JP2008039761A - Evaluating method of square layer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluating method of a square layer capable of improving measurement accuracy and analysis accuracy of order degree and fluidity of fat between cells of the square layer. <P>SOLUTION: The evaluating method of the square layer comprises at least a process of peeling the square layer 2 from a skin 0 using a support body 1 adhered with an adhesive 3, a process of leaving the support body 1 adhered with the square layer 2 for a predetermined time under a predetermined condition, a process of treating the square layer 2 adhered on the support body 1 with a spin probe agent, and a process of measuring the electron spin resonance spectrum using the support body 1 adhered with the square layer 2 treated with the spin probe agent. In treating with the spin probe agent, solution obtained by dissolving the spin probe agent in a water-soluble solvent and then diluting it with water is used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for evaluating a stratum corneum.

  The stratum corneum, which is about 10 to 20 μm in thickness and is the outermost tissue of the skin, is the main body of the skin barrier function that holds essential water in the living body and prevents the entry of harmful substances from outside the living body. Among them, intercellular lipids formed from ceramide, cholesterol and fatty acids are considered to play an important role in the barrier mechanism. As a method for analyzing the structure of the intercellular lipid, a calorimetric (DSC) method, an X-ray diffraction method, an infrared absorption method and the like are known. When these methods are used, the state of the intercellular lipid can be roughly grasped, but cannot be analyzed in association with the barrier property.

  Thus, attention has been paid to electron spin resonance (ESR) that can evaluate the structure of intercellular lipids in the stratum corneum by treating the stratum corneum with a spin probe agent (spin labeling agent). The fluidity of intercellular lipids in the stratum corneum measured using ESR is expressed as the degree of order S (see Non-Patent Document 1). However, the degree of order S obtained from the ex vivo ESR spectrum using a geometric technique has a small amount of change even when the stratum corneum is treated with a surfactant, and the data varies greatly.

In contrast, the degree of order S ₀ obtained by analyzing the ESR spectrum of the stratum corneum using a computer simulation method is capable of detecting subtle changes in the fluidity of intercellular lipids. Has been reported (see Non-Patent Document 2). However, further improvement in measurement accuracy of the degree of order S and S ₀ of the intercellular lipids of the stratum corneum is required.

On the other hand, a cultured skin (epidermal) model is known as an evaluation kit that substitutes for test animals such as rabbits and guinea pigs used in skin irritation tests, transdermal absorption tests, basic skin science research, and the like (Non-patent Document 3). reference). As a method for evaluating the structure of the skin (epidermal) tissue of such a cultured skin (epidermal) model, optical microscope observation, transmission electron microscope observation, etc. using H & E staining and immunostaining are known (Non-Patent Documents). 4). However, the state of intercellular lipids in the stratum corneum of skin (epidermis) tissue cannot be analyzed in association with barrier properties.
Int. J. et al. Pharm. 197: 193-202, 2000. SPECTROCHIMICA ACTA PART A 63 816-820, 2006 FRAGRANCE JOURNAL, 56-60, 2006-1 Arch Dermatol Res 285: 466-474, 1993

  An object of the present invention is to provide a method for evaluating a stratum corneum capable of improving the measurement accuracy and analysis accuracy of intercellular lipids in the stratum corneum and fluidity in view of the problems of the above-described conventional technology. And

  The invention according to claim 1 is a method for evaluating a stratum corneum, the step of attaching the stratum corneum to a support, the step of treating the stratum corneum attached to the support with a spin probe agent (spin label agent), At least a step of measuring an electron spin resonance spectrum using a support to which the stratum corneum treated with the spin probe agent is attached, and when the spin probe agent is treated with the spin probe agent, the spin probe agent is dissolved in a water-soluble solvent. A solution obtained by diluting with water after being dissolved is used.

  According to a second aspect of the present invention, in the method for evaluating a stratum corneum according to the first aspect, in the step of attaching the stratum corneum to the support, the stratum corneum is formed using the support having an adhesive attached thereto. It is characterized by having a step of peeling and a step of leaving the support to which the peeled stratum corneum is adhered for a predetermined time under predetermined conditions.

  The invention described in claim 3 is the stratum corneum evaluation method described in claim 2, wherein the adhesive contains cyanoacrylate.

