JP2012229924A - Quantitative analysis method of lipoperoxide - Google Patents
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本発明は、顔の所定部位における過酸化脂質の量を測定するための過酸化脂質の定量分析方法に関する。 The present invention relates to a lipid peroxide quantitative analysis method for measuring the amount of lipid peroxide in a predetermined part of the face.
近年の研究によれば、紫外線が皮膚に過度に照射されると、フリーラジカルや活性酸素を生じ、これらの物質が皮脂を酸化することで過酸化脂質が生成されることが報告されている。過酸化脂質は、アトピー性皮膚炎や接触皮膚炎、乾癬などの皮膚疾患に関与すると考えられており、いったん過酸化脂質が生成されると、酸化は連鎖的に進行し、皮膚表面に刺激を与えるだけにとどまらず、さらには分解して反応性の高いアルデヒドとなり、それが角層のタンパク質と結合することでカルボニル基を有する酸化タンパク質が生成し角質層の奥まで入り込んで細胞にダメージを与える。これらの角層酸化タンパク質は、皮膚の加齢に伴う老化や光老化を引き起こす要因であると考えられている。 According to recent studies, it has been reported that when ultraviolet rays are excessively irradiated to the skin, free radicals and active oxygen are generated, and these substances oxidize sebum to produce lipid peroxide. Lipid peroxide is thought to be involved in skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis. Once lipid peroxide is generated, oxidation proceeds in a chain and stimulates the skin surface. Not only does it give, but it decomposes into a highly reactive aldehyde, which binds to the protein in the stratum corneum to produce an oxidized protein with a carbonyl group that penetrates deep into the stratum corneum and damages the cells. . These stratum corneum oxidized proteins are considered to be factors that cause aging and photoaging with aging of the skin.
このように過酸化脂質は、ヒトの皮膚の老化現象に影響を与えることが知られており、顔の表皮に存在する過酸化脂質の量を精度良く測定し、過酸化脂質の量と皮膚の各種現象との関連性を検討することは皮膚科学分野において重要な課題となっている。 In this way, lipid peroxide is known to affect the aging phenomenon of human skin, and the amount of lipid peroxide present in the epidermis of the face is accurately measured to determine the amount of lipid peroxide and the amount of skin Examining the relationship with various phenomena is an important issue in the field of dermatology.
顔の表皮に存在する過酸化脂質を測定する分析方法として、本発明者らは高感度の測定が可能なジフェニル−１−ピレニルホスフィンを蛍光試薬に用いた蛍光検出法を採用するが、過酸化脂質を抽出する過程においては、従来より、所定の溶媒を用いて被験者の皮膚から表皮物質を抽出し、かかる溶媒を濃縮乾固し、その後濃縮乾固物を再溶解して分析が可能な程度にまで過酸化脂質の濃度を高め(濃縮乾固及び再溶解)、これを検体とする方法が確立されていた。 As an analysis method for measuring lipid peroxide present in the epidermis of the face, the present inventors adopt a fluorescence detection method using diphenyl-1-pyrenylphosphine as a fluorescent reagent, which can be measured with high sensitivity. In the process of extracting lipid oxide, it is conventionally possible to extract epidermis material from the skin of a subject using a predetermined solvent, concentrate the solvent to dryness, and then redissolve the concentrated dry matter to perform analysis. A method has been established in which the concentration of lipid peroxide is increased to a certain extent (concentration to dryness and redissolution) and this is used as a specimen.
しかしながら、本発明者らの研究から溶媒の濃縮乾固過程においては一部の過酸化脂質が分解し、前記の分析方法では過酸化脂質の定量に誤差が生じる可能性があることが確認された(図２)。また、濾紙で表皮物質を吸着しこれを溶媒で抽出する際、使用する溶媒によっては蛍光強度に誤差が生じ得ることも確認された(図３)。 However, the present inventors confirmed that some lipid peroxides were decomposed in the process of solvent concentration and solidification, and that the analysis method described above may cause errors in the quantification of lipid peroxides. (Figure 2). It was also confirmed that when the epidermis substance was adsorbed with filter paper and extracted with a solvent, an error could occur in the fluorescence intensity depending on the solvent used (FIG. 3).
さらに、表皮物質(過酸化脂質)の量は、個人による差が大きく(図１)、過酸化脂質の定量においては多数の被験者を測定する必要があるが、抽出溶媒の濃縮乾固等の操作には長時間を要することから、定量分析を効率的に行うことは困難であった。 Furthermore, the amount of epidermal substance (lipid peroxide) varies greatly depending on individuals (Fig. 1), and it is necessary to measure a large number of subjects in the quantification of lipid peroxide. It takes a long time to perform quantitative analysis efficiently.
本発明は、過酸化脂質を測定する定量分析法として蛍光検出法を採用し、従来における抽出方法の問題点を解消しつつ、特に顔の皮膚表面に存在する過酸化脂質の量を精度良く、効率的に測定するための定量分析方法を確立するものである。 The present invention adopts a fluorescence detection method as a quantitative analysis method for measuring lipid peroxide, and solves the problems of conventional extraction methods, while accurately measuring the amount of lipid peroxide present particularly on the skin surface of the face, It establishes a quantitative analysis method for efficient measurement.
本発明は、顔の皮膚表面に存在する過酸化脂質の量を精度良くかつ効率的に測定するための定量分析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 quantitative analysis method for accurately and efficiently measuring the amount of lipid peroxide present on the skin surface of the face.
