大きな領域（０〜１２μｍ付近）と、水分濃度がほぼ一定値を示す領域（１２〜２０μｍ）の２領域を、それぞれ直線で表現できることがわかる。
また、重水浸透後の軽水分布も同様に、２本の直線で近似できるプロファイルを示した。表層に近い濃度勾配の大きな領域（重水処理後では０〜１８μｍ）が、ほぼ直線性を維持したままその領域を広げたことは、（１）重水処理により角層が膨潤して厚くなったこと、および（２）重水が浸透してももともと皮膚内部に存在していた軽水の分布にはほとんど影響を及ぼしていないこと、を意味すると考えられる。
さらに、重水浸透後の重水分布から、重水が皮膚表面〜１３μｍ付近までの範囲に浸透している様子が観測できた。なお、皮膚表面０〜４μｍ付近での表層に向けた重水濃度の低下は、重水浸透処理からラマン測定までの間に、重水が揮発したためと推察される。
このように、重水分布と軽水分布を区別して観察することにより、皮膚における水分の挙動に関する情報量が飛躍的に向上する。
The distribution of light water before heavy water penetration in FIG. 6 is a straight line between two regions, a region having a large concentration gradient (near 0 to 12 μm) and a region having a substantially constant water concentration (12 to 20 μm). It can be expressed.
Similarly, the distribution of light water after heavy water infiltration showed a profile that can be approximated by two straight lines. A region with a large concentration gradient close to the surface layer (0-18 μm after heavy water treatment) was expanded while maintaining almost linearity. (1) The stratum corneum was swollen and thickened by heavy water treatment. And (2) It is considered that it means that even if heavy water permeates, it has little influence on the distribution of light water originally present in the skin.
Furthermore, from the distribution of heavy water after permeating heavy water, it was observed that heavy water permeated into the range from the skin surface to around 13 μm. In addition, it is guessed that the fall of the heavy water density | concentration toward the surface layer in the skin surface 0-4micrometer vicinity was because heavy water volatilized between the heavy water penetration process and the Raman measurement.
Thus, by distinguishing and observing the heavy water distribution and the light water distribution, the amount of information regarding the behavior of moisture in the skin is dramatically improved.
図７に示す重水浸透後の水分（軽水＋重水）分布は、軽水と重水の拡散係数が同じと近似しているため、重水ではなく軽水を皮膚に浸透させた場合に予想される水分分布に相当する。図７からは、皮膚外部から浸透した水（重水）と、もともと皮膚内部に存在していた水（軽水）とを区別することができない。
図７の結果からは、皮膚表面から５μｍにかけての水分濃度が上昇していることは確認できるが、それ以上の考察には困難である。
The water (light water + heavy water) distribution after heavy water penetration shown in FIG. 7 approximates that the diffusion coefficient of light water and heavy water is the same. Therefore, the water distribution is expected when light water penetrates the skin instead of heavy water. Equivalent to. From FIG. 7, it is not possible to distinguish between water permeated from outside the skin (heavy water) and water (light water) originally present inside the skin.
Although it can be confirmed from the results in FIG. 7 that the moisture concentration increases from the skin surface to 5 μm, it is difficult to consider further.
以上の結果から、本発明によれば、皮膚外部から内部へと水が浸透する様子を明瞭に観察することができる。また、重水の浸透に伴い角層が膨潤し、重水浸透前に存在していた軽水の濃度分布が皮膚のより内部側に伸長している様子が確認できる。
このように、重水を用いて、体内にもともと存在していた水と、体外から進入した水とを区別することにより、皮膚内部における水の動態を詳細に把握することができる。
From the above results, according to the present invention, it is possible to clearly observe how water permeates from the outside to the inside of the skin. In addition, the stratum corneum swells with the penetration of heavy water, and it can be confirmed that the concentration distribution of light water that existed before the penetration of heavy water extends to the inner side of the skin.
Thus, by using heavy water to distinguish between water originally present in the body and water that has entered from outside the body, it is possible to grasp in detail the dynamics of water inside the skin.