  The invention according to claim 4 is the stratum corneum evaluation method according to any one of claims 1 to 3, wherein the spin probe agent is a doxyl derivative of a long-chain fatty acid having 8 to 25 carbon atoms. It is characterized by being.

  The invention according to claim 5 is the stratum corneum evaluation method according to any one of claims 1 to 4, wherein the support is selected from the group consisting of quartz glass, glass, acrylic resin, and hard resin. It is made of a nonmagnetic material.

  The invention according to claim 6 is the stratum corneum evaluation method according to any one of claims 1 to 5, wherein the water-soluble solvent has 1 to 4 carbon atoms and 1 or more hydroxyl groups. It contains 3 or less alcohols.

  The invention according to claim 7 is the stratum corneum evaluation method according to any one of claims 1 to 6, wherein the degree of order of intercellular lipids in the stratum corneum is determined from the measured electron spin resonance spectrum. It further has a process.

  The invention according to claim 8 is the stratum corneum evaluation method according to any one of claims 1 to 7, further comprising a step of analyzing the measured electron spin resonance spectrum using a computer simulation method. It is characterized by having.

  The invention according to claim 9 is the stratum corneum evaluation method according to any one of claims 1 to 8, further comprising a step of detecting sebum in the stratum corneum from the measured electron spin resonance spectrum. It is characterized by having.

  The invention described in claim 10 is the stratum corneum evaluation method according to any one of claims 1 to 8, wherein the stratum corneum is formed by culture.

  The invention described in claim 11 is the stratum corneum evaluation method described in claim 10, wherein a drug is added during the culture.

  ADVANTAGE OF THE INVENTION According to this invention, the evaluation method of the stratum corneum which can improve the measurement precision and analysis precision of the degree of intercellular lipid of a stratum corneum and fluidity | liquidity can be provided.

  Next, the best mode for carrying out the present invention will be described with reference to the drawings.

  The method for evaluating a stratum corneum according to the present invention includes a step of attaching a stratum corneum to a support, a step of treating the stratum corneum attached to the support with a spin probe agent (spin label agent), and a corner treated with the spin probe agent. Using at least a step of measuring an electron spin resonance spectrum using a support to which a layer is attached, and using a solution obtained by dissolving a spin probe agent in a water-soluble solvent and then diluting with water. Treat the stratum corneum attached to.

  The spin probe agent preferably has a structure similar to lipid in order to introduce into the intercellular lipid in the stratum corneum or lipid coexisting with the stratum corneum and determine the state thereof. Examples thereof include spin probes having a structure similar to lipids as components used as lipids, cosmetics, or external preparations. Among these, a long-chain fatty acid doxyl derivative having 8 to 25 carbon atoms is preferable. More preferably, a long-chain fatty acid having 10 to 18 carbon atoms, particularly 18 carbon atoms is suitable. Specifically, 1-doxyl stearic acid, 2-doxyl stearic acid, 3-doxyl stearic acid, 4-doxyl stearic acid, 5-doxyl stearic acid, 6-doxyl stearic acid, 7-doxyl stearic acid, 8-doxyl Stearic acid, 9-doxyl stearic acid, 10-doxyl stearic acid, 11-doxyl stearic acid, 12-doxyl stearic acid, 13-doxyl stearic acid, 14-doxyl stearic acid, 15-doxyl stearic acid, 16-doxyl stearic acid , 17-doxyl stearic acid, 18-doxyl stearic acid and the like, and can be appropriately selected depending on the purpose.

  The support is preferably made of a nonmagnetic material selected from the group consisting of quartz glass, glass, acrylic resin and hard resin. In particular, when measuring ESR, it is particularly preferable to use a high-purity nonmagnetic material such as quartz glass so that a background signal from impurities is not generated. The shape of the support is not particularly limited as long as it can adhere the stratum corneum, but is preferably flat. Thereby, when measuring the ESR of the intercellular lipid in the stratum corneum, the directions of the stratum corneum are easily aligned, and the measurement accuracy can be improved. The size of the support is not particularly limited as long as it can be inserted into the cavity resonator of the ESR measurement device.

  In the present invention, the step of attaching the stratum corneum to the support includes the step of peeling the stratum corneum using the support to which the adhesive is attached and the support to which the peeled stratum corneum is attached under predetermined conditions. It can comprise the process of leaving for a predetermined time.

