JPWO2018193565A1 - Method for evaluating the effect of a test substance or article on the skin - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
被験物質又は物品をヒトの皮膚に接触させ、
接触前後の皮膚の水分量又は水分分布の変化量を、基準値と比較し、
比較結果から、前記被験物質又は物品が、皮膚バリア機能に与える影響を評価する、
被験物質又は物品の評価方法であって、
水の負荷量を変えての皮膚への水負荷試験により測定された、皮膚の水分量又は水分分布と、皮膚のバリア機能とが関連付けられ、
皮膚の水分量又は水分分布と皮膚のバリア機能との関連性から、皮膚のバリア機能に変化が確認されるときの皮膚の水分量又は水分分布が、前記基準値として設定されたものである、
被験物質又は物品の評価方法。
Bringing a test substance or article into contact with human skin,
Compare the amount of change in moisture content or moisture distribution of the skin before and after contact with the reference value,
Evaluating the influence of the test substance or the article on the skin barrier function from the comparison result
A method of evaluating a test substance or article, comprising
Water content or distribution of the skin, as measured by water load test on the skin with varying water load, is associated with the barrier function of the skin,
The water content or the water distribution of the skin when a change in the skin barrier function is confirmed is set as the reference value from the relation between the water content or the water distribution of the skin and the barrier function of the skin.
Evaluation method of test substance or article.
Description
本発明は、被験物質又は物品が皮膚に与える影響を評価する方法に関する。 The present invention relates to a method of evaluating the influence of a test substance or an article on the skin.
皮膚は、体内からの水分や生体成分の損失、並びに外部からの異物の進入を防ぐ機能を有している。このような機能は一般に、「皮膚のバリア機能」と呼ばれている。皮膚のバリア機能は主に、皮膚の最外層に存在する角層が担っている。 The skin has a function to prevent the loss of water and biological components from the body and the entry of foreign matter from the outside. Such a function is generally called "skin barrier function". The barrier function of the skin is mainly carried by the stratum corneum present in the outermost layer of the skin.
健常な皮膚の一般的性質として、角層の水分量は皮膚表面で約２０％〜３０％であることが知られている。深さ方向へ進むにつれ徐々に水分量は増加し、表皮では水分量は約７０％となる。そして、角層は人の目に触れる部分であるため、化学物質などの異物が皮膚に侵入した場合、炎症などの肌トラブルが一目瞭然で認識される。
また、おむつ、生理用品などの吸収性物品や、創傷保護材、マスク、ゴム手袋などで皮膚を被覆又は接触状態にすると、皮膚トラブルが生じる場合がある。実際に多くの女性は、生理用品の使用期間中に、かゆみやかぶれ等の皮膚トラブルに悩んでいる。また、赤ちゃんのおむつかぶれも頻度高く認められている。
これとは逆に、医薬品、医薬部外品、化粧料を皮膚に塗布してケアする場合、これらに含まれる有効成分を角層に十分に浸透させることが重要である。
このように、角層に対する物質の浸透性は、医療分野や美容分野において非常に重要な要素の１つである。As a general property of healthy skin, it is known that the water content of the stratum corneum is about 20% to 30% at the skin surface. The amount of water gradually increases as it moves in the depth direction, and the amount of water in the epidermis is about 70%. And, since the stratum corneum is a portion that human eyes see, when a foreign substance such as a chemical substance intrudes into the skin, skin troubles such as inflammation are recognized at a glance.
In addition, when the skin is covered or brought into contact with an absorbent article such as a diaper or a sanitary product, a wound protection material, a mask, a rubber glove or the like, skin problems may occur. In fact, many women suffer from skin problems such as itching and rash during the period of use of catamenial products. In addition, baby diaper rash is also frequently observed.
On the contrary, when applying medicines, quasi-drugs and cosmetics to the skin for care, it is important that the active ingredient contained in them is sufficiently penetrated into the stratum corneum.
Thus, the permeability of the substance to the stratum corneum is one of the very important elements in the medical field and the cosmetic field.
これまでに、ヒトの前腕内側に過剰な水分を含むガーゼやおむつを貼付した場合、皮膚の水分量が増加して角層が膨潤し、その結果ニコチン酸メチルの物質透過性が亢進することが知られている（非特許文献１参照）。このような現象に基づき、皮膚のバリア機能の評価方法として、ニコチン酸メチルを皮膚に塗布し評価する方法が汎用されている（例えば、非特許文献１〜３参照）。この方法では、ニコチン酸メチルが有する血管拡張作用により誘導される紅斑に基づき、皮膚のバリア機能を評価する。
しかし紅斑は、血流、外気温度、体温上昇、体温降下、身体の動き、発汗、被験者の皮膚色など、皮膚機能以外の要因によっても影響を受ける。よって、前述の方法ではバリア機能の評価結果が様々な要因（制御された環境や状況を除く）に左右されるおそれがあった。また、ニコチン酸メチルは血管拡張作用を有するため、被験者の皮膚にニコチン酸メチルを塗布すること自体、負担である可能性も考えられる。So far, when gauze or diapers containing excess water are attached to the inside of the forearm of a human, the water content of the skin increases and the stratum corneum swells, as a result, the substance permeability of methyl nicotinate is enhanced It is known (refer nonpatent literature 1). Based on such a phenomenon, as a method of evaluating the barrier function of the skin, a method of applying methyl nicotinate to the skin and evaluating it is widely used (see, for example, non-patent documents 1 to 3). In this method, the barrier function of the skin is evaluated based on the erythema induced by the vasodilating action of methyl nicotinate.
However, erythema is also influenced by factors other than skin function such as blood flow, ambient temperature, temperature rise, temperature drop, body movement, sweating, skin color of the subject, etc. Therefore, in the method described above, the evaluation result of the barrier function may be influenced by various factors (except for the controlled environment and situation). In addition, since methyl nicotinate has a vasodilator action, applying methyl nicotinate to the skin of a subject may itself be a burden.
本発明は、被験物質又は物品をヒトの皮膚に接触させ、接触前後の皮膚の水分量又は水分分布の変化量を、基準値と比較し、比較結果から、前記被験物質又は物品が、皮膚バリア機能に与える影響を評価する、被験物質又は物品の評価方法であって、
水の負荷量を変えての皮膚への水負荷試験により測定された、皮膚の水分量又は水分分布と、皮膚のバリア機能とが関連付けられ、皮膚の水分量又は水分分布と皮膚のバリア機能との関連性から、皮膚のバリア機能に変化が確認されるときの皮膚の水分量又は水分分布が、前記基準値として設定されたものである、被験物質又は物品の評価方法に関する。In the present invention, a test substance or article is brought into contact with human skin, and the amount of change in moisture content or distribution of skin before and after contact is compared with a reference value, and the comparison result shows that the test substance or article is a skin barrier A method for evaluating a test substance or article, which evaluates an influence on function, comprising:
The water content or distribution of the skin and the barrier function of the skin, which are measured by the water load test on the skin by changing the water load, are related to the water content or distribution of the skin and the skin barrier function The present invention relates to a method for evaluating a test substance or an article, wherein the amount or distribution of water in the skin at the time when a change in the barrier function of the skin is confirmed from the relevance of
また本発明は、被験物質又は物品をヒトの皮膚に接触させた後のヒトの皮膚の深さごとの水分量を測定して、被験物質又は物品の接触後の水分分布を取得し、被験物質又は物品の接触後の水分分布と基準水分分布とを比較することで、被験物質又は物品が、ヒトの皮膚のバリア機能に与える影響を評価する、被験物質又は物品の評価方法であって、
水又は水溶液のヒトの皮膚への負荷前に、ヒトの皮膚の深さ毎の水分量を測定して初期水分分布を取得し、水又は水溶液のヒトの皮膚への負荷終了直後に、ヒトの皮膚の深さ毎の水分量の測定による水又は水溶液の負荷後の水分分布の取得と、指標物質を皮膚に適用して該指標物質の浸透度の測定を行い、初期水分分布から水又は水溶液の負荷終了直後の水分分布への変化と、指標物質の浸透度から、指標物質の浸透度と水又は水溶液の負荷量との関連性を決定し、前記関連性が変化したときのヒトの皮膚の深さ毎の水又は水溶液の負荷後の水分分布を、前記基準水分分布とする、被験物質又は物品の評価方法に関する。The present invention also measures the amount of water per depth of human skin after bringing a test substance or article into contact with human skin, and acquires the water distribution after contact of the test substance or article, Or a method for evaluating a test substance or article, which evaluates the influence of a test substance or article on the barrier function of human skin by comparing the distribution of water after contact of the article with the reference water distribution.
Before loading water or an aqueous solution on human skin, measure the water content per depth of human skin to obtain an initial water distribution, and immediately after termination of water or aqueous solution loading on human skin, Acquisition of water distribution after loading of water or aqueous solution by measurement of water content for each depth of skin and measurement of permeability of the indicator substance by applying indicator substance to skin, water or aqueous solution from initial water distribution The relationship between the permeability of the indicator substance and the load of water or aqueous solution is determined from the change to the water distribution immediately after the end of loading and the permeability of the indicator substance, and human skin when the relation changes. Water distribution after loading of water or an aqueous solution for each depth is the reference water distribution, the evaluation method of a test substance or an article.
さらに本発明は、被験物質又は物品が、ヒトの皮膚のバリア機能に与える影響を評価する方法であって、
被験物質又は物品のヒトの皮膚への接触前と接触後の皮膚の深さ毎の水分量を測定して水分分布を取得し、接触前と接触後の水分分布における、接触前の初期の角層厚よりも深部の水分量の差を指標として、被験物質又は物品がヒトの皮膚のバリア機能に与える影響を評価する、被験物質又は物品の評価方法に関する。Furthermore, the present invention is a method of evaluating the influence of a test substance or article on the barrier function of human skin,
The water content is measured by measuring the amount of water per depth of the skin before and after contact of the test substance or article with human skin to obtain the water distribution, and the initial angle before contact in the water distribution before and after contact The present invention relates to a method for evaluating a test substance or an article, which evaluates the influence of the test substance or the article on the barrier function of human skin by using a difference in the amount of water deeper than the layer thickness as an index.
本発明の上記及び他の特徴及び利点は、適宜添付の図面を参照して、下記の記載からより明らかになるであろう。 The above and other features and advantages of the present invention will become more apparent from the following description with reference to the accompanying drawings as appropriate.
本発明は、非侵襲的で、被験者に対して負担が少なく安全、簡便かつ短時間に、被験物質又は物品が皮膚バリア機能に与える影響を評価する、被験物質又は物品の評価方法を提供する。 The present invention provides a method for evaluating a test substance or article which is non-invasive, evaluates the influence of the test substance or article on the skin barrier function in a low burden, safely, conveniently and in a short time to the subject.
従来より、皮膚の最表層に位置する角層の水分による膨潤状態（水和状態）が皮膚のバリア機能に影響し、それに伴い皮膚への物質の透過性も変化することが考えられていた。よって、被験物質又は物品が皮膚と接触することによって生じる角層の水和状態を把握できれば、被験物質又は物品が、皮膚のバリア機能にどのような影響を与えるかを、被験者に負担が少なく、様々な要因による影響を受けずに評価できるものと期待される。
しかし、皮膚への物質透過性変化と、その際の皮膚の水分量の増減や皮膚の深さ方向における水分分布との関係性について、詳細には解明されていなかった。Heretofore, it has been considered that the water swelling state (hydration state) of the stratum corneum located on the outermost layer of the skin affects the barrier function of the skin, and the permeability of the substance to the skin also changes accordingly. Therefore, if the hydration state of the stratum corneum caused by the test substance or article coming into contact with the skin can be grasped, the subject is less burdened by the test substance or article on the barrier function of the skin. It is expected that they can be evaluated without being affected by various factors.
However, the relationship between the change in substance permeability to the skin, and the increase or decrease in the amount of water in the skin and the water distribution in the depth direction of the skin at that time has not been elucidated in detail.
そこで本発明者らは、生きたヒト皮膚における物質の透過性と、皮膚の最表面に位置する角層の水分量や深さ方向における水分分布との間の関連性について検討を行った。その結果、角層の水分量や深さ方向における水分分布と、皮膚への物質の透過性との間で高い関連性があることを生きたヒトにおいて初めて見出した。そして、被験物質又は物品をヒトの皮膚に接触させた前後での、非侵襲的に測定した生きたヒトにおける皮膚水分量や深さ方向における水分分布の変化から、被験物質又は物品が皮膚のバリア機能に与える影響を評価できることを見い出した。
本発明はこれらの知見に基づき完成されるに至ったものである。Therefore, the present inventors examined the relationship between the permeability of a substance in living human skin and the water content in the stratum corneum located on the outermost surface of the skin and the water distribution in the depth direction. As a result, it was found for the first time in living humans that there is a high correlation between the moisture content in the stratum corneum and the water distribution in the depth direction, and the permeability of substances to the skin. Then, the test substance or article is a skin barrier because of changes in skin moisture content and depth distribution in a non-invasively measured living human before and after contacting the test substance or article with human skin. It has been found that the impact on function can be evaluated.
The present invention has been completed based on these findings.
本発明では、皮膚の水分量や水分分布と皮膚のバリア機能との関連性に基づき、被験物質又は物品の接触前後の皮膚の水分量や水分分布から、皮膚のバリア機能を評価する。よって本発明によれば、被験物質又は物品が皮膚に与える影響を安全、簡便かつ短時間に知ることができる。 In the present invention, the barrier function of the skin is evaluated from the moisture content and the moisture distribution of the skin before and after the contact of the test substance or the article based on the relationship between the moisture content and the moisture distribution of the skin and the barrier function of the skin. Therefore, according to the present invention, the influence of the test substance or the article on the skin can be known safely, simply and in a short time.
本発明では、図１に示すように、被験物質又は物品をヒトの皮膚に接触させ（S1）、被験物質又は物品の接触前後に測定した皮膚の水分分量又は水分分布の変化量や、接触後の皮膚の水分分布を、基準値又は基準水分分布と比較し（S2）、基準値又は基準水分分布との比較結果から、被験物質又は物品が皮膚のバリア機能に与える影響を評価する（S3）。ここで、皮膚のバリア機能に与える影響の解析と評価は、皮膚の水分量又は水分分布の変化量と、皮膚のバリア機能との関連性に基づいて行われる。
以下、本発明について詳細に説明する。In the present invention, as shown in FIG. 1, the test substance or article is brought into contact with human skin (S1), and the amount of change in moisture content or distribution of skin measured before and after contact with the test substance or article The water distribution of the skin of the skin is compared with the reference value or the reference water distribution (S2), and the influence of the test substance or the article on the barrier function of the skin is evaluated from the comparison result with the reference value or the reference water distribution (S3) . Here, analysis and evaluation of the effect on the barrier function of the skin are performed based on the relationship between the amount of change in the amount of moisture or the distribution of water in the skin and the barrier function of the skin.
Hereinafter, the present invention will be described in detail.
本明細書において「バリア機能」とは、皮膚外部から内部への物質の浸透性を指す。
本明細書において「被験物質」とは、皮膚との接触や皮膚への適用により、皮膚内へ浸透する物質（例えば、化合物、化学物質、色素、生体由来の分泌物や***物、保湿剤、外用剤、化粧料）をいう。なお「被験物質」及び後述する「指標物質」には、水溶性成分や疎水性成分も包含される。物質の具体例としては、水若しくは水蒸気、生理食塩水、リン酸緩衝生理食塩水、ニコチン酸メチル、ニコチン酸ヘキシル、乳酸、黄色4号（タートラジン）、赤色215号（ローダミンＢステアレート）、カフェイン、ユビナール、カロテノイド、レチノール、尿、汗、経血、便、擬似尿、擬似経血、擬似便、並びに、皮膚保湿剤、皮膚外用剤、皮膚化粧料若しくは蒸気温熱具に含まれる有効成分や薬効成分が挙げられる。As used herein, "barrier function" refers to the permeability of a substance from the outside to the inside of the skin.
In the present specification, the “test substance” refers to a substance that penetrates into the skin upon contact with the skin or application to the skin (for example, a compound, a chemical substance, a pigment, secretion or excrement of biological origin, a moisturizer, External preparation, cosmetics). The “test substance” and the “indicator substance” described later also include water-soluble components and hydrophobic components. Specific examples of the substance include water or water vapor, physiological saline, phosphate buffered saline, methyl nicotinate, hexyl nicotinate, lactic acid, yellow No. 4 (turtradine), red No. 215 (rhodamine B stearate), cafe In, ubinal, carotenoid, retinol, urine, sweat, menstrual blood, feces, simulated urine, simulated menstrual blood, simulated feces, as well as skin moisturizers, external preparations for skin, active ingredients contained in skin cosmetics or vapor heating tools, It includes medicinal ingredients.
まず、被験物質又は物品の皮膚への接触前後で、バリア機能の評価を行う部位の皮膚水分量又は水分分布を測定する。皮膚水分量又は水分分布の測定は、非侵襲的に測定することが好ましい。この場合、被験者に対して負担が少なく、安全に皮膚のバリア機能を評価できる。
角層内の水分量又は水分分布を非侵襲的に測定する方法は、常法から適宜選択することができる。例えば、ラマン分光法、皮膚静電容量測定手段、高周波電流測定手段、赤外分光法、又は近赤外分光法などを用いる方法が挙げられる。First, before and after the contact of the test substance or the article with the skin, the skin moisture content or the moisture distribution of the site where the barrier function is to be evaluated is measured. The measurement of skin moisture content or moisture distribution is preferably non-invasively measured. In this case, the burden on the subject is small, and the barrier function of the skin can be evaluated safely.
