JP2011128062A - Method for determining skin function - Google Patents
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Abstract
Description
本発明は皮膚機能、特に皮膚の保湿機能、バリア機能状態を評価・判定する方法に関する。 The present invention relates to a method for evaluating / determining skin function, particularly skin moisturizing function and barrier function state.
皮膚は、生体の最外層を覆い、外界との境界を形成して外界物質の侵入防止、水分蒸散防止などの生命維持に必須なバリア機能を有している。角質の細胞間には、セラミド、コレステロール及び脂肪酸を主成分とする脂質が多層状構造（ラメラ構造）を形成し、当該ラメラ構造が水分透過バリアとして重要な役割を果たしている。 The skin covers the outermost layer of the living body, forms a boundary with the outside world, and has a barrier function essential for life support such as prevention of entry of outside substances and prevention of water evaporation. Between the keratinous cells, lipids mainly composed of ceramide, cholesterol and fatty acids form a multilayer structure (lamellar structure), and the lamellar structure plays an important role as a moisture permeation barrier.
特に角質におけるセラミドは、上記バリアの形成に必須な成分であると考えられている。しかし、このセラミドの代謝は、加齢、紫外線曝露、乾燥肌、荒れ肌、アトピー性皮膚炎、老人性乾皮症、乾癬などによって、健全な状態が損われることがある。その結果、角層中のセラミド量が減少して、例えば、皮膚の保湿機能およびバリア機能の低下を引き起こすことが数多く報告されている。 In particular, ceramide in the keratin is considered to be an essential component for the formation of the barrier. However, this ceramide metabolism may be impaired by aging, UV exposure, dry skin, rough skin, atopic dermatitis, senile psoriasis, psoriasis, and the like. As a result, it has been reported many times that the amount of ceramide in the stratum corneum is reduced, for example, causing a decrease in the moisture retention function and barrier function of the skin.
従って、角層中のセラミド量を測定し、皮膚の保湿機能またはバリア機能を評価・判定することは、健全な皮膚状態を把握すると共に、乾燥肌、荒れ肌、アトピー性皮膚炎等を防止する上でも重要である。 Therefore, measuring the amount of ceramide in the stratum corneum and evaluating / determining the moisturizing function or barrier function of the skin helps to grasp a healthy skin state and prevent dry skin, rough skin, atopic dermatitis, etc. But it is important.
角層中のセラミドは、表皮細胞においてセリンとパルミトイルCoAを出発原料に、de novo合成されて生成するとされている。従来、角層中のセラミド量を測定するには、対象とする部位の角層を採取して分析していた。しかしながら、角層の採取ができない場合は、セラミド量を測定することができない、という問題があった。 Ceramide in the stratum corneum is supposed to be generated by de novo synthesis using serine and palmitoyl CoA as starting materials in epidermal cells. Conventionally, in order to measure the amount of ceramide in the stratum corneum, the stratum corneum at the target site has been collected and analyzed. However, when the stratum corneum cannot be collected, there is a problem that the amount of ceramide cannot be measured.
一方、セラミドは生体において各臓器や細胞でde novo合成されており、肝臓からはセラミドが血中に分泌されることが報告されている。実際にセラミドは人の血液にも存在し、アポリポタンパク質に結合した状態で血液中を輸送されていることが報告されている。斯かる血中セラミドについては、例えば、Gaucher病等の遺伝病において、血漿セラミド量が上昇すること（非特許文献１）や、アテローム性動脈硬化症において血漿総セラミドがリスクファクターとなり得ること（非特許文献２）等が報告されている。
しかし、血中セラミド量が角層中のセラミド量と相関するという報告はこれまでに全く存在しない。
On the other hand, ceramide is synthesized de novo in each organ and cell in the living body, and it is reported that ceramide is secreted into the blood from the liver. In fact, ceramide is also present in human blood and has been reported to be transported through blood in a state of being bound to apolipoprotein. Regarding such ceramide in blood, for example, the amount of plasma ceramide increases in genetic diseases such as Gaucher disease (Non-patent Document 1), and that plasma total ceramide can be a risk factor in atherosclerosis (non- Patent Document 2) and the like have been reported.
However, there has never been a report that the blood ceramide level correlates with the ceramide level in the stratum corneum.
本発明は、迅速で簡便に、皮膚の保湿機能、バリア機能等の皮膚機能を評価・判定する方法、及び皮膚機能改善剤の評価又は選択方法を提供することに関する。 The present invention relates to providing a method for evaluating and judging skin functions such as a skin moisturizing function and a barrier function, and a method for evaluating or selecting a skin function improving agent quickly and easily.