前記課題を解決するために本発明者らが検討を行った結果、顔の皮膚表面にある表皮物質を濾紙で吸着し、かかる濾紙をメタノールに浸漬し、その後該メタノールにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加え、生成したジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を測定することにより、効率的かつ精度良く過酸化脂質の量を分析できることを見出し、本発明を完成するに至った。 As a result of studies conducted by the present inventors in order to solve the above-mentioned problems, the epidermis substance on the surface of the face is adsorbed with a filter paper, and the filter paper is immersed in methanol, and then diphenyl-1-pyrenyl is immersed in the methanol. By adding phosphine (DPPP) and measuring the fluorescence intensity of the generated diphenyl-1-pyrenylphosphine oxide (DPPP = O), it was found that the amount of lipid peroxide can be analyzed efficiently and accurately, and the present invention was developed. It came to be completed.
すなわち本発明は、顔の所定部位における過酸化脂質の量を測定する方法であって、顔の所定部位に密着させ皮膚表面にある表皮物質を吸着させた濾紙をメタノールに浸漬し、その後、該メタノールにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加え、生成したジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を測定することにより過酸化脂質の量を測定することを特徴とする過酸化脂質の定量分析方法である。 That is, the present invention is a method for measuring the amount of lipid peroxide in a predetermined part of the face, wherein a filter paper that adheres to a predetermined part of the face and adsorbs an epidermis substance on the skin surface is immersed in methanol, and then the The amount of lipid peroxide is measured by adding diphenyl-1-pyrenylphosphine (DPPP) to methanol and measuring the fluorescence intensity of the generated diphenyl-1-pyrenylphosphine oxide (DPPP = O). This is a method for quantitative analysis of lipid peroxide.
さらに本発明は、メタノールが、純度が９９．８質量％以上で、水分含有量が０．０５質量％以下、不揮発成分の含有量が２質量ｐｐｍ以下であることを特徴とする過酸化脂質の定量分析方法である。 Furthermore, the present invention provides a lipid peroxide, characterized in that the methanol has a purity of 99.8% by mass or more, a water content of 0.05% by mass or less, and a nonvolatile component content of 2% by mass or less. It is a quantitative analysis method.
さらに本発明は、額部分の過酸化脂質の量を分析するときは、表皮物質を吸着させた濾紙を、濾紙１ｃｍ２ あたり２００〜１０００μＬ、特に好ましくは２００〜４００μＬに相当する量のメタノールに浸漬することを特徴とする過酸化脂質の定量分析方法である。 Further, in the present invention, when analyzing the amount of lipid peroxide in the forehead part, the filter paper on which the skin substance is adsorbed is immersed in 200 to 1000 μL, particularly preferably 200 to 400 μL of methanol per 1 cm 2 of the filter paper. A method for quantitative analysis of lipid peroxide.
また本発明は、鼻部分の過酸化脂質の量を分析するときは、表皮物質を吸着させた濾紙を、濾紙１ｃｍ２ あたり２００〜１６００μＬ、特に好ましくは２００〜５００μＬに相当する量のメタノールに浸漬することを特徴とする過酸化脂質の定量分析方法である。 In the present invention, when analyzing the amount of lipid peroxide in the nasal portion, the filter paper on which the epidermis substance is adsorbed is immersed in 200 to 1600 μL, particularly preferably 200 to 500 μL of methanol per 1 cm 2 of the filter paper. A method for quantitative analysis of lipid peroxide.
また本発明は、頬部分の過酸化脂質の量を分析するときは、表皮物質を吸着させた濾紙を、濾紙１ｃｍ２ あたり２００〜１２００μＬ、特に好ましくは２００〜６００μＬに相当する量のメタノールに浸漬することを特徴とする過酸化脂質の定量分析方法である。 In the present invention, when analyzing the amount of lipid peroxide in the cheek portion, the filter paper on which the epidermis substance is adsorbed is immersed in 200 to 1200 μL, particularly preferably 200 to 600 μL of methanol per 1 cm 2 of the filter paper. A method for quantitative analysis of lipid peroxide.
さらに本発明は、濾紙を浸漬した後のメタノールを濃縮乾固することなく、これにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加えることを特徴とする過酸化脂質の定量分析方法である。 Furthermore, the present invention is a method for quantitative analysis of lipid peroxide, characterized in that diphenyl-1-pyrenylphosphine (DPPP) is added thereto without concentrating and drying methanol after immersing the filter paper.
さらに本発明は、濾紙の厚さが、０．１８〜０．２３ｍｍであることを特徴とする過酸化脂質の定量分析方法である。 Furthermore, the present invention is a method for quantitative analysis of lipid peroxide, wherein the filter paper has a thickness of 0.18 to 0.23 mm.
本発明の定量分析方法によれば、顔の所定部位における過酸化脂質の量を精度良く定量分析することができる。 According to the quantitative analysis method of the present invention, the amount of lipid peroxide in a predetermined part of the face can be quantitatively analyzed with high accuracy.
また、所定の純度を有するメタノールで過酸化脂質を抽出することにより、蛍光強度の誤差を無くし精度良い定量分析が可能となる。 Further, by extracting lipid peroxide with methanol having a predetermined purity, errors in fluorescence intensity can be eliminated and accurate quantitative analysis can be performed.
