JP4261296B2 - Noninvasive determination of collagen - Google Patents
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Description
本発明は、コラーゲンの存在量の定量法に関し、更に詳細には、動物の皮膚の真皮コラーゲンの存在量の定量法に関する。 The present invention relates to a method for quantifying the amount of collagen present, and more particularly to a method for quantifying the amount of dermal collagen present in animal skin.
コラーゲンは、広く食品、飲料、化粧料などの皮膚外用剤に使用される原料であり、その定量は、コラーゲンがアミノ酸の連なった、天然の水溶性高分子と言う特質と、構成するアミノ酸組成などに様々なバリエーションが存する現状から、侵襲的な方法であっても、非常に困難であることが知られている。その一方、皮下に於ける真皮コラーゲン量は、加齢、光老化などにより大きく変わることが知られており、しわの発生可能性も真皮コラーゲン量を的確に測定できれば、かなり早期に検知することが可能であると推測されている。この様な老化モニタリングや、老化の予防措置実施の為にも、皮膚の真皮コラーゲンを測定することは意義深いことであり、該測定には、非侵襲的な方法が望まれている。生体関連のコラーゲンの測定技術としては、例えば、体液中のテロペプチドを定量して、生体組織でのコラーゲンの分解量を鑑別する方法（例えば、特許文献１、特許文献２を参照）、皮膚に近赤外線を照射し、それによって励起させて、コラーゲンを発光させ、その度合いより皮膚癌を鑑別する方法（例えば、特許文献３を参照）、皮膚に近赤外線を照射し、それによって皮膚温を上昇させ、その上昇の程度より、真皮コラーゲン量を定量する方法(例えば、特許文献４を参照)、６００〜８００ｎｍの光と、８００〜１０００ｎｍの２波長の光を照射し、その反射光の比より、真皮コラーゲン量を測定する方法（例えば、特許文献５を参照）などが既に知られているが、コラーゲンの存在状態と近似の環境下において、コラーゲンの存在量の明らかな標準検体を作成し、該標準サンプルの近赤外吸収スペクトルを計測し、計測した近赤外吸収スペクトルデータを統計的に解析し、回帰式を得、該回帰式を用い、コラーゲンの存在量を定量すべき検体より計測されて得た近赤外吸収スペクトルデータから算出する、真皮コラーゲンの存在量の定量法は未だ知られていないし、この様な測定法により、皮膚真皮コラーゲン量を、非侵襲的に、正確に測定できることも全く知られていない。 Collagen is a raw material that is widely used in skin preparations such as foods, beverages, and cosmetics. The amount of collagen is a natural water-soluble polymer in which collagen is linked to amino acids and the amino acid composition of the constituents. However, it is known that even an invasive method is very difficult. On the other hand, the amount of dermal collagen under the skin is known to change significantly with aging, photoaging, etc., and the possibility of wrinkles can be detected very early if the amount of dermal collagen can be accurately measured. It is speculated that it is possible. It is significant to measure dermal collagen of the skin for such aging monitoring and aging prevention measures, and a non-invasive method is desired for the measurement. Examples of biological-related collagen measurement techniques include a method of quantifying telopeptides in body fluids and distinguishing the amount of collagen degradation in biological tissues (see, for example, Patent Document 1 and Patent Document 2), A method of irradiating near-infrared rays, thereby exciting them to emit collagen and distinguishing skin cancer based on the degree (see, for example, Patent Document 3), irradiating the skin with near-infrared rays, thereby raising the skin temperature The amount of dermal collagen is quantified based on the degree of the rise (see, for example, Patent Document 4), light of 600 to 800 nm and light of two wavelengths of 800 to 1000 nm are irradiated, and the ratio of the reflected light the method of measuring the dermis collagen content (e.g., Patent Document 5 reference), but like already known, in an environment of approximation with the presence state of collagen, abundance of collagen Create a clear standard sample, measure the near-infrared absorption spectrum of the standard sample, statistically analyze the measured near-infrared absorption spectrum data, obtain a regression equation, and use the regression equation to determine the presence of collagen There is not yet known a method for quantifying the abundance of dermal collagen, which is calculated from the near-infrared absorption spectrum data obtained by measuring from the sample whose amount is to be quantified. It is not known at all that it can measure accurately non-invasively.