（４）重水浸透後の皮膚内部の軽水分布および重水分布のイメージング
健常男性（４０歳代）の前腕内側部に重水（４０℃）を満たした円筒状のカップを３０分間接触させた。その後カップを外し、皮膚上に残っている重水を軽くタオルで除いた後、速やかに前腕内側のラマンスペクトルを深さ方向（Ｚ方向）と水平方向（Ｘ方向）の２次元にて連続スキャンで測定した。ラマンスペクトル測定条件は以下のとおりである。
＜測定条件＞
装置：顕微ラマン分光器 ナノファインダー（商品名、東京インスツルメンツ製）
対物レンズ：１００×、ＮＡ１．３
レーザー：６３３ｎｍ、８ｍＷ（試料上）
積算時間：１ｓ／１点
ピンホール径：１５０μｍ
回折格子：１５０ｇｒ／ｍｍ
深さ方向（Ｚ）のスキャン間隔：２μｍ
深さ方向（Ｚ）のスキャン幅：４０μｍ（２１点）
水平方向（Ｘ）のスキャン間隔：２μｍ
水平方向（Ｘ）のスキャン幅：４０μｍ（２１点）
合計測定点数：４４１点
総積算時間：約８分
(4) Imaging of light water distribution and heavy water distribution inside skin after permeation of heavy water A cylindrical cup filled with heavy water (40 ° C.) was brought into contact with the inner side of the forearm of a healthy male (40s) for 30 minutes. After removing the cup and lightly removing heavy water remaining on the skin with a towel, the Raman spectrum inside the forearm can be quickly scanned in two dimensions in the depth direction (Z direction) and in the horizontal direction (X direction). It was measured. The Raman spectrum measurement conditions are as follows.
<Measurement conditions>
Apparatus: Microscopic Raman spectrometer Nano finder (trade name, manufactured by Tokyo Instruments)
Objective lens: 100 ×, NA 1.3
Laser: 633 nm, 8 mW (on sample)
Integration time: 1 s / 1 point Pinhole diameter: 150 μm
Diffraction grating: 150 gr / mm
Scanning interval in the depth direction (Z): 2 μm
Scan width in the depth direction (Z): 40 μm (21 points)
Horizontal (X) scan interval: 2 μm
Horizontal (X) scan width: 40 μm (21 points)
Total measurement points: 441 points Total integration time: Approximately 8 minutes
測定したラマンスペクトルに基づき、軽水分量および重水分量を算出し、得られたデータを基に軽水分布および重水分布のイメージングを行った。その結果をそれぞれ図８及び図９に示す。なお、図８及び図９はデータ処理ソフトＩｇｏｒ ｖｅｒ．５．０（商品名）により作成した。図８及び図９に示すデータは、皮膚を蛋白質、軽水、重水の３成分で近似して算出したものであり、重水分量は軽水換算量である。 Based on the measured Raman spectrum, light water content and heavy water content were calculated, and light water distribution and heavy water distribution were imaged based on the obtained data. The results are shown in FIGS. 8 and 9, respectively. 8 and 9 show data processing software Igor ver. 5.0 (trade name). The data shown in FIGS. 8 and 9 are calculated by approximating the skin with three components of protein, light water, and heavy water, and the amount of heavy water is a light water equivalent amount.
図８及び図９の結果から、以下のことがわかった。
まず、軽水は深部ほど濃度が高く、重水は表層ほど濃度が高いことがわかった。また、重水の多い部位では軽水が少ないことがわかった。これは、皮膚外部から重水を浸透させたことを考えれば当然の結果である。さらに、重水が皮膚の表面から深さ４０μｍ付近まで浸透している領域と、２０μｍ程度までしか浸透していない領域があることがわかった。
From the results of FIGS. 8 and 9, the following was found.
First, it was found that the concentration of light water is higher at the deeper part and the concentration of heavy water is higher at the surface layer. In addition, it was found that there was little light water in the heavy water area. This is a natural result considering that heavy water has permeated from the outside of the skin. Furthermore, it has been found that there are regions where heavy water penetrates from the surface of the skin to a depth of around 40 μm and regions where it penetrates only up to about 20 μm.
本実施例で示すように、重水を用いることにより、皮膚内での水の動態解析が行えることがわかった。本発明によれば、軽水のスペクトルに基づいて測定する従来の皮膚水分量の測定方法よりもはるかに詳細な情報を与えた。そのため、より明確かつ定量的に皮膚内での水の動態を知ることができる。さらに、本発明により、皮膚表面部位における水の動態も解析できることがわかった。 As shown in this example, it was found that the use of heavy water can analyze the dynamics of water in the skin. According to the present invention, much more detailed information is given than the conventional method for measuring skin moisture content, which is measured based on the spectrum of light water. Therefore, the dynamics of water in the skin can be known more clearly and quantitatively. Furthermore, it turned out that the dynamics of the water in the skin surface site | part can also be analyzed by this invention.
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| CN114166822A (en) * | 2021-12-03 | 2022-03-11 | 中南林业科技大学 | Method for measuring water content of wood cell wall based on Raman difference spectrum technology |
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