顔の所定部位に応じて所定量のメタノールで抽出することにより定量限度を上回る濃度の抽出溶媒を得ることができ、溶媒を濃縮乾固することなく分析することができるため、過酸化脂質が分解することなく正確に測定することができる。また、溶媒を濃縮乾固及び再溶解する工程が不要であることから分析時間を短縮することができ、効率的な定量分析が可能となる。 Extraction with a predetermined amount of methanol according to a predetermined part of the face can provide an extraction solvent with a concentration exceeding the limit of quantification, and analysis can be performed without concentrating the solvent to dryness. It is possible to measure accurately without doing. Moreover, since the process of concentrating, drying, and re-dissolving the solvent is unnecessary, the analysis time can be shortened and efficient quantitative analysis can be performed.
顔に密着させる濾紙を所定の大きさにすることにより、顔の皮膚に密着させやすく、また、メタノールに浸漬する際には、小さい容量のバイアル瓶の底に沈め、必要最少量のメタノールに浸漬することができる。このため、溶媒中の過酸化脂質の濃度を高めるための濃縮乾固・再溶解工程を採る必要が無く、精度の高い分析を行うことができる。 The filter paper that adheres to the face is sized to facilitate contact with the skin of the face, and when immersed in methanol, it is submerged in the bottom of a small-capacity vial and immersed in the minimum amount of methanol required. can do. For this reason, it is not necessary to take a concentration-drying / re-dissolution step for increasing the concentration of lipid peroxide in the solvent, and a highly accurate analysis can be performed.
本発明の実施の形態を以下に詳しく説明する。 Embodiments of the present invention will be described in detail below.
表皮物質の採取は、濾紙を皮膚に密着させ、濾紙に表皮物質を吸着させることにより行う。濾紙で表皮物質を採取することとしたのは、濾紙が実験用素材であり品質が一定であること、皮膚への影響が少ない天然素材であるセルロース繊維からなること、また、表皮物質を吸着しやすく、採取した表皮物質を溶媒を用いて抽出する際、溶媒と相互作用を生じることが少ないためである。 The skin material is collected by bringing the filter paper into close contact with the skin and adsorbing the skin material on the filter paper. The reason for collecting the epidermis material with filter paper is that the filter paper is an experimental material and the quality is constant, it is made of cellulose fiber, which is a natural material that has little effect on the skin, and it absorbs the epidermis material. This is because, when the collected epidermis substance is extracted using a solvent, it hardly causes an interaction with the solvent.
濾紙は、化学分析に使用できるものであれば、特に限定されるものではなく、市販品を使用することができる。市販品としては、例えばＡＤＶＡＮＴＥＣ社製の濾紙「定量分析用５種Ｃ」があり、これを所定の大きさに裁断して使用することができる。 The filter paper is not particularly limited as long as it can be used for chemical analysis, and a commercially available product can be used. As a commercially available product, for example, there is a filter paper “5 types C for quantitative analysis” manufactured by ADVANTEC, which can be used after being cut into a predetermined size.
顔に密着させる濾紙の面積は、測定する部位や目的に応じて、０．１〜１０ｃｍ２ の範囲で適宜選定することが好ましい。また、実施例に示すように、過酸化脂質を採取した濾紙を１ｃｍ２あたり、額部分であれば２００〜１０００μＬの溶媒に浸漬(鼻部分であれば２００〜１６００μＬ、頬部分であれば２００〜１２００μＬの溶媒に浸漬)することにより、蛍光検出法による定量分析が可能な濃度の過酸化脂質の抽出溶媒を得ることができる。 The area of the filter paper to be in close contact with the face is preferably appropriately selected within the range of 0.1 to 10 cm 2 according to the site to be measured and the purpose. Moreover, as shown in the Examples, the filter paper from which lipid peroxide was collected was immersed in 200 to 1000 μL of solvent per cm 2 for the forehead portion (200 to 1600 μL for the nose portion, 200 to 200 for the cheek portion). By immersing in 1200 μL of solvent, a lipid peroxide extraction solvent having a concentration capable of quantitative analysis by a fluorescence detection method can be obtained.
このようにして抽出溶媒を得ることで、一般的に行われる溶媒の濃縮乾固及び再溶解を行う必要が無く、定量分析の効率化を図ることができると共に、濃縮乾固による過酸化脂質の分解が無く、精度の高い定量を行うことが可能となる。 By obtaining the extraction solvent in this way, it is not necessary to perform concentration drying and re-dissolution of the solvent that is generally performed, and it is possible to improve the efficiency of quantitative analysis, and the lipid peroxide by concentration to dryness can be improved. There is no decomposition, and accurate quantification can be performed.