本発明は、この様な状況下為されたものであり、非侵襲的なコラーゲンの存在量の定量法に関し、更に詳細には、しわの進度予測などに有用な皮膚真皮コラーゲンの存在量の定量に好適な、非侵襲的なコラーゲンの存在量の定量法を提供することを課題とする。 The present invention has been made under such circumstances, and relates to a non-invasive method for quantitatively determining the abundance of collagen, and more specifically, the abundance of cutaneous dermal collagen useful for predicting the progress of wrinkles. It is an object of the present invention to provide a noninvasive method for quantitatively determining the abundance of collagen.
この様な状況に鑑みて、本発明者らは、非侵襲的なコラーゲンの存在量の定量法を求めて、鋭意研究努力を重ねた結果、人以外の動物の皮膚を脱脂処理した後、制御された量のコラーゲンを添加し、ホモジナイズすることにより作製されたコラーゲンの存在量の明らかな標準検体について、５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルを計測し、
計測した近赤外吸収スペクトルデータを、ＰＬＳ分析又は主成分分析により解析し、
コラーゲンの存在量と関係する因子を決定することにより得られた回帰式に、
被験動物の皮膚真皮の５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルの測定値を代入する方法により、被験動物の皮膚真皮のコラーゲンの存在量が非侵襲的に定量出来ることを見出し、発明を完成させるに至った。即ち、本発明は、以下に示す技術に関するものである。
（１）皮膚真皮のコラーゲンの存在量を定量するための回帰式を得る方法であって、人以外の動物の皮膚を脱脂処理した後、制御された量のコラーゲンを添加し、ホモジナイズすることにより作製されたコラーゲンの存在量の明らかな標準検体について、５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルを計測し、計測した近赤外吸収スペクトルデータを、ＰＬＳ分析又は主成分分析により解析し、コラーゲンの存在量と関係する因子を決定することを特徴とする、方法。
（２）前記近赤外吸収スペクトルが、フーリエ変換近赤外吸収スペクトルであることを特徴とする、（１）に記載の方法。
（３）前記近赤外吸収スペクトルデータを、ＳＮＶ処理、二次微分、及び平均化処理することを特徴とする、（１）又は（２）に記載の方法。
（４）被験動物の皮膚真皮のコラーゲンの存在量を定量する方法であって、（１）〜（３）の何れかに記載の方法により得られた回帰式に、被験動物の皮膚真皮の５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルデータを代入することを特徴とする、方法。
（５）被験動物が人であることを特徴とする、（４）に記載の方法。
In view of such a situation, the present inventors sought a non-invasive method for quantifying the amount of collagen present, and as a result of intensive research efforts, after defatting the skin of animals other than humans, For a standard sample with an apparent amount of collagen produced by adding and homogenizing the amount of collagen, a near-infrared absorption spectrum in the wavelength region of 5000 to 7000 cm −1 is measured,
Analyze the measured near infrared absorption spectrum data by PLS analysis or principal component analysis,
In the regression equation obtained by determining the factors related to the abundance of collagen,
By substituting the measured value of the near-infrared absorption spectrum of the skin dermis of the test animal in the wavelength region of 5000 to 7000 cm −1 , it was found that the abundance of collagen in the skin dermis of the test animal can be quantified non-invasively, The invention has been completed. That is, this invention relates to the technique shown below.
(1) A method for obtaining a regression equation for quantifying the abundance of collagen in the skin dermis by degreasing the skin of an animal other than a human, and then adding a controlled amount of collagen and homogenizing it. for obvious reference specimen abundance of the prepared collagen, and measuring the near infrared absorption spectrum in the wavelength region of 5000～7000Cm -1, near-infrared absorption spectrum data measured by PLS analysis or principal component analysis Analyzing and determining factors related to collagen abundance.
(2) The method according to (1), wherein the near infrared absorption spectrum is a Fourier transform near infrared absorption spectrum.
(3) The method according to (1) or (2), wherein the near-infrared absorption spectrum data is subjected to SNV processing, second-order differentiation, and averaging processing.
(4) A method for quantifying the abundance of collagen in the skin dermis of a test animal, wherein the regression equation obtained by the method according to any one of (1) to ( 3 ) is used to calculate 5000 of the skin dermis of the test animal. Substituting near-infrared absorption spectrum data in a wavelength region of ˜7000 cm −1 .