  The adhesive preferably does not generate a background signal when measuring ESR. Moreover, since the measurement precision of ESR may fall when there is little quantity of the stratum corneum adhering on a support body, it is preferable that an adhesive agent has big adhesive force. Specific examples of such adhesives include cyanoacrylate adhesives, benzol gum adhesives, epoxy adhesives, and emulsion adhesives. Among these, a cyanoacrylate-based adhesive is preferable because the measurement accuracy of ESR can be improved.

Cyanoacrylate has the general formula CH ₂ ═C (CN) COOR
In general, it is used as an instantaneous adhesive, but can be used to peel off the stratum corneum from the skin because it is polymerized using water as a catalyst. Specific examples of the functional group R in the above general formula include a methyl group, an ethyl group, a 2-methoxyethyl group, an n-butyl group, and an n-octyl group.

  FIG. 1 shows a method for peeling the stratum corneum from the skin. In the method shown in FIG. 1, after the adhesive 3 is attached to the support 1, the surface of the support 1 to which the adhesive 3 is attached is brought into contact with the human skin 0 (see FIG. 1 (a)). The stratum corneum 2 can be fixed on the support 1 nondestructively (non-invasively) (see FIG. 1B). Moreover, when measuring ESR, the installation direction of the stratum corneum with respect to the magnetic field can be set to a predetermined direction.

  In the present invention, by leaving the support to which the stratum corneum is attached, the adhesive is cured, the generation of the background signal derived from the adhesive can be suppressed, and the measurement accuracy of ESR can be improved. The leaving conditions and the leaving time are not particularly limited, but are preferably 10 to 14 days at room temperature and humidity. Normal temperature and normal humidity are conditions in a general laboratory and are usually 10 to 30 ° C. and 10 to 80% RH, preferably 15 to 27 ° C. and 30 to 60% RH. If left at a high temperature exceeding 40 ° C. for a long period of time, lipid oxidation, protein denaturation, etc. may occur, which may prevent correct evaluation.

  In FIG. 2, the time-dependent change of the ESR spectrum of the cyanoacrylate which added 5-doxyl stearic acid is shown. 2 (a) to 2 (g) show that they were left at room temperature and normal humidity for 30 minutes, 1 hour, 3 hours, 5 hours, 3 days, 7 days, and 10 days, respectively. FIG. 2 shows that when the standing time is short, curing is insufficient, and thus a peak derived from cyanoacrylate is detected. However, when the standing time is left for 10 days, this peak almost disappears.

  In the present invention, when the stratum corneum attached on the support is treated with the spin probe agent, a solution obtained by dissolving the spin probe agent in a water-soluble solvent and then diluting with water is added to the stratum corneum. Thus, the ESR spectrum shown in FIG. 3A is obtained, and the ESR measurement accuracy can be improved as compared with the case where the spin probe agent shown in FIG. 3B is directly dissolved in water. . The weight ratio of the spin probe agent to the water-soluble solvent is preferably 0.0001-1. If this weight ratio exceeds 1, the change in the state of the stratum corneum adhered on the support may become large, and if it is less than 0.0001, the effect of improving the measurement accuracy of ESR may not be obtained. The concentration of the spin probe agent in the spin probe agent solution is preferably 0.00001 to 0.1% by weight. If this concentration exceeds 0.1% by weight, the amount of spin probe added will increase, and the change in the state of the stratum corneum adhered on the support may increase, and is less than 0.00001% by weight. In addition, since the amount of spin probe agent added decreases, it may be difficult to uniformly treat the stratum corneum with the spin probe agent.

  Examples of the water-soluble solvent include 1 to 3 alcohols having 1 to 4 carbon atoms, ketones such as acetone and methyl ethyl ketone, amides such as dimethylformamide, and the like. Of these, 1 to 3 alcohols having 1 to 4 carbon atoms are preferable, and ethanol is particularly preferable.

  When measuring ESR using a support to which a stratum corneum treated with a spin probe agent is attached, it is preferable to use a sample holder shown below, although it is not particularly limited.

  FIG. 4 shows an example of a sample holder used in the present invention. 4A and 4B show a front view and a side view, respectively. In the sample holder shown in FIG. 4, the stratum corneum 2 is bonded onto the support 1 using the adhesive 3, and the support 1 is detachably fixed to the support fixing member 5 by the presser fitting 4. Yes.