また、実施例で濾紙を短辺５ｍｍ×長辺１０ｍｍの長方形の切片としこれを２枚使用したのは、小型のバイアル瓶の中で溶媒に浸漬することを可能とするためである。すなわち、小型のバイアル瓶の口頸の内径は約７ｍｍであり、底面直径は約１５ｍｍであることから、バイアル瓶の底面付近にある少量の溶媒に浸漬することができるよう、口頸を通過し底に沈めることができる形状としたものである。このように少量の溶媒に浸漬することにより、本発明者らが用いた蛍光検出法の定量限界値である０．０６ｐｐｍを超える濃度の抽出溶媒を濃縮乾固及び再溶解することなく得ることが可能となる。 In the examples, the filter paper was made into a rectangular section having a short side of 5 mm and a long side of 10 mm, and the two pieces were used in order to be able to be immersed in a solvent in a small vial. That is, since the inside diameter of the mouth and neck of a small vial is about 7 mm and the bottom diameter is about 15 mm, it passes through the mouth and neck so that it can be immersed in a small amount of solvent near the bottom of the vial. The shape can be submerged in the bottom. By soaking in a small amount of solvent in this way, it is possible to obtain an extraction solvent having a concentration exceeding 0.06 ppm, which is the quantification limit value of the fluorescence detection method used by the present inventors, without concentrating to dryness and redissolving. It becomes possible.
このように顔に密着させる濾紙は、バイアル瓶の大きさに応じて適宜裁断し、これを複数枚使用するなどして表皮物質を採取することが好ましい。 Thus, it is preferable that the filter paper to be brought into close contact with the face is appropriately cut according to the size of the vial, and the epidermis substance is collected by using a plurality of the paper.
濾紙の厚さは、０．１８〜０．２３ｍｍであることが好ましい。０．１８ｍｍより薄いと、表皮物質を十分に吸着できない可能性がある。一方、０．２３ｍｍより厚いと浸漬するメタノールの量を増やす必要があるため検体の濃度が低くなり、分析に必要な濃度の検体が得られない可能性がある。 The thickness of the filter paper is preferably 0.18 to 0.23 mm. If it is thinner than 0.18 mm, the epidermis substance may not be sufficiently adsorbed. On the other hand, if it is thicker than 0.23 mm, it is necessary to increase the amount of methanol to be immersed, so that the concentration of the sample is lowered, and there is a possibility that the sample having the concentration necessary for analysis cannot be obtained.
濾紙で吸着した過酸化脂質は、濾紙をメタノールに浸漬し、メタノールにより抽出する。メタノールは、純度が９９．８質量％以上で、不揮発成分の含有量が２ｐｐｍ以下、水分含有量が０．０５％以下であることが好ましい。このような純度の高いメタノールは、本発明で使用するジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)やジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)等の試薬との相互作用が無く、正確な分析を行うことができる。また、皮膚に密着する濾紙及びバイアル瓶を予め純度の高い前記のメタノールで予め洗浄しておけば、さら精度の高い分析値が得られることが期待される。メタノールの純度に関する試験結果は実施例にて詳述する。 Lipid peroxide adsorbed by the filter paper is extracted by immersing the filter paper in methanol. Methanol preferably has a purity of 99.8% by mass or more, a non-volatile component content of 2 ppm or less, and a moisture content of 0.05% or less. Such high-purity methanol has no interaction with reagents such as diphenyl-1-pyrenylphosphine (DPPP) and diphenyl-1-pyrenylphosphine oxide (DPPP = O) used in the present invention, and is accurate. Analysis can be performed. Further, if the filter paper and the vial that are in close contact with the skin are previously washed with the high-purity methanol, it is expected that an analytical value with higher accuracy can be obtained. The test results relating to the purity of methanol are described in detail in the examples.
純度が９９．８質量％以上で、不揮発成分の含有量が２ｐｐｍ以下、水分含有量が０．０５％以下のメタノールは、環境分析用として市販されているものを使用することができ、例えば、和光純薬工業株式会社製の環境分析用メタノール(純度が９９．８質量％以上、水分含有量が０．０５質量％以下、不揮発成分の含有量が１質量ｐｐｍ以下を規格とする)や純正化学株式会社製の製品名「メタノール５０００」(純度が９９．８質量％以上、水分含有量が０．０５質量％以下、不揮発成分の含有量が２質量ｐｐｍ以下を規格とする)等を使用することができる。 A methanol having a purity of 99.8% by mass or more, a non-volatile component content of 2 ppm or less, and a moisture content of 0.05% or less can be used for environmental analysis, for example, Methanol for environmental analysis manufactured by Wako Pure Chemical Industries, Ltd. (purity is 99.8% by mass or more, moisture content is 0.05% by mass or less, nonvolatile component content is 1 ppm by mass or less) or genuine The product name “Methanol 5000” manufactured by Kagaku Co., Ltd. (purity is 99.8% by mass or more, moisture content is 0.05% by mass or less, and nonvolatile component content is 2 mass ppm or less) is used. can do.
表皮物質を吸着させた濾紙をメタノールに浸漬し、表皮物質(過酸化脂質)を抽出したメタノールにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加えて過酸化脂質のヒドロキシペルオキシドと反応させ、得られた蛍光化合物ジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を測定することにより定量する。 It is obtained by immersing the filter paper adsorbed with epidermis substance in methanol, adding diphenyl-1-pyrenylphosphine (DPPP) to methanol from which epidermis substance (lipid peroxide) is extracted, and reacting with hydroxy peroxide of lipid peroxide. The fluorescent compound diphenyl-1-pyrenyl phosphine oxide (DPPP = O) was quantified by measuring the fluorescence intensity.
ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)は、過酸化物と化学量論的に反応して、蛍光性を有するジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)を生成する。ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)は蛍光性を有しないが、ジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)は蛍光性を有するので、その蛍光強度を測定することにより、検体中の過酸化物の量を測定することができる。 Diphenyl-1-pyrenylphosphine (DPPP) reacts stoichiometrically with peroxide to produce diphenyl-1-pyrenylphosphine oxide (DPPP = O) having fluorescence. Diphenyl-1-pyrenylphosphine (DPPP) does not have fluorescence, but diphenyl-1-pyrenylphosphine oxide (DPPP = O) has fluorescence. Therefore, by measuring the fluorescence intensity, The amount of peroxide can be measured.
ジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)を検出するには、励起光として３５２ｎｍの光を照射し、３８０ｎｍ付近にピークを有する蛍光のピーク強度を検出する。蛍光強度(ピーク強度)は、市販の適当な検出器を用いて測定すればよい。蛍光強度は被検体の過酸化脂質の存在量を表す指標として評価することができる。 In order to detect diphenyl-1-pyrenyl phosphine oxide (DPPP = O), 352 nm light is irradiated as excitation light, and the peak intensity of fluorescence having a peak near 380 nm is detected. The fluorescence intensity (peak intensity) may be measured using a commercially available appropriate detector. The fluorescence intensity can be evaluated as an index representing the abundance of lipid peroxide in the subject.
本発明で用いるジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)自体は公知化合物であり、市販品などから容易に入手できる。市販品としては、例えば、同仁化学社製「ＤＰＰＰ」等がある。本発明では検体中の過酸化脂質と反応させ、得られたジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を定量することにより検体中にある過酸化脂質の量を測定する。ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を含む試薬の形態は特に限定されず、固体でも液体（溶液、懸濁液など)でもよい。液体の場合には適当な溶媒(好ましくはジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)が一定の溶解度を示す有機溶媒など)に溶解または懸濁することによって試薬を調製することができる。 Diphenyl-1-pyrenylphosphine (DPPP) itself used in the present invention is a known compound and can be easily obtained from commercial products. Examples of commercially available products include “DPPP” manufactured by Dojin Chemical Co., Ltd. In the present invention, the amount of lipid peroxide in the sample is measured by reacting with lipid peroxide in the sample and quantifying the fluorescence intensity of the obtained diphenyl-1-pyrenylphosphine oxide (DPPP = O). The form of the reagent containing diphenyl-1-pyrenylphosphine (DPPP) is not particularly limited, and may be solid or liquid (solution, suspension, etc.). In the case of a liquid, the reagent can be prepared by dissolving or suspending in an appropriate solvent (preferably an organic solvent in which diphenyl-1-pyrenylphosphine (DPPP) exhibits a certain solubility).
図１は、女性被験者２４名の顔から採取した過酸化脂質の濃度を示すものである。濾紙(ＡＤＶＡＮＴＥＣ社製「定量分析用５種Ｃ」)を短辺５ｍｍ×長辺１０ｍｍの長方形の切片に裁断し、この切片２枚を被験者の顔に密着させて表皮物質を吸着させた後、メタノール２００μＬに浸漬・抽出した。このようにして得た抽出液を検体とし、これにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)(同仁化学社製「ＤＰＰＰ」)を加え、生成したジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を測定したものである。尚、ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)は、ＨＰＬＣ用メタノールに２．５ｍｇ／Ｌになるように溶解したものであり、フローインジェクション分析システムを用い、検体とシステム中で反応させた。 FIG. 1 shows the concentration of lipid peroxide collected from the faces of 24 female subjects. After cutting a filter paper (“5 types for quantitative analysis” manufactured by ADVANTEC) into rectangular sections having a short side of 5 mm × long side of 10 mm, the two pieces were brought into close contact with the face of the subject to adsorb the epidermis substance, It was immersed and extracted in 200 μL of methanol. The extract thus obtained was used as a specimen, and diphenyl-1-pyrenylphosphine (DPPP) (“DPPP” manufactured by Dojindo) was added thereto, and the resulting diphenyl-1-pyrenylphosphine oxide (DPPP = O ) Was measured. Diphenyl-1-pyrenylphosphine (DPPP) was dissolved in methanol for HPLC so as to have a concentration of 2.5 mg / L, and was reacted with a sample in the system using a flow injection analysis system.
図１に示すように過酸化脂質の量は個人差が大きく、皮膚の各種現象との関連性について有意性のあるデータを取得するためには相当数のサンプルが必要である。本発明者らは、高感度で測定できる蛍光検出法を採用するが、本法による定量限界は０．０６ｐｐｍであるため、この数値を下回る被験者は定量データとして採用することはできない。 As shown in FIG. 1, the amount of lipid peroxide varies greatly from person to person, and a considerable number of samples are required to obtain data that is relevant to various skin phenomena. The present inventors employ a fluorescence detection method that can be measured with high sensitivity. However, since the limit of quantification by this method is 0.06 ppm, subjects below this value cannot be employed as quantification data.
検体の濃度を高める方法としては、従来より抽出溶媒(メタノール)を減圧下で遠心し、溶媒を揮散させて濃縮乾固し、これを再溶解して対象物質の濃度を高め(濃縮乾固及び再溶解)、これを検体とする方法がある。 As a method for increasing the concentration of the specimen, conventionally, the extraction solvent (methanol) is centrifuged under reduced pressure, the solvent is evaporated, the solution is concentrated to dryness, and this is redissolved to increase the concentration of the target substance (concentration to dryness and There is a method of using this as a specimen.
