JP5460528B2 - Method for measuring skin moisture content - Google Patents
Method for measuring skin moisture content Download PDFInfo
- Publication number
- JP5460528B2 JP5460528B2 JP2010196879A JP2010196879A JP5460528B2 JP 5460528 B2 JP5460528 B2 JP 5460528B2 JP 2010196879 A JP2010196879 A JP 2010196879A JP 2010196879 A JP2010196879 A JP 2010196879A JP 5460528 B2 JP5460528 B2 JP 5460528B2
- Authority
- JP
- Japan
- Prior art keywords
- raman spectrum
- skin
- protein
- lipid
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims description 25
- 238000001237 Raman spectrum Methods 0.000 claims description 168
- 150000002632 lipids Chemical class 0.000 claims description 76
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 63
- 102000004169 proteins and genes Human genes 0.000 claims description 54
- 108090000623 proteins and genes Proteins 0.000 claims description 54
- 238000011088 calibration curve Methods 0.000 claims description 10
- 238000000691 measurement method Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 210000003491 skin Anatomy 0.000 description 128
- 210000000434 stratum corneum Anatomy 0.000 description 34
- 238000005259 measurement Methods 0.000 description 20
- 210000002374 sebum Anatomy 0.000 description 20
- 238000001228 spectrum Methods 0.000 description 19
- 206010013786 Dry skin Diseases 0.000 description 13
- 210000000245 forearm Anatomy 0.000 description 13
- 238000001069 Raman spectroscopy Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 9
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 8
- 239000011521 glass Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000012937 correction Methods 0.000 description 5
- 230000037336 dry skin Effects 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 2
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229940106189 ceramide Drugs 0.000 description 2
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 2
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 2
- SASYSVUEVMOWPL-NXVVXOECSA-N decyl oleate Chemical compound CCCCCCCCCCOC(=O)CCCCCCC\C=C/CCCCCCCC SASYSVUEVMOWPL-NXVVXOECSA-N 0.000 description 2
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229940031439 squalene Drugs 0.000 description 2
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000003841 Raman measurement Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229920003350 Spectratech® Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 229910002056 binary alloy Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000000794 confocal Raman spectroscopy Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012888 cubic function Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000010291 electrical method Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Description
本発明は、皮膚水分量の測定方法に関する。 The present invention relates to a method for measuring skin moisture content.
皮膚の水分量は、皮膚の形状・光学特性・力学特性に直接的な影響を及ぼす因子であるため、皮膚の状態や機能を制御する技術の開発においては、皮膚の水分量を正確に測定する技術が必要になる。また、皮膚の水分量は、個々人の皮膚の状態や環境により大きく変動するため、皮膚の状態の個人差や部位差を特性化する上でも、皮膚の水分量を適切に把握することが重要である。 Skin moisture is a factor that directly affects skin shape, optical properties, and mechanical properties. Therefore, in the development of technologies that control skin conditions and functions, skin moisture is accurately measured. Technology is needed. In addition, since the moisture content of the skin varies greatly depending on the skin condition and environment of each individual, it is important to properly grasp the moisture content of the skin in characterizing individual differences and site differences in the skin condition. is there.
皮膚の水分量を測定する方法としては、従来からスキコンやコルネオメーターを始めとする電気的な手法が広く用いられてきている。これらの手法は水分量の変動を鋭敏に且つ簡便にとらえることができる長所を有する一方、測定深度が不明瞭なこと、水分量を絶対量として産出できないこと、水以外の因子の影響を受けやすいこと等の短所も指摘されている。 As a method for measuring the moisture content of the skin, an electrical method such as a ski control or a corneometer has been widely used. While these methods have the advantage of being able to catch fluctuations in moisture content sensitively and easily, the measurement depth is unclear, the moisture content cannot be produced as an absolute amount, and it is easily affected by factors other than water. Some disadvantages are pointed out.
このような従来型の水分測定法の課題を解決すべく、共焦点ラマン分光法を用いた水分測定法も開発されてきている。この方法は、皮膚内の特定部位にレーザー光を集光し、集光位置から発生するラマン散乱光を検出してラマンスペクトルとして解析することにより、集光部位における水分量を計測する方法である（例えば特許文献１）。特許文献１に記載の方法は、皮膚角層の成分組成をタンパク質と水の二成分系で近似し、皮膚のラマンスペクトルにおいて、ＣＨ伸縮振動とＯＨ伸縮振動の信号強度比からタンパク質量に対する水分質量の比率を算出し、この比率に基づいて皮膚中の水分量を推定するものである。
しかしながら、実際の皮膚はタンパク質と水の他に、脂質（細胞間脂質や皮脂等）が第三の主要成分として存在する。ラマンスペクトルにおいて、脂質由来のＣＨ伸縮振動のピークはタンパク質由来のＣＨ伸縮振動のピークに近接して存在するため、タンパク質由来のＣＨ伸縮振動の信号を検出すると、脂質由来のＣＨ伸縮振動の信号も共雑信号として検出してしまう。したがって、ラマンスペクトルのデータからタンパク質由来のＣＨ伸縮振動の信号のみを正確に読み取ることができず、皮膚水分量を正確に測定することは困難であった。
In order to solve the problem of the conventional moisture measurement method, a moisture measurement method using confocal Raman spectroscopy has been developed. This method is a method of measuring the amount of water in the condensing site by condensing laser light at a specific site in the skin, detecting Raman scattered light generated from the condensing position, and analyzing it as a Raman spectrum. (For example, patent document 1). The method described in
However, in actual skin, in addition to protein and water, lipids (such as intercellular lipids and sebum) are present as the third main component. In the Raman spectrum, since the peak of CH stretching vibration derived from lipid exists close to the peak of CH stretching vibration derived from protein, when the signal of CH stretching vibration derived from protein is detected, the signal of CH stretching vibration derived from lipid also appears. It will be detected as a busy signal. Accordingly, it is difficult to accurately read only the signal of CH stretching vibration derived from protein from Raman spectrum data, and it is difficult to accurately measure the skin moisture content.
本発明は、ラマン分光法により皮膚の水分量を正確かつ簡便に測定する方法を提供することを課題とする。 An object of the present invention is to provide a method for accurately and simply measuring the moisture content of the skin by Raman spectroscopy.
本発明者等は上記課題に鑑み鋭意検討を行った結果、皮膚試料をタンパク質、水及び脂質の三成分系で近似し、脂質由来のラマンスペクトルの寄与分を、皮膚の実測ラマンスペクトルから差し引くことで、タンパク質由来のＣＨ伸縮振動の信号強度を正確に読み取り、その結果皮膚水分量を正確かつ簡便に測定できることを見い出した。本発明はこれらの知見に基づいて完成させたものである。 As a result of intensive studies in view of the above problems, the present inventors approximate a skin sample with a ternary system of protein, water and lipid, and subtract the contribution of the lipid-derived Raman spectrum from the measured Raman spectrum of the skin. Thus, it was found that the signal intensity of protein-derived CH stretching vibration was accurately read, and as a result, the skin moisture content could be measured accurately and simply. The present invention has been completed based on these findings.
本発明は、少なくとも１種のモデル脂質のラマンスペクトルを測定する工程、
皮膚試料のラマンスペクトルを測定する工程、
前記の皮膚試料のラマンスペクトルから、前記少なくとも１種のモデル脂質のラマンスペクトルにおける該モデル脂質の寄与分を差し引いた補正ラマンスペクトルを得る工程、及び
前記補正ラマンスペクトルにおいて、タンパク質のＣＨ伸縮振動由来の信号強度と水のＯＨ伸縮振動由来との信号強度の比率に基づき皮膚水分量を測定する工程
を含む、皮膚水分量の測定方法に関する。
The present invention comprises measuring a Raman spectrum of at least one model lipid,
Measuring the Raman spectrum of the skin sample;
Obtaining a corrected Raman spectrum obtained by subtracting the contribution of the model lipid in the Raman spectrum of the at least one model lipid from the Raman spectrum of the skin sample; and in the corrected Raman spectrum, derived from the CH stretching vibration of the protein. The present invention relates to a method for measuring skin moisture content, including a step of measuring skin moisture content based on a ratio of signal strength to signal strength derived from OH stretching vibration of water.