本発明者らは、皮膚機能を評価するためのマーカーについて種々検討したところ、ヒト血液中のセラミド量が、角層セラミド量及び経皮水分蒸散量（ＴＥＷＬ）やコンダクタンス値と相関し、血中セラミド量を測定することにより、皮膚の保湿機能、バリア機能等の皮膚機能を評価できること、また皮膚機能改善剤を評価・選択できることを見出した。 As a result of various studies on markers for evaluating skin function, the present inventors have found that the amount of ceramide in human blood correlates with the amount of horny layer ceramide, the amount of transdermal water transpiration (TEWL) and the conductance value. It was found that by measuring the amount of ceramide, it was possible to evaluate skin functions such as skin moisturizing function and barrier function, and to evaluate and select skin function improving agents.
すなわち、本発明は、以下の１）〜４）に係るものである。
１）被験者から採取した血液中のセラミド量を測定し、その測定値に基づいて皮膚バリア機能を判定することを含む、皮膚機能の判定方法。
２）皮膚機能が、皮膚バリア機能又は保湿機能である上記１）の判定方法。
３）血液中のセラミド量の測定値から、角層セラミド量、ＴＥＷＬ値及びコンダクタンス値から選ばれる数量を推定し、それに基づき皮膚機能を判定する、上記１）又は２）の判定方法。
４）以下の（Ａ）及び（Ｂ）の工程を含むことを特徴とする、皮膚機能改善剤の評価又は選択方法。
（Ａ）被験物質を被験者又は被験動物に投与する工程
（Ｂ）血液中のセラミド量を測定し、その変化を評価する工程
That is, the present invention relates to the following 1) to 4).
1) A method for determining skin function, comprising measuring an amount of ceramide in blood collected from a subject and determining a skin barrier function based on the measured value.
2) The determination method according to 1) above, wherein the skin function is a skin barrier function or a moisturizing function.
3) The determination method according to 1) or 2) above, wherein a quantity selected from a stratum corneum amount, a TEWL value, and a conductance value is estimated from a measured value of the amount of ceramide in blood, and skin function is determined based on the estimated amount.
4) A method for evaluating or selecting a skin function improving agent, comprising the following steps (A) and (B):
(A) A step of administering a test substance to a subject or a test animal (B) A step of measuring the amount of ceramide in blood and evaluating the change
本発明によれば、血清などの血中のセラミド量を測定することにより、特別な装置を用いることなく、迅速で簡便に、保湿機能、バリア機能等の皮膚機能を評価・判定することが可能になり、また皮膚機能改善剤をスクリーニングすることができる。 According to the present invention, by measuring the amount of ceramide in blood such as serum, it is possible to evaluate and determine skin functions such as a moisturizing function and a barrier function quickly and easily without using a special device. And a skin function improving agent can be screened.
本発明の方法において、測定される「セラミド」とは、スフィンゴシン塩基のアミノ基に脂肪酸が酸アミド結合した構造をもつ化合物を意味する。従って、スフィンゴシン塩基と、脂肪酸との組合せにより種々のセラミド・クラスを包含するものであり、特定のセラミド・クラスを示すものではない。すなわち、本発明において測定されるセラミド量は、種々のクラスのセラミドを総じて含むセラミド量（総セラミド量）であるのが好ましい。 In the method of the present invention, “ceramide” to be measured means a compound having a structure in which a fatty acid is bonded to an amino group of a sphingosine base with an acid amide bond. Accordingly, various ceramide classes are included depending on the combination of sphingosine base and fatty acid, and do not indicate a specific ceramide class. That is, the amount of ceramide measured in the present invention is preferably the amount of ceramide (total amount of ceramide) containing various classes of ceramide as a whole.
本発明における血液としては、医療機関等においてヒトから採取されるものであればよく、血清、血漿等を用いることができ、血清が好適である。
また、マウス等の哺乳動物においては、血漿中のセラミド量が生理的要因で変動することが報告されていることから（THE JOURNAL OF BIOLOGICAL CHEMISTRY ,VOL283,NO20,PP13538-13548）、皮膚機能改善剤の評価又は選択においては、当該動物から採取される血液を用いることもできる。
The blood in the present invention may be any blood collected from a human in a medical institution or the like, and serum, plasma and the like can be used, and serum is preferred.
In mammals such as mice, the amount of ceramide in plasma has been reported to vary due to physiological factors (THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL283, NO20, PP13538-13548). In the evaluation or selection, blood collected from the animal can also be used.