  The shape, material, etc. of the presser fitting 4 are not particularly limited as long as the support 1 can detachably fix the support 1 to the support fixing member 5. In FIG. 4, the presser fitting 4 is fixed to the support fixing member 5 using screws 6, but other means may be used.

  The shape of the support fixing member 5 is not particularly limited as long as it can be fixed in the cavity resonator of the ESR measurement device. The material of the support fixing member 5 is not particularly limited, and examples thereof include resins such as Teflon (registered trademark).

  In FIG. 4, the presser fitting 4 is used as means for detachably fixing the support 1 to the support fixing member 5, but other means may be used, for example, as shown in FIG. 5. The holding screw 7 can be mentioned. The material of the holding screw 7 is not particularly limited, and examples thereof include Teflon (registered trademark), acrylic resin, hard plastic, metal (nonmagnetic material), and the like. Further, a member such as rubber may be provided and fixed between the support 1 and the holding screw 7.

  In addition, as shown in FIG. 5, you may provide the mark 8 in a sample holder so that the position of the support body 1 can be known within the cavity resonator of an ESR measuring device.

  In the present invention, the order S of intercellular lipids can be determined from the ESR spectrum of intercellular lipids in the stratum corneum. The order S of intercellular lipids can be obtained from the following equation.

S = [A (parallel) -A (perpendicular)] / [A zz -1/2 (A xx + A yy)] · (a 0 '/ a 0)
a ₀ ′ = (A _xx + A _yy + A _zz ) / 3
(A _xx , A _yy , A _zz ) = (6.1, 6.1, 32.4) Gauss
a ₀ = [A (parallel) + 2A (perpendicular)] / 3
In addition, A (parallel) and A (perpendicular) are calculated | required from an ESR spectrum, as shown in FIG. At this time, the degree of order S of the intercellular lipid is 0 to 1. However, as the degree of order S of the intercellular lipid is larger, the regularity of the structure of the intercellular lipid is higher as shown in FIG. FIG. 8 shows ESR spectra of intercellular lipids having different degrees of order S. 8A, 8B, and 8C show that S is 0.6, 0.57, and 0.19, respectively.

In the present invention, it is preferable to analyze the ESR spectrum of the intercellular lipid using a computer simulation method. Thereby, the precision of the degree of order S of intercellular lipid can further be improved. Examples of the computer simulation method include a method using an NLLS (Non-linear least-squares) fitting program. The degree of order S ₀ of the intercellular lipid obtained in this way is expressed by the equation

で表され、スピンプローブ剤として、５−ドキシルステアリン酸を用いた場合、図９に示すように、ニトロキシドの回転拡散の角度の広がりが求められる。なお、γは、回転拡散対称軸とｚ軸のなす角であり、Ωは、状態数、Ｕは、内部エネルギー、ｋは、ボルツマン定数、Ｔは、絶対温度を表す。

In the case where 5-doxyl stearic acid is used as the spin probe agent, as shown in FIG. 9, the angular spread of the rotational diffusion of nitroxide is required. Note that γ is an angle formed between the rotational diffusion symmetry axis and the z axis, Ω is the number of states, U is the internal energy, k is the Boltzmann constant, and T is the absolute temperature.

  In the present invention, as the stratum corneum, those formed by culturing for a predetermined time under predetermined conditions can be used. When the stratum corneum is formed by culturing, it is not particularly limited, and an epidermal tissue having only an epidermal layer and a skin tissue having an artificial dermis layer such as ceramide and collagen can be used. When culturing, FBS (growth factor) may be added as necessary. Moreover, in order to improve the degree of order of the intercellular lipid, it is preferable to add drugs such as SK-Influx and SymRepair. At this time, the drug may be added to the medium, but is preferably added directly to the stratum corneum. Examples of the cultured epidermis model cultured using epidermal tissue include LABCYTE EPI-MODEL (manufactured by J-TEC). Examples of the cultured skin model cultured using skin tissue include TESTSKIN (manufactured by Toyobo), Neoderm. -ED (made by TEGO Science) etc. are mentioned. For example, the epidermal tissue used in LABCYTE EPI-MODEL is cultured and layered using normal human skin cells, and has a structure that is morphologically similar to human skin, A spiny layer, a granular layer, and a stratum corneum are sequentially laminated.