本発明によれば、ラマン分光法により皮膚の水分量を正確かつ簡便に測定することができる。 According to the present invention, the moisture content of the skin can be measured accurately and simply by Raman spectroscopy.
以下、本発明を図面に基づいて詳細に説明する。
図１において、Ａは皮膚試料（健常男性の前腕）のラマンスペクトルを示し、Ｂは脱脂乾燥皮膚試料のラマンスペクトルを示し、Ｃはモデル皮脂のラマンスペクトルを示し、Ｄはモデル細胞間脂質のラマンスペクトルを示し、Ｅは水のラマンスペクトルを示す。
図１からも明らかなように、脱脂乾燥皮膚試料等のタンパク質由来のＣＨ伸縮振動のピークと、皮脂や細胞間脂質等の脂質由来のＣＨ伸縮振動のピークは、一部が重複した波数領域で観測される。そのため、タンパク質、水及び脂質を構成成分とする皮膚試料のラマンスペクトルを測定すると、図１のＡに示すように、タンパク質、水及び脂質をそれぞれ由来とするピークが重畳したスペクトルが得られる。従って、ラマン分光法を利用し、皮膚試料をタンパク質及び水の二成分系で近似して行う従来の方法では、タンパク質量を実際よりも多く見積もってしまい、その結果水分量を少なく見積もってしまうため、正確な皮膚水分量を測定することができない。
この問題に対して、皮膚試料をタンパク質、水及び脂質の三成分系で近似し、図１のＢに示すような皮膚試料のラマンスペクトルからラマンスペクトルにおける脂質の寄与分を差し引いた補正ラマンスペクトルを作成し、補正ラマンスペクトルにおけるタンパク質のＣＨ伸縮振動由来の信号強度に対する水のＯＨ伸縮振動由来の信号強度の比率に基づき皮膚水分量を測定する。このようにして皮膚水分量の測定することで、正確に皮膚水分量を測定することができることを本発明者等は見出した。本発明はこの知見に基くものである。
Hereinafter, the present invention will be described in detail with reference to the drawings.
In FIG. 1, A shows the Raman spectrum of a skin sample (forearm of a healthy man), B shows the Raman spectrum of a defatted and dried skin sample, C shows the Raman spectrum of model sebum, and D shows the Raman of model intercellular lipids. A spectrum is shown, E shows the Raman spectrum of water.
As is clear from FIG. 1, the peak of CH stretching vibration derived from protein such as defatted and dried skin sample and the peak of CH stretching vibration derived from lipid such as sebum and intercellular lipid are in a wave number region where a part overlaps. Observed. Therefore, when a Raman spectrum of a skin sample containing protein, water, and lipid as constituents is measured, a spectrum in which peaks derived from protein, water, and lipid are superimposed is obtained as shown in FIG. Therefore, in the conventional method in which a skin sample is approximated by a two-component system of protein and water using Raman spectroscopy, the amount of protein is estimated more than the actual amount, and as a result, the amount of water is estimated less. Unable to measure the accurate skin moisture content.
To solve this problem, approximate a skin sample with a ternary system of protein, water, and lipid, and obtain a corrected Raman spectrum obtained by subtracting the lipid contribution in the Raman spectrum from the Raman spectrum of the skin sample as shown in FIG. The moisture content of the skin is measured based on the ratio of the signal intensity derived from the OH stretching vibration of water to the signal intensity derived from the CH stretching vibration of the protein in the corrected Raman spectrum. The present inventors have found that the skin moisture content can be accurately measured by measuring the skin moisture content in this manner. The present invention is based on this finding.
本発明の皮膚水分量の測定方法を具体的に説明する。
本発明において、少なくとも１種のモデル脂質のラマンスペクトル（図２ｃ及びｄ参照）及び測定対象である皮膚試料のラマンスペクトル（図２ａ参照）を測定する。次に、皮膚試料のラマンスペクトルから、モデル脂質のラマンスペクトルにおける該モデル脂質の寄与分を差し引いた補正ラマンスペクトルを得る（図２ｆ参照）。そして、前記補正ラマンスペクトルにおいて、タンパク質のＣＨ伸縮振動由来の信号強度と水のＯＨ伸縮振動由来の信号強度との比率に基づき皮膚水分量を測定する。
The method for measuring the skin moisture content of the present invention will be specifically described.
In the present invention, the Raman spectrum (see FIGS. 2c and d) of at least one model lipid and the Raman spectrum (see FIG. 2a) of the skin sample to be measured are measured. Next, a corrected Raman spectrum is obtained by subtracting the contribution of the model lipid in the Raman spectrum of the model lipid from the Raman spectrum of the skin sample (see FIG. 2f). In the corrected Raman spectrum, the skin moisture content is measured based on the ratio between the signal intensity derived from the CH stretching vibration of the protein and the signal intensity derived from the OH stretching vibration of water.
本発明は、ラマン分光法を用いて皮膚の水分量を測定する方法である。本発明の測定方法は、採取した皮膚試料を測定対象としてもよいし（侵襲法）、生体を覆う皮膚をそのままの状態で測定対象としてもよい（非侵襲法）が、肌の状態を直接測定できる非侵襲法が好ましい。皮膚試料の由来はヒトの他、マウス、モルモット、ラット、ブタ、イヌなどの動物であってもよい。また、皮膚の由来部位についても特に制限はない。皮膚試料のラマンスペクトルは一般的な方法で得ることができるが、顕微ラマン法により得ることが好ましい。ラマンスペクトルの測定装置に特に制限はなく、通常の装置を用いることができるが、水分量の測定箇所が狭い場合には、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。 The present invention is a method of measuring the moisture content of skin using Raman spectroscopy. In the measurement method of the present invention, the collected skin sample may be a measurement target (invasive method), or the skin covering the living body may be the measurement target as it is (non-invasive method), but the skin state is directly measured. A non-invasive method that can be used is preferred. The skin sample may be derived from animals such as mice, guinea pigs, rats, pigs, and dogs in addition to humans. Moreover, there is no restriction | limiting in particular also about the origin part of skin. The Raman spectrum of the skin sample can be obtained by a general method, but is preferably obtained by a microscopic Raman method. There is no particular limitation on the Raman spectrum measuring apparatus, and a normal apparatus can be used. However, when the water content measurement location is narrow, it is preferable to use a measuring apparatus having a confocal optical system.
ラマンスペクトルの測定における光源はレーザー光源であることが好ましい。光源の波長に特に制限はないが、５００〜１１００ｎｍが好ましく、６００〜９００ｎｍがより好ましい。光源から発せられる照射光の皮膚表面に対する入射角は０°〜６０°が好ましく、０°〜３０°がより好ましい。ここで、「入射角」とは、皮膚表面に対して垂直な法線からの角度を意味する。 The light source in the measurement of the Raman spectrum is preferably a laser light source. Although there is no restriction | limiting in particular in the wavelength of a light source, 500-1100 nm is preferable and 600-900 nm is more preferable. The incident angle of the irradiation light emitted from the light source with respect to the skin surface is preferably 0 ° to 60 °, and more preferably 0 ° to 30 °. Here, the “incident angle” means an angle from a normal line perpendicular to the skin surface.