セラミドの測定方法としては、公知の方法、すなわち、ガスクロマトグラフ質量分析計（GC-MS）、TLC、液体クロマトグラフ質量分析計（LC-MSあるいはLC-MS/MS）等を単独あるいは組み合わせて測定することができる。以下に、その一例を示す。
１）採取した血清を、リン酸緩衝液（PBS）等の緩衝液で希釈し、これにクロロホルム、メタノールを加え、Bligh & Dyer法による脂質の抽出を行なう。
２）次いで、セラミドを含む画分に内部標準物質を添加し、LC-MSにより血清1mLあたりのセラミド量を定量する。
As a method for measuring ceramide, a known method, that is, gas chromatograph mass spectrometer (GC-MS), TLC, liquid chromatograph mass spectrometer (LC-MS or LC-MS / MS), etc. is used alone or in combination. can do. An example is shown below.
1) Dilute the collected serum with a buffer solution such as phosphate buffer (PBS), add chloroform and methanol to it, and extract lipids by the Bligh & Dyer method.
2) Next, an internal standard substance is added to the fraction containing ceramide, and the amount of ceramide per mL of serum is determined by LC-MS.
斯かる方法により、健常者の血液中のセラミド量を測定したところ、後記実施例に示すように、血液中のセラミド量は、同時に測定した角層セラミド量、特に膝裏、手の甲、額、頬における角層セラミド量と高い相関を示した（実施例１）。更に、脂肪酸摂取により角層セラミド量は増加し、ＴＥＷＬ値は低下して、皮膚バリア機能が向上することが知られているが（特開2009-084244号公報）、当該脂肪酸摂取時における血中セラミドの変化量と、角層セラミド量及びＴＥＷＬ値の変化量との相関性を解析したところ、角層セラミド量及びコンダクタンス値の変動量とは正の相関があり、ＴＥＷＬ値の変動量とは負の相関があることが示された（実施例２）。従って、血液中のセラミド量は、角層セラミド量及び皮膚バリア機能や保湿機能等の皮膚機能を把握するための指標となり、これを測定することにより皮膚機能を判定することができる。 When the amount of ceramide in the blood of a healthy person was measured by such a method, as shown in the examples below, the amount of ceramide in the blood was measured simultaneously with the amount of horny layer ceramide, particularly the back of the knee, back of the hand, forehead, cheek. (Example 1) which showed a high correlation with the amount of stratum corneum in. Furthermore, it is known that the amount of cerebral ceramide increases, the TEWL value decreases, and the skin barrier function is improved by fatty acid intake (Japanese Patent Laid-Open No. 2009-084244). When the correlation between the amount of change of ceramide and the amount of change of horny layer ceramide and TEWL value was analyzed, the amount of change of horny layer ceramide and conductance value had a positive correlation, and the amount of change of TEWL value was A negative correlation was shown (Example 2). Therefore, the amount of ceramide in the blood serves as an index for grasping the amount of stratum corneum and the skin functions such as the skin barrier function and the moisturizing function, and the skin function can be determined by measuring this.
判定は、例えば、血清1mLあたりのセラミド量が１μg未満の場合（好ましくは0.6μg以下の場合）、角層のセラミドが少なく、バリア機能や保湿機能の低下した状態と判断される。この場合、額や頬および手の甲のような部位では特にこれらの機能が低下した状態であると判断される。 For example, when the amount of ceramide per 1 mL of serum is less than 1 μg (preferably 0.6 μg or less), it is determined that the ceramide in the stratum corneum is small and the barrier function or the moisture retention function is deteriorated. In this case, it is determined that these functions are particularly deteriorated at the parts such as the forehead, cheek and back of the hand.
このように、本発明の皮膚機能の判定方法によれば、角層セラミド量及び皮膚バリア機能や保湿機能の測定値がない場合でも、血中セラミド値から皮膚機能を判定することができるため、健康診断や他の採血を伴う試験で採取した血液を利用して、その個人の皮膚機能を判定することができる。たとえば、何らかの生理作用変化に対する生体の変動を観察する試験において（ストレス負荷や睡眠、生活習慣等）、その生理作用に対する血液中のセラミド量に変化が生じた場合、角層セラミド量及び皮膚バリア機能や保湿機能に対する影響の指標となる。この場合の判定は、例えば、生理的変動の前後や、異なる群間において、血清中のセラミド濃度が5%以上、好ましくは10%以上変動した場合に、角層セラミド量及び皮膚バリア機能や保湿機能に対する影響がある、と判断される。一方、血清中のセラミド濃度が増加した場合は角層セラミド量の増加や皮膚バリア機能の改善や保湿機能の改善が生じ、血清中のセラミド濃度が減少した場合は、角層セラミド量の減少や皮膚バリア機能の低下や保湿機能の低下が生じた、と判断される。 Thus, according to the skin function determination method of the present invention, skin function can be determined from the blood ceramide value even when there is no measurement value of horny layer ceramide amount and skin barrier function or moisturizing function, The blood function of the individual can be determined using blood collected in medical examinations and other tests involving blood collection. For example, in a test that observes changes in the living body due to some physiological action change (stress load, sleep, lifestyle, etc.), if there is a change in the amount of ceramide in the blood for that physiological action, the amount of stratum corneum and skin barrier function It becomes an index of the influence on the moisture retention function. The determination in this case is, for example, when the ceramide concentration in serum fluctuates 5% or more, preferably 10% or more before and after physiological fluctuations, or between different groups, the amount of stratum corneum ceramide and the skin barrier function or moisture retention. It is judged that there is an influence on the function. On the other hand, an increase in serum ceramide concentration results in an increase in the amount of horny layer ceramide, an improvement in skin barrier function, and an improvement in moisturizing function, and a decrease in serum ceramide concentration results in a decrease in the amount of horny layer ceramide. It is judged that the skin barrier function and the moisturizing function are reduced.