FIG. 15 shows a method of attaching the cultured skin (epidermal) tissue to the support. In the method shown in FIG. 15, the cultured skin (epidermal) tissue 11 is taken out with tweezers 12 (see FIG. 15A) and placed on the support 1 to place the skin (epidermal) tissue 11 on the support 1. It can be fixed (see FIG. 15B). Thus, since the skin (epidermal) tissue 11 can be fixed without using an adhesive, it is not necessary to leave it to cure the adhesive. When measuring the ESR spectrum, although not particularly limited, a sample holder similar to that shown in FIG. 4 or FIG. 5 is used except that the skin (skin) tissue 11 is used instead of the stratum corneum 2 and the adhesive 3. It is preferable to use it. Further, the degree of order S and S ₀ can be obtained in the same manner as described above. At this time, the degree of order S of the skin (skin) tissue 11 and the stratum corneum peeled from the skin (skin) tissue 11 are the same within the experimental error range.

  The stratum corneum evaluation method of the present invention can be applied to the stratum corneum before and after the cosmetic is applied, thereby evaluating the effect of the cosmetic, changes in the stratum corneum due to irradiation with light such as ultraviolet rays, and temperature changes.

  Using 5 sheets of Aron Alpha A Sankyo (manufactured by Sankyo Pharmaceutical Co., Ltd.) as a cyanoacrylate adhesive 3 on a flat plate (support 1) made of quartz glass of 70 mm × 8 mm × 0.5 mm, As shown in FIG. 1, five layers of the stratum corneum 2 were collected from the skin of the inner side of the forearm of male A. Next, the support 1 to which each stratum corneum 2 was adhered was allowed to stand at room temperature and humidity for 10 days, and then immersed in 1 mg / dl of 5-doxyl stearic acid solution at 37 ° C. for 1 hour. Further, excess 5-doxyl stearic acid was removed by washing with distilled water, and a sample holder as shown in FIG. 4 was prepared using the presser fitting 4, the support fixing member 5, and the screw 6. At this time, a 1 mg / dl 5-doxyl stearic acid solution was prepared by dissolving 1 mg of 5-doxyl stearic acid in 100 μl of ethanol and then diluting with distilled water.

  The obtained sample holder was inserted into a cavity resonator of an X-band ESR measurement device JES-REIX type (manufactured by JEOL Ltd.), a central magnetic field 336 mT, a sweep width 15 mT, a sweep time 8 minutes, and a magnetic field modulation width 0. ESR measurement was performed at room temperature under the conditions of 2 mT, a time constant of 1 second, a magnification of 1.25 × 1000, a microwave frequency of 9 GHz, and a microwave output of 10 mW. The obtained ESR spectrum is shown by a dotted line in FIG. 10A to 10E show the surface layers (first layer) to fifth layer of the peeled stratum corneum, respectively. Furthermore, the degree of order S of the intercellular lipid was determined. The results are shown in Table 1.

次に、ＮＬＬＳフィッティングプログラムを用いて、コンピューターシミュレーション法により、ＥＳＲスペクトルを解析した。その結果を図１０に実線で示す。さらに、細胞間脂質の秩序度Ｓ_０を求めた。この結果を表１に示す。

Next, the ESR spectrum was analyzed by the computer simulation method using the NLLS fitting program. The result is shown by a solid line in FIG. Furthermore, the degree of order S ₀ of the intercellular lipid was determined. The results are shown in Table 1.

In addition, before and after the surface layer 2 of each stratum corneum was collected, the transdermal moisture transpiration (TEWL) was measured with a moisture transpiration meter (manufactured by Vapometer Delfin). Figure 11 shows the relationship between _{S 0} and TEWL.

  In the ESR spectrum indicated by the dotted line in FIG. 10, it can be seen that (a) is characterized by a peak indicated by an arrow as compared with (b) to (e). Therefore, when ESR measurement was performed in the same manner as described above except that the surface layer of the stratum corneum 2 was collected from the inner forearm portion of male B and the forehead portion before and after face washing, an ESR spectrum as shown in FIG. 12 was obtained. FIGS. 12A to 12C respectively show the forearm inner part, the forehead part before face washing, and the forehead part after face washing. Also in FIG. 12, since the peak shown by the arrow is seen as in FIG. 10, this peak is considered to be derived from sebum. The reason for this is that a strong peak is observed in the forehead part (b) before washing the face with a lot of sebum, and that this peak is reduced in the forehead part (c) after face washing. For this reason, when evaluating the structure of the intercellular lipid in the stratum corneum, it is preferable to analyze by excluding the ESR spectrum having a peak derived from sebum.