(5) The method according to ( 4 ), wherein the test animal is a human.
本発明によれば、非侵襲的なコラーゲンの存在量の定量法を提供することが出来る。 According to the present invention, it is possible to provide a non-invasive method for quantifying the abundance of collagen.
本発明のコラーゲンの存在量の定量法は、コラーゲンの存在量を定量する方法であって、コラーゲンの存在状態と近似の環境下において、コラーゲンの存在量の明らかな標準検体を作成し、該標準サンプルの近赤外吸収スペクトルを計測し、計測した近赤外吸収スペクトルデータを統計的に解析し、回帰式を得、該回帰式を用い、コラーゲンの存在量を定
量すべき検体より計測されて得た近赤外吸収スペクトルデータから算出することを特徴する。かかる方法で定量される対象となるコラーゲンとしては、コラーゲンが一様に存在している対象であれば特段の限定はされず、例えば、皮膚など生体に存在するコラーゲン、飲料、錠剤、化粧料など人工的に製造されたコラーゲン含有組成物等が好ましく例示できる。特に好ましいものは、非侵襲的にコラーゲンを測定する必要の高い対象であり、生体に於ける皮膚のコラーゲンなどが特に好ましく例示できる。
The method for quantifying the abundance of collagen according to the present invention is a method for quantifying the abundance of collagen, wherein a standard specimen with a clear abundance of collagen is prepared in an environment similar to the presence of collagen. Measure the near-infrared absorption spectrum of the sample, statistically analyze the measured near-infrared absorption spectrum data, obtain a regression equation, and use the regression equation to measure the amount of collagen present from the specimen to be quantified. It is characterized by calculating from the obtained near-infrared absorption spectrum data. The collagen of interest to be quantified in such a way, if the subject collagen exists uniformly particular limitation is not the sole, such as collagen present in a biological and skin, beverages, tablets, cosmetics, etc. A collagen-containing composition produced artificially can be preferably exemplified. Particularly preferred is a subject that needs to measure collagen non-invasively, and skin collagen in a living body can be particularly preferably exemplified.
本発明の定量法に於ける、標準検体としては、測定試料と類似の環境にあるコラーゲンであって、その存在量が明確なものを用いることが好ましい。また、かかるコラーゲンの存在量の明確な標準検体としては、コラーゲンの存在量の異なる数種類のものを用意することが好ましい。これは、コラーゲンの存在量と、近赤外吸収スペクトルの吸収ピークの強度の回帰を向上させるためである。かかる標準検体の数としては、１０以上が好ましく、２０以上が特に好ましい。また、再現性を見る意味で、ほぼ同一の存在量に調整した標準検体を複数用意することも好ましい。例えば、皮膚など生体に存在するコラーゲンであれば、同一乃至は別種の動物の皮膚などをエーテル等を用いて脱脂した後に、濃度の異なる数種のコラーゲンなどを添加し、ホモジナイズすることで良く拡散させ、しかる後に、近赤外吸収スペクトルを計測し、コラーゲン濃度と吸収率より、回帰式を求める様な方法が例示でき、飲料、錠剤、化粧料など人工的に製造されたコラーゲン含有組成物の場合には、同様にコラーゲン含有量の異なる組成物を作成し、これの近赤外吸収スペクトルを計測し、回帰式を求めるような方法が好ましく例示できる As the standard sample in the quantification method of the present invention, it is preferable to use collagen that is in an environment similar to the measurement sample and has a clear abundance. Moreover, it is preferable to prepare several types of standard specimens with a clear amount of collagen present, with different amounts of collagen present. This is to improve the regression of the abundance of collagen and the intensity of the absorption peak of the near infrared absorption spectrum. The number of such standard samples is preferably 10 or more, particularly preferably 20 or more. In view of reproducibility, it is also preferable to prepare a plurality of standard samples adjusted to almost the same abundance. For example, in the case of collagen existing in a living body such as skin, it is possible to diffuse well by defatting the skin of the same or different animal using ether etc. and then adding several types of collagen with different concentrations and homogenizing. After that, it is possible to exemplify a method for measuring a near infrared absorption spectrum and obtaining a regression equation from the collagen concentration and absorption rate. For example, beverages, tablets, cosmetics and other collagen-containing compositions produced artificially. In this case, a method in which compositions having different collagen contents are similarly prepared, the near infrared absorption spectrum thereof is measured, and a regression equation is obtained can be preferably exemplified.