本発明の皮膚機能改善剤の評価又は選択方法は、（Ａ）被験物質を被験者又は被験動物に投与する工程、（Ｂ）血液中のセラミド量を測定し、その変化を評価する工程、を含むものである。
この方法は、例えば、何らかの機能性成分（例えば老化や紫外線に対する抵抗性を高める皮膚機能改善を目指した機能性成分や、生活習慣病や肥満、疲労等の皮膚機能以外の改善を目指した機能性成分等）を含む機能性食品や医薬品（被験物質）の摂取試験等において採取した血液を用いて、これらの被験物質の摂取による血液中のセラミドの変化量を測定し、その被験物質が皮膚バリア機能や保湿機能に対して有効か否かを判断することにより行うことができる。また斯かる皮膚機能改善剤の評価又は選択は、前述した動物を用いて行うことでもよい。
被験物質が皮膚機能改善作用を有するか否かの評価は、例えば、摂取の前後や、対照群と摂取群との群間において、血清中のセラミド濃度が5%以上、好ましくは10%以上変動した場合に、皮膚機能が変動したと判断でき、血清中のセラミド濃度が増加した場合は皮膚機能改善剤として有用であると評価される。
The method for evaluating or selecting a skin function improving agent of the present invention includes (A) a step of administering a test substance to a subject or a test animal, and (B) a step of measuring the amount of ceramide in blood and evaluating the change thereof. It is a waste.
For example, this method can be used for any functional component (for example, a functional component aimed at improving skin function that increases resistance to aging or ultraviolet rays, or a function aimed at improving other than skin functions such as lifestyle-related diseases, obesity, and fatigue). Using blood collected in the intake test of functional foods and pharmaceuticals (test substances) containing components, etc., the amount of change in ceramide in the blood due to the intake of these test substances is measured. This can be done by determining whether it is effective for the function and the moisture retention function. In addition, the evaluation or selection of such a skin function improving agent may be performed using the animals described above.
Evaluation of whether the test substance has a skin function improving effect, for example, before and after ingestion, between the control group and the ingestion group, the serum ceramide concentration varies by 5% or more, preferably 10% or more In this case, it can be determined that the skin function has changed, and if the serum ceramide concentration increases, it is evaluated as being useful as a skin function improving agent.
１．実験方法
１）被験者
インフォームドコンセントが得られた健常皮膚を有する10名（男性９名、女性１名、21〜29才、平均24.2歳）。
1. Experimental method 1) Subjects 10 subjects with healthy skin with informed consent (9 men, 1 woman, 21-29 years old, average 24.2 years old).
２）採血および血清の採取
12時間の絶食後、採血を行い、20mLの血液を採取した。室温で1時間静置した後、遠心分離を行い（3000rpm、5min、RT）、血清を採取した。血清は−20℃で保存した。
2) Blood collection and serum collection
After 12 hours of fasting, blood was collected and 20 mL of blood was collected. After standing at room temperature for 1 hour, centrifugation was performed (3000 rpm, 5 min, RT), and serum was collected. Serum was stored at -20 ° C.
３）角層採取部位
下記に示した13部位
頭皮、額、頬、肩、体側、腰、背中、膝裏、脛、足裏、上腕内側、前腕内側、手の甲
3) The stratum corneum
４）被験部位の洗浄
額、頬、上腕内側、前腕内側については、洗浄剤で洗浄し、水洗した後、水分を拭い5分間馴化した。
上記の4部位以外の部位に関しては、70%エタノールを含ませた脱脂綿で軽く拭き取り、5分間馴化した。
4) Cleaning the test area The forehead, cheek, upper arm inner side and forearm inner side were cleaned with a cleaning agent, washed with water, then wiped off with water and acclimatized for 5 minutes.
About parts other than said 4 site | parts, lightly wiped with the absorbent cotton soaked with 70% ethanol, and acclimatized for 5 minutes.