しかしながら、過酸化脂質を抽出した抽出溶媒(メタノール)を濃縮乾固することにより含有する過酸化脂質が分解され、定量測定値に誤差が生じる可能性があることを見出した。図２は、標準の過酸化脂質を濾紙にしみ込ませ、これをメタノールに浸漬して抽出し、抽出したメタノールを濃縮乾固せずにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加えてジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)を生成し、蛍光強度を測定して回収率を測定したものと、抽出したメタノールを減圧下で遠心し溶媒を揮散させて濃縮乾固し、これをメタノール１００μＬで再溶解して検体とし、これにジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加え、回収率を測定したものとを比較した結果である。尚、標準の過酸化脂質とは、リノール酸メチルに紫外線(ＵＶ)を照射してその一部を過酸化脂質とし、溶媒に溶解し、濃度を検定したものである(過酸化脂質濃度０．７７ｐｐｍ)。また、ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)は、メタノールに溶解し、２．５質量ｐｐｍのメタノール溶液として添加した。 However, it has been found that the lipid peroxide contained therein is decomposed by concentrating and drying the extraction solvent (methanol) from which the lipid peroxide has been extracted, resulting in an error in the quantitative measurement value. FIG. 2 shows a case where a standard lipid peroxide is soaked in a filter paper, extracted by immersing it in methanol, and diphenyl-1-pyrenylphosphine (DPPP) is added to the extracted methanol without concentrating to dryness. 1-pyrenyl phosphine oxide (DPPP = O) was produced, the fluorescence intensity was measured and the recovery rate was measured, and the extracted methanol was centrifuged under reduced pressure to evaporate the solvent and concentrated to dryness. This is a result of comparison with a sample obtained by redissolving in 100 μL of methanol to prepare a sample, and adding diphenyl-1-pyrenylphosphine (DPPP) to the sample and measuring the recovery rate. The standard lipid peroxide is obtained by irradiating methyl linoleate with ultraviolet rays (UV), converting a part thereof to lipid peroxide, dissolving in a solvent, and examining the concentration (lipid peroxide concentration of 0. 77 ppm). Further, diphenyl-1-pyrenylphosphine (DPPP) was dissolved in methanol and added as a 2.5 mass ppm methanol solution.
図２の結果から、溶媒の濃縮乾固過程により、過酸化脂質の回収率は低下することが確認された(回収率約６０％)。この結果から、過酸化脂質の定量分析において、その精度を高めるためには抽出溶媒を濃縮乾固することなく、ジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加えることが重要であることがわかる。 From the results of FIG. 2, it was confirmed that the recovery rate of lipid peroxide was reduced by the concentration and drying process of the solvent (recovery rate of about 60%). From this result, it can be seen that in quantitative analysis of lipid peroxide, it is important to add diphenyl-1-pyrenylphosphine (DPPP) without concentrating and drying the extraction solvent in order to increase the accuracy.
つぎに、過酸化脂質を抽出する溶媒の種類について検討した。
図３は、各溶媒で洗浄した未使用の濾紙(１ｃｍ２)を、新たな各溶媒１ｍＬに２４時間浸漬し、浸漬後の各溶媒にジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加え、蛍光強度を測定したものである。ここで、特級メタノールとは、純度９９．８質量％以上で、水分含有量が０．１質量％以下、不揮発物が５質量ｐｐｍ以下のメタノール(和光純薬工業株式会社製の試薬特級メタノール)であり、環境分析用メタノールとは、純度が９９．８質量％以上で、水分含有量が０．０５質量％以下、不揮発成分の含有量が１質量ｐｐｍ以下のメタノール(和光純薬工業株式会社製の試薬特級メタノール)である。尚、図３中、「洗浄なし」とは、濾紙を溶媒で洗浄することなく、環境分析用メタノールに浸漬したものである。
Next, the type of solvent for extracting lipid peroxide was examined.
FIG. 3 shows that unused filter paper (1 cm 2 ) washed with each solvent is immersed in 1 mL of each new solvent for 24 hours, diphenyl-1-pyrenylphosphine (DPPP) is added to each solvent after immersion, and fluorescence is obtained. The strength is measured. Here, the special grade methanol is a methanol having a purity of 99.8% by mass or more, a water content of 0.1% by mass or less, and a nonvolatile content of 5 mass ppm or less (reagent special grade methanol manufactured by Wako Pure Chemical Industries, Ltd.). The environmental analysis methanol is methanol having a purity of 99.8% by mass or more, a water content of 0.05% by mass or less, and a nonvolatile component content of 1 mass ppm or less (Wako Pure Chemical Industries, Ltd.). Reagent-grade methanol). In FIG. 3, “no washing” means that the filter paper is immersed in environmental analysis methanol without washing with a solvent.