本発明において光源から発せられる照射光は、集光レンズにより測定部位で焦点を結ぶことが好ましい。皮膚水分量の測定は、皮膚表面から真皮組織までのいずれかの深さでの水分量を測定するのが好ましい。したがって、皮膚表面からの焦点深度は０〜１０００μｍであることが好ましく、０〜２００μｍであることがより好ましい。
集光レンズとして対物レンズを使用する場合において、その倍率、開口数（Ｎ．Ａ．）に特に制限はない。例えば、４０〜１００倍の倍率で、開口数を０．９〜１．５とすることで高い空間分解能での測定ができるし、１０〜４０倍の倍率で開口数を０．３〜０．９とすれば、水平方向及び深さ方向により広範囲の測定を行うことが可能になる。
In the present invention, the irradiation light emitted from the light source is preferably focused at the measurement site by the condenser lens. Measurement of the moisture content of the skin preferably measures the moisture content at any depth from the skin surface to the dermal tissue. Therefore, the depth of focus from the skin surface is preferably 0 to 1000 μm, and more preferably 0 to 200 μm.
When an objective lens is used as the condenser lens, there are no particular restrictions on the magnification and numerical aperture (NA). For example, measurement with high spatial resolution can be performed by setting the numerical aperture to 0.9 to 1.5 at a magnification of 40 to 100 times, and the numerical aperture from 0.3 to 0.00 at a magnification of 10 to 40 times. If it is 9, it becomes possible to perform a wide range of measurements in the horizontal direction and the depth direction.
本発明におけるモデル脂質は、皮膚に含まれうる脂質であれば特に制限はなく、１種又は２種以上の脂質を本発明におけるモデル脂質として用いてもよい。本発明においては、モデル脂質として液体脂及び／又は固体脂を用いることが好ましい。液体脂としては、オレイン酸、トリオレイン酸、オレイン酸デシルもしくはスクワレン又はこれらの混合物などが挙げられる。また、固体脂としては、細胞間脂質が挙げられ、例えば、セラミドを用いることができる。 The model lipid in the present invention is not particularly limited as long as it can be contained in the skin, and one or more lipids may be used as the model lipid in the present invention. In the present invention, it is preferable to use liquid fat and / or solid fat as model lipids. Examples of the liquid fat include oleic acid, trioleic acid, decyl oleate or squalene, or a mixture thereof. Moreover, as solid fat, an intercellular lipid is mentioned, For example, a ceramide can be used.
本発明の皮膚水分量の測定方法について具体的に説明する。しかし、本発明はこれに制限するものではない。
本発明の測定方法において、測定対象である皮膚試料のラマンスペクトルをそれぞれ測定する。そして、皮膚試料のラマンスペクトルから、別途測定した少なくとも１種のモデル脂質のラマンスペクトルから算出したモデル脂質の寄与分を差し引いた補正ラマンスペクトルを作成する。このようにして得られた補正ラマンスペクトルにおける、２９００ｃｍ-1付近のラマン信号強度Ｉprotein（主にタンパク質のＣＨ伸縮振動に由来する。以下、タンパク質のＣＨ伸縮振動由来の信号強度ともいう。）と３４００ｃｍ-1付近のラマン信号強度ＩOH（主に水のＯＨ伸縮振動に由来する。以下、水のＯＨ伸縮振動由来の信号強度ともいう。）との比率に基づいて、皮膚水分量を測定する。
The skin moisture content measuring method of the present invention will be specifically described. However, the present invention is not limited to this.
In the measurement method of the present invention, the Raman spectrum of the skin sample to be measured is measured. Then, a corrected Raman spectrum is created by subtracting the contribution of the model lipid calculated from the Raman spectrum of at least one model lipid separately measured from the Raman spectrum of the skin sample. In the corrected Raman spectrum thus obtained, the Raman signal intensity I protein near 2900 cm −1 (mainly derived from the CH stretching vibration of the protein. Hereinafter also referred to as the signal intensity derived from the CH stretching vibration of the protein). The skin moisture content is measured based on the ratio to the Raman signal intensity I OH in the vicinity of 3400 cm −1 (mainly derived from the OH stretching vibration of water, hereinafter also referred to as the signal intensity derived from the OH stretching vibration of water). .
前記補正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具体的に説明するが、本発明はこれに限定されるものではない。
前記補正ラマンスペクトルにおいて、皮膚の角層の主要成分のうち脂質の寄与分を排除し、水とタンパク質の二成分系で近似している。したがって、前記補正ラマンスペクトルにおける、Ｉprotein、ＩOH、及び皮膚試料中の水とタンパク質の質量比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 corrected Raman spectrum will be specifically described, but the present invention is not limited to this.
In the corrected Raman spectrum, the contribution of lipid is excluded from the main components of the stratum corneum of the skin and approximated by a binary system of water and protein. Therefore, the following relationship is given between I protein , I OH , and the water to protein mass ratio R H2O / protein in the corrected Raman spectrum.
ここで、Ａ及びＢは、水分量既知のタンパク質（例えば、脱脂皮膚試料）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及び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 moisture content (for example, a degreased skin sample).
In formula (1), I OH and I protein can be represented by the following formulas.
本発明において、予め脱脂皮膚試料のラマンスペクトルを調湿下で測定し、このようにして得られた脱脂皮膚試料のラマンスペクトルにおけるタンパク質のＣＨ伸縮振動由来の信号強度に対する水のＯＨ伸縮振動由来の信号強度の比率と、該脱脂皮膚試料に含まれる水分量との関係を示す検量線を作成し、該検量線を用いて前記補正ラマンスペクトルから皮膚水分量を測定することが好ましい。 In the present invention, the Raman spectrum of the defatted skin sample was measured in advance under humidity control, and the water spectrum derived from the OH stretching vibration of water relative to the signal intensity derived from the CH stretching vibration of the protein in the Raman spectrum of the defatted skin sample thus obtained. It is preferable to prepare a calibration curve indicating the relationship between the signal intensity ratio and the amount of moisture contained in the defatted skin sample, and measure the skin moisture content from the corrected Raman spectrum using the calibration curve.
上記検量線は、水分量既知の脱脂皮膚試料を複数用意し、ラマンスペクトルを測定し、前記式（１）の関係に従い、ＩproteinとＩOHとの比率と、該脱脂皮膚試料の総重量に対する該脱脂皮膚試料に含まれる水分量の比率をプロットすることで得られる。上記水分量既知の脱脂皮膚試料に制限はないが、水分量既知の角層であることが好ましい。 The calibration curve is prepared by preparing a plurality of defatted skin samples having a known water content, measuring the Raman spectrum, and according to the relationship of the above formula (1), the ratio of I protein to I OH and the total weight of the defatted skin sample. It is obtained by plotting the ratio of the amount of water contained in the defatted skin sample. The defatted skin sample with a known moisture content is not limited, but is preferably a stratum corneum with a known moisture content.
本発明において前記Ｉproteinは、補正ラマンスペクトルの好ましくは２８００〜３０５０ｃｍ-1、より好ましくは２８００〜３０３０ｃｍ-1の波数範囲におけるピーク面積であることが好ましい。また、前記ＩOHは、補正ラマンスペクトルの好ましくは３１００〜３８００ｃｍ-1、より好ましくは３１００〜３７５０ｃｍ-1の波数範囲におけるピーク面積であることが好ましい。
The I protein in the present invention, it is preferable preferably corrected
本発明の皮膚水分量の測定方法の具体的態様について説明する。
まず、脱脂皮膚試料のラマンスペクトルにおけるＩproteinに対するＩOHの比率と、該脱脂皮膚試料の総質量に対する該脱脂皮膚試料に含まれる水分量の比率との関係を示す検量線を作成する。次に、測定対象である皮膚試料のラマンスペクトルを測定する。次に、前記皮膚試料のラマンスペクトルから、脂質の寄与分を差し引いた補正ラマンスペクトルを作成する。次に、前記補正ラマンスペクトルにおける、Ｉproteinに対するＩOHの比率を算出する。そして、Ｉproteinに対するＩOHの比率から、検量線を用いて、皮膚水分量を測定する。
A specific embodiment of the method for measuring skin moisture content of the present invention will be described.