５）角層採取
頭皮を除く12ヶ所の被験部位からは、いずれも2.5cm×6.4cmのPPSテープ（ニチバン）で同部位から連続10枚ストリッピングすることにより角層を採取した。頭皮に関しては採取部位1cm四方の毛髪を短くカットし、1.0cm×1.0cmのPPSテープで連続10枚ストリッピングした。得られた角層は直ちに冷凍保存し、解析まで−20℃で保存した。
5) Collection of stratum corneum The stratum corneum was collected from 12 test sites other than the scalp by continuously stripping 10 sheets from the same site with 2.5 cm x 6.4 cm PPS tape (Nichiban). For the scalp, the hair of 1 cm square was cut short and the strips were stripped continuously with 1.0cm x 1.0cm PPS tape. The resulting stratum corneum was immediately stored frozen and stored at -20 ° C until analysis.
６）血清からの脂質抽出
採取した血清量を計測し、合計10.5mlとなるようにPBSで希釈した。各血清サンプルを3.5mlずつ3本に分注する。それぞれのサンプルにクロロホルム4.375ml、メタノール8.75mlを加え、20分間振とうした。その後、遠心分離（1500rpm、5分間、RT）を行い、上清を回収した。上清にクロロホルム 4.4ml、PBS 4.4mlを加え20分間振とうし、遠心分離後（1500rpm、5分間、RT）、下相を回収した。3本に分注した下相を1本にまとめ、乾燥窒素気流で溶媒を留去した。
6) Lipid extraction from serum The amount of collected serum was measured and diluted with PBS to a total of 10.5 ml. Dispense 3.5 ml of each serum sample. To each sample, 4.375 ml of chloroform and 8.75 ml of methanol were added and shaken for 20 minutes. Thereafter, centrifugation (1500 rpm, 5 minutes, RT) was performed, and the supernatant was collected. To the supernatant, 4.4 ml of chloroform and 4.4 ml of PBS were added and shaken for 20 minutes. After centrifugation (1500 rpm, 5 minutes, RT), the lower phase was recovered. The lower phase dispensed into three was combined into one, and the solvent was distilled off with a dry nitrogen stream.
７）セラミド定量
（i）血清抽出脂質の前処理方法
ヒト血清からの脂質抽出物をクロロホルム/メタノール＝2/1(v/v)に溶解し、10nmolの内部標準物質を添加し、窒素気流下で乾固した。その後、5mLのクロロホルム/メタノール＝99.5/0.5(v/v)に溶解し、予め10mLのクロロホルム/メタノール＝99.5/0.5でコンディショニングした固相抽出カートリッジ(Waters Sep-Pak Vac RC 500mg Silica Cartriges)に添加した。初めに10mLのクロロホルム/メタノール＝99.5/0.5で溶出して溶出液を廃棄し、その後10mLのクロロホルム/メタノール＝95/5、90/10、40/60、10/90(v/v)で順次溶出して溶出液を同一のスクリュー管に回収した。この溶出液を窒素気流下で乾固し、10mLのクロロホルム/メタノール＝2/1(v/v)に溶解してねじ口遠沈管に移した。この溶液を再度窒素気流下で乾固し、5mLのヘキサン/2-プロパノール＝95/5に溶解し、順相LC-MS測定に供した。
LC-MS測定は既報と同様に実施した(Jounal of Lipid Research 2008. 49: 1466-1476）。
7) Ceramide quantification (i) Pretreatment method of serum-extracted lipid Dissolve lipid extract from human serum in chloroform / methanol = 2/1 (v / v), add 10 nmol of internal standard substance, and under nitrogen stream To dry. Then, dissolve in 5 mL of chloroform / methanol = 99.5 / 0.5 (v / v) and add to a solid-phase extraction cartridge (Waters Sep-
LC-MS measurement was performed in the same manner as previously reported (Jounal of Lipid Research 2008. 49: 1466-1476).
（ii）テープの前処理方法
得られたテープストリッピング角層をすべて半分に切断し、2.25cm2×10枚をLC-MSによるセラミド定量に用いた。角層からの脂質の抽出、内部標準の添加、固相抽出、LC-MS測定は既報と同様に実施した(Jounal of Lipid Research 2008. 49: 1466-1476）。
得られたセラミド定量値は、標準物質により感度補正を行い、総炭素数の異なる成分ごとに絶対量を得た。続いて総炭素数の異なる成分を同じセラミドクラス毎に総和し、クラス別の定量値を得た。さらに全クラスの定量値を総和して総セラミド量を得た。また採取した角層の量でセラミド量を補正するため、残り半分（2.25cm2×10枚）の角層から可溶性タンパク質を抽出し、タンパク質定量にて得られた値で補正した。
(Ii) Tape pretreatment method The obtained tape stripping stratum corneum was all cut in half, and 2.25 cm 2 × 10 sheets were used for ceramide determination by LC-MS. Lipid extraction from the stratum corneum, addition of internal standard, solid phase extraction, and LC-MS measurement were performed in the same manner as previously reported (Jounal of Lipid Research 2008. 49: 1466-1476).