図３の結果から、未使用の濾紙でありながら、アセトンあるいは特級メタノールで洗浄し浸漬すると、浸漬したこれらの溶媒からは過酸化脂質の含有を示す蛍光が検出された。また、洗浄しない未使用の濾紙も浸漬した環境分析用メタノールから過酸化脂質の含有を示す蛍光が検出された。一方、環境分析用メタノールで洗浄し浸漬すると、浸漬した環境分析用メタノールからは蛍光は検出されなかった。このことから、環境分析用メタノール(純度が９９．８質量％以上で、水分含有量が０．０５質量％以下、不揮発成分の含有量が１質量ｐｐｍ以下のメタノール)で濾紙を洗浄した後、環境分析用メタノールで抽出することにより、濾紙あるいは溶媒に由来する過酸化脂質濃度の分析誤差を解消することができることが確認された。 From the results shown in FIG. 3, when the filter paper was unused, it was washed and immersed in acetone or special grade methanol, and fluorescence indicating the presence of lipid peroxide was detected from these soaked solvents. In addition, fluorescence indicating the inclusion of lipid peroxide was detected from methanol for environmental analysis in which unused filter paper that was not washed was also immersed. On the other hand, when washed and immersed in environmental analysis methanol, no fluorescence was detected from the immersed environmental analysis methanol. From this, after washing the filter paper with methanol for environmental analysis (purity is 99.8% by mass or more, water content is 0.05% by mass or less, nonvolatile component content is 1 mass ppm or less), It was confirmed that the analysis error of lipid peroxide concentration derived from filter paper or solvent can be eliminated by extraction with methanol for environmental analysis.
これらの結果から過酸化脂質の定量分析の精度を高めるためには、抽出溶媒を濃縮乾固することなく定量分析すること、また溶媒としては所定の純度のメタノールを使用することが重要であることが確認された。そして抽出溶媒を濃縮乾固せずに定量分析するためには、抽出した過酸化脂質の濃度が本発明者らが用いた蛍光検出法による定量限界濃度(０．０６ｐｐｍ)を超える必要がある。このため、濾紙から過酸化脂質を抽出するための溶媒の適切な量について検討した。 From these results, in order to improve the accuracy of quantitative analysis of lipid peroxide, it is important to quantitatively analyze the extraction solvent without concentrating to dryness, and to use methanol of the specified purity as the solvent. Was confirmed. In order to perform quantitative analysis without concentrating and drying the extraction solvent, the concentration of the extracted lipid peroxide needs to exceed the limit of quantification (0.06 ppm) determined by the fluorescence detection method used by the present inventors. For this reason, the appropriate amount of the solvent for extracting lipid peroxide from filter paper was examined.
図４は、濾紙(短辺５ｍｍ×長辺１０ｍｍの長方形の切片２枚)に標準品(０．７７ｐｐｍ)１０μＬを吸着させ、これを環境分析用メタノールに浸漬し、標準品の回収率を測定した結果を示すものである。 FIG. 4 shows that 10 μL of a standard product (0.77 ppm) is adsorbed on a filter paper (two rectangular sections having a short side of 5 mm and a long side of 10 mm), immersed in methanol for environmental analysis, and the recovery rate of the standard product is measured. The results are shown.
図４に示すように、２００μＬ以上の溶媒で抽出することにより、標準品を１００％回収できたが、１００μＬでは数値のバラツキが大きく、回収率が低い結果であった。この結果から、抽出する溶媒(環境分析用メタノール)の量としては２００μＬ以上が必要であることが確認された。尚、１００μＬで回収率が低い原因としては、濾紙が溶媒に完全に浸漬せず、標準品を十分に抽出できなかったことによるものと考察される。 As shown in FIG. 4, 100% of the standard product could be recovered by extracting with 200 μL or more of the solvent, but 100 μL showed a large variation in numerical values and a low recovery rate. From this result, it was confirmed that the amount of solvent (methanol for environmental analysis) to be extracted requires 200 μL or more. The reason why the recovery rate is low at 100 μL is considered to be that the filter paper was not completely immersed in the solvent and the standard product could not be sufficiently extracted.
つぎにヒトの各部位(額、鼻、頬、内腕)における過酸化脂質の量を測定した。 Next, the amount of lipid peroxide in each human part (forehead, nose, cheek, inner arm) was measured.
「試験方法」
定量分析の概略工程を図６に示す。
１）濾紙を裁断して得た短辺５ｍｍ×長辺１０ｍｍの切片を複数用意し、環境分析用メタノールで洗浄し乾燥した。
２）採取する各部位を洗浄し、２時間経過後に、濾紙の切片２枚を測定部位の皮膚に長辺が向かい合うように並べ、５０ｇで荷重し表皮物質を採取した(各部位につき被験者３名)。
３）採取後は、小型のバイアル瓶に濾紙切片２枚を収容し、環境分析用メタノール２００μＬを添加して過酸化脂質を抽出した。
４）抽出溶媒は、濃縮することなくジフェニル−１−ピレニルホスフィン(ＤＰＰＰ)を加え、過酸化脂質との反応により生成したジフェニル−１−ピレニルホスフィンオキシド(ＤＰＰＰ＝Ｏ)の蛍光強度を測定した。
"Test method"
A schematic process of quantitative analysis is shown in FIG.
1) A plurality of sections each having a short side of 5 mm and a long side of 10 mm obtained by cutting the filter paper were prepared, washed with environmental analysis methanol, and dried.
2) Wash each site to be collected, and after 2 hours, place two pieces of filter paper so that the long sides face the skin of the measurement site, load 50 g, and collect epidermis material (3 subjects per site) ).
3) After collection, two pieces of filter paper sections were placed in a small vial, and 200 μL of environmental analysis methanol was added to extract lipid peroxide.
4) Diphenyl-1-pyrenylphosphine (DPPP) is added to the extraction solvent without concentrating, and the fluorescence intensity of diphenyl-1-pyrenylphosphine oxide (DPPP = O) produced by reaction with lipid peroxide is measured. did.