First, a calibration curve indicating the relationship between the ratio of I OH to I protein in the Raman spectrum of the degreased skin sample and the ratio of the amount of water contained in the degreased skin sample to the total mass of the degreased skin sample is prepared. Next, the Raman spectrum of the skin sample to be measured is measured. Next, a corrected Raman spectrum is created by subtracting the lipid contribution from the Raman spectrum of the skin sample. Next, the ratio of I OH to I protein in the corrected Raman spectrum is calculated. Then, the skin moisture content is measured from the ratio of I OH to I protein using a calibration curve.
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.
（試験例１）補正ラマンスペクトルの妥当性評価
［脱脂乾燥角層の調製］
健常男性（４０歳代）のかかとより角層片（３．０〜５．０ｍｇ）をナイフで切除後、クロロホルム−メタノール（１：１）液に一昼夜浸漬した。その後角層片試料を取り出し、自然乾燥後、イオン交換水に一昼夜浸漬した。その後角層片試料を取り出し、自然乾燥させることで、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脱脂乾燥角層を調製した。
(Test Example 1) Validity evaluation of corrected Raman spectrum [Preparation of degreased and dried stratum corneum]
A horny layer piece (3.0 to 5.0 mg) was excised with a knife from the heel of a healthy male (40s), and then immersed in a chloroform-methanol (1: 1) solution overnight. Thereafter, the sample of the stratum corneum was taken out, naturally dried, and then immersed in ion-exchanged water for one day. Thereafter, the stratum corneum piece sample was taken out and naturally dried to prepare a degreased and dried stratum corneum from which oil-soluble components such as lipids and water-soluble components such as amino acids were removed.
［モデル皮脂の調製］
下記組成のモデル皮脂を調製した。
オレイン酸（関東化学製） １７．４質量％
トリオレイン酸（関東化学製） ４３．４質量％
オレイン酸デシル（和光純薬製） ２６．５質量％
スクワレン（関東化学製） １２．７質量％
[Preparation of model sebum]
A model sebum having the following composition was prepared.
Oleic acid (Kanto Chemical) 17.4% by mass
Trioleic acid (manufactured by Kanto Chemical Co.) 43.4% by mass
Decyl oleate (Wako Pure Chemical Industries, Ltd.) 26.5% by mass
Squalene (manufactured by Kanto Chemical) 12.7% by mass
上記の脱脂乾燥角層及びモデル皮脂、並びにモデル細胞間脂質（シグマ社製non-hydroxy fatty ceramide）及び水（イオン交換水）の各試料についてラマンスペクトルを測定した。ラマンスペクトルの測定条件を以下に示す。
装置：顕微ラマン分光器 Ｎａｎｏｆｉｎｄｅｒ３０（商品名、東京インスツルメンツ）
レーザー：Ｈｅ−Ｎｅレーザー（励起波長：６３２．８ｎｍ）
回析格子：１５０ｇｒ／ｍｍ
対物レンズ：１００倍、油浸、ＮＡ＝１．３（ニコン）
ピンホール直径：１５０μｍ
各試料のラマンスペクトルの測定方法を以下に示す。
脱脂乾燥角層については、脱脂乾燥角層を五酸化りんをセットした容器に入れ、容器底面のガラス窓越しにレーザーを照射し、絶乾状態での脱脂乾燥角層のラマンスペクトルを測定した。モデル皮脂、モデル細胞間脂質及び水については、ガラス製の試料台に試料を置き、ガラス越しにレーザーを照射し、ラマンスペクトルを測定した。
Raman spectra were measured for each sample of the defatted and dried stratum corneum and model sebum, model intercellular lipid (non-hydroxy fatty ceramide manufactured by Sigma) and water (ion-exchanged water). The measurement conditions of the Raman spectrum are shown below.
Apparatus: Microscopic Raman spectrometer Nanofinder 30 (trade name, Tokyo Instruments)
Laser: He-Ne laser (excitation wavelength: 632.8 nm)
Diffraction grating: 150 gr / mm
Objective lens: 100 times, oil immersion, NA = 1.3 (Nikon)
Pinhole diameter: 150 μm
The method for measuring the Raman spectrum of each sample is shown below.
For the degreased and dried stratum corneum, the degreased and dried stratum corneum was placed in a container in which phosphorus pentoxide was set, and a laser was irradiated through the glass window on the bottom of the container, and the Raman spectrum of the degreased and dried stratum corneum was measured. For model sebum, model intercellular lipid and water, a sample was placed on a glass sample stage, a laser was irradiated through the glass, and a Raman spectrum was measured.
このようにして得られた脱脂乾燥角層、モデル皮脂、モデル細胞間脂質及び水のラマンスペクトルを、それぞれ図１中のＢ〜Ｅに示す。 The Raman spectra of the degreased and dried stratum corneum, model sebum, model intercellular lipid and water thus obtained are shown in B to E in FIG.
さらに、健常男性（４０歳代）の前腕の皮膚のラマンスペクトル（皮膚表面からの深さ３μｍ）を測定した。その結果を、図１中のＡに示す。
Furthermore, the Raman spectrum of the skin of the forearm of a healthy male (40's) (
次に、図２中のａに示す、前記条件下で測定した皮膚試料のラマンスペクトルから、図２中のｃ及びｄに示す脂質の寄与分のラマンスペクトルを差し引いて得られる補正ラマンスペクトルの妥当性について検討した。
なお、合成ラマンスペクトルは下記の通りに作成した。
Next, the validity of the corrected Raman spectrum obtained by subtracting the Raman spectrum of the lipid contribution shown in c and d in FIG. 2 from the Raman spectrum of the skin sample measured under the above conditions, shown in a in FIG. The sex was examined.
The synthetic Raman spectrum was prepared as follows.
（１）ベースラインの蛍光補正
脱脂乾燥角層及び皮膚のラマンスペクトルでは蛍光によるベースラインのゆがみが認められた。そこで、２０００〜２８００ｃｍ-1および３８００〜４２００ｃｍ-1の波数領域のデータより、最小二乗法を用いた三次関数近似に基づきベースラインを予測し、これを差し引くことにより蛍光の影響を排除したラマンスペクトルに補正する。
(1) Baseline fluorescence correction Baseline distortion due to fluorescence was observed in the degreased dry stratum corneum and the Raman spectrum of the skin. Therefore, the Raman spectrum from the data of the wavenumber region of 2000～2800Cm -1 and 3800～4200Cm -1, predicting a baseline based on cubic function approximation using the least square method to eliminate the influence of fluorescence by subtracting this To correct.
（２）ラマンスペクトルの重ね合わせ
蛍光の影響を排除した補正後の脱脂乾燥角層のラマンスペクトルをＩSC(ω)、モデル皮脂のラマンスペクトルをＩSEB(ω)、モデル細胞間脂質のラマンスペクトルをＩCER(ω)、水のラマンスペクトルをＩWAT(ω)とし、これらを重ね合わせて算出されるラマンスペクトルを合成ラマンスペクトルＩCAL(ω)とする。ＩCAL(ω)は下記式（４）で表すことができる。
(2) Superposition of Raman spectra I SC (ω) for the corrected degreased dry stratum corneum excluding the influence of fluorescence, I SEB (ω) for the Raman spectrum of model sebum, and the Raman spectrum of model intercellular lipids Is I CER (ω), the Raman spectrum of water is I WAT (ω), and the Raman spectrum calculated by superimposing these is the combined Raman spectrum I CAL (ω). I CAL (ω) can be expressed by the following formula (4).