The method for non-invasively measuring the water content or water distribution in the stratum corneum can be appropriately selected from conventional methods. For example, methods using Raman spectroscopy, skin capacitance measuring means, high frequency current measuring means, infrared spectroscopy, near infrared spectroscopy, etc. may be mentioned.
本発明では、皮膚に対して被験物質又は物品を負荷した場合の皮膚水分量又は水分分布の変化を、ラマン分光法、皮膚静電容量測定手段などに従い皮膚水分量又は水分分布を測定できる装置を用いて測定する。そして、その変化量から、該物質又は物品が皮膚のバリア機能に及ぼす影響を評価する。
後述の実施例で示したように、ヒトの皮膚に過剰な水分を負荷すると物質透過性が亢進し、皮膚のバリア機能は低下する。
このような知見に対して、本発明者らは、皮膚に対して被験物質又は物品を負荷した場合の皮膚水分量又は水分分布の変化値や基準水分分布を基準に、皮膚のバリア機能を評価できることをはじめて見出した。後述の実施例でも示すように、皮膚の水分量がある一定値までは物質浸透性は亢進しない。しかし、皮膚の水分量が一定値以上増加すると、物質浸透性が亢進する。なお、後述の実施例では、指標物質の代表例として、水溶性物質であるニコチン酸メチルの浸透性の評価を行った。水溶性物質は皮膚の水分量が増えると皮膚内へ物質が分配されやすくなり、皮膚により浸透する可能性が考えられる。In the present invention, a device capable of measuring skin moisture content or moisture distribution according to Raman spectroscopy, skin capacitance measurement means, etc., for changes in skin moisture content or moisture distribution when a test substance or article is loaded on the skin Use and measure. Then, from the amount of change, the influence of the substance or the article on the barrier function of the skin is evaluated.
As shown in the following examples, when the human skin is loaded with excess water, the material permeability is enhanced and the barrier function of the skin is reduced.
With respect to such findings, the present inventors evaluate the barrier function of the skin based on the change value of the skin moisture amount or the moisture distribution or the reference moisture distribution when the test substance or the article is loaded on the skin. I found for the first time what I could do. As shown in the following examples, the substance permeability does not increase until the water content of the skin reaches a certain value. However, if the amount of water in the skin increases beyond a certain value, substance permeability will be enhanced. In Examples described later, the permeability of methyl nicotinate, which is a water-soluble substance, was evaluated as a representative example of the indicator substance. The water-soluble substance is likely to be distributed into the skin as the amount of water in the skin increases, and it is considered that the substance may penetrate into the skin.
本発明において、被験物質又は物品が、皮膚バリア機能に与える影響を評価するための基準値や基準水分分布は適宜設定することができる。以下、本発明における基準値や基準水分分布の設定方法ついて、具体例に基づき説明する。しかし、本発明はこれに制限するものではない。
まず、水の負荷量を変えて皮膚への水負荷試験を行い、各水負荷量での皮膚の水分量又は水分分布を常法により測定する。次に、測定した皮膚の水分量又は水分分布と、皮膚のバリア機能との関連付けを行う。In the present invention, the reference value and reference water distribution for evaluating the influence of the test substance or the article on the skin barrier function can be set appropriately. Hereinafter, the method of setting the reference value and the reference moisture distribution in the present invention will be described based on a specific example. However, the present invention is not limited to this.
First, the water loading test is performed by changing the water loading amount, and the water content or water distribution of the skin at each water loading amount is measured by an ordinary method. Next, the measured moisture content or moisture distribution of the skin is associated with the barrier function of the skin.
本発明の第１の実施態様において、皮膚の水分量又は水分分布と皮膚のバリア機能との関連付けは、皮膚のバリア機能に変化が確認されるときの皮膚の水分分量又は水分分布を「基準値」として設定することで行う。
「基準値」の設定方法としては、皮膚外部から内部への前記指標物質の浸透性から設定する方法が挙げられる。前述の皮膚への水負荷試験に続いて皮膚への指標物質の負荷試験を行って指標物質の浸透性を常法により測定し、指標物質の浸透性に変化がみられたときの皮膚の水分量又は水分分布を「基準値」とすることができる。なお、本実施態様においては、負荷した水分量と、指標物質の浸透性との間で関連に変化が確認されたときや、指標物質の浸透性が一定の状態に達したときを基準値とすることができる。In the first embodiment of the present invention, the association between the moisture content or moisture distribution of the skin and the barrier function of the skin refers to the moisture content or moisture distribution of the skin when changes in the barrier function of the skin are confirmed. It does by setting as ".
As a method of setting the "reference value", a method of setting from the permeability of the indicator substance from the outside to the inside of the skin can be mentioned. Following the above-mentioned water load test on the skin, the load test of the indicator substance on the skin is carried out to measure the permeability of the indicator substance by a conventional method, and the moisture of the skin when the permeability of the index substance is changed The amount or moisture distribution can be taken as the "reference value". In the present embodiment, when a change in the relationship between the amount of water loaded and the permeability of the indicator substance is confirmed, or when the permeability of the indicator substance reaches a constant state, can do.
本発明の第２の実施態様において、皮膚の水分量又は水分分布と皮膚のバリア機能との関連付けは、指標物質の浸透度と水又は水溶液の負荷量との関連性が変化したときの、ヒトの皮膚の深さ毎の水又は水溶液の負荷後の水分分布を、「基準水分分布」として設定することで行う。
本実施態様ではまず、水又は水溶液のヒトの皮膚への負荷前に、ヒトの皮膚の深さ毎の水分量を測定して初期水分分布を取得する。
さらに、水又は水溶液をヒトの皮膚に一定時間負荷し、負荷終了直後にヒトの皮膚の深さ毎の水分量を測定して水又は水溶液の負荷後の水分分布を取得する。続けて指標物質を皮膚に適用して該指標物質の浸透度を測定する。
そして、指標物質の浸透度と水又は水溶液の負荷量との関連性が変化するまで、水又は水溶液の負荷量を増加又は減少させながら前記測定を繰り返す。この測定結果に基づいて、指標物質の浸透度と水又は水溶液の負荷量との関連性が変化したときのヒトの皮膚の深さ毎の水又は水溶液の負荷後の水分分布を、前記基準水分分布とすることができる。In the second embodiment of the present invention, the association between skin moisture content or moisture distribution and skin barrier function is determined by changing the relationship between the permeability of the indicator substance and the load of water or an aqueous solution. The water distribution after loading of water or an aqueous solution for each skin depth is set as the “reference water distribution”.
In the present embodiment, first, before the load of water or an aqueous solution on human skin, the amount of water at each depth of human skin is measured to obtain an initial water distribution.
Furthermore, water or an aqueous solution is loaded on human skin for a certain period of time, and immediately after the end of loading, the amount of water at each depth of human skin is measured to obtain the water distribution after loading of water or an aqueous solution. Subsequently, the indicator substance is applied to the skin to measure the permeability of the indicator substance.
Then, the measurement is repeated while increasing or decreasing the loading amount of water or an aqueous solution until the relationship between the permeability of the indicator substance and the loading amount of water or an aqueous solution changes. The reference moisture distribution after loading of water or an aqueous solution for each depth of human skin when the relationship between the permeability of the indicator substance and the loading amount of water or an aqueous solution changes based on the measurement result, It can be distributed.
本発明の第１及び第２の実施態様において、皮膚への浸透性を評価するための指標物質は、適宜選択することができる。例えば、ニコチン酸メチル、ニコチン酸ヘキシル、乳酸、黄色4号、赤色215号、カフェイン、ユビナール、カロテノイド、レチノール、並びに、皮膚保湿剤、皮膚外用剤、皮膚化粧料若しくは蒸気温熱具に含まれる有効成分や薬効成分が挙げられる。 In the first and second embodiments of the present invention, an index substance for evaluating skin permeability can be selected as appropriate. For example, methyl nicotinate, hexyl nicotinate, lactic acid, yellow No. 4, red No. 215, caffeine, ubinal, carotenoids, retinol and skin moisturizers, external preparations for skin, skin cosmetics or steam heating devices are effective. Ingredients and medicinal ingredients can be mentioned.
本発明の第３の実施態様では、初期の角層厚よりも深部の水分量と、皮膚のバリア機能とを関連付ける。
本実施態様では、被験物質又は物品をヒトの皮膚に接触させる前と接触させた後の皮膚の深さ毎の水分量を測定して水分分布を取得する。そして、接触前後の水分分布を比較し、接触前の初期の角層厚よりも深部の水分量の差を指標として、被験物質又は物品の評価を行う。In the third embodiment of the present invention, the water barrier function is associated with the amount of water deeper than the initial angular layer thickness.
In this embodiment, the moisture distribution is obtained by measuring the amount of water at each depth of the skin before and after the test substance or the article is brought into contact with human skin. Then, the water distribution before and after the contact is compared, and the evaluation of the test substance or the article is performed by using the difference in the amount of water in the deeper portion than the initial angular layer thickness before the contact.
本発明では、角層表層から顆粒層に達するまでのいずれかの領域又は部位での水分量又は水分分布を測定してもよい。あるいは、本発明で用いる水分量又は水分分布の測定装置の特性、例えば皮膚の内部に照射した励起波長によるラマン散乱の強度などを考慮し、皮膚表面から所定の深さまでの領域の水分量若しくは水分分布、又は皮膚表面から所定の深さの部位の水分量を測定してもよい。例えば、皮膚のバリア機能を評価するために、皮膚表面から初期の角層厚における水分量又は水分分布を測定することが好ましい。 In the present invention, the water content or water distribution in any region or site from the stratum corneum surface layer to the granule layer may be measured. Alternatively, considering the characteristics of the water content or water distribution measurement device used in the present invention, for example, the intensity of Raman scattering by the excitation wavelength irradiated to the inside of the skin, the water content or water content in the region from the skin surface to a predetermined depth The distribution or the amount of water at a predetermined depth from the skin surface may be measured. For example, in order to evaluate the barrier function of the skin, it is preferable to measure the amount of water or the distribution of water at an initial angular layer thickness from the skin surface.
なお本明細書において、「初期の角層厚」とは、水分、被験物質又は物品負荷前の角層の厚さを指す。なお、ラマンスペクトルからの初期の角層厚の測定は、IFSCC Magazine, 2009, vol. 12(1), p. 9-15に記載の方法に準じて行うことができる。常法に従い測定したラマンスペクトルから各深さにおける水分量を算出し、深さ方向に対して一定距離（例えば1μm）ごとに近似的に算出した水分量の数値をプロットしたグラフを作成し、水分分布プロファイルを取得する。そして、取得した水分プロファイルに対して２本の直線で近似し、その交点までの深さを、角層表層からの角層厚と定義することができる。
また、ラマンスペクトルで皮膚の水分量を測定する場合、タンパク質のＣＨ伸縮振動由来の信号強度は、対物レンズの窓材と皮膚の境界よりやや皮膚側で最大値を示した後に、深部にいくほど信号強度が低下する。皮膚では深部にいくほど光の拡散などによる信号の減衰が生じるためである。窓材にはタンパク質のＣＨ伸縮振動由来の信号は存在しないため、レーザーの焦点が窓材の内にあるときはＣＨ伸縮振動由来の信号強度は０になる。一方、皮膚内にレーザーの焦点があるときのＣＨ伸縮振動由来の信号強度を１００とすると、窓材と皮膚の界面に焦点がきた場合、ＣＨ伸縮振動由来の信号強度は５０となる。そこで、ＣＨ伸縮振動由来の信号強度が最大値の半値を示すところを皮膚と窓材の境界と定義している。ただし、実際には窓材にはＣＨ伸縮振動由来の信号強度が弱いながらも検出されることがある。そのため、特開２０１２−５０７３９号公報に記載の方法に従って水分量を計算、解析すると、高い水分量を示すことになり、皮膚と窓材の界面、すなわち皮膚の最表面の水分量に関しては適切ではないデータであるということが理論上明らかとなっている。さらに、本条件に用いた共焦点ラマン分光装置の深さ方向の空間分解能は２μｍで、解析の際に１μｍ毎の水分量を補間して算出している。よって、皮膚表面の水分量は、データ解析からは省くことが好ましい。また、共焦点ラマンの装置によってそれぞれ分解能が異なることを考慮し、皮膚表面水分量を求める際には、本発明においては、実際の皮膚表面から最低５μｍまでのデータを省き、皮膚表面から５μｍ以上の部位をデータ解析上の「皮膚表面」とすることが好ましく、実際の皮膚表面から最低２μｍまでのデータを省き、皮膚表面から２μｍ以上の部位をデータ解析上の「皮膚表面」とすることがより好ましく、実際の皮膚表面から２μｍまでのデータを省き、皮膚表面から２μｍの部位をデータ解析上の「皮膚表面」とすることがさらに好ましい。
共焦点ラマン分光顕微鏡を用いた測定で得られたラマンスペクトルから水分量を近似的に算出する方法は、特開２０１２−５０７３９号公報に記載の方法を参照して行うことができる。In the present specification, the "initial angular layer thickness" refers to the thickness of the stratum corneum prior to loading of moisture, a test substance or an article. The measurement of the initial angular layer thickness from the Raman spectrum can be performed according to the method described in IFSCC Magazine, 2009, vol. 12 (1), p. 9-15. The amount of water at each depth is calculated from the Raman spectrum measured according to the conventional method, and a graph plotting the numerical value of the amount of water approximately calculated every fixed distance (for example, 1 μm) in the depth direction is created. Get distribution profile. Then, the obtained water profile can be approximated by two straight lines, and the depth up to the intersection can be defined as the angular layer thickness from the surface layer of the stratum corneum.
In addition, when measuring the water content of the skin by Raman spectrum, the signal intensity derived from CH stretching vibration of protein shows a maximum value slightly on the skin side from the boundary between the window material of the objective lens and the skin, and then goes deeper Signal strength decreases. This is because the skin is apt to attenuate the signal due to the diffusion of light as it gets deeper. Since there is no signal derived from CH stretching vibration of protein in the window material, the signal intensity derived from CH stretching vibration becomes zero when the focal point of the laser is within the window material. On the other hand, assuming that the signal strength derived from CH stretching vibration when the focus of the laser is in the skin is 100, the signal strength originating from CH stretching vibration is 50 when the interface is at the interface between the window material and the skin. Therefore, the place where the signal intensity derived from CH stretching vibration shows a half value of the maximum value is defined as the boundary between the skin and the window material. However, in fact, although the signal strength derived from CH stretching vibration may be weak in the window material, it may be detected. Therefore, when the water content is calculated and analyzed according to the method described in JP 2012-50739 A, a high water content will be shown, and the interface between the skin and the window material, that is, the water content on the outermost surface of the skin is appropriate. It is theoretically clear that there is no data. Furthermore, the spatial resolution in the depth direction of the confocal Raman spectroscopy apparatus used for this condition is 2 μm, and the water content in each 1 μm is calculated by interpolation at the time of analysis. Therefore, it is preferable to omit the amount of water on the skin surface from data analysis. In addition, considering the difference in resolution depending on the confocal Raman devices, when determining the skin surface water content, in the present invention, data of at least 5 μm from the actual skin surface is omitted, and 5 μm or more from the skin surface In the data analysis, it is preferable to use the "skin surface" in data analysis, and to omit data of at least 2 μm from the actual skin surface, and use the "skin surface" in data analysis to be 2 μm or more from the skin surface More preferably, data up to 2 μm from the actual skin surface are omitted, and a site 2 μm from the skin surface is taken as the “skin surface” for data analysis.
The method of approximately calculating the water content from the Raman spectrum obtained by the measurement using the confocal Raman spectroscopy microscope can be performed with reference to the method described in JP-A-2012-50739.
ラマン分光法にて測定した初期の角層厚は通常、およそ１０μｍ〜２０μｍ、平均で約１３μｍである。そこで、本発明において、皮膚の水分量は、実際の皮膚表面からの深さ２〜４０μｍの深さまで、好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より好ましくは皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで、を測定し、解析するのが好ましい。 The initial angular layer thickness measured by Raman spectroscopy is usually about 10 μm to 20 μm, and about 13 μm on average. Therefore, in the present invention, the moisture content of the skin is from 2 to 40 μm from the actual skin surface, preferably from 2 to 35 μm from the skin surface, more preferably from the skin surface It is preferable to measure and analyze up to a depth of 2 to 20 μm, more preferably to a depth of 9 to 15 μm from the skin surface, and more preferably to a depth of about 13 μm from the skin surface.