The obtained ceramide quantitative value was subjected to sensitivity correction with a standard substance, and an absolute amount was obtained for each component having a different total carbon number. Subsequently, components with different total carbon numbers were summed for each ceramide class, and quantitative values for each class were obtained. Furthermore, the total amount of ceramide was obtained by summing up the quantitative values of all classes. In addition, in order to correct the amount of ceramide with the amount of the stratum corneum collected, soluble protein was extracted from the remaining half (2.25 cm 2 × 10) stratum corneum and corrected with the value obtained by protein quantification.
２．結果
（i）血清中のセラミド解析
被験者10名の血清中に存在する総セラミド量を算出したところ、個人間で総セラミド量は異なることが確認された（2930±0.42ng/ml）。これまでに血漿中には平均5500ng/mlのセラミドが存在していることが報告されている(W. Drobnik, et al., Journal of Lipid Research 44, 754-761, (2003)）。
2. Results (i) Ceramide analysis in serum When the total amount of ceramide present in the serum of 10 subjects was calculated, it was confirmed that the total amount of ceramide was different among individuals (2930 ± 0.42 ng / ml). So far, it has been reported that an average of 5500 ng / ml of ceramide is present in plasma (W. Drobnik, et al., Journal of Lipid Research 44, 754-761, (2003)).
（ii）角層セラミドと血清セラミドの相関解析
被験者10名の身体各13部位（頭皮、額、頬、肩、体側、背中、腰、膝裏、脛、足裏、上腕内側、前腕内側、手の甲）における角層セラミド量と血清のセラミド量について、それぞれ相関を解析した。
角層セラミド量と血清セラミド量との相関性を解析した結果を図１に示した。
６部位〔額（R=0.71）、頬（R=0.54）、前腕内側（R=0.49）、膝裏（R=0.76）、肩（R=0.30）、手の甲（R=0.73）において角層セラミド量と血清セラミド量の間に高い相関が認められた。
(Ii) Correlation analysis between stratum corneum and
The result of analyzing the correlation between the amount of stratum corneum and the amount of serum ceramide is shown in FIG.
6 sites of stratum corneum in the forehead (R = 0.71), cheeks (R = 0.54), inner forearm (R = 0.49), back of the knee (R = 0.76), shoulder (R = 0.30), back of hand (R = 0.73) A high correlation was found between the amount and serum ceramide level.
実施例2
１．実験方法
１）被験者
インフォームドコンセントが得られた健常皮膚を有する男性８名（22〜27才、平均24.2歳）。
Example 2
1. Experimental method 1) Subjects Eight males (22 to 27 years old, average 24.2 years old) with healthy skin from whom informed consent was obtained.
２）被験品
表1に示す処方の錠剤を製造した。
ｉ）ルナックBA（登録商標）含有錠：1錠（350mg）あたりルナックBA（花王株式会社；ベヘン酸（C22:0）／90.1％、リグノセリン酸（C24:0）／1.9％、アラキジン酸（C20:0）／6.9％、ステアリン酸（C18:0）／0.3％）を125mg含有。
ii）プラセボ錠：上記錠剤からルナックBAを除いて調製したもの
i) Lunac BA (registered trademark) -containing tablets: Lunac BA (Kao Corporation; behenic acid (C22: 0) /90.1%, lignoceric acid (C24: 0) /1.9%, arachidic acid (C20) per tablet (350 mg) 0) /6.9% and stearic acid (C18: 0) /0.3%).
ii) Placebo tablet: prepared from the above tablet without lunac BA
３）被験品の摂取期間と方法
ｉ）摂取期間：被験品を4錠/日の割合で2回に分けて（朝2錠、夕方2錠）、連続8週間摂取した。
ii）摂取方法：摂取の際は2錠あたりコップ1杯（約100ml）の水とともに摂取した。
iii）被験品摂取：1回目の採血が終了した直後、試験参加者に被験品を2錠摂取してもらい、被験品の摂取を開始した。
3) Ingestion period and method of test product i) Ingestion period: The test product was divided into 4 tablets / day twice (2 tablets in the morning and 2 tablets in the evening) and continuously ingested for 8 weeks.
ii) Ingestion method: Ingestion was taken with 1 cup (about 100 ml) of water per 2 tablets.
iii) Ingestion of test product: Immediately after the end of the first blood collection, the test participants ingested 2 tablets of the test product, and ingestion of the test product was started.