ここで式（４）における係数Ｃ1〜Ｃ4は、合成ラマンスペクトルの作成に用いた各ラマンスペクトルの寄与分に関するパラメーターである。
蛍光の影響を排除した補正後の皮膚のラマンスペクトルＩOBS(ω)を、上記合成スペクトルをＩCAL(ω)によりできるだけ良く再現できるように、２５００〜４０００ｃｍ-1の波数領域で、最小二乗法によって、パラメーターＣ1〜Ｃ4を決定した。なお各パラメーターの算出値が負の数となった場合は、そのパラメーターをゼロに固定した上で、再度最小二乗近似を行った。
具体的には、２５００〜４０００ｃｍ-1の各波数における合成ラマンスペクトルＩCAL(ω)と実測した皮膚のラマンスペクトルＩOBS(ω)との差の二乗の和が最小となるようにパラメーターＣ1〜Ｃ4を決定することにより、各成分の寄与分を決定した。
Here, the coefficients C 1 to C 4 in the equation (4) are parameters relating to the contribution of each Raman spectrum used to create the synthetic Raman spectrum.
The least squares method in the wavenumber region of 2500 to 4000 cm −1 so that the corrected Raman spectrum I OBS (ω) excluding the influence of fluorescence can be reproduced as well as possible by the above-mentioned synthetic spectrum by I CAL (ω). Determined the parameters C 1 to C 4 . In addition, when the calculated value of each parameter became a negative number, after fixing the parameter to zero, the least square approximation was performed again.
Specifically, the parameter C 1 is set so that the sum of the squares of the difference between the synthesized Raman spectrum I CAL (ω) at each wave number of 2500 to 4000 cm −1 and the actually measured skin Raman spectrum I OBS (ω) is minimized. by determining -C 4, it was determined the contribution of each component.
本発明における、皮膚試料のラマンスペクトルから脂質の寄与分を差し引いた補正ラマンスペクトルＩOBS―LIP(ω)は、下記式（５）で表すことができる。 The corrected Raman spectrum I OBS-LIP (ω) obtained by subtracting the lipid contribution from the Raman spectrum of the skin sample in the present invention can be expressed by the following formula (5).
さらに、皮膚試料のラマンスペクトルから、脂質の寄与分に加えて、水の寄与分も差し引いた補正ラマンスペクトルＩOBS―LIP-WAT（ω）は、下記式（６）で表すことができる。 Furthermore, the corrected Raman spectrum I OBS-LIP-WAT (ω) obtained by subtracting the contribution of water in addition to the contribution of lipid from the Raman spectrum of the skin sample can be expressed by the following formula (6).
このようにして得られた、補正ラマンスペクトルＩOBS―LIP(ω)及び補正ラマンスペクトルＩOBS―LIP-WAT（ω）を図２中ｆ及びｇに示す。 The corrected Raman spectrum I OBS-LIP (ω) and the corrected Raman spectrum I OBS-LIP-WAT (ω) thus obtained are shown in f and g in FIG.
図２から明らかなように、皮膚試料のラマンスペクトルから脂質及び水の寄与分を差し引いて得られた補正ラマンスペクトルｇのパターンは、脱脂乾燥角層のラマンスペクトルｂのパターンと非常によく類似していた。従って、上記工程により、皮膚試料に含まれる水分量を正確に反映した補正ラマンスペクトルが得られることが示された。 As is apparent from FIG. 2, the pattern of the corrected Raman spectrum g obtained by subtracting the lipid and water contributions from the Raman spectrum of the skin sample is very similar to the pattern of the Raman spectrum b of the defatted dry stratum corneum. It was. Therefore, it was shown that a corrected Raman spectrum that accurately reflects the amount of water contained in the skin sample can be obtained by the above process.
（試験例２）補正ラマンスペクトルの妥当性評価
レーザー光の焦点の位置を変化させた以外は試験例１と同様の条件で健常男性（４０歳代）の前腕の皮膚のラマンスペクトルを測定し、測定深度の変化に伴うラマンスペクトルの変化を調べた。結果を図３に示す。
(Test Example 2) Appropriate evaluation of the corrected Raman spectrum The Raman spectrum of the skin of the forearm of a healthy male (40s) was measured under the same conditions as in Test Example 1 except that the focal position of the laser beam was changed. The change of the Raman spectrum with the change of the measurement depth was investigated. The results are shown in FIG.
図３中、ｈは皮膚表面よりも１μｍ外側に照射光の焦点をあわせたときのラマンスペクトルである。この条件における深さ方向の空間分解能は１．８μｍなので、皮膚表面より１μｍ外側に焦点を合わせると皮膚からの信号が微弱ながら観測される。図３のｈに示すスペクトルでは、皮脂に由来するピーク（２８８０ｃｍ-1付近のショルダー形状のピーク）が観測された。
図３中、ｉ及びｊはそれぞれ皮膚表面からの深さ１μｍ及び３μｍの位置に照射光の焦点をあわせたときのラマンスペクトルであり、角層のラマンスペクトルに相当する。図３中、ｋは皮膚表面からの深さ１１μｍの位置に照射光の焦点をあわせたときのラマンスペクトルであり、ＣＨ伸縮振動のピーク面積（２８００〜３０００ｃｍ-1）に対するＯＨ伸縮振動のピーク面積（３１００〜３８００ｃｍ-1）が十分に大きいことから、顆粒層またはそれより深い領域を測定しているものと考えられる。
In FIG. 3, h is a Raman spectrum when the irradiation light is focused 1 μm outside the skin surface. Since the spatial resolution in the depth direction under this condition is 1.8 μm, when focusing on the outer side of the skin surface by 1 μm, the signal from the skin is observed while being weak. In the spectrum shown in h of FIG. 3, a peak derived from sebum (a shoulder-shaped peak near 2880 cm −1 ) was observed.
In FIG. 3, i and j are Raman spectra when the irradiation light is focused at positions of 1 μm and 3 μm in depth from the skin surface, respectively, and correspond to the Raman spectrum of the stratum corneum. In FIG. 3, k is a Raman spectrum when the irradiation light is focused at a depth of 11 μm from the skin surface, and the peak area of OH stretching vibration relative to the peak area of CH stretching vibration (2800 to 3000 cm −1 ). Since (3100-3800 cm −1 ) is sufficiently large, it is considered that the granular layer or a deeper region is measured.
次に、前記皮膚表面からの各距離におけるラマンスペクトルを測定し、試験例１と同じように脱脂乾燥角層、モデル皮脂、モデル細胞間脂質及び水（イオン交換水）のラマンスペクトルを用いて脂質の寄与分を決定し、皮膚試料のラマンスペクトルから脂質の寄与分を差し引いた補正ラマンスペクトルＩOBS―LIP(ω)並びに皮膚試料のラマンスペクトルから脂質及び水の寄与分も差し引いた補正ラマンスペクトルＩOBS―LIP-WAT（ω）を作成した。その結果を図４（ａ）〜（ｄ）に示す。なお、図４（ａ）は皮膚表面外側１μｍにおける前腕の皮膚のラマンスペクトルを示し、図４（ｂ）は皮膚表面から深さ１μｍにおける前腕の皮膚のラマンスペクトルを示し、図４（ｃ）は皮膚表面から深さ３μｍにおける前腕の皮膚のラマンスペクトルを示し、図４（ｄ）は皮膚表面から深さ１１μｍにおける前腕の皮膚のラマンスペクトルを示す。また、図４（ａ）〜（ｄ）中、ａは皮膚試料の実測のラマンスペクトルを示す。また、図４（ａ）〜（ｄ）中、ｂ〜ｅは、式（４）を用いてａのスペクトルを再現した際の、各素スペクトルの寄与分に相当する。即ちｂは脱脂乾燥皮膚試料のラマンスペクトルの寄与分を示し、ｃはモデル皮脂のラマンスペクトルの寄与分を示し、ｄはモデル細胞間脂質のラマンスペクトルの寄与分を示し、ｅは水のラマンスペクトルの寄与分を示す。また、図４（ａ）〜（ｄ）中、ｆは前記ａの皮膚試料のラマンスペクトルから脂質の寄与分（前記ｃ及びｄの各スペクトル）を差し引いた補正ラマンスペクトルを示し、ｇは前記ａの皮膚試料のラマンスペクトルから脂質の寄与分（前記ｃ及びｄの各スペクトル）及び水の寄与分（前記ｅのスペクトル）を差し引いた補正ラマンスペクトルを示す。 Next, the Raman spectrum at each distance from the skin surface is measured, and the lipid is obtained using the Raman spectrum of degreased and dried stratum corneum, model sebum, model intercellular lipid and water (ion-exchanged water) as in Test Example 1. of determining the contribution, corrected Raman spectra I OBS-LIP (ω) as well as correction Raman spectrum I in which skin samples from the Raman spectra by subtracting also the contribution of the lipid and water minus the contribution of lipid from the Raman spectra of skin samples OBS-LIP-WAT (ω) was created. The results are shown in FIGS. 4A shows the Raman spectrum of the forearm skin at 1 μm outside the skin surface, FIG. 4B shows the Raman spectrum of the forearm skin at a depth of 1 μm from the skin surface, and FIG. FIG. 4D shows the Raman spectrum of the forearm skin at a depth of 3 μm from the skin surface, and FIG. 4D shows the Raman spectrum of the forearm skin at a depth of 11 μm from the skin surface. Further, in FIGS. 4A to 4D, “a” represents an actually measured Raman spectrum of the skin sample. 4A to 4D, b to e correspond to contributions of the respective elementary spectra when the spectrum of a is reproduced using the equation (4). That is, b represents the contribution of the Raman spectrum of the defatted dry skin sample, c represents the contribution of the Raman spectrum of the model sebum, d represents the contribution of the Raman spectrum of the model intercellular lipid, and e represents the Raman spectrum of water. The contribution of 4 (a) to 4 (d), f represents a corrected Raman spectrum obtained by subtracting the lipid contribution (each spectrum of c and d) from the Raman spectrum of the skin sample of a, and g represents the a. 5 shows a corrected Raman spectrum obtained by subtracting the contribution of lipid (each spectrum of c and d) and the contribution of water (spectrum of e) from the Raman spectrum of the skin sample.