この様な測定に使用される近赤外吸収スペクトルの波長領域としては、４０００〜１００００ｃｍ−１が好ましく例示でき、更に好ましくは５０００〜７０００ｃｍ−１である。これは、この波長域に、コラーゲンの特異吸収が多く存するからである。又、近赤外吸収スペクトルとしては、ダイオードアレーでも、フーリエ変換吸収スペクトルでも構わないが、好ましいものはフーリエ変換吸収スペクトルである。これは、より精度高く回帰式が求められるからである。測定された近赤外吸収スペクトルは、好ましくはフーリエ変換された後、前記コラーゲン含有量とともに統計化学的分析にかけられ、その因果関係を数量化される。この数量関係と測定すべき検体のスペクトルとの対比より、測定すべき検体中のコラーゲンの存在量が算出される。 The wavelength region of the near infrared absorption spectrum used in such measurements, 4000～10000Cm -1 can be preferably exemplified, more preferably from 5000~7000cm -1. This is because there are many specific absorptions of collagen in this wavelength region. Further, the near infrared absorption spectrum may be a diode array or a Fourier transform absorption spectrum, but a Fourier transform absorption spectrum is preferable. This is because the regression equation is obtained with higher accuracy. The measured near-infrared absorption spectrum is preferably subjected to Fourier transform and then subjected to statistical chemical analysis together with the collagen content, and the causal relationship is quantified. The abundance of collagen in the sample to be measured is calculated from the comparison between the quantity relationship and the spectrum of the sample to be measured.
前記多変量解析と別称されている統計化学的分析とは、分光データなどの化学的な特性と物性などの特性値との関係を計量学的な処理によって関係づけ、解析する手法であり、重回帰分析或いは主成分分析などが知られている。この内、重回帰分析としてはＰＬＳ分析が好適に例示できる。このＰＬＳ分析であるが、この分析法は特定の試料に於ける波長などの連続的な因子の変化に対して、吸光度などの変数の出現する分光スペクトルパターンと当該試料のある示性値の間の関係を分析する場合において、各示性値と因子ごとの変数の変化を分析する手技として確立されているものである。又、主成分分析は、同様な分析において、変動に寄与する第一主成分を分析し、しかる後この第一主成分軸に対して直交する第二主成分軸を分析し、この２つの主成分軸がつくる座標におけるパターン変化で物性を比較、推定する方法である。この様なＰＬＳ分析或いは主成分分析と言った、多変量解析は、市販されているソフトウェアを使用して行うことができる。この様な多変量解析用のソフトウェアとしては、例えば、ＧＬサイエンス社より販売されている、ピロエット（ＰＩＲＯＵＥＴＴ）、サイバネットシステム社より販売されている、マットラボ（ＭＡＴＬＡＢ）横川電気株式会社より販売されている、アンスクランブラーＩＩ（ＵｎｓｃｒａｎｂｌｅｒＩＩ）、セパノヴァ（ＳＥＰＡＮＯＶＡ）社より販売されているシムカ（ＳＩＭＣＡ）等のソフトウェアが例示できる。又、これらに加えてシムカ（ＳＩＭＣＡ）と言われるアルゴリズムを加えることができる。かかるアルゴリズムは前記ソフトウェア中に組み込まれている場合が多く、主成分分析の表示に有用である。これらのソフトウェアを利用して、近赤外吸収スペクトルを解析し、その結果を本発明の定量法で用いる場合、大凡の処理ステップは次に示す手順による。この時、使用するフーリエ変換近赤外吸収スペクトルは測定して得られた原スペクトルでも良いし、前記原スペクトルをデータ加工したものでも良い。データ加工の方法としては、例えば、一次微分値、二次微分値、三次微分値などの多次微分値や平滑化（Smoothing）、ノーマライズ（Normalize）、MSC（Multiplicative Scatter Correction）、SNV（Standard Normal Variate）、平均化（Mean-Center）、オートスケール（Autoscale）などが好ましく例示できる。 Statistical chemical analysis, also referred to as multivariate analysis, is a technique for associating and analyzing the relationship between chemical characteristics such as spectroscopic data and characteristic values such as physical properties by a quantitative process. Regression analysis or principal component analysis is known. Of these, PLS analysis can be suitably exemplified as the multiple regression analysis. This PLS analysis is based on the spectral spectrum pattern in which a variable such as absorbance appears and a certain characteristic value of the sample with respect to a continuous change in a factor such as a wavelength in a specific sample. This is an established technique for analyzing the change of each characteristic value and the variable for each factor. In the principal component analysis, the first principal component contributing to the fluctuation is analyzed in the same analysis, and then the second principal component axis orthogonal to the first principal component axis is analyzed. In this method, physical