４）採血および血清の採取
摂取前、摂取４週間後、摂取８週間後において、それぞれ12時間の絶食後、採血を行い、20mLずつの血液を採取した。室温で1時間静置した後、遠心分離を行い（3000rpm、5min、RT）、血清を採取した。血清は−20℃で保存した。
4) Blood collection and serum collection Before ingestion, 4 weeks after ingestion, and 8 weeks after ingestion, blood was collected after 12 hours of fasting, and 20 mL of blood was collected. After standing at room temperature for 1 hour, centrifugation was performed (3000 rpm, 5 min, RT), and serum was collected. Serum was stored at -20 ° C.
５）皮膚測定
採血を行った日に、被験部位（頬）を洗浄剤で洗浄した後、20℃、40%RHの環境下で10分間の馴化を行った（摂取前、摂取４週間後、摂取８週間後）。
5) Skin measurement On the day of blood collection, the test site (cheek) was washed with a detergent and then conditioned for 10 minutes in an environment of 20 ° C and 40% RH (before ingestion, 4 weeks after ingestion, 8 weeks after ingestion).
６）経皮水分蒸散量測定
頬部において、3点ずつTEWL（TEWAMETER TM300, CK electronic GmbH社製）の測定を行った（20℃、40%RHの環境下）。
6) Measurement of transdermal water transpiration amount TEWL (TEWAMETER TM300, manufactured by CK electronic GmbH) was measured at three points on the cheek (under an environment of 20 ° C. and 40% RH).
７）角層水分量測定
頬部において、10点ずつコンダクタンス（SKICON-200EX, IBS社製）の測定を行った（20℃、40%RHの環境下）。
7) Measurement of moisture content in stratum corneum Conductance (SKICON-200EX, manufactured by IBS) was measured at 10 points on the cheek (20 ° C, 40% RH).
８）角層採取
採血および上記皮膚測定を行った日に、皮膚測定に続いて頬部からテープストリッピングにより角層を採取した（摂取前、摂取４週間後、摂取８週間後）。PPSテープを用いて（6.4cm×2.5cm）、深さ方向に同一部位から連続5枚採取した。得られた角層は直ちに冷凍保存し、解析まで−20℃で保存した。
8) Collection of stratum corneum On the day of blood collection and skin measurement, the stratum corneum was collected from the cheek by tape stripping following the skin measurement (before ingestion, 4 weeks after ingestion, 8 weeks after ingestion). Using a PPS tape (6.4 cm × 2.5 cm), five consecutive samples were collected from the same site in the depth direction. The resulting stratum corneum was immediately stored frozen and stored at -20 ° C until analysis.
９）セラミド解析
ｉ）血清からの脂質抽出
採取した血清1.0mlにメタノール2.5ml、クロロホルム1.25mlを加え、20分間振とうした。その後、遠心分離（1500rpm、5分間、RT）を行い、上清を回収した。上清にクロロホルム 1.25ml、PBS 1.25mlを加え20分間振とうし、遠心分離後（1500rpm、5分間、RT）、下相を回収した。下相を乾燥窒素気流で溶媒を留去した。
9) Ceramide analysis i) Lipid extraction from serum To 2.5 ml of collected serum, 2.5 ml of methanol and 1.25 ml of chloroform were added and shaken for 20 minutes. Thereafter, centrifugation (1500 rpm, 5 minutes, RT) was performed, and the supernatant was collected. To the supernatant, 1.25 ml of chloroform and 1.25 ml of PBS were added and shaken for 20 minutes. After centrifugation (1500 rpm, 5 minutes, RT), the lower phase was recovered. The solvent was distilled off from the lower phase with a dry nitrogen stream.
ii）血清抽出脂質の前処理方法
ヒト血清からの脂質抽出物をクロロホルム/メタノール＝2/1(v/v)に溶解し、10nmolの内部標準物質を添加し、窒素気流下で乾固した。その後、5mLのクロロホルム/メタノール＝99.5/0.5(v/v)に溶解し、予め10mLのクロロホルム/メタノール＝99.5/0.5でコンディショニングした固相抽出カートリッジ(Waters Sep-Pak Vac RC 500mg Silica Cartriges)に添加した。初めに10mLのクロロホルム/メタノール＝99.5/0.5で溶出して溶出液を廃棄し、その後10mLのクロロホルム/メタノール＝95/5、90/10、40/60、10/90(v/v)で順次溶出して溶出液を同一のスクリュー管に回収した。この溶出液を窒素気流下で乾固し、10mLのクロロホルム/メタノール＝2/1(v/v)に溶解してねじ口遠沈管に移した。この溶液を再度窒素気流下で乾固し、5mLのヘキサン/2-プロパノール＝95/5に溶解し、順相LC-MS測定に供した。
LC-MS測定は既報と同様に実施した(Jounal of Lipid Research 2008. 49: 1466-1476）。
ii) Pretreatment method of serum-extracted lipid A lipid extract from human serum was dissolved in chloroform / methanol = 2/1 (v / v), 10 nmol of internal standard substance was added, and the mixture was dried under a nitrogen stream. Then, dissolve in 5 mL of chloroform / methanol = 99.5 / 0.5 (v / v) and add to a solid-phase extraction cartridge (Waters Sep-
LC-MS measurement was performed in the same manner as previously reported (Jounal of Lipid Research 2008. 49: 1466-1476).