後述の実施例でも示すように、ラマン分光法や皮膚静電容量測定手段などで測定した皮膚の水分量又は水分分布と、皮膚のバリア機能とは強い関連性を有する。具体的には、被験物質又は物品の接触により皮膚の水分量の変化量が一定値以上増加すると、ニコチン酸メチルなどの物質の皮膚への浸透性が亢進する。
本発明では、上述のような皮膚の水分量又は水分分布と、バリア機能との関連性に基づいて、非侵襲的に測定した皮膚の水分量又は水分分布の変化量から、被験物質又は物品が、皮膚のバリア機能に及ぼす影響を評価する。具体的には、被験物質又は物品の接触後の皮膚の水分量が、所定の基準値以上の変化量で増加した場合、バリア機能を低下させる（例えば、皮膚外部から内部への物質の浸透性を亢進させる）と評価することができる。一方、被験物質又は物品の接触後の皮膚の水分量が、基準値より小さい変化量で変化した場合、バリア機能は変化してないと評価することができる。As also shown in the examples described later, the moisture content or moisture distribution of the skin measured by Raman spectroscopy, skin capacitance measurement means or the like has a strong relationship with the barrier function of the skin. Specifically, when the amount of change in the amount of water in the skin increases by a certain value or more due to the contact of a test substance or an article, the permeability of a substance such as methyl nicotinate to the skin is enhanced.
In the present invention, the test substance or article is obtained from the change in the amount or distribution of moisture in the skin non-invasively measured based on the relationship between the amount or distribution of moisture in the skin as described above and the barrier function. Evaluate the effect on skin barrier function. Specifically, when the water content of the skin after contact with the test substance or article increases by a change amount equal to or more than a predetermined reference value, the barrier function is reduced (for example, the permeability of the substance from the outside to the inside of the skin) Can be evaluated as On the other hand, when the moisture content of the skin after the contact of the test substance or the article changes by a smaller change than the reference value, it can be evaluated that the barrier function is not changed.
例えば、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、皮膚静電容量測定装置（静電容量から計算された水分量の相対値(A.U.)を算出する装置）を用いて測定した、被験物質又は物品の接触前後の皮膚の水分量の変化量の基準値を、８(A.U.)、好ましくは１２(A.U.)、より好ましくは１６(A.U.)に設定する。そして、実際に測定した被験物質又は物品の接触後の皮膚の水分量が、前記基準値以上増加した場合に、皮膚外部から内部への物質の浸透性が亢進し、被験物質又は物品の接触によりバリア機能は低下したと評価することができる。これに対して、水分量の変化量が前記基準値より小さい場合、被験物質又は物品の接触によってもバリア機能は変化していないと評価できる。 For example, the skin capacitance is measured after cleaning the skin and acclimating for at least 20 minutes to the skin on the inside of the forearm of a healthy subject, and calculating the skin capacitance measuring device The reference value of the amount of change in skin moisture before and after contact with a test substance or article, as measured using a measurement device, is set to 8 (AU), preferably 12 (AU), more preferably 16 (AU). Do. And when the moisture content of the skin after contact of the test substance or article actually measured increases more than the reference value, the permeability of the substance from the outside to the inside of the skin is enhanced, and the contact of the test substance or article The barrier function can be evaluated as degraded. On the other hand, when the amount of change in the amount of water is smaller than the reference value, it can be evaluated that the barrier function is not changed even by the contact of the test substance or the article.
加えて、被験物質又は物品の接触前後の皮膚の水分分布を比較し、初期の角層厚を基準として、被験物質又は物品の接触により初期の角層厚よりも深いところの皮膚の水分量が増加した場合、皮膚外部から内部への物質の浸透性を亢進しやすくなり、バリア機能は低下したと評価できる。
具体的には、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、実際の皮膚表面から所定の深さまで（好ましくは皮膚表面からの深さ４０μｍの深さまで、より好ましくは皮膚表面からの深さ３５μｍの深さまで、より好ましくは皮膚表面からの深さ２０μｍの深さまで）における、被験物質又は物品の接触前後の皮膚の水分分布をラマン分光法により測定する。そして、被験物質又は物品の接触前後の皮膚の水分分布を比較し、被験物質又は物品の接触により、初期の角層厚より深部における皮膚の水分量が増加した場合に、皮膚外部から内部への物質の浸透性が亢進し、被験物質又は物品の接触によりバリア機能は低下したと評価することができる。In addition, the moisture distribution of the skin before and after contact with the test substance or article is compared, and based on the initial angular layer thickness, the skin moisture content deeper than the initial angular layer thickness by contact with the test substance or article If it is increased, the permeability of the substance from the outside to the inside of the skin is likely to be enhanced, and the barrier function can be evaluated to be reduced.
Specifically, the skin is washed and acclimated for a minimum of 20 minutes to target the inner skin of the forearm of a healthy subject to a predetermined depth from the actual skin surface (preferably 40 μm deep from the skin surface) Measurement of the moisture distribution of the skin before and after contact with the test substance or article at a depth of 35 μm deep from the skin surface, more preferably 20 μm deep from the skin surface, by Raman spectroscopy Do. Then, the water distribution of the skin before and after the contact of the test substance or article is compared, and when the water content of the skin at a deeper portion than the initial angular layer thickness increases due to the contact of the test substance or article, The permeability of the substance is enhanced, and it can be evaluated that the barrier function is reduced by the contact of the test substance or the article.
また、被験物質又は物品の接触前後の皮膚表面の水分量を測定し、被験物質又は物品の接触による、皮膚表面の水分量の変化量からも、バリア機能に及ぼす影響を評価できる。
具体的には、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、皮膚表面における、被験物質又は物品の接触前後の皮膚の水分量をラマン分光法により測定し、被験物質又は物品の接触前後の皮膚の水分量を比較する。本発明においては例えば、ラマン分光法より得られた皮膚表面水分量の被験物質又は物品の接触前後の変化量の基準値を２．０倍、より好ましくは２．５倍、さらに好ましくは３．０倍に設定する。そして、実際に測定した被験物質又は物品の接触後の、皮膚の表面水分量が前記基準値以上に増加した場合に、皮膚外部から内部への物質の浸透性が亢進し、被験物質又は物品の接触によりバリア機能は低下したと評価することができる。これに対して、皮膚の表面水分量の変化量が前記基準値より小さい場合、被験物質又は物品の接触によってもバリア機能は変化していないと評価できる。In addition, the amount of water on the skin surface before and after contact with the test substance or article can be measured, and the effect on the barrier function can also be evaluated from the amount of change in the water level on the skin surface due to the contact with the test substance or article.
Specifically, the skin on the inside of the forearm of a healthy subject after washing the skin and acclimating for at least 20 minutes is targeted, and the amount of water on the skin surface before and after contact with the test substance or article is measured by Raman spectroscopy. Measure and compare the moisture content of the skin before and after contact with the test substance or article. In the present invention, for example, the reference value of the amount of change in skin surface water content obtained by Raman spectroscopy before and after contact with a test substance or article is 2.0 times, more preferably 2.5 times, still more preferably 3. Set to 0 times. And, when the surface moisture content of the skin after contact of the test substance or article actually measured increases to the above standard value, the permeability of the substance from the outside to the inside of the skin is enhanced, and the test substance or article It can be evaluated that the barrier function is lowered by the contact. On the other hand, when the amount of change in the surface water content of the skin is smaller than the reference value, it can be evaluated that the barrier function is not changed even by the contact of the test substance or the article.
また、被験物質又は物品の接触前後の初期の角層厚に相当する深さの水分量を測定し、被験物質又は物品の接触による、初期の角層厚に相当する深さの水分量の変化量からも、バリア機能に及ぼす影響を評価できる。
具体的には、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、初期の角層厚に相当する部位における、被験物質又は物品の接触前後の皮膚の水分量をラマン分光法により測定し、被験物質又は物品の接触前後の皮膚の水分量を比較する。本発明においては例えば、ラマン分光法より得られた初期の角層厚に相当する部位の水分量の被験物質又は物品の接触前後の変化量の基準値を８％、好ましくは１１％、さらに好ましくは１４％に設定する。そして、実際に測定した被験物質又は物品の接触後の、初期の角層厚に相当する部位の水分量が前記基準値以上増加した場合に、皮膚外部から内部への物質の浸透性を亢進し、被験物質又は物品の接触によりバリア機能は低下したと評価することができる。これに対して、初期の角層厚に相当する部位の水分量の変化量が前記基準値より小さい場合、被験物質又は物品の接触によってもバリア機能は変化していないと評価できる。In addition, the amount of water at a depth corresponding to the initial angular layer thickness before and after contact with the test substance or article is measured, and the change in the amount of water at a depth corresponding to the initial angular layer thickness by contact with the test substance or article The amount can also assess the impact on barrier function.
Specifically, for the skin on the inside of the forearm of a healthy subject after cleansing the skin and acclimating for a minimum of 20 minutes, the skin before and after contact with the test substance or article at the site corresponding to the initial angular layer thickness. The water content is measured by Raman spectroscopy and the water content of the skin before and after contact with the test substance or article is compared. In the present invention, for example, the reference value of the amount of change in the water content of the part corresponding to the initial angular layer thickness obtained by Raman spectroscopy before and after contact with the test substance or article is 8%, preferably 11%, more preferably Is set to 14%. Then, when the amount of water at a site corresponding to the initial angular layer thickness after contact of the test substance or article actually measured increases by the above-mentioned reference value or more, the permeability of the substance from the outside to the inside of the skin is enhanced It can be evaluated that the barrier function has been reduced by the contact of the test substance or the article. On the other hand, when the amount of change in the amount of water at a portion corresponding to the initial angular layer thickness is smaller than the reference value, it can be evaluated that the barrier function is not changed by the contact of the test substance or article.
あるいは、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、ラマン分光法により測定した、皮膚表面からの所定の深さまで（好ましくは皮膚表面からの深さ２〜４０μｍの深さまで、より好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より好ましくは皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで）における、被験物質又は物品の接触前後の皮膚の水分量の曲線下面積(Area under the curve of water profile)を求め、被験物質又は物品の接触前後の曲線下面積を比較する。本発明においては例えば、ラマン分光法より得られた被験物質又は物品の接触前後の曲線下面積の変化量の基準値を、１．４倍、好ましくは１．６倍、さらに好ましくは１．８倍に設定する。そして、実際に測定した被験物質又は物品の接触後の、皮膚表面から所定の深さまでにおける曲線下面積が前記基準値以上に増加した場合に、皮膚外部から内部への物質の浸透性が亢進し、被験物質又は物品の接触によりバリア機能は低下したと評価することができる。これに対して、曲線下面積の変化量が前記基準値より小さい場合、被験物質又は物品の接触によってもバリア機能は変化していないと評価できる。なお本明細書において「面積下曲線」は、角層における水分量を示すものであって、深さごとの水分量を深さ方向に積分することによって求められる。 Alternatively, to the skin on the inside of the forearm of a healthy subject after washing the skin and acclimating for a minimum of 20 minutes, to a predetermined depth from the skin surface (preferably the depth from the skin surface) measured by Raman spectroscopy To a depth of 2 to 40 μm, more preferably to a depth of 2 to 35 μm from the skin surface, more preferably to a depth of 2 to 20 μm from the skin surface, more preferably to a depth of 9 to Determine the area under the curve of water content of the skin before and after contact with the test substance or article at a depth of 15 μm, more preferably to a depth of about 13 μm from the skin surface, The area under the curve before and after contact of the test substance or article is compared. In the present invention, for example, the reference value of the amount of change in the area under the curve before and after contact of the test substance or article obtained by Raman spectroscopy is 1.4 times, preferably 1.6 times, more preferably 1.8. Set to double. Then, when the area under the curve from the skin surface to the predetermined depth after contact of the test substance or article actually measured increases to the above-mentioned reference value or more, the permeability of the substance from the skin to the inside of the skin is enhanced. It can be evaluated that the barrier function has been reduced by the contact of the test substance or the article. On the other hand, when the amount of change in the area under the curve is smaller than the reference value, it can be evaluated that the barrier function is not changed even by the contact of the test substance or the article. In the present specification, the "area-under-area curve" indicates the water content in the stratum corneum, and is obtained by integrating the water content for each depth in the depth direction.
ラマン分光法による皮膚の水分量の測定は、特開２０１２−５０７３９号公報に記載の方法を参照して行うことができる。本方法は、皮膚試料をタンパク質、水、およ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いた補正ラマンスペクトルを作成し、タンパク質由来のＣＨ伸縮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方法である。以下、ラマン分光法による皮膚の水分量の測定方法について詳細に説明する。しかし、本発明はこれに制限するものではない。 The measurement of the moisture content of the skin by Raman spectroscopy can be performed with reference to the method described in JP-A-2012-50739. This method approximates a skin sample with a ternary system of protein, water, and lipid, and generates a corrected Raman spectrum by subtracting the contribution of the lipid-derived Raman spectrum from the measured Raman spectrum of the skin, and CH derived from protein This is a method of measuring the amount of skin moisture based on the ratio of the signal intensity derived from the OH stretching vibration of water to the stretching signal intensity. Hereinafter, the method of measuring the water content of the skin by Raman spectroscopy will be described in detail. However, the present invention is not limited to this.
まず、ベースラインの蛍光の補正をおこなう。測定した皮膚のラマンスペクトルから蛍光によるベースラインのゆがみを排除するため、２０００〜２８００cm-1および３８００〜４２００cm-1の波数領域のデータより、最小二乗法をもち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。
蛍光の影響を排除した補正後の皮膚のスペクトルを、特開２０１２−５０７３９号公報記載の式（４）を用いて脱脂乾燥角層、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４つの重ねあわせで近似する。すなわち、最小二乗法により前記式（４）における係数Ｃ1〜Ｃ4を決定する。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。以下に示すモデル脂質のラマンスペクトルにおけるモデル脂質の寄与量を最小二乗法で見積もる。
そして、皮脂の寄与分及び細胞間脂質の寄与分を差し引いた特開２０１２−５０７３９号公報記載の式（５）で表される補正ラマンスペクトルを得る。次に、式（５）で表される補正ラマンスペクトルにおけるＣＨ伸縮信号（波数領域：２８００〜３０３０cm-1）のピーク面積に対するＯＨ伸縮振動のピーク面積（波数領域：３１００〜３７５０cm-1）の比率を算出する。そして、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タンパク質量の比とをプロットして得られる検量線（例えば、特開２０１２−５０７３９号公報の図５参照）に基づいて皮膚の水分量（質量％）を算出する。First, baseline fluorescence correction is performed. In order to eliminate the distortion of the baseline due to fluorescence from the measured Raman spectrum of the skin, the baseline is based on the cubic approximation using the least squares method from the data of the wavenumber region of 2000-2800 cm -1 and 3800-4200 cm -1. Predict and subtract this.
The spectrum of the skin after correction excluding the influence of the fluorescence is shown in FIG. 4 using the formula (4) described in JP-A-2012-50739, which is a four-layer Raman spectrum of a defatted dried stratum corneum, model sebum, model intercellular lipid and water. Approximate together. That is, the coefficients C 1 to C 4 in the equation (4) are determined by the least squares method. At this time, C 2 I SEB (ω) is a contribution of sebum, and C 3 I CER (ω) is a contribution of intercellular lipid. The amount of contribution of the model lipid in the Raman spectrum of the model lipid shown below is estimated by the method of least squares.
And the correction | amendment Raman spectrum represented by Formula (5) of Unexamined-Japanese-Patent No. 2012-50739 which deducted the contribution of sebum and the contribution of intercellular lipid is obtained. Then, CH stretch signal (wavenumber region: 2800~3030cm -1) in the correction Raman spectra of the formula (5) the peak area of the OH stretching vibration to the peak area of the (wavenumber region: 3100~3750cm -1) Ratio of Calculate Then, calibration obtained by plotting 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 Raman spectrum and the ratio of the amount of water to the amount of protein contained in the defatted skin sample The moisture content (mass%) of the skin is calculated based on the line (see, for example, FIG. 5 of JP 2012-50739A).
前述の脱脂乾燥角層、モデル皮脂、モデル細胞間脂質の調製方法の詳細を示す。しかし、本発明はこれに制限するものではない。
脱脂乾燥角層
健常男性（４０代）のかかとより角層片をナイフで切除後、クロロホルムーメタノール（１：１）に一昼夜浸漬する。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、アミノ酸などの水溶性成分を除去した脱脂乾燥角層を調製する。
モデル皮脂
下記成分を混合し、モデル皮脂を調製する。
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％
モデル細胞間脂質
シグマ社製non-hydroxy fatty cermaideをモデル細胞間脂質とする。The details of the method of preparing the above-described defatted dried stratum corneum, model sebum, and model intercellular lipid are shown. However, the present invention is not limited to this.
After cutting off the stratum corneum with a knife from the heel of a defatted dried stratum corneum healthy male (40's), it is immersed in chloroform-methanol (1: 1) overnight. After that, the stratum corneum piece sample is taken out and naturally dried to prepare a defatted dried stratum corneum from which oil-soluble components such as lipids and water-soluble components such as amino acids are removed.
Model sebum The following ingredients are mixed to prepare model sebum.
Oleic acid (Kanto Chemical Co., Ltd.): 17.4% by mass
Trioleic acid (Kanto Chemical Co., Ltd.): 43.4% by mass
Decyl oleate (Wako Pure Chemical Industries): 26.5% by mass
Squaren (Kanto Chemical Co., Ltd.): 12.7 mass%
Model intercellular lipid A non-hydroxy fatty cermaide manufactured by Sigma is used as a model intercellular lipid.