  Moreover, in FIG. 10, in (a), although the difference considered to originate in the peak derived from sebum is seen between the ESR spectrum shown by a dotted line, and the ESR spectrum analyzed by the computer simulation method shown by a solid line, (b) In (e), such a difference is not seen. This is presumably because 5-doxyl stearic acid is well taken up into intercellular lipids.

Furthermore, it can be seen from Table 1 that (a) is smaller in S ₀ than in (b) to (e), and from FIG. 11, from the middle layer to the lower layer of the stratum corneum where S ₀ is constant at a high value. It can be seen that TEWL rises (barrier breakdown). In addition, since it has been reported by the electron microscope that the lipid bilayer disappears in the surface layer of the horny layer (see Proc 17th IFSCC, 2: 865-880, 1992), the above results are supported.

Next, S ₀ and TEWL were obtained in the same manner as above except that six stratum corneum 2 were collected from the skin of the inner side of the forearm of male B, and the results shown in FIG. 13 were obtained. In general, the cornified outer membrane (conified envelope; CE) is a protein-like bag that wraps the horny layer cells, which changes from hydrophilic to hydrophobic in the stratum corneum. It is believed that intercellular lipids are fixed on the basis of This change starts slowly from the lower layer, the change in the hydrophobicity middle has been found to be complete, the is S ₀ increases at middle, the CE of intercellular lipid of horny layer was changed to hydrophobic This is considered to be because the mobility is lowered compared to the lower layer. From the middle layer to the lower layer, the regularity of intercellular lipids increases and S ₀ increases, but the motility of intercellular lipids that are insufficiently joined to CE increases because the CE surface is immature. Te, it is considered to _{S 0} is reduced. For this reason, it is thought that the intercellular lipid fixed on CE matured in the middle layer of the stratum corneum may contribute to the barrier function of the skin.

Incidentally, factors defining the S ₀ are considered two there. The first is the regularity of the lipid chains, the regularity is increased, S ₀ is increased. The second is a motile, if motility by drying decreases, S ₀ is increased. A specific example will be described below. S ₀ was determined in the same manner as above except that three stratum corneum 2 were collected from the skin of the inner side of the forearm of male C, and the results shown before drying in FIG. 14 were obtained. Furthermore, when S ₀ was determined after freeze-drying for 6 hours, the results shown in FIG. 14 after drying were obtained. In addition, when the stratum corneum is in a dry state, it has been reported that S ₀ increases due to a decrease in motility even though the structure of intercellular lipids in the stratum corneum is disordered (J. Japan Cosmetic Science Science). Society, 25: 130-135, 2001), thus supporting the above results. Actually, in the stratum corneum, it is considered that the moisture gradually decreases from the lower layer to the upper layer. Therefore, if only the influence of moisture, that is, the motility is taken into consideration, S ₀ may be increased. However, since the stratum corneum is finally peeled off, it is thought that the structure of the intercellular lipid is broken as the desmosome and intercellular lipid are decomposed by the enzyme and move upward. In the results shown in Table 1, the tendency that S _{0 of} (b) is slightly lower than that of (c) is recognized because of the decrease in mobility due to the decrease in the amount of water in the stratum corneum. This is thought to be because the decline exceeded the value.

The following operation was performed aseptically in a clean bench using a cultured epidermis model LABCYTE EPI-MODEL (manufactured by J-TEC).
(1) Warm the medium in a 37 ° C. water bath.
(2) Dispense 1 ml of warmed medium into each well of a 12-well plate.
(3) Remove the culture cup containing human epidermal tissue with sterilized tweezers and transfer it to each well of a 12-well plate.
(4) Cover the 12-well plate, place it in a CO ₂ incubator, and take it out the next day.
(5) Aspirate the medium outside the culture cup of each well.
(6) Dispense 1 ml of the warmed medium to the outside of the culture cup in each well to exchange the medium.
(7) Thereafter, the operations (4) to (6) are repeated.