properties are compared and estimated by pattern changes in the coordinates created by the component axes. Multivariate analysis such as PLS analysis or principal component analysis can be performed using commercially available software. As such software for multivariate analysis, for example, sold by GL Science, Pirouett, sold by Cybernet Systems, sold by Matlab Yokogawa Electric Co., Ltd. Software such as Simsca (SIMCA) sold by Unscrambler II and Sepanova (SEPANOVA) can be exemplified. In addition to these, an algorithm called SIMCA can be added. Such an algorithm is often incorporated in the software and is useful for displaying principal component analysis. When these softwares are used to analyze near-infrared absorption spectra and the results are used in the quantification method of the present invention, the general processing steps are as follows. At this time, the Fourier transform near-infrared absorption spectrum to be used may be an original spectrum obtained by measurement, or may be obtained by processing the original spectrum. Data processing methods include, for example, primary differential values, secondary differential values, tertiary differential values, etc., smoothing (Smoothing), normalization (Normalize), MSC (Multiplicative Scatter Correction), SNV (Standard Normal) Preferred examples include Variate, Average (Mean-Center), and Autoscale.
ＰＬＳ分析の場合
（１）皮膚や製剤などの標準検体の分散型或いはダイオードアレイタイプの近赤外吸収スペクトル或いはそれらのフーリエ変換スペクトルやフーリエ変換スペクトルを所望により、二次微分等データ加工を行い、波長と近赤外吸収スペクトル乃至はその加工データとの行列を作成する。
（２）前記行列と標準検体に含有されるコラーゲン量との行列を作成し、コラーゲンの存在量の動きに対して、動きの大きい近赤外吸収スペクトル乃至はその加工データを抽出し、その波長を特定する。
（３）抽出した近赤外吸収スペクトル乃至はその加工データと示性値より検量線を作成する。同時に、コラーゲンの存在量ごとに検量線上へのプロットを作成しておく。
（４）試験試料のフーリエ変換近赤外吸収スペクトルを測定し、所望により二次微分等のデータ加工する。
（５）（４）のデータより（２）で特定された波長のデータを抽出する。
（６）（５）で抽出されたデータを検量線上への写像を作成する。或いは、データを検量線上へプロットする。
（７）（３）の示性値ごとのプロットと（５）の写像乃至はプロットとを比較し、測定試料のコラーゲン存在量を推測する。
尚、（２）以下の作業はコンピューターソフトウェアを利用することにより行うことができる。
For PLS analysis (1) Dispersion of standard specimens such as skin and preparations, or near-infrared absorption spectrum of diode array type or their Fourier transform spectrum or Fourier transform spectrum, if desired, data processing such as second derivative, A matrix of the wavelength and the near-infrared absorption spectrum or the processed data is created.
(2) Create a matrix of the matrix and the amount of collagen contained in the standard specimen, extract a near-infrared absorption spectrum or processed data having a large movement with respect to movement of the amount of collagen, and wavelength Is identified.
(3) A calibration curve is created from the extracted near-infrared absorption spectrum or the processed data and the characteristic value. At the same time, a plot on the calibration curve is created for each amount of collagen present.
(4) The Fourier transform near-infrared absorption spectrum of the test sample is measured, and data such as second derivative is processed as desired.
(5) Extract the data of the wavelength specified in (2) from the data of (4).
(6) Create a map of the data extracted in (5) onto the calibration curve. Alternatively, the data is plotted on a calibration curve.
(7) The plot for each characteristic value in (3) is compared with the mapping or plot in (5) to estimate the amount of collagen present in the measurement sample.