「試験結果」
図５に示すように、額部では、平均で約０．３ｐｐｍの過酸化脂質を測定することができた。また、最も少ない被験者は、約０．１２ｐｐｍであった。この数値は、溶媒２００μＬ中の濃度を示すものであり、蛍光検出法による定量限界値は０．０６ｐｐｍであることを考慮すると、例えば溶媒の量を４００μＬとしても、すべての被験者のデータが取得可能である(最少の被験者の濃度であっても０．０６ｐｐｍを超えることができる)。さらに溶媒の量を１０００μＬとしても、平均の過酸化脂質量であれば定量可能であり、このことは、被験者の過酸化脂質量が正規分布に従うと仮定すると、約半数の被験者のデータを取得できることを意味する。したがって、図４の結果とあわせると、額部の過酸化脂質を定量するためには、濾紙１ｃｍ２あたり環境分析用メタノール２００〜１０００μＬで抽出することが好適であり、特に２００〜４００μＬで抽出することが好適であることがわかる。
"Test results"
As shown in FIG. 5, in the forehead part, an average of about 0.3 ppm of lipid peroxide could be measured. The smallest number of subjects was about 0.12 ppm. This value indicates the concentration in 200 μL of solvent, and considering that the quantitative limit value by the fluorescence detection method is 0.06 ppm, for example, even if the amount of solvent is 400 μL, data of all subjects can be acquired. (Even a minimum subject concentration can exceed 0.06 ppm). Furthermore, even if the amount of the solvent is 1000 μL, it can be quantified as long as the average amount of lipid peroxide is obtained, and this means that about half of the subjects' data can be obtained assuming that the amount of lipid peroxide of the subjects follows a normal distribution. Means. Therefore, in combination with the results of FIG. 4, in order to quantify the lipid peroxide in the forehead, it is preferable to extract with 200 to 1000 μL of environmental analysis methanol per 1 cm 2 of filter paper, particularly with 200 to 400 μL. It turns out that it is suitable.
鼻部についてみると、過酸化脂質の濃度は平均で約０．４８ｐｐｍである。また、最少値は約０．１５ｐｐｍである。額部と同様の考察をすれば、鼻部の過酸化脂質を定量するためには、濾紙１ｃｍ２あたり環境分析用メタノール２００〜１６００μＬで抽出することが好適であり、特に２００〜５００μＬで抽出することが好適である。 Looking at the nose, the average lipid peroxide concentration is about 0.48 ppm. The minimum value is about 0.15 ppm. Considering the same consideration as the forehead part, in order to quantify the lipid peroxide in the nasal part, it is preferable to extract with 200 to 1600 μL of environmental analysis methanol per 1 cm 2 of filter paper, and particularly with 200 to 500 μL. Is preferred.
頬部では、過酸化脂質の濃度は平均で約０．３６ｐｐｍである。また、最少値０．１８ｐｐｍである。前記と同様の考察をすれば、頬部の過酸化脂質を定量するためには、濾紙１ｃｍ２あたり環境分析用メタノール２００〜１２００μＬで抽出することが好適であり、特に２００〜６００μＬで抽出することが好適である。 In the cheek, the lipid peroxide concentration averages about 0.36 ppm. The minimum value is 0.18 ppm. Considering the same consideration as above, in order to quantify lipid peroxide in the cheek, it is preferable to extract with 200 to 1200 μL of environmental analysis methanol per 1 cm 2 of filter paper, and particularly with 200 to 600 μL. Is preferred.
尚、内腕部の過酸化脂質は、蛍光検出法における定量限界値より低い濃度であるため、本発明による定量分析法により定量することは不適であることが確認できる。 Since the lipid peroxide in the inner arm has a concentration lower than the quantification limit value in the fluorescence detection method, it can be confirmed that quantification by the quantitative analysis method according to the present invention is inappropriate.
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| JP2020110752A (en) * | 2019-01-09 | 2020-07-27 | 株式会社あじかん | Peroxide lipid absorbent, food product, and production method of peroxide lipid absorbent |
| JPWO2020213743A1 (en) * | 2019-04-19 | 2020-10-22 |
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| JP2020110752A (en) * | 2019-01-09 | 2020-07-27 | 株式会社あじかん | Peroxide lipid absorbent, food product, and production method of peroxide lipid absorbent |
| JP7250269B2 (en) | 2019-01-09 | 2023-04-03 | 株式会社あじかん | Lipid peroxide adsorbent and method for producing lipid peroxide adsorbent |
| JPWO2020213743A1 (en) * | 2019-04-19 | 2020-10-22 | ||
| WO2020213743A1 (en) * | 2019-04-19 | 2020-10-22 | 株式会社 資生堂 | Method and device for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibiting agent, method for screening anti-photoaging and/or dermal pigmentation inhibiting agent, method for evaluating anti-photoaging and/or dermal pigmentation inhibiting cosmetic treatment, and method for evaluating photoaging and/or dermal pigmentation |
| JP7439059B2 (en) | 2019-04-19 | 2024-02-27 | 株式会社 資生堂 | Methods and devices for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibitors, screening methods for anti-photoaging and/or dermal pigmentation inhibitors, and anti-photoaging and/or dermal pigmentation inhibitors. Evaluation method of cosmetic treatment for suppressing photoaging and/or dermal pigmentation, and evaluation method of photoaging and/or dermal pigmentation degree |
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