  In the above operation, on the 7th to 13th days, 0.1 ml of the drug shown in Table 2 (no addition means 0.1 ml of water not containing the drug) is added on the epidermal tissue every day on the 14th day. The degree of order S was measured in the eyes (see Table 2 (a)). Further, in the above operation, on the 7th to 13th days, 0.1 ml of the drug shown in Table 2 was added to 1 ml of the warmed medium every day, and the degree of order S was measured on the 14th day (see Table 2 (b)). ). Further, in the above operation, 0.1 ml of the drug shown in Table 2 was added onto the epidermal tissue on the 13th day, and the degree of order S was measured on the 14th day (see Table 2 (c)).

  When measuring the degree of order S, first, the cultured epidermal tissue was attached to a flat plate (support 1) made of quartz glass of 70 mm × 8 mm × 0.5 mm as shown in FIG. Next, it was immersed in a 1 mg / dl 5-doxyl stearic acid solution at 37 ° C. for 1 hour. Further, excess 5-doxyl stearic acid was removed by washing with distilled water, and a sample holder similar to that shown in FIG. 4 was prepared using the presser fitting 4, the support fixing member 5, and the screw 6. At this time, a 1 mg / dl 5-doxyl stearic acid solution was prepared by dissolving 1 mg of 5-doxyl stearic acid in 100 μl of ethanol and then diluting with distilled water.

  The obtained sample holder was inserted into a cavity resonator of an X-band ESR measurement device JES-REIX type (manufactured by JEOL Ltd.), a central magnetic field 336 mT, a sweep width 15 mT, a sweep time 8 minutes, and a magnetic field modulation width 0. ESR measurement was performed at room temperature under the conditions of 2 mT, a time constant of 1 second, a magnification of 1.25 × 1000, a microwave frequency of 9 GHz, and a microwave output of 10 mW. Furthermore, the degree of order S of the intercellular lipid was determined.

なお、ＳＫ−Ｉｎｆｌｕｘ（Ｄｅｇｕｓｓａ社製）の組成は、脂質成分６．０％、水ｑ．ｓ．１００％、乳酸エステル１０％、カーボマー０．３％、キサンタンガム０．３％、メチルパラベン０．３％及びプロピルパラベン０．２％からなる。また、脂質成分６．０％は、セラミド複合体１．５％、コレステロール０．５％、遊離脂肪酸３．５％及びフィトスフィンゴシン０．５％からなり、セラミド複合体１．５％は、０．００１％のセラミドＩ、０．５％のセラミドＩＩＩ、０．５％のセラミドＩＩＩＢ及び０．５％のセラミドＶＩからなる。

The composition of SK-Influx (manufactured by Degussa) is 6.0% lipid component, water q. s. 100%, 10% lactate, 0.3% carbomer, 0.3% xanthan gum, 0.3% methylparaben and 0.2% propylparaben. In addition, 6.0% of the lipid component consists of ceramide complex 1.5%, cholesterol 0.5%, free fatty acid 3.5% and phytosphingosine 0.5%, and ceramide complex 1.5% is 0%. .001% ceramide I, 0.5% ceramide III, 0.5% ceramide IIIB and 0.5% ceramide VI.

  On the 7th to 13th days, 0.1 ml of the drug shown in Table 3 was added to 1 ml of the warmed medium every day, and the degree of order S was measured on the 14th day. The degree of order S was determined. Three samples were prepared for each drug.

また、図１６に、表３の無添加（１）（図１６（ａ）参照）及び１％ＳＫ−Ｉｎｆｌｕｘ水溶液（１）（図１６（ｂ）参照）のＥＳＲスペクトルを示す。表３より、ＳＫ−Ｉｎｆｌｕｘ及びＳｙｍＲｅｐａｉｒ（Ｓｙｍｐｒｉｓｅ社製）は、細胞間脂質の秩序度Ｓを向上させる効果を有することがわかる。なお、ＳｙｍＲｅｐａｉｒは、ヘキシルデカノール、ビサボロール、セチルヒドロキシプロリンパルミタミド、ステアリン酸及びアブラナ種子ステロールからなる。

FIG. 16 shows the ESR spectra of the additive-free (1) (see FIG. 16 (a)) and 1% SK-Influx aqueous solution (1) (see FIG. 16 (b)) in Table 3. From Table 3, it can be seen that SK-Influx and SymRepair (manufactured by Symprise) have an effect of improving the order S of intercellular lipids. SymRepair consists of hexyl decanol, bisabolol, cetyl hydroxyprolymph mitamide, stearic acid and rapeseed sterol.