(2) The following operations can be performed using computer software.
主成分分析の場合
（１）皮膚の分散型或いはダイオードアレイタイプの近赤外吸収スペクトル或いはそれらのフーリエ変換スペクトルやフーリエ変換スペクトルを所望により、二次微分等データ加工を行い、波長と近赤外吸収スペクトル乃至はその加工データとの行列を作成する。
（２）前記行列について主成分分析を行い、第一主成分軸を作成する。
（３）第一主成分と直交する第二主成分軸を作成する。
（４）第一主成分軸と第二主成分軸が作る平面上に（１）のスペクトルの第一主成分と第二主成分が作る点をプロットする。
（５）所望によりシムカなどのアルゴリズムを用いてグルーピングを行う。
（６）（１）と同様に試験試料の近赤外スペクトルを測定し、（４）と同様のプロットを行う。
（７）（４）のプロット乃至は（５）のグルーピングを指標に試験試料の鑑別を行う。
In the case of principal component analysis (1) The near-infrared absorption spectrum of the skin dispersion type or diode array type, or the Fourier transform spectrum or Fourier transform spectrum thereof, if desired, is subjected to data processing such as second derivative, and the wavelength and near infrared An absorption spectrum or a matrix with the processed data is created.
(2) A principal component analysis is performed on the matrix to create a first principal component axis.
(3) Create a second principal component axis orthogonal to the first principal component.
(4) The points formed by the first principal component and the second principal component of the spectrum of (1) are plotted on the plane formed by the first principal component axis and the second principal component axis.
(5) Grouping is performed using an algorithm such as shimuka as desired.
(6) Measure the near-infrared spectrum of the test sample in the same manner as (1), and perform the same plot as in (4).
(7) The test sample is identified using the plot in (4) or the grouping in (5) as an index.
以下に、実施例を挙げて、本発明について更に詳細に説明を加えるが、本発明が、かかる実施例にのみ限定されないことは言うまでもない。 Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.
＜実施例１＞
ヘアレスマウス皮膚を用いて，皮膚由来成分が共存する条件下においてコラーゲンの定量実験をin vitroにて行った．即ち，ホモジナイズしたヘアレスマウス皮膚にエーテルを加え脱脂し，これにコラーゲンの濃度を段階的に添加し、再度ホモジナイズした．これを凍結乾燥させ円柱状に成型した後，フーリエ変換型近赤外吸収スペクトルを計測した。コラーゲン添加濃度としては，０（無添加），０．２０，０．４４，０．６３ｗｔ％の４点を用いた。得られた吸収スペクトルを，SNV（Standard Normal Variate），二次微分，平均化（Mean-Center）処理し，そのスペクトルとコラーゲン添加濃度をピロエットのＰＬＳ分析にかけ、ＰＬＳモデルを求めた。結果を表１に示す．これより、主因子数は２個の相関係数は０．９６７であり，ＳＥＶは０．０６２であり，処理した近赤外吸収スペクトルとコラーゲン添加濃度との間に良好な相関性があることがわかる。近赤外吸収スペクトルのフーリエ変換後のスペクトルを図１に、グラフ上に回帰状況を図示したものを図２に示す。これより、かかるＰＬＳモデルを用いることにより、皮膚由来成分に妨害を受けることなく皮膚内のコラーゲンの存在量を定量出来ることが判る。
<Example 1>
In hairless mouse skin, collagen was quantified in vitro in the presence of skin-derived components. That is, ether was added to the homogenized hairless mouse skin for defatting, and then the collagen concentration was added stepwise and homogenized again. This was freeze-dried and formed into a cylindrical shape, and then a Fourier transform type near infrared absorption spectrum was measured. As the collagen addition concentration, four points of 0 (no addition), 0.20, 0.44, and 0.63 wt% were used. The obtained absorption spectrum was subjected to SNV (Standard Normal Variate), second-order differentiation, and averaging (Mean-Center) treatment, and the spectrum and collagen addition concentration were subjected to Pyroet PLS analysis to obtain a PLS model. The results are shown in Table 1. Thus, the number of main factors is 0.967, the correlation coefficient is 0.967, SEV is 0.062, and there is a good correlation between the processed near infrared absorption spectrum and the collagen addition concentration. I understand. The spectrum after Fourier transform of the near-infrared absorption spectrum is shown in FIG. 1, and the regression diagram is shown on the graph in FIG. From this, it can be seen that by using such a PLS model, the abundance of collagen in the skin can be quantified without being disturbed by skin-derived components.