図４（ａ）〜（ｄ）から明らかなように、皮膚試料のラマンスペクトルから脂質及び水の寄与分を差し引いて得られた補正ラマンスペクトルｇのパターンは、脱脂乾燥角層のラマンスペクトルｂのパターンと非常によく類似していた。さらに、図４（ａ）〜（ｄ）から、皮膚表面からの距離によって、ラマンスペクトルにおける脂質（細胞間脂質及び皮脂）由来の信号強度が異なっていることがわかる。従って、上記工程により、ラマンスペクトルにおける脂質の寄与分を考慮して差し引くことで、皮膚試料に含まれる水分量を正確に反映した補正ラマンスペクトルが得られることが示された。 As is clear from FIGS. 4A to 4D, the pattern of the corrected Raman spectrum g obtained by subtracting the contribution of lipid and water from the Raman spectrum of the skin sample is the Raman spectrum b of the degreased dried stratum corneum. It was very similar to the pattern. Furthermore, it can be seen from FIGS. 4A to 4D that the signal intensity derived from lipids (intercellular lipids and sebum) in the Raman spectrum differs depending on the distance from the skin surface. Therefore, it was shown that a corrected Raman spectrum that accurately reflects the amount of water contained in the skin sample can be obtained by subtracting the contribution of lipid in the Raman spectrum by the above process.
また、図４の結果から、皮膚表面の外側のラマンスペクトルではモデル皮脂の寄与分が認められるのに対し、皮膚内部（深さ１、３、１１μｍ）のラマンスペクトルではモデル皮脂の寄与分が認められなかった（寄与分がゼロと算出された）。この結果は、皮膚内部の脂質が液体脂ではなく、一定の構造を有する固体脂であることと一致する。
また、図４の結果は、角層に相当する深さ１μｍ及び３μｍのラマンスペクトルではモデル細胞間脂質の寄与分が大きいのに対し、顆粒層以下に相当する深さ１１μｍのラマンスペクトルではモデル細胞間脂質の寄与分が小さいことを示している。この結果は、角層内には細胞間脂質の量が多く存在するのに対し、顆粒層以下では細胞間脂質はほとんど存在していないことと一致する。
In addition, from the results shown in FIG. 4, the contribution of model sebum is recognized in the Raman spectrum outside the skin surface, whereas the contribution of model sebum is recognized in the Raman spectrum inside the skin (
In addition, the results of FIG. 4 show that the contribution of model intercellular lipids is large in the 1 μm and 3 μm deep Raman spectra corresponding to the stratum corneum, while the model cell is 11 μm deep in the Raman spectrum corresponding to the granular layer and below. It shows that the contribution of interlipid is small. This result is consistent with the fact that there is a large amount of intercellular lipid in the stratum corneum, whereas there is almost no intercellular lipid below the granular layer.
実施例
（１）検量線の作成
健常男性（４０歳代）のかかとより角層片（３．０〜５．５ｍｇ）をナイフで切除後、クロロホルム−メタノール（１：１）液に一昼夜浸漬した。その後角層片試料を取り出し、自然乾燥後、イオン交換水に一昼夜浸漬した。その後角層片試料を取り出し、自然乾燥させることで、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脱脂乾燥角層を調製した。
Example (1) Preparation of calibration curve After cutting a horny layer piece (3.0-5.5 mg) with a knife from the heel of a healthy male (40's), it was immersed in chloroform-methanol (1: 1) solution overnight. . Thereafter, the sample of the stratum corneum was taken out, naturally dried, and then immersed in ion exchange water for a whole day and night. Thereafter, the stratum corneum piece sample was taken out and naturally dried to prepare a degreased and dried stratum corneum from which oil-soluble components such as lipids and water-soluble components such as amino acids were removed.
前記脱脂乾燥角層を調湿剤の入ったガラスボトムディッシュに一昼夜以上放置後、ガラスボトムディッシュに入れたまま、下記調湿剤を用いた調湿環境下でのラマンスペクトルの測定を行った。
調湿剤１：五酸化りん（関東化学、特級、乾燥状態で使用）
調湿剤２：シリカゲル（関東化学、乾燥用、乾燥状態で使用）
調湿剤３：塩化ナトリウム（和光純薬、特級、飽和水溶液で使用、平衡湿度75%R.H.）
調湿剤４：臭化カリウム（スペクトラテック、KBｒ錠剤用、平衡湿度84%R.H.）
調湿剤５：硫酸ナトリウム（関東化学、特級、飽和水溶液で使用、平衡湿度93%R.H.）
調湿剤６：リン酸水素二ナトリウム（和光純薬、特級、飽和水溶液で使用、平衡湿度95%R.H.）
The degreased and dried stratum corneum was allowed to stand in a glass bottom dish containing a humidity control agent for more than one day and night, and the Raman spectrum was measured in a humidity control environment using the following humidity control agent while being left in the glass bottom dish.
Humidifier 1: Phosphorus pentoxide (Kanto Chemical, special grade, used in dry condition)
Humidifier 2: Silica gel (Kanto Chemical, for drying, used in a dry state)
Conditioner 3: Sodium chloride (Wako Pure Chemicals, special grade, used in saturated aqueous solution, equilibrium humidity 75% RH)
Conditioning agent 4: Potassium bromide (Spectratech, for KBr tablets, equilibrium humidity 84% RH)
Humidifier 5: Sodium sulfate (Kanto Chemical, special grade, used in saturated aqueous solution, equilibrium humidity 93% RH)
Humidifier 6: Disodium hydrogen phosphate (Wako Pure Chemicals, special grade, used in saturated aqueous solution, equilibrium humidity 95% RH)
ラマンスペクトル測定後、調湿環境下で平衡化した脱脂角層をガラスボトムデッシュより取り出し、速やかに精密天秤を用いて重量を測定した。五酸化りん平衡時の角層の含水量をゼロとみなし、かつ上記脱脂角層は水とタンパク質の２成分で近似できるとみなし、各湿度平衡時の水とタンパク質の質量比ＲH2O/proteinを式（７）を用いて算出した。 After the Raman spectrum measurement, the degreased horny layer equilibrated in the humidity-controlled environment was taken out from the glass bottom dish, and the weight was quickly measured using a precision balance. The water content of the stratum corneum at the time of phosphorus pentoxide equilibrium is considered to be zero, and the degreasing stratum corneum is considered to be approximated by two components of water and protein, and the mass ratio R H2O / protein of water and protein at each humidity equilibrium is determined. It calculated using Formula (7).