It is a figure which shows the method of peeling a stratum corneum from skin. It is a figure which shows a time-dependent change of the ESR spectrum (center magnetic field 336mT) of the cyanoacrylate which added 5-doxyl stearic acid. It is a figure which shows the ESR spectrum (center magnetic field 336mT) explaining the effect by dissolving a spin probe agent in a water-soluble solvent. It is sectional drawing which shows an example of the sample holder used by this invention, (a) is a front view, (b) is a side view. It is sectional drawing which shows the other example of the sample holder used by this invention, (a) is a front view, (b) is a side view. It is a figure explaining the method of calculating | requiring A (parallel) and A (perpendicular). It is a figure explaining the relationship between the order degree S of an intercellular lipid, and the regularity of the structure of an intercellular lipid. It is a figure which shows the ESR spectrum (central magnetic field 336mT) of the intercellular lipid from which order degree S differs. Is a diagram illustrating the order parameter S ₀ of intercellular lipids. It is a figure which shows the ESR spectrum (central magnetic field 336mT) obtained in Example 1. FIG. It is a diagram showing the relationship between S ₀ and TEWL. It is a figure which shows the ESR spectrum (central magnetic field 336mT) obtained in Example 1. FIG. It is a diagram showing the relationship between S ₀ and TEWL. Drying is a diagram illustrating the S ₀ before and after. It is a figure which shows the method of making the culture | cultivation skin (epidermis) tissue adhere to a support body. It is a figure which shows the ESR spectrum (central magnetic field 336mT) obtained in Example 3.

Explanation of symbols

DESCRIPTION OF SYMBOLS 0 Skin 1 Support body 2 Corneal layer 3 Adhesive 4 Holding metal 5 Holder 6 Screw 7 Holding screw 8 Mark 9 Intercellular lipid 10 Spin probe 11 Skin (epidermis) tissue 12 Tweezers

Claims (11)

Attaching the stratum corneum to the support;
Treating the stratum corneum attached to the support with a spin probe agent (spin label agent);
At least a step of measuring an electron spin resonance spectrum using a support to which a stratum corneum treated with the spin probe agent is attached;
A method for evaluating a stratum corneum, comprising using a solution obtained by dissolving the spin probe agent in a water-soluble solvent and then diluting with water when treating with the spin probe agent. The step of attaching the stratum corneum to the support is
Using the support to which the adhesive is attached, peeling the stratum corneum;
The method for evaluating a stratum corneum according to claim 1, further comprising a step of leaving the support to which the peeled stratum corneum is adhered for a predetermined time under a predetermined condition.   The method for evaluating a stratum corneum according to claim 2, wherein the adhesive contains cyanoacrylate.   The stratum corneum evaluation method according to any one of claims 1 to 3, wherein the spin probe agent is a doxyl derivative of a long-chain fatty acid having 8 to 25 carbon atoms.   The method for evaluating a stratum corneum according to any one of claims 1 to 4, wherein the support is made of a nonmagnetic material selected from the group consisting of quartz glass, glass, acrylic resin, and hard resin. .   The stratum corneum according to any one of claims 1 to 5, wherein the water-soluble solvent contains an alcohol having 1 to 4 carbon atoms and 1 to 3 hydroxyl groups. Evaluation method.   The stratum corneum according to any one of claims 1 to 6, further comprising a step of determining the degree of order (fluidity) of intercellular lipids in the stratum corneum from the measured electron spin resonance spectrum. Evaluation methods.   The stratum corneum evaluation method according to any one of claims 1 to 7, further comprising a step of analyzing the measured electron spin resonance spectrum using a computer simulation method.   The stratum corneum evaluation method according to any one of claims 1 to 8, further comprising a step of detecting sebum from the measured electron spin resonance spectrum.   The stratum corneum evaluation method according to any one of claims 1 to 8, wherein the stratum corneum is formed by culture.   The method for evaluating a stratum corneum according to claim 10, wherein a drug is added during the culture.

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