＜実施例２＞
実施例１で示した実験と同様に，ホモジナイズしたヘアレスマウス皮膚にエーテルを加え脱脂し，これにコラーゲンの濃度を既知量添加し、再度ホモジナイズした．これを凍結乾燥させ円柱状に成型した後，フーリエ変換型近赤外吸収スペクトルを計測した。得られた吸収スペクトルを，SNV（Standard Normal Variate），二次微分，平均化（Mean-Center）処理し，そのスペクトルを実施例１で求めたＰＬＳモデルに代入しコラーゲン予測値を求めた．得られた予測値と実際に添加したコラーゲン値との相関性は，相関係数が０．９８９，ＳＥＰが０．４５１であり，回帰式 ｙ＝０．３４１ｘ ＋０．１２２で表されることがわかった．これより，実施例１で作製したＰＬＳモデルは，皮膚由来成分に妨害を受けることなく皮膚内のコラーゲンの存在量を定量出来ることが判明した．
<Example 2>
In the same manner as in the experiment shown in Example 1, ether was added to the homogenized hairless mouse skin for defatting, and a known amount of collagen was added thereto, followed by homogenization again. This was freeze-dried and formed into a cylindrical shape, and then a Fourier transform type near infrared absorption spectrum was measured. The obtained absorption spectrum was subjected to SNV (Standard Normal Variate), second-order differentiation, and averaging (Mean-Center) processing, and the spectrum was substituted into the PLS model obtained in Example 1 to obtain a collagen predicted value. The correlation between the obtained predicted value and the actually added collagen value is 0.989, SEP is 0.451, and is expressed by a regression equation y = 0.341x + 0.122. all right. From this, it was found that the PLS model produced in Example 1 can quantify the abundance of collagen in the skin without being disturbed by skin-derived components.
＜実施例３＞
人のボランティアを用いて、顔の各部位の近赤外吸収スペクトルを計測し、これを実施例１のＰＬＳモデルに代入して皮下のコラーゲンの存在量を定量した。この値は、シワの多い部位ではコラーゲンの存在量が少なく、シワの少ない部位では存在量が多く、妥当な測定結果であった。
<Example 3>
Using human volunteers, near-infrared absorption spectra of each part of the face were measured, and this was substituted into the PLS model of Example 1 to quantify the abundance of subcutaneous collagen. This value was a reasonable measurement result because the abundance of collagen was small in a site with many wrinkles and a large amount was present in a site with few wrinkles.
本発明は、生体などの皮下に存在するコラーゲン量を非侵襲的に定量する方法に応用できる。 The present invention can be applied to a method for noninvasively quantifying the amount of collagen existing subcutaneously in a living body or the like.
Claims (5)
人以外の動物の皮膚を脱脂処理した後、制御された量のコラーゲンを添加し、ホモジナイズすることにより作製されたコラーゲンの存在量の明らかな標準検体について、５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルを計測し、
計測した近赤外吸収スペクトルデータを、ＰＬＳ分析又は主成分分析により解析し、
コラーゲンの存在量と関係する因子を決定することを特徴とする、方法。 A method for obtaining a regression equation for quantifying the abundance of collagen in the dermal skin ,
After degreasing the animal skin non-human, adding a controlled amount of collagen, the obvious reference specimen abundance of collagen made by homogenizing, in the wavelength region of 5000～7000Cm -1 Measure the near infrared absorption spectrum,
Analyze the measured near infrared absorption spectrum data by PLS analysis or principal component analysis,
Determining a factor related to the abundance of collagen.
請求項１〜３の何れか一項に記載の方法により得られた回帰式に、
被験動物の皮膚真皮の５０００〜７０００ｃｍ-1の波長領域の近赤外吸収スペクトルデータを代入することを特徴とする、方法。 A method for quantifying the abundance of collagen in the skin dermis of a test animal,
In the regression equation obtained by the method according to any one of claims 1 to 3 ,
A method comprising substituting near-infrared absorption spectrum data in a wavelength region of 5000 to 7000 cm −1 of a skin dermis of a subject animal.
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