式（７）において、ＲH2O/proteinは湿度Ｈにおける含水率を表し、Ｗ(Ｈ)は湿度Ｈにおける角層重量を表し、Ｗdryは五酸化りん平衡時の角層重量を表す。 In formula (7), R H2O / protein represents the moisture content at humidity H, W (H) represents the stratum corneum weight at humidity H, and W dry represents the stratum corneum weight at the time of phosphorus pentoxide equilibrium.
また、上記調湿角層より得られたラマンスペクトルの、タンパク質の信号強度Ｉprotein及びＯＨの信号強度ＩOHを、前記式（２）及び式（３）より算出した。次に、タンパク質の信号強度Ｉprotein及びＯＨの信号強度ＩOHの信号強度比Ｉ（OH/CH）を式（８）を用いて算出した。 Further, the signal intensity I protein of protein and the signal intensity I OH of OH in the Raman spectrum obtained from the humidity control layer were calculated from the above formulas (2) and (3). Next, the signal intensity ratio I (OH / CH) of the signal intensity I protein of protein and the signal intensity I OH of OH was calculated using equation (8).
以上のようにして算出した、各湿度平衡時の水とタンパク質の質量比ＲH2O/proteinと、タンパク質の信号強度Ｉprotein及びＯＨの信号強度ＩOHの信号強度比Ｉ（OH/CH）との関係を図５に示す。なお図５中の直線のＸ軸との切片は、便宜上ＯＨのみに由来するピークとみなした3100〜3750cm-1の領域における、タンパク質のNH伸縮振動の寄与分に相当する。 The mass ratio R H2O / protein of water and protein at each humidity equilibrium and the signal intensity ratio I (OH / CH) of the protein signal intensity I protein and OH signal intensity I OH calculated as described above. The relationship is shown in FIG. In addition, the intercept with the X-axis of the straight line in FIG. 5 is equivalent to the contribution of NH stretching vibration of protein in the region of 3100 to 3750 cm −1 regarded as a peak derived from OH only for convenience.
（２）ラマンスペクトルの測定
健常女性（２０〜３０歳代）６名の前腕のラマンスペクトルを皮膚表面からの深さ方向の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 Ｎａｎｏｆｉｎｄｅｒ３０（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：３ｓ／１点
ピンホール径：１５０μｍ
回折格子：１５０ｇｒ／ｍｍ
深さ方向のスキャン間隔：２μｍ
合計測定点数：２１点
総積算時間：約１分
(2) Measurement of Raman spectrum The Raman spectrum of the forearm of 6 healthy women (20-30 years old) was measured by continuous scanning in the depth direction from the skin surface. The Raman spectrum measurement conditions are as follows.
<Measurement conditions>
Apparatus: Microscopic Raman spectrometer Nanofinder 30 (trade name, manufactured by Tokyo Instruments)
Objective lens: 100 ×, NA1.3
Laser: 633 nm, 8 mW (on sample)
Integration time: 3 s / 1 point Pinhole diameter: 150 μm
Diffraction grating: 150 gr / mm
Depth scan interval: 2 μm
Total measurement points: 21 points Total integration time: Approximately 1 minute
（３）ラマンスペクトルにおける、皮膚試料の脂質の寄与分の算出
上記の１２６スペクトル（２１スペクトル×６名）に対して、試験例１と同様にベースラインの蛍光補正を行った。次にこの蛍光補正後のスペクトルを、前記式（４）を用いて４つのラマンスペクトル（脱脂乾燥角層、モデル皮脂、モデル細胞間脂質、水）の重ねあわせで表現することを試みた。ここでの４種のラマンスペクトルを得た測定試料、測定条件は試験例１と同一である。最小二乗法により前記式（４）における係数Ｃ1〜Ｃ4を決定した。このときＣ2ＩSEB（ω）が皮脂の寄与分、Ｃ3ＩCER（ω）が細胞間脂質の寄与分となる。
(3) Calculation of contribution of lipid in skin sample in Raman spectrum Baseline fluorescence correction was performed in the same manner as in Test Example 1 on the above 126 spectrum (21 spectra × 6 names). Next, an attempt was made to express this fluorescence-corrected spectrum by superimposing four Raman spectra (defatted and dried stratum corneum, model sebum, model intercellular lipid, and water) using the above equation (4). The measurement samples and measurement conditions obtained from the four types of Raman spectra here are the same as those in Test Example 1. The coefficients C 1 to C 4 in the above equation (4) were determined by the least square method. At this time, C 2 I SEB (ω) is the contribution of sebum, and C 3 I CER (ω) is the contribution of the intercellular lipid.
（４）補正ラマンスペクトルの作成
前記（２）で得られた皮膚表面からの深さ方向の連続スキャンで測定したラマンスペクトルから、前記（３）で得られた各皮膚表面からの深さにおける脂質の寄与分Ｃ2ＩSEB（ω）とＣ3ＩCER（ω）を差し引いて、補正ラマンスペクトルを作成した。
(4) Preparation of corrected Raman spectrum Lipid at depth from each skin surface obtained in (3) from Raman spectrum measured by continuous scanning in depth direction from skin surface obtained in (2) C 2 I SEB (ω) and C 3 I CER (ω) were subtracted from each other to create a corrected Raman spectrum.
（５）水分量の測定
前記（４）で得られた補正ラマンスペクトルにおけるＩprotein（波数領域：２８００〜３０３０ｃｍ-1）に対するＩOH（波数領域：３１００〜３７５０ｃｍ-1）の比率を算出した。このように算出した比率から、前記（１）で作成した検量線に基づいて皮膚の水分量（質量％）を算出した。その結果を図６の曲線Ａに示す。また前記（４）に示す補正ラマンスペクトルを作成せずにＩproteinとＩOHを算出し、前記（１）で作成した検量線に基づいて皮膚の水分量（質量％）を算出した。その結果を図６の曲線Ｂに併せて示す。
(5) water content measuring the (4) I protein (wavenumber region: 2800~3030cm -1) in the obtained corrected Raman spectra I OH (wavenumber region: 3100~3750cm -1) for was calculated ratio of. From the ratio calculated in this manner, the moisture content (% by mass) of the skin was calculated based on the calibration curve created in (1) above. The result is shown by curve A in FIG. Further, I protein and I OH were calculated without preparing the corrected Raman spectrum shown in (4), and the moisture content (mass%) of the skin was calculated based on the calibration curve prepared in (1). The results are shown together with curve B in FIG.
図６の結果から、曲線Ａ、Ｂ共に、皮膚表面から深部に向かって水分量が上昇し、皮膚表面からの深さが１０〜１５μｍ以下の領域で７０％程度の一定値に達している。一般にこの水分上昇が見られる領域が角層領域、水分が一定値を示す領域が顆粒層以下の領域と考えられている。一般に角層では水分等の喪失や刺激物質の侵入を防ぐために、細胞間に細胞間脂質と呼ばれる脂質層が出現する。
脂質の寄与分を補正していない曲線Ｂでは、タンパク質の信号を過剰に見積もっているために、いずれの被験者の場合でも水分量が過少に見積もられてしまう。そのため角層領域では、曲線Ｂは脂質補正後の曲線Ａよりも低値を示している。一方、角層以下の領域では細胞間脂質層が形成されていないため、角層よりも脂質量は十分に少ない。そのため皮膚表面からの深さが１０〜１５μｍ以下の領域では、脂質の寄与分の補正の有無に関わらず、水分量は７０％程度を示したと考えられる。以上より、脂質の寄与分の補正により得られた水分プロファイルは、皮膚構造に関する既知の知見と合致した。
From the result of FIG. 6, in both curves A and B, the amount of water increases from the skin surface toward the deep part, and reaches a constant value of about 70% in the region where the depth from the skin surface is 10 to 15 μm or less. In general, it is considered that the region where the water increase is observed is the stratum corneum region, and the region where the water has a constant value is the region below the granular layer. In general, in the stratum corneum, a lipid layer called intercellular lipid appears between cells in order to prevent loss of moisture and the like and invasion of stimulating substances.