iii）テープストリッピング角層のセラミド定量
得られたテープストリッピング角層をすべて半分に切断し、2.25cm2×10枚をLC-MSによるセラミド定量に用いた。角層からの脂質の抽出、内部標準の添加、固相抽出、LC-MS測定は既報と同様に実施した(Jounal of Lipid Research 2008. 49: 1466-1476）。得られたセラミド定量値は、標準物質により感度補正を行い、総炭素数の異なる成分ごとに絶対量を得た。続いて総炭素数の異なる成分を同じセラミドクラス毎に総和し、クラス別の定量値を得た。さらに全クラスの定量値を総和して総セラミド量を得た。また採取した角層の量でセラミド量を補正するため、残り半分（2.25cm2×10枚）の角層から可溶性タンパク質を抽出し、タンパク質定量にて得られた値で補正した。
iii) Determination of ceramide in tape stripping stratum corneum The tape stripping stratum corneum obtained was all cut in half, and 2.25 cm 2 × 10 sheets were used for ceramide determination by LC-MS. Lipid extraction from the stratum corneum, addition of internal standard, solid phase extraction, and LC-MS measurement were performed in the same manner as previously reported (Jounal of Lipid Research 2008. 49: 1466-1476). The obtained ceramide quantitative value was subjected to sensitivity correction with a standard substance, and an absolute amount was obtained for each component having a different total carbon number. Subsequently, components with different total carbon numbers were summed for each ceramide class, and quantitative values for each class were obtained. Furthermore, the total amount of ceramide was obtained by summing up the quantitative values of all classes. In addition, in order to correct the amount of ceramide with the amount of the stratum corneum collected, soluble protein was extracted from the remaining half (2.25 cm 2 × 10) stratum corneum and corrected with the value obtained by protein quantification.
２．結果
脂肪酸摂取前の角層セラミド量と血清セラミド量との相関性を解析した結果を図２に示した。
脂肪酸摂取群毎の摂取前に対する摂取４週後、摂取８週後の変動量の相関を解析した結果を図３に示した。
脂肪酸摂取前においても血清中のセラミド濃度は角層セラミド濃度及び皮膚コンダクタンスと正の相関が、TEWL値と負の相関が認められた。一方、脂肪酸摂取による血清中のセラミド濃度の変動量は、角層セラミド濃度の変動量と正の相関が、TEWL値の変動量との負の相関が認められた。
2. Results The results of analysis of the correlation between the amount of stratum corneum before fatty acid intake and the amount of serum ceramide are shown in FIG.
FIG. 3 shows the result of analyzing the correlation of the fluctuation amount after 4 weeks of intake and after 8 weeks of intake with respect to the intake before each fatty acid intake group.
Even before fatty acid intake, serum ceramide concentration was positively correlated with stratum corneum ceramide concentration and skin conductance, and negatively correlated with TEWL value. On the other hand, the fluctuation amount of serum ceramide concentration due to fatty acid intake was positively correlated with the fluctuation amount of stratum corneum ceramide concentration, and negatively correlated with the fluctuation amount of TEWL value.
Claims (4)
（Ａ）被験物質を被験者又は被験動物に投与する工程
（Ｂ）血液中のセラミド量を測定し、その変化を評価する工程 A method for evaluating or selecting a skin function improving agent, comprising the following steps (A) and (B):
(A) A step of administering a test substance to a subject or a test animal (B) A step of measuring the amount of ceramide in blood and evaluating the change
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| JP2013250174A (en) * | 2012-06-01 | 2013-12-12 | Kao Corp | Evaluation method for moisture permeation barrier function |
| JP2014013201A (en) * | 2012-07-04 | 2014-01-23 | Shiseido Co Ltd | Apparatus, method, and program for visualizing ingredient distribution |
| JP2015210199A (en) * | 2014-04-28 | 2015-11-24 | 株式会社ミルボン | Evaluation method for degree of carbonylation |
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