本発明で用いる物品としては、例えば、吸収性物品（脱脂綿、生理用ナプキン、紙おむつ、布おむつ、トレーニングパンツ、成人用おむつ、汗止めパット、尿取りパット、産褥パット、パンティーライナー、吸収パットなど）、蒸気温熱具、創傷保護材（絆創膏、ガーゼ、包帯など）、マスク、ゴム手袋などが挙げられる。
また、前記被験物質としては、水若しくは水蒸気、化粧料、保湿剤若しくは外用剤、蒸気温熱具に含まれる有効成分や薬効成分、生理食塩水、リン酸緩衝生理食塩水、及び生体由来の分泌物や***物である尿、汗、経血、便などが挙げられる。なお、本発明において、擬似尿、疑似汗、擬似経血、擬似便などは生体由来の分泌物や***物に含まれる。
さらに、皮膚への被験物質又は物品の接触時間も適宜設定することができる。例えば、前記被験物質又は物品をヒトの皮膚に接触させる時間は、１時間以上６時間以下、好ましくは２時間以上４時間以下、より好ましくは２．５時間以上３．５時間以下、さらに好ましくは３時間程度である。The articles used in the present invention include, for example, absorbent articles (cotton, sanitary napkins, paper diapers, cloth diapers, training pants, diapers for adults, sweat pads, urine pads, puerperal pads, panty liners, absorbent pads, etc.) , Steam heaters, wound protection materials (such as plasters, gauze, bandages), masks, rubber gloves, etc.
Moreover, as the test substance, water or water vapor, cosmetics, a moisturizing agent or an external preparation, an active ingredient or medicinal active ingredient contained in a steam heating tool, physiological saline, phosphate buffered saline, and secretions derived from a living body And excrements such as urine, sweat, menstrual blood and stool. In the present invention, simulated urine, simulated sweat, simulated menstrual blood, simulated stool and the like are included in secretions and excretions derived from a living body.
Furthermore, the contact time of the test substance or article with the skin can also be set appropriately. For example, the time for which the test substance or article is in contact with human skin is 1 hour or more and 6 hours or less, preferably 2 hours or more and 4 hours or less, more preferably 2.5 hours or more and 3.5 hours or less, more preferably It is about 3 hours.
本発明の評価方法は前述のように、皮膚水分量又は水分分布を非侵襲的に測定し、物品又は被験物質の接触による皮膚の水分量又は水分分布の変化量と皮膚のバリア機能との関連性に基づいて、測定した皮膚の水分量の変化量から皮膚のバリア機能、特に物質の浸透性を評価する。 As described above, the evaluation method of the present invention non-invasively measures skin moisture content or moisture distribution, and relates the change in the moisture content or moisture distribution of the skin upon contact with an article or a test substance with the barrier function of the skin. Based on the sex, the barrier function of the skin, particularly the permeability of the substance, is evaluated from the change in the amount of water in the skin measured.
また、前述の、被験物質又は物品の接触による皮膚の水分量又は水分分布の変化量とバリア機能との関連性に基づいて、非侵襲的に測定した皮膚の水分量又は水分分布の変化量から、角層の状態を評価することができる。
例えば、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、皮膚静電容量測定装置を用いて測定した、被験物質又は物品負荷前後の皮膚の水分量の変化量の基準値を、８(A.U.)、好ましくは１２(A.U.)、より好ましくは１６(A.U.)に設定する。そして、実際に測定した被験物質又は物品負荷後の皮膚の水分量が、前記基準値以上増加した場合に、角層のバリア機能が低下したと評価することができる。
加えて、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、皮膚表面から所定の深さまで（好ましくは皮膚表面からの深さ４０μｍの深さまで、より好ましくは皮膚表面からの深さ３５μｍの深さまで、より好ましくは皮膚表面からの深さ２０μｍの深さまで）における、被験物質又は物品の接触前後の皮膚の水分分布をラマン分光法により測定する。そして、被験物質又は物品の接触前後の皮膚の水分分布を比較し、被験物質又は物品の接触により、初期の角層厚より深部における皮膚の水分量が増加した場合に、角層のバリア機能は低下した、と評価できる。
また、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、皮膚表面からの深さ２μｍにおける、被験物質又は物品の接触前後の皮膚の表面水分量をラマン分光法により測定する。そして、被験物質又は物品の接触前後の皮膚の表面水分量を比較する。そして、得られた皮膚表面水分量の被験物質又は物品の接触前後の変化量の基準値を２．０倍、より好ましくは２．５倍、さらに好ましくは３．０倍に設定する。そして、実際に測定した被験物質又は物品の接触後の皮膚の表面水分量が、前記基準値以上に増加した場合に、角層のバリア機能は低下した、と評価できる。
また、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、初期の角層厚に相当する部位における、被験物質又は物品の接触前後の皮膚の水分量をラマン分光法により測定する。そして、被験物質又は物品の接触前後の皮膚の水分量を比較する。そして、ラマン分光法より得られた初期の角層厚に相当する部位の水分量の被験物質又は物品の接触前後の変化量の基準値を８％、好ましくは１１％、さらに好ましくは１４％に設定する。そして、実際に測定した被験物質又は物品の接触後の初期の角層厚に相当する部位の水分量が、前記基準値以上増加した場合に、バリア機能は低下したと評価できる。
あるいは、皮膚を洗浄し、最低２０分間順化した後の健常者の前腕内側の皮膚を対象とし、ラマン分光法により測定した、皮膚表面からの所定の深さまで（好ましくは皮膚表面からの深さ２〜４０μｍの深さまで、より好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より好ましくは皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで）における、皮膚の水分量の曲線下面積(Area under the curve of water profile)を求める。被験物質又は物品の接触前の皮膚の水分量の曲線下面積に対する被験物質又は物品の一定時間接触後の皮膚の水分量の曲線下面積の変化量の基準値を、１．４倍、好ましくは１．６倍、さらに好ましくは１．８倍に設定する。そして、実際に測定した被験物質又は物品負荷後の、皮膚表面から所定の深さまでにおける皮膚の水分量の曲線下面積が前記基準値以上に増加した場合に、角層のバリア機能は低下したと評価できる。Also, based on the change in the amount of moisture in the skin or the distribution of water non-invasively measured based on the relationship between the change in the amount of moisture or the distribution of moisture in the skin due to contact with the test substance or article and the barrier function described above. The condition of the stratum corneum can be evaluated.
For example, after the skin has been cleaned and acclimated for a minimum of 20 minutes, the skin of the inside of the forearm of a healthy subject is targeted and measured with a skin capacitance measuring device, of the amount of water in the skin before and after loading the test substance or article. The reference value of the variation is set to 8 (AU), preferably 12 (AU), more preferably 16 (AU). And when the moisture content of the skin after the test substance or goods load actually measured increased more than the said reference value, it can be evaluated that the barrier function of the stratum corneum fell.
In addition, the skin is washed and acclimated for a minimum of 20 minutes to target the inner skin of the forearm of a healthy subject to a predetermined depth from the skin surface (preferably to a depth of 40 μm from the skin surface, preferably) The water distribution of the skin before and after contact with the test substance or article at a depth of 35 μm from the skin surface, more preferably a depth of 20 μm from the skin surface, is measured by Raman spectroscopy. Then, the moisture distribution of the skin before and after the contact of the test substance or article is compared, and when the water content of the skin at a deeper portion than the initial angular layer thickness increases due to the contact of the test substance or article, the barrier function of the stratum corneum is It can be evaluated that it has fallen.
In addition, the skin of the inside of the forearm of a healthy subject after washing the skin and acclimating for a minimum of 20 minutes is targeted, and the surface water content of the skin before and after contact with the test substance or article at a depth of 2 μm from the skin surface Measure by spectroscopy. Then, the surface moisture content of the skin before and after the contact of the test substance or the article is compared. Then, the reference value of the amount of change of the skin surface moisture amount before and after contact with the test substance or article obtained is set to 2.0 times, more preferably 2.5 times, still more preferably 3.0 times. And when the surface moisture content of the skin after the contact of the to-be-tested substance or article actually measured increases to the said reference value or more, it can be evaluated that the barrier function of the stratum corneum fell.
In addition, target the skin on the inside of the forearm of a healthy subject after cleansing the skin and acclimating for a minimum of 20 minutes, and measuring the water content of the skin before and after contact with the test substance or article at the site corresponding to the initial angular layer thickness. Measured by Raman spectroscopy. Then, the water content of the skin before and after the contact of the test substance or the article is compared. And the reference value of the amount of change in the moisture content of the part corresponding to the initial angular layer thickness obtained from Raman spectroscopy before and after contact with the test substance or article is 8%, preferably 11%, more preferably 14%. Set The barrier function can be evaluated to have decreased when the water content of the portion corresponding to the initial angular layer thickness after contact of the test substance or article actually measured increases by the above-mentioned reference value or more.
Alternatively, to the skin on the inside of the forearm of a healthy subject after washing the skin and acclimating for a minimum of 20 minutes, to a predetermined depth from the skin surface (preferably the depth from the skin surface) measured by Raman spectroscopy To a depth of 2 to 40 μm, more preferably to a depth of 2 to 35 μm from the skin surface, more preferably to a depth of 2 to 20 μm from the skin surface, more preferably to a depth of 9 to The area under the curve of water content of skin is determined up to a depth of 15 μm, more preferably to a depth of about 13 μm from the skin surface. The reference value of the change in the area under the curve of the water content of the skin after contact for a given period of time with the area under the curve of the water content of the skin before contact with the test substance or article is 1.4 times, preferably It is set to 1.6 times, more preferably 1.8 times. The barrier function of the stratum corneum is lowered when the area under the curve of the moisture content of the skin from the skin surface to a predetermined depth after loading of the test substance or article actually measured increases above the reference value It can be evaluated.
本発明の評価方法を利用して、吸収性物品（脱脂綿、生理用ナプキン、紙おむつ、布おむつ、トレーニングパンツ、成人用おむつ、汗止めパット、尿取りパット、産褥パット、パンティーライナー、吸収パットなど）、皮膚化粧料若しくは外用剤、創傷保護材（絆創膏、ガーゼ、包帯など）、マスク若しくはゴム手袋、蒸気温熱具などの製品の性能を評価することができる。
例えば、吸収性物品、創傷保護材、並びにマスク若しくはゴム手袋については、これらの着用前後の皮膚のバリア機能を本発明の評価方法を利用して評価することで、これらの製品の性能（例えば、水分（濡れ）の吸収性、水蒸気（蒸れ）の外部への発散性、皮膚トラブルの起こしやすさ）を、安全で簡便に短時間で評価できる。なお、吸収性物品の評価の際には、生理食塩水や擬似尿などを含ませた吸収性物品を評価に用いてもよい。
本発明により物品の着用や適用後の皮膚の水分量又は水分分布を評価すれば、ヒトの皮膚に本実施例で使用したニコチン酸メチルなどの物質を塗布する必要なく、物質の透過性を評価することができる。具体的には、吸収性物品、創傷保護材、並びにマスク若しくはゴム手袋について、現行品と改良品との比較の際に、装着前後の皮膚の水分量又は水分分布から物質透過性を推し量ることができる。このようにすれば、吸収性物品などの開発を効率よく行うことができる。
また、皮膚の水和状態を利用して、化粧料、外用剤などに含まれる有効成分を皮膚に浸透させることができることが報告されている（例えば、J. Invest. Dermatol., 1963, vol. 41, p. 307-311参照）。よって、化粧料、外用剤、蒸気温熱具などの皮膚への塗布、貼付又は着用前後の皮膚のバリア機能を本発明の評価方法を利用して評価することで、これらの製品に含まれる有効成分などの皮膚への透過性を、安全で簡便に短時間で評価できる。Absorbent articles (cotton wool, sanitary napkins, paper diapers, cloth diapers, training pants, adult diapers, sweat pads, urine pads, puerperal pads, panty liners, absorbent pads, etc.) using the evaluation method of the present invention It is possible to evaluate the performance of products such as skin cosmetics or external preparations, wound protection materials (adhesives, gauze, bandages, etc.), masks or rubber gloves, and steam heaters.
For example, for absorbent articles, wound protection materials, and masks or rubber gloves, the performance of these products can be evaluated by evaluating the barrier function of the skin before and after wearing them using the evaluation method of the present invention (for example, The ability to absorb moisture (wet), wick the water vapor (steam) to the outside, and cause skin problems can be evaluated safely and easily in a short time. In addition, in the case of evaluation of an absorbent article, you may use the absorbent article which contained physiological saline, artificial urine, etc. for evaluation.
By evaluating the moisture content or moisture distribution of the skin after wearing or applying the article according to the present invention, it is possible to evaluate the permeability of the substance without having to apply the substance such as methyl nicotinate used in this example to human skin. can do. Specifically, for absorbent articles, wound protection materials, and masks or rubber gloves, when comparing the current product with the improved product, it is possible to infer material permeability from the moisture content or moisture distribution of the skin before and after wearing it can. In this way, development of an absorbent article etc. can be performed efficiently.
In addition, it has been reported that active ingredients contained in cosmetics, external preparations and the like can be made to penetrate the skin by utilizing the hydration state of the skin (for example, J. Invest. Dermatol., 1963, vol. 41, p. 307-311). Therefore, the active ingredient contained in these products by evaluating the barrier function of the skin before and after application to the skin such as cosmetics, external preparations, steam heating tools, sticking or wearing, using the evaluation method of the present invention Etc. can be evaluated safely and easily in a short time.
本発明の、被験物質又は物品が、皮膚のバリア機能に与える影響を評価するためのシステムは、被験物質又は物品の接触による皮膚の水分量又は水分分布の変化量と皮膚のバリア機能との関連性に関する情報を格納し、関連性に関する情報に基づき、被験物質又は物品の接触による皮膚の水分量又は水分分布の変化量から、前記影響を評価する。
本発明のシステムはいわゆるコンピュータであり、例えば、バスで相互に接続される、ＣＰＵ（Central Processing Unit）、メモリ、入出力インタフェース等を有する。メモリは、ＲＡＭ（Random Access Memory）、ＲＯＭ（Read Only Memory）、ハードディスク、可搬型記憶媒体等である。入出力インタフェースは、表示装置や入力装置等のようなユーザインタフェース装置と接続される。入出力インタフェースは、ネットワークを介して他のコンピュータと通信を行う通信装置等と接続されてもよい。The system for evaluating the influence of the test substance or article on the barrier function of the skin according to the present invention relates to the relationship between the change in the amount of water or the distribution of water in the skin due to contact with the test substance or article and the barrier function of the skin. Information on sex is stored, and based on the information on relevance, the effect is evaluated from the amount of change in moisture content or moisture distribution of skin due to contact with a test substance or article.
The system of the present invention is a so-called computer, and has, for example, a CPU (Central Processing Unit), a memory, an input / output interface and the like mutually connected by a bus. The memory is a random access memory (RAM), a read only memory (ROM), a hard disk, a portable storage medium, or the like. The input / output interface is connected to a user interface device such as a display device or an input device. The input / output interface may be connected to a communication device or the like that communicates with another computer via a network.
本発明の評価方法及び評価システムは、ヒトの皮膚、好ましくは、被験物質又は物品を負荷していない状態で正常なバリア機能を有するヒト（健常者）の皮膚、を対象とすることが好ましい。 The evaluation method and the evaluation system of the present invention are preferably directed to human skin, preferably human (healthy person) skin having a normal barrier function in the state where no test substance or article is loaded.
上述した実施形態に関し、本発明はさらに以下の評価方法、及び評価システムを開示する。 The present invention further discloses the following evaluation method and evaluation system regarding the embodiment mentioned above.
＜１＞被験物質又は物品をヒトの皮膚に接触させ、
接触前後の皮膚の水分量又は水分分布の変化量を、基準値と比較し、
比較結果から、前記被験物質又は物品が、皮膚バリア機能に与える影響を評価する、
被験物質又は物品の評価方法であって、
水の負荷量を変えての皮膚への水負荷試験により測定された、皮膚の水分量又は水分分布と、皮膚のバリア機能とが関連付けられ、
皮膚の水分量又は水分分布と皮膚のバリア機能との関連性から、皮膚のバリア機能に変化が確認されるときの皮膚の水分量又は水分分布が、前記基準値として設定されたものである、
被験物質又は物品の評価方法。<1> bringing a test substance or article into contact with human skin,
Compare the amount of change in moisture content or moisture distribution of the skin before and after contact with the reference value,
Evaluating the influence of the test substance or the article on the skin barrier function from the comparison result
A method of evaluating a test substance or article, comprising
Water content or distribution of the skin, as measured by water load test on the skin with varying water load, is associated with the barrier function of the skin,
The water content or the water distribution of the skin when a change in the skin barrier function is confirmed is set as the reference value from the relation between the water content or the water distribution of the skin and the barrier function of the skin.
Evaluation method of test substance or article.
＜２＞皮膚のバリア機能の変化を確認するために、皮膚外部から内部への指標物質の浸透性がさらに測定され、指標物質の浸透性の結果から前記基準値が設定されたものである、前記＜１＞項に記載の方法。
＜３＞前記被験物質又は物品の接触後の皮膚水分量が、基準値以上の変化量で増加した場合、バリア機能が低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１＞又は＜２＞項に記載の方法。
＜４＞前記被験物質又は物品の接触後の皮膚水分量の変化量が、基準値より小さい場合、バリア機能は変化していない、好ましくは皮膚外部から内部への物質の浸透性は亢進していない、と評価する、前記＜１＞〜＜３＞のいずれか１項に記載の方法。<2> In order to confirm the change in the barrier function of the skin, the permeability of the indicator substance from the outside to the inside of the skin is further measured, and the reference value is set from the result of the permeability of the indicator substance, The method according to the above <1>.
<3> When the skin moisture content after contact with the test substance or article is increased by a change amount equal to or higher than the reference value, the barrier function is reduced, preferably the permeability of the substance from the outside to the inside of the skin is enhanced The method according to <1> or <2> above, which is evaluated.
<4> When the amount of change in skin moisture after contact with the test substance or article is smaller than the reference value, the barrier function is not changed, preferably the permeability of the substance from the outside to the inside of the skin is enhanced The method according to any one of the above <1> to <3>, which is evaluated as not present.
＜５＞被験物質又は物品をヒトの皮膚に接触させた後のヒトの皮膚の深さごとの水分量を測定して、被験物質又は物品の接触後の水分分布を取得し、
被験物質又は物品の接触後の水分分布と基準水分分布とを比較することで、被験物質又は物品が、ヒトの皮膚のバリア機能に与える影響を評価する、被験物質又は物品の評価方法であって、
水又は水溶液のヒトの皮膚への負荷前に、ヒトの皮膚の深さ毎の水分量を測定して初期水分分布を取得し、
水又は水溶液のヒトの皮膚への負荷終了直後に、ヒトの皮膚の深さ毎の水分量の測定による水又は水溶液の負荷後の水分分布の取得と、指標物質を皮膚に適用して該指標物質の浸透度の測定を行い、
初期水分分布から水又は水溶液の負荷終了直後の水分分布への変化と、指標物質の浸透度から、指標物質の浸透度と水又は水溶液の負荷量との関連性を決定し、
前記関連性が変化したときのヒトの皮膚の深さ毎の水又は水溶液の負荷後の水分分布を、前記基準水分分布とする、
被験物質又は物品の評価方法。<5> Measure the amount of water per depth of human skin after bringing the test substance or article into contact with human skin, and acquire the water distribution after the contact of the test substance or article,
A method for evaluating a test substance or article, which evaluates the influence of the test substance or article on the barrier function of human skin by comparing the distribution of water after contact of the test substance or article with the reference water distribution. ,
Before applying water or an aqueous solution to human skin, measure the amount of water at each depth of human skin to obtain an initial water distribution,
Immediately after the end of the load of water or aqueous solution on human skin, acquisition of water distribution after load of water or aqueous solution by measurement of the amount of water per depth of human skin and application of the indicator substance to the skin, the index Measure the permeability of the substance,
From the change from the initial water distribution to the water distribution immediately after the end of the water or aqueous solution load and the permeability of the indicator substance, determine the relationship between the permeability of the indicator substance and the water or aqueous solution load,
The water distribution after loading of water or an aqueous solution for each depth of human skin when the relevance changes is taken as the reference water distribution.
Evaluation method of test substance or article.
＜６＞被験物質又は物品の接触前と接触後のヒトの皮膚の所定の深さ位置の水分量を測定し、接触前の水分量と接触後の水分量との差を取得し、
取得した水分量の差と、前記基準水分分布とに基づき、皮膚バリア機能に与える影響を評価する、前記＜５＞項に記載の評価方法。<6> The amount of water at a predetermined depth position of human skin before and after contact with the test substance or article is measured, and the difference between the amount of water before contact and the amount of water after contact is obtained,
The evaluation method according to <5>, wherein the influence on the skin barrier function is evaluated based on the acquired difference in the amount of water and the reference water distribution.
＜７＞前記指標物質が、ニコチン酸メチル、ニコチン酸ヘキシル、乳酸、黄色4号、赤色215号、カフェイン、ユビナール、カロテノイド、レチノール、並びに、皮膚保湿剤、皮膚外用剤、皮膚化粧料若しくは蒸気温熱具に含まれる有効成分若しくは薬効成分からなる群より選ばれる少なくとも１種である、前記＜２＞〜＜６＞のいずれか１項に記載の方法。
＜８＞皮膚の水分量を皮膚静電容量測定手段により測定し解析する、前記＜１＞〜＜７＞のいずれか１項に記載の方法。
＜９＞健常者の皮膚を評価対象とし、被験物質又は物品の接触後の皮膚の水分量を皮膚静電容量測定手段により測定し、被験物質又は物品の接触後の皮膚の水分量が、被験物質又は物品の接触前の皮膚の水分量に対して、８(A.U.)以上、好ましくは１２(A.U.)以上、より好ましくは１６(A.U.)以上、の変化量で増加した場合、バリア機能は低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜８＞項に記載の方法。
＜１０＞健常者の皮膚を評価対象とし、被験物質又は物品の接触後の皮膚の水分量を皮膚静電容量測定手段により測定し、被験物質又は物品の接触後の皮膚の水分量が、被験物質又は物品の接触前の皮膚の水分量に対して、１６(A.U.)増加より小さい変化量、好ましくは１２(A.U.)増加より小さい変化量、より好ましくは８(A.U.)増加より小さい変化量、の場合、バリア機能は変化していない、好ましくは皮膚外部から内部への物質の浸透性は亢進していない、と評価する、前記＜８＞項に記載の方法。
＜１１＞皮膚表面からの深さ２〜４０μｍの深さまで、好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より好ましくは皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで、の水分量又は水分分布をラマン分光法により測定し解析する、前記＜１＞〜＜７＞のいずれか１項に記載の方法。
＜１２＞健常者の皮膚を評価対象とし、皮膚表面から所定の深さまで、好ましくは皮膚表面からの深さ４０μｍの深さまで、より好ましくは皮膚表面からの深さ３５μｍの深さまで、より好ましくは皮膚表面からの深さ２０μｍの深さまで、における、被験物質又は物品の接触前後の皮膚の水分分布をラマン分光法により測定し、被験物質又は物品の接触前後の皮膚の水分分布を比較し、被験物質又は物品の接触により、初期の角層厚より深部における皮膚の水分量が増加した場合、バリア機能が低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１１＞項に記載の方法。
＜１３＞健常者の皮膚を評価対象とし、ラマン分光法より得られた被験物質又は物品の接触後の皮膚表面水分量が、被験物質又は物品の接触前の皮膚表面水分量に対して、２．０倍以上、好ましくは２．５倍以上、より好ましくは３．０倍以上、に増加した場合、バリア機能は低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１１＞項に記載の方法。
＜１４＞健常者の皮膚を評価対象とし、ラマン分光法より得られた被験物質又は物品の接触後の皮膚表面水分量が、被験物質又は物品の接触前の皮膚表面水分量に対して、３．０倍増加より小さい場合、好ましくは２．５倍より小さい場合、より好ましくは２．０倍より小さい場合、バリア機能は変化していない、好ましくは皮膚外部から内部への物質の浸透性は亢進していない、と評価する、前記＜１１＞項に記載の方法。
＜１５＞健常者の皮膚を評価対象とし、ラマン分光法より得られた、被験物質又は物品の接触後の初期の角層厚に相当する部位の水分量が、被験物質又は物品の接触前の初期の角層厚に相当する部位の水分量に対して、８％以上、好ましくは１１％以上、より好ましくは１４％以上、増加した場合、バリア機能は低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１１＞項に記載の方法。
＜１６＞健常者の皮膚を評価対象とし、ラマン分光法より得られた、被験物質又は物品の接触後の初期の角層厚に相当する部位の水分量が、被験物質又は物品の接触前の初期の角層厚に相当する部位の水分量に対して、１４％増加より小さい場合、好ましくは１１％増加より小さい場合、さらに好ましくは８％増加より小さい場合、バリア機能は変化していない、好ましくは皮膚外部から内部への物質の浸透性は亢進していない、と評価する、前記＜１１＞項に記載の方法。
＜１７＞健常者の皮膚を評価対象とし、ラマン分光法より得られた、皮膚表面からの所定の深さまで、好ましくは皮膚表面からの深さ２〜４０μｍの深さまで、より好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで、における、被験物質又は物品の接触後の皮膚の水分量の曲線下面積を求め、被験物質又は物品の接触後の皮膚の水分量の曲線下面積が、被験物質又は物品の接触前の皮膚の水分量の曲線下面積に対して１．４倍以上、好ましくは１．６倍以上、さらに好ましくは１．８倍以上、に増加した場合、バリア機能は低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１１＞項に記載の方法。
＜１８＞健常者の皮膚を評価対象とし、ラマン分光法より得られた、皮膚表面からの所定の深さまで、好ましくは皮膚表面からの深さ２〜４０μｍの深さまで、より好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで、における、被験物質又は物品の接触後の皮膚の水分量の曲線下面積を求め、被験物質又は物品の接触後の皮膚の水分量の曲線下面積が、被験物質又は物品の接触前の皮膚の水分量の曲線下面積に対して１．８倍より小さい場合、好ましくは１．６倍より小さい場合、さらに好ましくは１．４倍より小さい場合、バリア機能は変化していない、好ましくは皮膚外部から内部への物質の浸透性は亢進していない、と評価する、前記＜１１＞項に記載の方法。<7> The indicator substance is methyl nicotinate, hexyl nicotinate, lactic acid, yellow No. 4, red No. 215, caffeine, ubinal, carotenoid, retinol, skin moisturizer, external preparation for skin, skin cosmetic or vapor The method according to any one of the above <2> to <6>, which is at least one selected from the group consisting of an active ingredient or a medicinal ingredient contained in a heating implement.
The method of any one of said <1>-<7> which measures and analyzes the water content of <8> skin by skin capacitance measurement means.
<9> The skin of a healthy subject is to be evaluated, the water content of the skin after contact with the test substance or article is measured by the skin capacitance measuring means, and the water content of the skin after contact with the test substance or article is The barrier function decreases when the water content of the skin before contact with the substance or article increases by a change of 8 (AU) or more, preferably 12 (AU) or more, more preferably 16 (AU) or more. The method according to the above <8>, which preferably evaluates that the permeability of the substance from the outside to the inside of the skin is enhanced.
<10> The skin of a healthy subject is to be evaluated, the water content of the skin after contact with the test substance or article is measured by the skin capacitance measuring means, and the water content of the skin after contact with the test substance or article is Change by less than 16 (AU) increase, preferably less than 12 (AU) increase, more preferably less than 8 (AU) increase, relative to the moisture content of the skin before contact of the substance or article In the case of the above <8>, it is evaluated that the barrier function is not changed, preferably the permeability of the substance from the outside to the inside of the skin is not enhanced.
<11> A depth of 2 to 40 μm from the skin surface, preferably a depth of 2 to 35 μm from the skin surface, more preferably a depth of 2 to 20 μm from the skin surface, more preferably The water content or water distribution is measured and analyzed by Raman spectroscopy to a depth of 9 to 15 μm from the skin surface, more preferably to a depth of about 13 μm from the skin surface, The method according to any one of 7>.
The skin of <12> healthy subjects is to be evaluated, to a predetermined depth from the skin surface, preferably to a depth of 40 μm from the skin surface, more preferably to a depth of 35 μm from the skin surface, more preferably The moisture distribution of the skin before and after contact with the test substance or article at a depth of 20 μm from the skin surface is measured by Raman spectroscopy, and the moisture distribution of the skin before and after contact with the test substance or article is compared Evaluate as the barrier function is reduced, preferably the permeability of the substance from the outside to the inside of the skin is enhanced, if the amount of water in the skin increases in depth than the initial angular layer thickness due to contact of the substance or article. The method according to <11>.
The skin surface water content after contact of the test substance or article obtained by Raman spectroscopy for skin of a <13> healthy subject is to be evaluated 2 with respect to the skin surface water content before contact of the test substance or article When the barrier function is decreased, preferably the permeability of the substance from the outside to the inside of the skin is enhanced, The method according to the above <11>, which is to be evaluated.
The skin surface water content after contact of the test substance or article obtained by Raman spectroscopy is for the skin of the healthy person <14> to be evaluated, and 3 with respect to the skin surface water level before contact of the test substance or article. The barrier function is not changed, preferably less than 2.5 times, preferably less than 2.5 times, more preferably less than 2.0 times, preferably the permeability of the substance from the outside to the inside of the skin is The method according to the above <11>, which is evaluated as not having been promoted.
The water content of the site corresponding to the initial angular layer thickness after contact of the test substance or article obtained by Raman spectroscopy is for the skin of a <15> healthy subject to be evaluated, before contact of the test substance or article. When the water content of the part corresponding to the initial angular layer thickness is increased by 8% or more, preferably 11% or more, more preferably 14% or more, the barrier function is decreased, preferably from outside to inside of the skin The method according to <11>, wherein the substance is evaluated as having enhanced permeability.
The water content of the site corresponding to the initial angular layer thickness after contact of the test substance or article obtained from Raman spectroscopy, with the skin of a <16> healthy subject evaluated, is before contact of the test substance or article With respect to the water content of the portion corresponding to the initial angular layer thickness, the barrier function has not changed if it is smaller than 14%, preferably smaller than 11%, more preferably smaller than 8%. The method according to the above <11>, which preferably evaluates that the permeability of the substance from the outside to the inside of the skin is not enhanced.
The skin of <17> healthy subjects is to be evaluated, to a predetermined depth from the skin surface obtained by Raman spectroscopy, preferably to a depth of 2 to 40 μm from the skin surface, more preferably from the skin surface Depth of 2 to 35 μm, more preferably 2 to 20 μm from the skin surface, more preferably 9 to 15 μm from the skin surface, more preferably 13 μm from the skin surface The area under the curve of the moisture content of the skin after the contact of the test substance or article at the depth of the degree is determined, and the area under the curve of the moisture content of the skin after the contact of the test substance or article is the contact of the test substance or article The barrier function decreased when the area under the curve of the water content of the previous skin was increased by 1.4 times or more, preferably 1.6 times or more, more preferably 1.8 times or more, preferably the skin was decreased From outside to inside The method according to the above <11>, wherein it is evaluated that the permeability of the substance to the skin is enhanced.
The skin of <18> healthy subjects is to be evaluated, to a predetermined depth from the skin surface obtained by Raman spectroscopy, preferably to a depth of 2 to 40 μm from the skin surface, more preferably from the skin surface Depth of 2 to 35 μm, more preferably 2 to 20 μm from the skin surface, more preferably 9 to 15 μm from the skin surface, more preferably 13 μm from the skin surface The area under the curve of the moisture content of the skin after the contact of the test substance or article at the depth of the degree is determined, and the area under the curve of the moisture content of the skin after the contact of the test substance or article is the contact of the test substance or article The barrier function has not changed if it is less than 1.8 times, preferably less than 1.6 times, more preferably less than 1.4 times the area under the curve of the water content of the previous skin. Preferred Or the method according to the above <11>, wherein it is evaluated that the permeability of the substance from the outside to the inside of the skin is not enhanced.
＜１９＞被験物質又は物品が、ヒトの皮膚のバリア機能に与える影響を評価する方法であって、
被験物質又は物品のヒトの皮膚への接触前と接触後の皮膚の深さ毎の水分量を測定して水分分布を取得し、
接触前と接触後の水分分布における、接触前の初期の角層厚よりも深部の水分量の差を指標として、被験物質又は物品がヒトの皮膚のバリア機能に与える影響を評価する、
被験物質又は物品の評価方法。<19> A method for evaluating the influence of a test substance or article on the barrier function of human skin,
Before the contact of the test substance or article with human skin and after the contact, measure the amount of water at each depth of the skin to obtain the water distribution,
Evaluate the effect of the test substance or article on the barrier function of human skin, using the difference in the amount of water deeper than the initial angular layer thickness before contact in the water distribution before and after contact as an indicator,
Evaluation method of test substance or article.
＜２０＞皮膚表面からの深さ２〜４０μｍの深さまで、好ましくは皮膚表面からの深さ２〜３５μｍの深さまで、より好ましくは皮膚表面からの深さ２〜２０μｍの深さまで、より好ましくは皮膚表面からの深さ９〜１５μｍの深さまで、より好ましくは皮膚表面からの深さ１３μｍ程度の深さまで、の水分量をラマン分光法により測定し解析する、前記＜１９＞項に記載の方法。
＜２１＞健常者の皮膚を評価対象とし、皮膚表面から所定の深さまでにおける、被験物質又は物品の接触前後の皮膚の水分分布をラマン分光法により測定し、被験物質又は物品の接触前後の皮膚の水分分布を比較し、被験物質又は物品の接触により、初期の角層厚より深部における皮膚の水分量が増加した場合、バリア機能が低下した、好ましくは皮膚外部から内部への物質の浸透性が亢進した、と評価する、前記＜１９＞又は＜２０＞項に記載の方法。<20> A depth of 2 to 40 μm from the skin surface, preferably a depth of 2 to 35 μm from the skin surface, more preferably a depth of 2 to 20 μm from the skin surface, more preferably The method according to the above <19>, wherein the amount of water is measured and analyzed by Raman spectroscopy to a depth of 9 to 15 μm from the skin surface, more preferably to a depth of about 13 μm from the skin surface .
<21> The skin of a healthy subject is to be evaluated, the moisture distribution of the skin before and after contact with the test substance or article from the skin surface to a predetermined depth is measured by Raman spectroscopy, and the skin before and after contact with the test substance or article When the water content of the skin is increased by the contact of the test substance or article and the skin water content in the deeper area than the initial angular layer thickness, the barrier function is reduced, preferably the permeability of the substance from the outside to the inside of the skin The method according to <19> or <20> above, wherein
＜２２＞皮膚水分量又は水分分布を非侵襲的に測定する、前記＜１＞〜＜２１＞のいずれか１項に記載の方法。
＜２３＞被験物質又は物品を負荷していない状態で正常なバリア機能を有するヒト（健常者）の皮膚で評価する、前記＜１＞〜＜２２＞のいずれか１項に記載の方法。
＜２４＞ラマン分光法、皮膚静電容量測定手段、高周波電流測定手段、赤外分光法、及び近赤外分光法からなる群より選ばれる少なくとも１つに従い皮膚の水分量を測定する、前記＜１＞〜＜７＞、＜１９＞、＜２２＞及び＜２３＞のいずれか１項に記載の方法。
＜２５＞前記被験物質が、水若しくは水蒸気、皮膚化粧料、皮膚保湿剤若しくは皮膚外用剤、蒸気温熱具に含まれる有効成分若しくは薬効成分、生理食塩水、リン酸緩衝生理食塩水、並びに生体由来の分泌物若しくは***物からなる群より選ばれる少なくとも１種である、前記＜１＞〜＜２４＞のいずれか１項に記載の方法。
＜２６＞前記物品が、吸収性物品（好ましくは脱脂綿、生理用ナプキン、紙おむつ、布おむつ、トレーニングパンツ、成人用おむつ、汗止めパット、尿取りパット、産褥パット、パンティーライナー、若しくは吸収パット）、蒸気温熱具、創傷保護材（好ましくは絆創膏、ガーゼ、若しくは包帯）、マスク、又はゴム手袋である、前記＜１＞〜＜２４＞のいずれか１項に記載の方法。
＜２７＞前記吸収性物品が生理食塩水又は擬似尿などを含有する、前記＜２６＞項に記載の方法。
＜２８＞前記被験物質又は物品をヒトの皮膚に接触させる時間が、１時間以上６時間以下、好ましくは２時間以上４時間以下、より好ましくは２．５時間以上３．５時間以下、さらに好ましくは３時間程度、である、前記＜１＞〜＜２７＞のいずれか１項に記載の方法。The method of any one of said <1>-<21> which measures <22> skin moisture content or moisture distribution noninvasively.
<23> The method according to any one of <1> to <22>, wherein the evaluation is performed on the skin of a human (healthy person) having a normal barrier function in the state of not loading a test substance or an article.
<24> The moisture content of the skin is measured according to at least one selected from the group consisting of Raman spectroscopy, skin capacitance measuring means, high frequency current measuring means, infrared spectroscopy, and near infrared spectroscopy, The method of any one of 1>-<7>, <19>, <22>, and <23>.
<25> The test substance is water or water vapor, skin cosmetics, skin moisturizers or skin external preparations, active ingredients or medicinal ingredients contained in a steam heating device, physiological saline, phosphate buffered saline, and biological origin The method according to any one of <1> to <24>, which is at least one selected from the group consisting of secretions or excretions of
<26> The article is an absorbent article (preferably absorbent cotton, sanitary napkins, paper diapers, cloth diapers, training pants, diapers for adults, sweat pads, urine pads, puerperal pads, panty liners or absorbent pads), The method according to any one of the above <1> to <24>, which is a steam heating device, a wound protection material (preferably a bandage, a gauze, or a bandage), a mask, or a rubber glove.
<27> The method according to <26>, wherein the absorbent article contains physiological saline or simulated urine.
<28> The time for which the test substance or article is in contact with human skin is 1 hour to 6 hours, preferably 2 hours to 4 hours, more preferably 2.5 hours to 3.5 hours, further preferably The method according to any one of <1> to <27>, wherein is about 3 hours.
＜２９＞被験物質又は物品のヒトの皮膚への接触による皮膚の水分量又は水分分布の変化量と皮膚のバリア機能との関連性に関する情報を格納し、関連性に関する情報に基づき、被験物質又は物品の接触後の皮膚の水分量又は水分分布から、被験物質又は物品が、皮膚のバリア機能に与える影響を評価するためのシステム。 <29> Information on the relationship between the amount of change in moisture content or moisture distribution of the skin and the barrier function of the skin due to contact of the test substance or article with human skin is stored, and based on the information on the relevance, the test substance or A system for evaluating the influence of a test substance or an article on the barrier function of the skin from the moisture content or moisture distribution of the skin after the contact of the article.
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 The present invention will be described in more detail based on examples given below, but the invention is not meant to be limited by these.
試験例１ 水分負荷前後のニコチン酸メチルの透過性と皮膚の水分量の変化の測定
（１）紅斑スコアの測定
健常な男性、女性１０名（２０代〜４０代）を被験者とした。前腕内側に異なる量の水分を負荷し、水分負荷後のニコチン酸メチルの透過性を評価した。測定は、２４〜２５℃、相対湿度４０〜５０％の環境可変室で、２０分順化した後に行った。Test Example 1 Measurement of Permeability of Methyl Nicotinate Before and After Water Load and Change of Skin Water Content (1) Measurement of Erythematous Score A test subject was a healthy male and 10 females (20's to 40's). Different amounts of water were loaded inside the forearm, and the permeability of methyl nicotinate after water loading was evaluated. The measurement was performed after conditioning for 20 minutes in an environmental variable chamber at 24 to 25 ° C. and a relative humidity of 40 to 50%.
２．５cm×２．５cmのメンバンカット綿（白十字社製）に、脱脂綿１cm2当たりの水分負荷量が０mL（０mL/cm2）、０．０２mL（０．０２mL/cm2）、０．０４mL（０．０４mL/cm2）、０．２０mL（０．２０mL/cm2）となるように蒸留水を含ませ、これを前腕内側にのせ透湿性絆創膏（商品名：スキナゲート）で周囲を固定し３時間貼付した。メンバンカット綿から水分が漏れるのを防ぐため、メンバンカット綿の皮膚と接しない面はドレッシング材（商品名：テガダームＴＭ トランスペアレント ドレッシング）で覆い、貼付中に皮膚が蒸れるのを防ぐためドレッシング材は注射針29G（TERUMO, Japan）で１cm2当たり８個、穴をあけた。メンバンカット綿を貼付している間、試験参加者には運動や屋外での長時間の作業は禁止した。The water load per cm 2 of the absorbent cotton is 0 mL (0 mL / cm 2 ), 0.02 mL (0.02 mL / cm 2 ), and 2.5 cm × 2.5 cm of the material of the maran cut cotton (manufactured by White Cross Co., Ltd.) .04mL (0.04mL / cm 2), 0.20mL included the (0.20mL / cm 2) and so as distilled water, which put moisture-permeable adhesive plaster to the inner forearm (trade name: Sukinageto) around in It fixed and stuck for 3 hours. To prevent the member down cut cotton moisture leakage, member emission cut skin and the surface which is not in contact cotton dressing (trade name: Tegaderm TM Transparent Dressing) covered with a dressing material to prevent skin that stuffy during application 8 holes per 1 cm 2 were drilled with an injection needle 29G (TERUMO, Japan). Participants were not allowed to exercise or work outdoors for extended periods while applying member cut cotton.
メンバンカット綿を除去した直後に、０．００７質量％ニコチン酸メチル水溶液５．０μＬを含ませた、直径５ｍｍにくり抜いたろ紙（商品名：Advantec No2）を試験部位に１５秒間負荷した。ニコチン酸メチルの負荷から３０分後に、生じた紅斑を下記の評価基準に従い目視判定を行った。
目視判定により得られた紅斑スコアから、各水分負荷量でのニコチン酸メチルの透過性を評価した。統計解析にはＩＢＭ ＳＰＳＳバージョン２３のソフトウェアを使用した。
その結果を表１に示す。
（評価基準）
スコア０ ：反応なし。
スコア０．５：若干紅い。
スコア１ ：ろ紙の面積の半分以上紅い。
スコア２ ：ろ紙を当てた面積がはっきり紅い。
スコア３ ：ろ紙の面積より大きく紅い。
スコア４ ：紅みがろ紙の面積より大きく、腫れている。Immediately after removing the member cut cotton, a filter paper (trade name: Advantec No 2) hollowed to a diameter of 5 mm containing 5.0 μL of a 0.007 mass% methyl nicotinate aqueous solution was loaded on the test site for 15 seconds. After 30 minutes from the load of methyl nicotinate, the resulting erythema was visually determined according to the following evaluation criteria.
Permeability of methyl nicotinate at each water loading was evaluated from the erythema score obtained by visual evaluation. IBM SPSS version 23 software was used for statistical analysis.
The results are shown in Table 1.
(Evaluation criteria)
Score 0: no response.
Score 0.5: slightly reddish.
Score 1: More than half of the area of the filter paper.
Score 2: The area covered with filter paper is clearly reddish.
Score 3: larger than the area of filter paper
Score 4: The area is larger than the area of red paper and is swollen.
表１に示すように、水分の負荷量が０．００mL/cm2又は０．０２mL/cm2の場合と比較して、０．０４mL/cm2又は０．２０mL/cm2の水分を負荷することで、ニコチン酸メチルによる紅斑スコアが有意に上昇した。すなわち、一定量以上の水分を皮膚に負荷することで、ニコチン酸メチルの皮膚への透過性が有意に亢進した。
本試験例の健常なヒトの前腕において、水分の負荷量が少なくとも０．０４mL/cm2の場合に皮膚バリア機能が低下していると判断することができる。よって、水分負荷量が０．０４mL/cm2の場合の皮膚の状態を、皮膚のバリア機能を評価するための基準状態とすることができる。As shown in Table 1, the load amount of water as compared with the case of 0.00mL / cm 2 or 0.02 mL / cm 2, to load a moisture 0.04 mL / cm 2 or 0.20 mL / cm 2 The erythema score by methyl nicotinate increased significantly. That is, the permeability of methyl nicotinate to the skin was significantly enhanced by loading the skin with a certain amount or more of water.
In the healthy human forearm of this test example, it can be judged that the skin barrier function is reduced when the water loading amount is at least 0.04 mL / cm 2 . Therefore, the condition of the skin when the water loading amount is 0.04 mL / cm 2 can be used as a reference condition for evaluating the barrier function of the skin.
負荷されたニコチン酸メチルが皮膚へ浸透又は通過し、末梢血管に働きかけ血管拡張作用を示すと、皮膚上で紅斑が観察される。表１に示すように、水分負荷前の皮膚（水分負荷量：０mL/cm2）では、ニコチン酸メチルを負荷しても紅斑は観察されにくい。これは、ニコチン酸メチルが皮膚内へ浸透されにくく、末梢血管まで到達できないためと考えられる。すなわち、水分負荷前の皮膚では、バリア機能が維持されていると推察できる。一方、一定量以上の水分負荷後の皮膚では、ニコチン酸メチルの負荷によって紅斑が観察される。この結果から、水分を負荷することでニコチン酸メチルが皮膚へ浸透又は通過した、すなわち皮膚のバリア機能が低下したと判断できる。When the loaded methyl nicotinate penetrates or passes through the skin and acts on peripheral blood vessels to exhibit vasodilator action, erythema is observed on the skin. As shown in Table 1, in the skin before water load (water load: 0 mL / cm 2 ), erythema is hardly observed even when methyl nicotinate is loaded. It is considered that this is because methyl nicotinate is difficult to penetrate into the skin and can not reach peripheral blood vessels. That is, in the skin before water load, it can be inferred that the barrier function is maintained. On the other hand, erythema is observed in the skin after a certain amount of water loading due to the loading of methyl nicotinate. From this result, it can be judged that methyl nicotinate permeated or passed through the skin by loading water, that is, the barrier function of the skin was reduced.
（２）皮膚静電容量測定装置を用いた皮膚水分量の測定
２．５cm×２．５cmのメンバンカット綿（白十字社製）に、脱脂綿１cm2当たりの水分負荷量が０mL（０mL/cm2）、０．０１mL（０．０１mL/cm2）、０．０２mL（０．０２mL/cm2）、０．０３mL（０．０３mL/cm2）、０．０４mL（０．０４mL/cm2）、０．０６mL（０．０６mL/cm2）、０．１０mL（０．１０mL/cm2）、０．２０mL（０．２０mL/cm2）となるように蒸留水を含ませ、これを前記被験者の前腕内側にのせ透湿性絆創膏（商品名：スキナゲート）で周囲を固定し３時間貼付した。メンバンカット綿から水分が漏れるのを防ぐため、メンバンカット綿の皮膚と接しない面はドレッシング材（商品名：テガダームＴＭ トランスペアレント ドレッシング）で覆い、貼付中に皮膚が蒸れるのを防ぐためドレッシング材は注射針29G（TERUMO, Japan）で１cm2当たり８個、穴をあけた。メンバンカット綿を貼付している間、試験参加者には運動や屋外での長時間の作業は禁止した。(2) Measurement of skin moisture content using a skin capacitance measuring device A 2.5 cm × 2.5 cm marin cut cotton (manufactured by White Cross) with a water loading amount of 0 mL (0 mL / 0 cm 2 ). cm 2), 0.01mL (0.01mL / cm 2), 0.02mL (0.02mL / cm 2), 0.03mL (0.03mL / cm 2), 0.04mL (0.04mL / cm 2 Containing distilled water so as to be 0.06 mL (0.06 mL / cm 2 ), 0.10 mL (0.10 mL / cm 2 ), and 0.20 mL (0.20 mL / cm 2 ). The subject was placed on the inside of the forearm of the subject, fixed with a moisture-permeable adhesive bandage (trade name: Skinagate), and affixed for 3 hours. To prevent the member down cut cotton moisture leakage, member emission cut skin and the surface which is not in contact cotton dressing (trade name: Tegaderm TM Transparent Dressing) covered with a dressing material to prevent skin that stuffy during application 8 holes per 1 cm 2 were drilled with an injection needle 29G (TERUMO, Japan). Participants were not allowed to exercise or work outdoors for extended periods while applying member cut cotton.
メンバンカット綿の貼付前と、メンバンカット綿の除去直後に、メンバンカット綿を貼付した部位の皮膚の水分量を皮膚静電容量測定装置（商品名：コルネオメーター ＣＭ-825、Courage+Khazaka Germany社製）で測定した。そして、メンバンカット綿の除去直後の値から、メンバンカット綿の貼付前の値を差し引いた値を算出し、角層水分量の変化量を測定した。統計解析にはＩＢＭ ＳＰＳＳバージョン２３のソフトウェアを使用した。
その結果を表２に示す。Skin capacitance measurement device (trade name: Corneometer CM-825, Courage +) for the moisture content of the skin of the site where the member-cut cotton is applied before the application of the member-cut cotton and immediately after the removal of the member-cut cotton It measured by Khazaka Germany company). Then, a value obtained by subtracting the value before application of the memberan cut cotton from the value immediately after removal of the memberan cut cotton was calculated, and the amount of change in the stratum corneum water content was measured. IBM SPSS version 23 software was used for statistical analysis.
The results are shown in Table 2.
表２に示すように、水分の負荷量が０mL/cm2から０．０６mL/cm2までの間は、皮膚の水分量は水分の負荷量に依存的に増加した。特に、水分の負荷量が０．０３mL/cm2以上の場合、皮膚の水分量は有意に増加した。一方、水分の負荷量が０．０６mL/cm2以上の場合は、皮膚の水分量はほぼ一定の数値となった。
以上の結果から、水分の負荷量が一定量に達するまでは、皮膚の水分量は水分の負荷量に依存して増加する。特に、水分の負荷量が一定値以上増えると皮膚の水分量は有意に増加する。
さらに前述のように、水分の負荷量が少なくとも０．０４mL/cm2の場合に皮膚バリア機能が低下していると判断でき、水分負荷量が０．０４mL/cm2の場合の皮膚の状態を皮膚のバリア機能を評価するための基準状態とした。よって、この基準状態での皮膚水分量の変化量を、本発明の評価方法における基準値とすることができる。As shown in Table 2, the water content of the skin increased depending on the water loading amount while the water loading amount was from 0 mL / cm 2 to 0.06 mL / cm 2 . In particular, when the water loading amount was 0.03 mL / cm 2 or more, the water content of the skin increased significantly. On the other hand, when the water loading amount was 0.06 mL / cm 2 or more, the water content of the skin was a nearly constant value.
From the above results, the amount of water in the skin increases depending on the amount of applied water until the amount of applied water reaches a certain amount. In particular, the amount of water in the skin increases significantly when the amount of water loading increases beyond a certain value.
Further, as described above, when the load amount of water of at least 0.04 mL / cm 2 can be determined that the skin barrier function is impaired, the state of the skin when the water loading amount of 0.04 mL / cm 2 This was a standard condition for evaluating the barrier function of the skin. Therefore, the amount of change in the amount of skin water in this reference state can be used as a reference value in the evaluation method of the present invention.
（３）共焦点ラマン分光顕微鏡を用いた皮膚水分量の測定
２．５cm×２．５cmのメンバンカット綿（白十字社製）に、脱脂綿１cm2当たりの水分負荷量が０mL（０mL/cm2）、０．０２mL（０．０２mL/cm2）、０．０４mL（０．０４mL/cm2）、０．２０mL（０．２０mL/cm2）となるように蒸留水を含ませ、これを前記被験者の前腕内側にのせ透湿性絆創膏（商品名：スキナゲート）で周囲を固定し３時間貼付した。メンバンカット綿から水分が漏れるのを防ぐため、メンバンカット綿の皮膚と接しない面はドレッシング材（商品名：テガダームＴＭ トランスペアレント ドレッシング）で覆い、貼付中に皮膚が蒸れるのを防ぐためドレッシング材は注射針29G（TERUMO, Japan）で１cm2当たり8個、穴をあけた。メンバンカット綿を貼付している間、試験参加者には運動や屋外での長時間の作業は禁止した。(3) Measurement of skin moisture content using a confocal Raman spectroscopy microscope The moisture load of 1 cm 2 of absorbent cotton is 0 mL (0 mL / cm) to 2.5 cm × 2.5 cm marin cut cotton (manufactured by White Cross). 2 ) include distilled water so that 0.02 mL (0.02 mL / cm 2 ), 0.04 mL (0.04 mL / cm 2 ), and 0.20 mL (0.20 mL / cm 2 ) The test subject was placed on the inner side of the forearm, fixed around with a moisture-permeable adhesive (trade name: Skinagate), and applied for 3 hours. To prevent the member down cut cotton moisture leakage, member emission cut skin and the surface which is not in contact cotton dressing (trade name: Tegaderm TM Transparent Dressing) covered with a dressing material to prevent skin that stuffy during application 8 holes per 1 cm 2 were drilled with an injection needle 29G (TERUMO, Japan). Participants were not allowed to exercise or work outdoors for extended periods while applying member cut cotton.
メンバンカット綿の貼付前と、メンバンカット綿の除去直後に、メンバンカット綿を貼付した部位の皮膚の水分量を共焦点ラマン分光顕微鏡（商品名：ナノファインダー30、東京インスツルメンツ社製）で測定した。なお測定条件は以下の通りである。
（測定条件）
励起波長：６３３nm
積算時間：３秒/１スペクトル
回析格子：１５０本/mm
対物レンズ：１００倍、油浸、ＮＡ＝１．３（ニコン）
深さ方向スキャン条件:２μm間隔で計２１点
１部位あたり５回測定The amount of moisture in the skin of the area where the member cut cotton is attached before and after the attachment of the member cut cotton is a confocal Raman spectroscopy microscope (trade name: Nanofinder 30, manufactured by Tokyo Instruments) It measured by. The measurement conditions are as follows.
(Measurement condition)
Excitation wavelength: 633 nm
Integration time: 3 seconds / 1 spectrum grating: 150 / mm
Objective lens: 100x, oil immersion, NA = 1.3 (Nikon)
Depth direction scan conditions: 5 times per 21 points in total at 2 μm intervals
共焦点ラマン分光顕微鏡を用いた測定で得られたラマンスペクトルから水分量を近似的に算出する方法は、特開２０１２−５０７３９号公報に記載の方法を参照して行った。
さらに、IFSCC Magazine, 2009, vol. 12(1), p. 9-15に記載の方法に準じて、ラマン分光法により取得した水分プロファイルを２本の１次直線で近似し、その交点までの深さを初期の角層厚（メンバンカット綿の貼付前の角層厚）として定義した。The method of approximately calculating the water content from the Raman spectrum obtained by the measurement using the confocal Raman spectroscopy microscope was performed with reference to the method described in JP-A-2012-50739.
Furthermore, according to the method described in IFSCC Magazine, 2009, vol. 12 (1), p. 9-15, the water profile obtained by Raman spectroscopy is approximated by two first-order straight lines, and The depth was defined as the initial angular layer thickness (angular layer thickness prior to application of the Meman cut cotton).
次に、深さ方向の皮膚内の水分量分布をグラフにプロットした。その結果を図２（ａ）〜（ｄ）に示す。図２（ａ）〜（ｄ）に示すグラフにおいて、Ｘ軸は皮膚の深さ（μｍ）、Ｙ軸は水分量（％）を示す。
図２（ａ）に示すグラフより求められた水分負荷前の平均の角層厚は１３μｍであった。そして、図２（ｂ）に示すように、水分負荷量が０．０２mL/cm2の場合、水分負荷前と比較して、水分負荷前の角層厚に相当する１３μｍまでの領域で、皮膚の水分量の増加を確認したが、水負荷前の角層厚よりも深いところの水分量の増加は確認されなかった。
一方で、紅斑スコアに関する前述の実験結果において皮膚のバリア機能が低下し、物質の浸透性の亢進が観察された、水負荷量が０．０４mL/cm2以上の場合、水分負荷前の初期の角層厚よりも深いところの皮膚の水分量が増加した（図２（ｃ）及び（ｄ）参照）。水分負荷前の角層厚よりも深いところの水分量が増加することは、角層が膨潤したためと推察でき、より物質が浸透しやすくなったと評価できる。
よって、皮膚のバリア機能に与える影響を評価するための指標として、水分分布を用いて初期の角層厚よりも深部の水分量の差を用いることができる。Next, the moisture distribution in the skin in the depth direction was plotted on a graph. The results are shown in FIGS. 2 (a) to 2 (d). In the graphs shown in FIGS. 2 (a) to 2 (d), the X axis indicates the depth of the skin (μm), and the Y axis indicates the water content (%).
The average angular layer thickness before the water load calculated | required from the graph shown to FIG. 2 (a) was 13 micrometers. And as shown in FIG.2 (b), when water load is 0.02 mL / cm < 2 >, compared with water load front, in the area | region to 13 micrometers corresponded to the angular layer thickness before water load, An increase in the amount of water was confirmed, but no increase in the amount of water deeper than the thickness before the water loading was confirmed.
On the other hand, the barrier function of the skin decreased in the above-mentioned experimental result about the erythema score, and the enhancement of the permeability of the substance was observed, and when the water loading amount is 0.04 mL / cm 2 or more, the initial stage before water loading The amount of water in the skin deeper than the stratum corneum layer increased (see FIGS. 2 (c) and (d)). The increase in the amount of water deeper than the stratum corneum thickness before the moisture loading can be inferred to be due to the swelling of the stratum corneum, and it can be evaluated that the substance is more easily permeated.
Therefore, as an index for evaluating the influence on the barrier function of the skin, it is possible to use the difference in the amount of water in the deep part rather than the initial angular layer thickness using the water distribution.
表面水分量（深さ２μｍにおける水分量）の変化量（Δ表面水分量）を算出した。
なお、対物レンズの窓材と皮膚の境界（皮膚のはじまり）を区別して皮膚の表面位置を決定するため、皮膚に含まれるタンパク質のＣＨ伸縮振動由来の信号を目安にした。以下、その詳細を説明する。
ラマンスペクトルのタンパク質のＣＨ伸縮振動由来の信号強度は、前述のように、対物レンズの窓材と皮膚の境界よりやや皮膚側で最大値を示した後に、深部にいくほど信号強度が低下する。窓材にはタンパク質のＣＨ伸縮振動由来の信号は存在しないため、レーザーの焦点が窓材の内にあるときはＣＨ伸縮振動由来の信号強度は０になる。一方、皮膚内にレーザーの焦点があるときのＣＨ伸縮振動由来の信号強度を１００とすると、窓材と皮膚の界面に焦点がきた場合、ＣＨ伸縮振動由来の信号強度は５０となる。ただし、実際には窓材にはＣＨ伸縮振動由来の信号強度が弱いながらも検出されることがある。よって、本発明で測定に用いた共焦点ラマン分光装置であるナノファインダーの空間分解能を考慮し、実際に測定した値である表面から深さ２μｍの部位の水分量を「表面水分量」とした。
その結果を表３に示す。The amount of change (Δ surface water content) of the surface water content (water content at a depth of 2 μm) was calculated.
In addition, in order to distinguish the window material of the objective lens and the boundary of the skin (the beginning of the skin) to determine the surface position of the skin, a signal derived from CH stretching vibration of a protein contained in the skin was used as a guide. The details will be described below.
As described above, the signal intensity derived from the CH stretching vibration of a protein in the Raman spectrum shows a maximum value slightly on the skin side of the boundary between the window material of the objective lens and the skin, and then the signal intensity decreases with depth. Since there is no signal derived from CH stretching vibration of protein in the window material, the signal intensity derived from CH stretching vibration becomes zero when the focal point of the laser is within the window material. On the other hand, assuming that the signal strength derived from CH stretching vibration when the focus of the laser is in the skin is 100, the signal strength originating from CH stretching vibration is 50 when the interface is at the interface between the window material and the skin. However, in fact, although the signal strength derived from CH stretching vibration may be weak in the window material, it may be detected. Therefore, considering the spatial resolution of the nanofinder, which is a confocal Raman spectroscopy device used in the measurement in the present invention, the amount of water at a site 2 μm deep from the surface, which is an actually measured value, was defined as “surface moisture”. .
The results are shown in Table 3.
表３に示すように、水負荷前後の表面水分量が変化した。具体的には、水負荷量が０．０２mL/cm2では、水分負荷後の表面水分量（％）は水分負荷前と比較して１．３９倍に増加した。同様に、前述の基準状態とした、水分負荷量が０．０４mL/cm2の場合では、水分負荷後の表面水分量（％）は水分負荷前と比較して２．４０倍に増加し、水分負荷量が０．２０mL/cm2では２．９９倍に増加した。
よって、本発明の評価方法における基準値として、皮膚表面の水分量も用いることができる。As shown in Table 3, the surface moisture content before and after water loading changed. Specifically, when the water loading amount was 0.02 mL / cm 2 , the surface water content (%) after the water loading was increased by 1.39 times compared to before the water loading. Similarly, when the water loading amount is 0.04 mL / cm 2 , which is the above-mentioned reference state, the surface water content (%) after water loading increases by 2.40 times compared to before water loading, The water loading increased to 2.99 times at 0.20 mL / cm 2 .
Therefore, the amount of water on the skin surface can also be used as a reference value in the evaluation method of the present invention.
さらに、水分負荷前後の、皮膚表面から深さ１３μｍの初期の角層厚に相当する深さの水分量の変化量（Δ深さ13μmの水分量）についても算出した。その結果を表４に示す。 Furthermore, the amount of change in the amount of water at a depth corresponding to an initial angular layer thickness of 13 μm deep from the skin surface before and after the water load (Δ water amount of 13 μm deep) was also calculated. The results are shown in Table 4.
表４に示すように、負荷する水分量に応じて、水負荷前の角層の厚さに相当する深さ１３μｍにおける水分量が変化した。水負荷前後の深さ１３μｍの水分量を比較した場合も物質浸透性が亢進した水負荷量0.04mL/cm2以降で増加し、すなわち、初期の角層厚に相当する部位の深さの水分量が増加したことがわかった。
よって、本発明の評価方法における基準値として、初期の角層厚に相当する深さの部位の水分量の変化量も用いることができる。As shown in Table 4, the water content at a depth of 13 μm, which corresponds to the thickness of the stratum corneum before water loading, changed in accordance with the water content to be loaded. When the water content of depth 13 μm before and after water loading is compared, it also increases at water loading amount 0.04 mL / cm 2 or more where the material permeability is enhanced, that is, the water at the depth of the part corresponding to the initial angular layer thickness It turned out that the quantity increased.
Therefore, as the reference value in the evaluation method of the present invention, it is also possible to use the amount of change in the amount of water at a portion at a depth corresponding to the initial angular layer thickness.
次に、皮膚表面からの深さ１μｍから水負荷前の初期の角層厚に相当する１３μｍの深さまでの水分量の曲線下面積（Area Under the Curve (AUC) of water profile １−１３μｍ）の結果を表５に示す。統計解析にはＩＢＭ ＳＰＳＳバージョン２３のソフトウェアを使用した。 Next, the area under the curve of the amount of water from a depth of 1 μm from the skin surface to a depth of 13 μm corresponding to the initial angular layer thickness before water loading (Area Under the Curve (AUC) of water profile 1-13 μm) The results are shown in Table 5. IBM SPSS version 23 software was used for statistical analysis.
表５に示すように、水負荷前後の皮膚表面からの深さ１μｍから１３μｍまでの深さの水分量の曲線下面積が変化した。具体的には、水負荷量が０．０２mL/cm2では、水分負荷後の水分量の曲線下面積は水分負荷前と比較して１．２０倍に増加した。同様に、前述の基準状態とした、水分負荷量が０．０４mL/cm2の場合では、水分負荷後の水分量の曲線下面積は水分負荷前と比較して１．５９倍に増加し、水分負荷量が０．２０mL/cm2では１．８３倍に増加した。
よって、本発明の評価方法における基準値として、皮膚表面からの所定の深さまでにおける皮膚の水分量の曲線下面積も用いることができる。As shown in Table 5, the area under the curve of the amount of water at a depth of 1 μm to 13 μm from the skin surface before and after water loading was changed. Specifically, when the water loading amount was 0.02 mL / cm 2 , the area under the curve of the water content after the water loading increased by 1.20 times compared to before the water loading. Similarly, when the water loading amount is 0.04 mL / cm 2 , which is the reference state described above, the area under the water content curve after water loading increases by 1.59 times compared to before water loading, The water loading increased to 1.83 times at 0.20 mL / cm 2 .
Therefore, the area under the curve of the water content of the skin up to a predetermined depth from the skin surface can also be used as a reference value in the evaluation method of the present invention.
表３〜５に示す結果より、物質の浸透性が亢進していない水負荷量０．０２mL/cm2の場合の表面水分量、深さ１３μｍの水分量又は水分量の曲線下面積の変化量と比べて、皮膚のバリア機能が低下し、物質浸透性が亢進した水負荷量０．０４mL/cm2以降では表面水分量、深さ１３μｍの水分量又は水分量の曲線下面積の変化量が大きく増加したことで、水溶性物質が分配されやすくなり、より浸透しやすくなった可能性が推察できた。From the results shown in Tables 3 to 5, according to the water loading amount of 0.02 mL / cm 2 in which the permeability of the substance is not enhanced, the surface water content, the water content at a depth of 13 μm, or the change of the area under the water content curve Compared to the above, the barrier function of the skin is reduced, and the water permeability at which the substance permeability is enhanced is 0.04 mL / cm 2 or later, the surface water content, the water content of 13 μm depth or the change amount of the area under the water content curve It was possible to infer that the water-soluble substance could be distributed more easily and penetrated more easily due to the large increase.
以上のように、コルネオメーターやラマン分光法などで非侵襲的に測定した皮膚の水分量や水分分布から、水負荷量がある一定値以上増加すると物質の透過性が亢進することがわかった。すなわち、コルネオメーターやラマン分光法などで非侵襲的に測定した皮膚の水分量や水分分布から、基準状態における皮膚水分量又は水分分布との比較により、物質の透過性、すなわち皮膚のバリア機能を評価することができる。 As described above, it was found from the moisture content and moisture distribution of the skin non-invasively measured by a corneometer, Raman spectroscopy, etc. that the permeability of the substance is enhanced when the water loading amount is increased by a certain value or more. That is, based on the moisture content and moisture distribution of the skin non-invasively measured with a corneometer or Raman spectroscopy, the permeability of the substance, ie, the barrier function of the skin, is compared with the moisture content or moisture distribution of the skin in the standard state. It can be evaluated.
試験例２ 吸収性物品の性能比較試験
（１）皮膚への水分の負荷
健常な女性１名（３０代）を被験者とし、吸収性物品が前腕内側の皮膚へ与える影響を評価した。測定は２４〜２５℃、相対湿度４０〜５０％の環境可変室で、２０〜３０分順化した後に行った。Test Example 2 Performance Comparison Test of Absorbent Articles (1) Water Load on Skin One healthy woman (30's) was used as a subject to evaluate the influence of the absorbent articles on the skin on the inside of the forearm. The measurement was performed after conditioning for 20 to 30 minutes in an environment variable chamber at 24 to 25 ° C. and a relative humidity of 40 to 50%.
（２）コルネオメーターを用いた皮膚水分量の測定
２種類の吸収性物品（吸収性物品Ａ及び吸収性物品Ｂ）を５．０cm×５．０cmに切断して試験片を調製し、それぞれに生理食塩水３．０mLを注入し(０．１２mL/cm2試験片面積)、それらを前腕内側に３時間貼付した。なお、吸収性物品Ａは市販の使い捨ておむつであり、吸収性物品Ｂは市販の綿製のおむつである。
試験片貼付前後の皮膚水分量を、試験例１と同様コルネオメーターで測定し、試験片貼付前後の皮膚水分量の変化量を算出した。その結果を表６に示す。(2) Measurement of skin moisture content using a corneometer Two types of absorbent articles (absorbent article A and absorbent article B) are cut into 5.0 cm x 5.0 cm to prepare test pieces, 3.0 mL of saline was injected (0.12 mL / cm 2 test strip area) and they were affixed to the inside of the forearm for 3 hours. In addition, the absorbent article A is a commercially available disposable diaper, and the absorbent article B is a commercially available cotton diaper.
The skin water content before and after the test piece application was measured with a corneometer as in Test Example 1, and the change amount of the skin water content before and after the test element application was calculated. The results are shown in Table 6.
表６に示すように、吸収性物品Ａを貼付した場合と比較して、吸収性物品Ｂを貼付した場合、皮膚水分量が増加していた。
また、吸収性物品Ａを貼付した場合の皮膚水分量の変化量は、試験例１において基準値とした、水分負荷量が０．０４mL/cm2の場合の皮膚水分量の変化量（19.8±8.6(A.U)）よりも少なかった。よって、吸収性物品Ａを貼付しても、バリア機能は変化していないと評価できる。一方、吸収性物品Ｂを貼付した場合の皮膚水分量の変化量は、前記基準値よりも多いので、吸収性物品Ｂを貼付するとバリア機能は低下すると評価できる。As shown in Table 6, compared with the case where the absorbent article A was attached, when the absorbent article B was attached, the skin moisture amount was increased.
In addition, the amount of change in skin moisture when the absorbent article A is applied is the amount of change in skin moisture (19.8 ±) when the water loading amount is 0.04 mL / cm 2 , which is the reference value in Test Example 1. Less than 8.6 (AU)). Therefore, even if the absorbent article A is attached, it can be evaluated that the barrier function is not changed. On the other hand, since the change amount of the skin moisture amount at the time of sticking the absorbent article B is larger than the said reference value, when the absorbent article B is stuck, it can be evaluated that a barrier function falls.
（３）共焦点ラマン分光顕微鏡を用いた皮膚水分量の測定
試験例１と同様に、皮膚表面から４０μｍまで、深さ方向に２μｍ間隔毎にラマンスペクトルを測定した。このようにして測定し特開２０１２−５０７３９号公報に記載の方法に従って水分量を近似的に算出し、深さ方向の皮膚内の水分量分布の値をグラフにプロットした。その結果を図３に示す。(3) Measurement of Skin Water Content Using Confocal Raman Spectroscopic Microscope Similar to Test Example 1, Raman spectra were measured at intervals of 2 μm in the depth direction up to 40 μm from the skin surface. The water content was approximately calculated according to the method described in JP-A-2012-50739, and the value of the water content distribution in the skin in the depth direction was plotted on a graph. The results are shown in FIG.
図３に示すように、吸収性物品Ａを貼付した場合と比較して、吸収性物品Ｂを貼付した場合、皮膚水分量が増加していた。また同様に、吸収性物品Ａを貼付した場合と比較して、吸収性物品Ｂを貼付した場合、水分負荷前の初期の角層厚よりも深いところの水分量が増加した。
よって、水分分布を用いて初期の角層厚よりも深部の水分量の差からも、吸収性物品Ａを貼付してもバリア機能は変化しないのに対し、吸収性物品Ｂを貼付するとバリア機能は低下する、と評価できる。As shown in FIG. 3, the skin moisture amount was increased when the absorbent article B was attached as compared with the case where the absorbent article A was attached. Similarly, compared with the case where the absorbent article A was attached, when the absorbent article B was attached, the amount of water at a deeper position than the initial angular layer thickness before the water load was increased.
Therefore, the barrier function does not change even when the absorbent article A is attached, even when the absorbent article A is attached, from the difference in the water content in the deep part from the initial angular layer thickness using the moisture distribution. Can be evaluated as decreasing.
さらに、図３に示す水分プロファイルから、表面水分量（皮膚表面からの深さ２μｍの部位の水分量）を算出した。その結果を表７に示す。 Further, the surface water content (the water content of a site 2 μm deep from the skin surface) was calculated from the water profile shown in FIG. The results are shown in Table 7.
表７に示すように、水負荷前後の表面水分量（％）は吸収性物品Ａの貼付後は貼付前と比較して、１．１９倍増加した。これに対して吸収性物品Ｂの貼付後は貼付前と比較して、２．８６倍増加した。
また、吸収性物品Ａを貼付した場合の表面水分量は、試験例１において基準値とした、水分負荷量が０．０４mL/cm2の場合の表面水分量の増加分（２．４０倍）よりも小さかった。よって、吸収性物品Ａを貼付しても、バリア機能は変化していないと評価できる。一方、吸収性物品Ｂを貼付した場合の表面水分量の増加分は、前記基準値よりも大きいので、吸収性物品Ｂを貼付するとバリア機能は低下すると評価できる。As shown in Table 7, the surface moisture content (%) before and after water loading was increased by 1.19 times after the application of the absorbent article A as compared to before the application. On the other hand, after sticking of the absorbent article B, it increased by 2.86 times compared with before sticking.
In addition, the surface water content when the absorbent article A is attached is the reference value in Test Example 1, and the increase in the surface water content when the water loading amount is 0.04 mL / cm 2 (2.40 times) It was smaller than that. Therefore, even if the absorbent article A is attached, it can be evaluated that the barrier function is not changed. On the other hand, since the increase in surface water content when the absorbent article B is attached is larger than the reference value, it can be evaluated that the barrier function is reduced when the absorbent article B is attached.
次に、図３に示す水分プロファイルから、皮膚表面から深さ１３μｍの初期の角層厚に相当する深さの水分量の変化量（Δ深さ13μmの水分量）を算出した。その結果を表８に示す。 Next, from the water profile shown in FIG. 3, the amount of change in the amount of water at a depth corresponding to an initial angular layer thickness of 13 μm from the skin surface (Δ water amount of 13 μm depth) was calculated. The results are shown in Table 8.
表８に示すように、吸収性物品Ａを貼付した場合と比較して、吸収性物品Ｂを貼付した場合、深さ１３μｍにおける水分量が大きく増加した。
また、吸収性物品Ａを貼付した場合の初期の角層厚に相当する深さの水分量の変化量は、試験例１において基準値とした、水分負荷量が０．０４mL/cm2の場合の初期の角層厚に相当する深さの水分量の変化量（9.21±9.2（％））よりも少なかった。よって、吸収性物品Ａを貼付しても、バリア機能は変化していないと評価できる。一方、吸収性物品Ｂを貼付した場合の初期の角層厚に相当する深さの水分量の変化量は、前記基準値よりも多いので、吸収性物品Ｂを貼付するとバリア機能は低下すると評価できる。As shown in Table 8, compared to the case where the absorbent article A was attached, when the absorbent article B was attached, the water content at a depth of 13 μm was greatly increased.
In addition, when the amount of change in the amount of water at a depth corresponding to the initial angular layer thickness when the absorbent article A is attached is 0.04 mL / cm 2 when the water loading amount is set as the reference value in Test Example 1 The amount of change in water content at the depth corresponding to the initial angular layer thickness (9.21 ± 9.2 (%)) was smaller. Therefore, even if the absorbent article A is attached, it can be evaluated that the barrier function is not changed. On the other hand, since the amount of change in the amount of water at a depth corresponding to the initial angular layer thickness when sticking the absorbent article B is larger than the reference value, it is evaluated that the barrier function decreases when the absorbent article B is stuck. it can.
さらに、図３に示す水分分布から、試験片貼付前後の皮膚表面からの深さ１μｍから１３μｍの深さまでの水分量の曲線下面積（AUC of water profile １−１３μｍ）を算出した。その結果を表９に示す。 Furthermore, the area under the curve (AUC of water profile 1-13 μm) of the amount of water from the depth of 1 μm to the depth of 13 μm from the skin surface before and after application of the test piece was calculated from the water distribution shown in FIG. The results are shown in Table 9.
表９に示すように、水負荷前後の皮膚表面からの深さ１μｍから１３μｍまでの深さの水分量の曲線下面積は吸収性物品Ａの貼付後は貼付前と比較して、１．０８倍増加した。これに対して吸収性物品Ｂの貼付後は貼付前と比較して、１．８９倍増加した。
また、吸収性物品Ａを貼付した場合の曲線下面積の変化量は、試験例１において基準値とした、水分負荷量が０．０４mL/cm2の場合の曲線下面積の増加分（１．５９倍）よりも小さかった。よって、吸収性物品Ａを貼付しても、バリア機能は変化していないと評価できる。一方、吸収性物品Ｂを貼付した場合の曲線下面積の増加分は前記基準値よりも大きいので、吸収性物品Ｂを貼付するとバリア機能は低下すると評価できる。As shown in Table 9, the area under the curve of the amount of water at a depth of 1 μm to 13 μm from the skin surface before and after water loading is 1.08 after application of the absorbent article A compared to before application. Doubled. On the other hand, after sticking of the absorbent article B, it increased by 1.89 times compared with before sticking.
In addition, the amount of change in the area under the curve when the absorbent article A is attached is the increase in the area under the curve when the water loading amount is 0.04 mL / cm 2 , which is the reference value in Test Example 1 (1. It was smaller than 59 times). Therefore, even if the absorbent article A is attached, it can be evaluated that the barrier function is not changed. On the other hand, since the increase in the area under the curve when the absorbent article B is attached is larger than the reference value, it can be evaluated that the barrier function is reduced when the absorbent article B is attached.
（４）物質透過性の評価
試験片を除去した直後に、０．００７質量％の濃度のニコチン酸メチル水溶液を１５秒、試験片を貼付した皮膚に負荷し、ニコチン酸メチル処理から２０分後の紅斑スコアを試験例１と同様にして目視判定した。
その結果を表１０に示す。(4) Evaluation of substance permeability Immediately after removing the test piece, a methyl nicotinate aqueous solution with a concentration of 0.007% by mass was loaded on the skin to which the test piece was applied for 15 seconds, and 20 minutes after the methyl nicotinate treatment The erythema score was visually determined in the same manner as in Test Example 1.
The results are shown in Table 10.
表１０に示すように、吸収性物品Ａを貼付した場合と比較して、吸収性物品Ｂを貼付した場合紅斑スコアが上昇し、ニコチン酸メチルの透過性が亢進していた。 As shown in Table 10, compared with the case where the absorbent article A was attached, the erythema score increased when the absorbent article B was attached, and the permeability of methyl nicotinate was enhanced.
上記結果に示すように、吸収性物品Ａを貼付した場合、コルネオメーターによる皮膚の水分量の増加、並びにラマン分光法で測定した皮膚の表面水分量の増加、深さ１３μｍにおける水分量の増加、及び皮膚表面からの深さ１μｍから１３μｍまでの水分量の曲線下面積の増加はいずれも、基準とする水分分布に比べて小さいと評価できた。よって、皮膚の水分量吸収性物品Ａを貼付した場合は、水溶性物質の透過性が低いと判断される。吸収性物品Ａを貼付して水溶性物質の透過性を実際に判定したところ、皮膚の外側から内側への水溶性物質の透過性が低かった。すなわち吸収性物品Ａは、上記結果から、水溶性物質の皮膚への透過性は亢進されず、皮膚トラブルを起こしにくい吸収性物品であると言える。 As shown in the above results, when the absorbent article A is applied, the increase of the water content of the skin by the corneometer, the increase of the surface water content of the skin measured by Raman spectroscopy, the increase of the water content at a depth of 13 μm, And the increase in the area under the curve of the water content from the skin surface to a depth of 1 μm to 13 μm can be evaluated to be smaller than the reference water distribution. Therefore, when the water content absorbent article A of the skin is attached, it is judged that the permeability of the water-soluble substance is low. When the absorbent article A was applied and the permeability of the water-soluble substance was actually judged, the permeability of the water-soluble substance from the outside to the inside of the skin was low. That is, from the above results, it can be said that the permeability of the water-soluble substance to the skin is not enhanced and the absorbent article A is an absorbent article that is less likely to cause skin problems.
一方、吸収性物品Ｂを貼付した場合、コルネオメーターによる皮膚の水分量の増加、並びにラマン分光法で測定した皮膚の表面水分量の増加、深さ１３μｍにおける水分量の増加、及び皮膚表面からの深さ１μｍから１３μｍまでの水分量の曲線下面積の増加はいずれも、基準とする水分分布に比べて大きいと評価できた。よって、吸収性物品Ｂを貼付した場合は、水溶性物質の透過性が高いと判断される。吸収性物品Ｂを貼付して水溶性物質の透過性を実際に判定したところ、驚くべきことに皮膚の外側から内側への水溶性物質の透過性が上昇していたことがわかった。すなわち吸収性物品Ｂは、水溶性物質の皮膚への透過性が亢進され、皮膚トラブルを起こす可能性がある吸収性物品であると言える。 On the other hand, when the absorbent article B is applied, the moisture content of the skin increases by the corneometer, the surface moisture content of the skin measured by Raman spectroscopy, the moisture content at a depth of 13 μm, and the surface of the skin The increase in the area under the curve of the water content from 1 μm to 13 μm in depth was evaluated to be larger than the reference water distribution. Therefore, when the absorbent article B is attached, it is judged that the permeability of the water-soluble substance is high. When the absorbent article B was applied and the permeability of the water-soluble substance was actually judged, it was found that the permeability of the water-soluble substance from the outside to the inside of the skin was surprisingly increased. That is, it can be said that the absorbent article B is an absorbent article in which the permeability of the water-soluble substance to the skin is enhanced and the skin trouble may occur.
以上より、本発明の方法に従って被験物質又は物品をヒトの皮膚に接触させ、接触前後の皮膚の水分量又は水分分布を測定した結果から、前記被験物質又は物品が、皮膚のバリア機能に与える影響を評価する方法として有効であることが確認できた。 As described above, the test substance or article is brought into contact with human skin according to the method of the present invention, and the result of measuring the water content or water distribution of the skin before and after contact shows the influence of the test substance or article on the barrier function of the skin. It has been confirmed that it is effective as a method of evaluating
本発明をその実施態様とともに説明したが、我々は特に指定しない限り我々の発明を説明のどの細部においても限定しようとするものではなく、添付の請求の範囲に示した発明の精神と範囲に反することなく幅広く解釈されるべきであると考える。 While the present invention has been described in conjunction with its embodiments, we do not intend to limit our invention in any detail of the description unless otherwise specified, which is contrary to the spirit and scope of the invention as set forth in the appended claims. I think that it should be interpreted broadly without.
Claims (29)
接触前後の皮膚の水分量又は水分分布の変化量を、基準値と比較し、
比較結果から、前記被験物質又は物品が、皮膚バリア機能に与える影響を評価する、
被験物質又は物品の評価方法であって、
水の負荷量を変えての皮膚への水負荷試験により測定された、皮膚の水分量又は水分分布と、皮膚のバリア機能とが関連付けられ、
皮膚の水分量又は水分分布と皮膚のバリア機能との関連性から、皮膚のバリア機能に変化が確認されるときの皮膚の水分量又は水分分布が、前記基準値として設定されたものである、
被験物質又は物品の評価方法。Bringing a test substance or article into contact with human skin,
Compare the amount of change in moisture content or moisture distribution of the skin before and after contact with the reference value,
Evaluating the influence of the test substance or the article on the skin barrier function from the comparison result
A method of evaluating a test substance or article, comprising
Water content or distribution of the skin, as measured by water load test on the skin with varying water load, is associated with the barrier function of the skin,
The water content or the water distribution of the skin when a change in the skin barrier function is confirmed is set as the reference value from the relation between the water content or the water distribution of the skin and the barrier function of the skin.
Evaluation method of test substance or article.
被験物質又は物品の接触後の水分分布と基準水分分布とを比較することで、被験物質又は物品が、ヒトの皮膚のバリア機能に与える影響を評価する、被験物質又は物品の評価方法であって、
水又は水溶液のヒトの皮膚への負荷前に、ヒトの皮膚の深さ毎の水分量を測定して初期水分分布を取得し、
水又は水溶液のヒトの皮膚への負荷終了直後に、ヒトの皮膚の深さ毎の水分量の測定による水又は水溶液の負荷後の水分分布の取得と、指標物質を皮膚に適用して該指標物質の浸透度の測定を行い、
初期水分分布から水又は水溶液の負荷終了直後の水分分布への変化と、指標物質の浸透度から、指標物質の浸透度と水又は水溶液の負荷量との関連性を決定し、
前記関連性が変化したときのヒトの皮膚の深さ毎の水又は水溶液の負荷後の水分分布を、前記基準水分分布とする、
被験物質又は物品の評価方法。Measuring the amount of water per depth of human skin after bringing the test substance or article into contact with human skin, and acquiring the water distribution after the contact of the test substance or article,
A method for evaluating a test substance or article, which evaluates the influence of the test substance or article on the barrier function of human skin by comparing the distribution of water after contact of the test substance or article with the reference water distribution. ,
Before applying water or an aqueous solution to human skin, measure the amount of water at each depth of human skin to obtain an initial water distribution,
Immediately after the end of the load of water or aqueous solution on human skin, acquisition of water distribution after load of water or aqueous solution by measurement of the amount of water per depth of human skin and application of the indicator substance to the skin, the index Measure the permeability of the substance,
From the change from the initial water distribution to the water distribution immediately after the end of the water or aqueous solution load and the permeability of the indicator substance, determine the relationship between the permeability of the indicator substance and the water or aqueous solution load,
The water distribution after loading of water or an aqueous solution for each depth of human skin when the relevance changes is taken as the reference water distribution.
Evaluation method of test substance or article.
取得した水分量の差と、前記基準水分分布とに基づき、皮膚バリア機能に与える影響を評価する、
請求項５に記載の評価方法。Measure the amount of water at a predetermined depth position of human skin before and after contact with the test substance or article, and obtain the difference between the amount of water before contact and the amount of water after contact,
Evaluate the effect on skin barrier function based on the difference between the obtained water content and the reference water distribution,
The evaluation method according to claim 5.
被験物質又は物品のヒトの皮膚への接触前と接触後の皮膚の深さ毎の水分量を測定して水分分布を取得し、
接触前と接触後の水分分布における、接触前の初期の角層厚よりも深部の水分量の差を指標として、被験物質又は物品がヒトの皮膚のバリア機能に与える影響を評価する、
被験物質又は物品の評価方法。A method for evaluating the influence of a test substance or article on the barrier function of human skin,
Before the contact of the test substance or article with human skin and after the contact, measure the amount of water at each depth of the skin to obtain the water distribution,
Evaluate the effect of the test substance or article on the barrier function of human skin, using the difference in the amount of water deeper than the initial angular layer thickness before contact in the water distribution before and after contact as an indicator,
Evaluation method of test substance or article.
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