In curve B in which the lipid contribution is not corrected, the protein signal is overestimated, so the water content is underestimated in any subject. Therefore, in the stratum corneum region, curve B shows a lower value than curve A after lipid correction. On the other hand, since the intercellular lipid layer is not formed in the region below the stratum corneum, the amount of lipid is sufficiently smaller than that of the stratum corneum. Therefore, in the region where the depth from the skin surface is 10 to 15 μm or less, it is considered that the water content showed about 70% regardless of whether or not the lipid contribution was corrected. From the above, the moisture profile obtained by correcting the lipid contribution was consistent with the known knowledge about the skin structure.
Claims (5)
皮膚試料のラマンスペクトルを測定する工程、
前記の皮膚試料のラマンスペクトルから、前記少なくとも１種のモデル脂質のラマンスペクトルにおける該モデル脂質の寄与分を差し引いた補正ラマンスペクトルを得る工程、及び
前記補正ラマンスペクトルにおいて、タンパク質のＣＨ伸縮振動由来の信号強度と水のＯＨ伸縮振動由来の信号強度との比率に基づき皮膚水分量を測定する工程
を含む、皮膚水分量の測定方法。 Measuring a Raman spectrum of at least one model lipid;
Measuring the Raman spectrum of the skin sample;
Obtaining a corrected Raman spectrum obtained by subtracting the contribution of the model lipid in the Raman spectrum of the at least one model lipid from the Raman spectrum of the skin sample; and in the corrected Raman spectrum, derived from the CH stretching vibration of the protein. A method for measuring skin moisture content, comprising a step of measuring skin moisture content based on a ratio between signal strength and signal strength derived from water OH stretching vibration.
The measurement method according to claim 4, wherein the signal intensity derived from the CH stretching vibration of the protein is a peak area in a wave number width of at least 100 cm −1 including a maximum peak of the signal of the CH stretching vibration derived from the protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010196879A JP5460528B2 (en) | 2010-09-02 | 2010-09-02 | Method for measuring skin moisture content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010196879A JP5460528B2 (en) | 2010-09-02 | 2010-09-02 | Method for measuring skin moisture content |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012050739A JP2012050739A (en) | 2012-03-15 |
| JP5460528B2 true JP5460528B2 (en) | 2014-04-02 |
Family
ID=45904802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2010196879A Active JP5460528B2 (en) | 2010-09-02 | 2010-09-02 | Method for measuring skin moisture content |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5460528B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10548517B2 (en) | 2014-11-07 | 2020-02-04 | Samsung Electronics Co., Ltd. | Spectroscopic apparatus for biological material and spectroscopic method using the same |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6128549B2 (en) * | 2013-03-19 | 2017-05-17 | 花王株式会社 | Skin age evaluation method and cosmetic evaluation method |
| KR101951758B1 (en) * | 2017-11-17 | 2019-02-25 | 광주과학기술원 | Method for quantitative analysis of pork lard in a fat mixture using raman spectroscopy and apparatus for the same |
| JP7025641B2 (en) * | 2018-03-28 | 2022-02-25 | アルケア株式会社 | An arithmetic processing device for evaluating skin moisture content, a program, an electronic device equipped with the arithmetic processing apparatus, and a method for evaluating skin moisture content. |
| CN109171662A (en) * | 2018-10-10 | 2019-01-11 | 河南农业大学 | The detector of moisture content of skin and the detection method of moisture content of skin |
| CN114166819A (en) * | 2021-11-26 | 2022-03-11 | 中南林业科技大学 | Method for measuring water content of wood cell wall based on Raman spectrum technology |
| 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 |
| CN114532987B (en) * | 2022-02-16 | 2023-12-19 | 北京市神经外科研究所 | Information processing method and device based on Raman spectrum and storage medium |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005111103A (en) * | 2003-10-10 | 2005-04-28 | Pola Chem Ind Inc | Method for identifying tendency of occurrence of comedo |
| JP4657175B2 (en) * | 2006-08-28 | 2011-03-23 | 花王株式会社 | Measuring method of protein fiber retention |
| JP5068555B2 (en) * | 2007-02-06 | 2012-11-07 | 株式会社 資生堂 | Beauty method evaluation method |
| JP4679596B2 (en) * | 2008-02-27 | 2011-04-27 | 花王株式会社 | Method for measuring moisture content in hair |
| JP2009244259A (en) * | 2008-03-12 | 2009-10-22 | Kao Corp | Liquid water content technique and contact agent |
-
2010
- 2010-09-02 JP JP2010196879A patent/JP5460528B2/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10548517B2 (en) | 2014-11-07 | 2020-02-04 | Samsung Electronics Co., Ltd. | Spectroscopic apparatus for biological material and spectroscopic method using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2012050739A (en) | 2012-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5460528B2 (en) | Method for measuring skin moisture content | |
| Mandurah et al. | Monitoring remineralization of enamel subsurface lesions by optical coherence tomography | |
| Dąbrowska et al. | In vivo confirmation of hydration-induced changes in human-skin thickness, roughness and interaction with the environment | |
| Caspers et al. | In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles | |
| WO2001051094A1 (en) | Diagnostic method using a deuterated agent for imaging specific tissue | |
| JP2014209094A (en) | Information acquisition device and information acquisition method for acquiring information on sample using terahertz wave | |
| Cals et al. | Raman spectroscopic analysis of the molecular composition of oral cavity squamous cell carcinoma and healthy tongue tissue | |
| Wang et al. | Monte Carlo simulation of in vivo Raman spectral Measurements of human skin with a multi‐layered tissue optical model | |
| Chuchuen et al. | Quantitative analysis of microbicide concentrations in fluids, gels and tissues using confocal Raman spectroscopy | |
| CN104067313B (en) | Imaging device | |
| US20090326359A1 (en) | Method of in vivo detection and/or diagnosis of cancer using fluorescence based dna image cytometry | |
| Sowa et al. | Precision of Raman depolarization and optical attenuation measurements of sound tooth enamel | |
| EP2689234A1 (en) | Method to determine the absorption coefficient in turbid media | |
| Tfayli et al. | Confocal Raman microspectroscopy on excised human skin: uncertainties in depth profiling and mathematical correction applied to dermatological drug permeation | |
| WO2000020843A1 (en) | Method and apparatus for measuring locally and superficially the scattering and absorption properties of turbid media | |
| JP4679596B2 (en) | Method for measuring moisture content in hair | |
| US20190343394A1 (en) | Cartilage-tissue analysis device | |
| JP2009244259A (en) | Liquid water content technique and contact agent | |
| JP5706226B2 (en) | Evaluation method for collagen state in skin and evaluation method for skin aging | |
| Tfaili et al. | Shedding light on the laser wavelength effect in Raman analysis of skin epidermises | |
| JP4657175B2 (en) | Measuring method of protein fiber retention | |
| Tai et al. | Illumination and fluorescence collection volumes for fiber optic probes in tissue | |
| JP2010210401A (en) | Method for measuring lipid quantity and contact agent | |
| Ando et al. | Imaging of demineralized enamel in intact tooth by epidetected stimulated Raman scattering microscopy | |
| JP5288912B2 (en) | Method for measuring skin moisture content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130611 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20131212 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20131217 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140114 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 5460528 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |