JP2015001476A - Coating film observation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of collecting a coating film of a skin external composition, coated on skin, for each horny layer and observing it.SOLUTION: A coating film observation method includes: a step (1) in which a support body having a base material provided with a through hole and an adhesive part formed on the skin connection surface side of the base material is bonded to a skin surface; a step (2) in which a skin external composition is coated on the through hole part of the bonded support body from the non-skin connection surface side of the support body; a step (3) in which the support body is peeled from skin, and a horny layer connected to the support body and the coating film of the skin external composition coated on the horny layer are collected together with the support body; and step (4) in which the coating film on the collected horny layer is observed.

Description

  The present invention relates to a method for observing a coating film of a composition for external application to the skin surface. The present invention also relates to a method for observing the epidermal stratum corneum to which a skin external composition is applied. Furthermore, this invention relates to the preparation method of the sample for observation used for the said method.

  The feeling of use of cosmetics and the sustainability of effects are important factors when consumers purchase cosmetics. In the development of cosmetics, this feeling of use and the like is mainly evaluated by sensory evaluation. However, since sensory evaluation is a method in which humans actually use products, evaluation is time-consuming and inefficient, and results include the evaluator's subjectivity. There are problems such as difficulty in obtaining results.

As an objective cosmetic evaluation method that does not rely on sensory evaluation, for example, humans use cosmetics and observe the state of cosmetics on the user's skin or measure physical properties. Yes. However, since this method is performed on the human body, the observation mode and measuring equipment are limited in consideration of the influence on the human body. In particular, morphological observation at the micro level is difficult.
On the other hand, methods using collected skin and cosmetics and methods using alternative skin have also been proposed. Cosmetics are used for the stratum corneum (the stratum corneum) that is the uppermost layer of the skin. Examples of methods for examining the state of the stratum corneum include Non-Patent Document 1 and Patent Document 1. Non-Patent Document 1 describes that stratum corneum cells are collected using a mesh grid and the structure analysis of intercellular lipids is performed. Patent Document 1 proposes a method for measuring physical properties such as elastic modulus of stratum corneum cells.
As a method for examining the state of cosmetics, for example, Patent Document 2 proposes a method in which cosmetics such as lipsticks are applied to plant leaves, and this is frozen and cut to observe the cross section of the cosmetic film. ing. In addition, cosmetics are applied on the surface of artificial skin or peeled skin (such as pig skin), and the state of the coating film is observed with a microscope.
Furthermore, Non-Patent Document 2 is a method for collecting and analyzing cosmetics after applying them on human skin. In this method, a replica agent is dropped onto a decorative film applied on the skin and cured, and then the replica agent and the decorative film are peeled off from the skin after a certain period of time, and the film is observed with a microscope. is there.

JP 2012-93144 A JP 2005-84005 A

G. S. K. Pilgram et al., Journal of Microscopy, Vol.189 (1), p.71-78, January 1998 Tanaka Kenichi et al., Establishment of microscopic evaluation method for cosmetic coatings and application to formulation development, Abstracts of 51st SCCJ Research Discussion Meeting, Japan Cosmetic Engineers Association, November 19, 2002, p.21- twenty four

In order to appropriately evaluate the feeling of use of cosmetics and the sustainability of effects, it is desirable to collect cosmetics and skin samples while maintaining and reproducing the state of use as much as possible. For that purpose, it is possible to collect and observe a cosmetic coating film including the stratum corneum to which the cosmetic is applied, that the collected sample is not deformed or altered from the state at the time of use, and the time course after the cosmetic application. It is preferable that observation is possible.
The said nonpatent literature 1 and patent document 1 are not the methods of analyzing the cosmetics on skin. Moreover, although the method of the said patent document 2 and the method using the peeled skin can evaluate the state at the time of cosmetics application to some extent, it is difficult to evaluate even a temporal change. Since the actual cosmetics are used by being applied onto human skin, the properties of the cosmetics change with the passage of time due to the influence of the flow of the coating film due to body temperature, moisture evaporation from the skin surface, and the like. Moreover, although the method of the said nonpatent literature 2 can observe the time-dependent change of a coating film, the structure of a coating film may deform | transform at the time of dripping or hardening of a replica agent. In addition, when the cured cosmetic coating film is peeled from the skin, the coating film structure on the peeling surface is destroyed, and a part of the coating film remains on the skin, and the entire structure of the coating film cannot be observed. Problems arise.

  It is an object of the present invention to provide a method capable of observing a coating film of a composition for external use such as a cosmetic applied on the skin in an extremely close state during actual use. Specifically, it is an object to provide a method capable of collecting and observing the stratum corneum of the epidermis without deforming or altering the coating film of the composition applied to the skin.

In view of the above problems, the present inventors have intensively studied a method for collecting and observing the skin stratum corneum while maintaining the state in which the external composition for skin is applied on the skin.
As a result, a support body provided with an adhesive portion on one side of a substrate having a through hole is adhered to the skin, and the composition for external use on the skin is applied to the skin under the through hole of the support body. The composition can be applied directly, and when the support is peeled off from the skin after the application, the stratum corneum under the support is peeled together by the action of the adhesive portion of the support, and the composition for external use on the peeled stratum corneum It was found that the paint film of the object can be collected together. Thereby, the whole coating film was able to be extract | collected, without impairing the coating film of a boundary surface with a stratum corneum. The present invention has been completed based on this finding.

That is, this invention relates to the coating-film observation method which has the process of following (1)-(4).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (4) The step of observing the coating film on the collected stratum corneum

Moreover, this invention relates to the observation method of a stratum corneum which has the process of following (1)-(3) and (5).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (5) A step of observing the collected stratum corneum

Moreover, this invention relates to the preparation method of the sample for coating-film observation which has the process of said (1)-(3).
Moreover, this invention relates to the preparation method of the sample for stratum corneum observation which has the process of said (1)-(3).

  Furthermore, this invention relates to the evaluation method of the external composition for skin which evaluates the performance of the external composition for skin based on the observation result obtained by the said observation method.

According to the method of the present invention, the coating film of the external composition for skin applied on the skin can be observed in an extremely close state during actual use. Moreover, according to the method of the present invention, it is possible to observe over time how the applied external composition for skin behaves on the skin. Furthermore, according to the method of the present invention, it is possible to observe the state of the epidermal stratum corneum to which the external composition for skin has been applied. In these methods, it is possible to observe the fine structure of the coating film and stratum corneum at the submicron level.
Furthermore, according to the method of the present invention, it is possible to prepare a sample for observing the coating film or stratum corneum of the composition for external use while maintaining a state close to the actual use state.
Furthermore, according to the method of the present invention, the performance of the external composition for skin can be evaluated based on the observation result of the coating film or the stratum corneum.

It is a figure which shows typically an example of the support body used for this invention. (A) is the top view which looked at the support body from the non-skin joint surface, (b) is AA sectional drawing of a support body. It is a figure which shows typically the state which stuck the support body of FIG. 1 to human skin. It is a figure which shows typically the state which apply | coated the skin external composition from on the support body of FIG. (A) is a state in which a support is attached to the inside of a human arm and a composition for external application to the skin is applied from above; (b) is a cross-sectional view of the support after the composition is applied in (a) Are shown respectively. It is a figure which shows typically the state which peeled the support body of FIG. 3 from the skin. It is a figure which shows typically the outer edge area | region and observation area | region of a support body. It is an enlarged photograph of the base-material through-hole used in the Example. It is a figure which shows the outline of the adhesion part formation process of the support body used in the Example. (A) is the apparatus figure which apply | coats the adhesive to a base-material center part, (b)-(d) is sectional drawing of the base-material part of the apparatus of (a), (b) is before adhesive application. The state, (c) shows the state immediately after application of the pressure-sensitive adhesive, and (d) shows the state after suction of the pressure-sensitive adhesive. It is a figure which shows the result of the X-ray diffraction of the coating film sample extract | collected in the Example. It is a scanning-type photomicrograph of the cross section of the sample for observation extract | collected in the Example. FIG. 10 is an enlarged scanning micrograph of the coating film portion of FIG. 9.

The observation sample preparation method of the present invention includes the following steps (1) to (3).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. The step of collecting the coating film of the composition for external use on the skin By using this method, a sample for observing the coating composition of the composition for external application on the skin and the sample for observing the stratum corneum applied with the composition for external application of the skin are used. Can be prepared. Since the observation sample prepared by the method of the present invention is a method of collecting a coating film including the stratum corneum, the entire applied coating film is collected without damaging the coating film on the boundary surface with the stratum corneum. be able to. In addition, since no chemical or physical treatment is performed on the coating film at the time of collection, alteration and deformation of the coating film can be suppressed. Furthermore, since it is a minimally invasive method in which the adhesive part of the support is attached to the skin and peeled off, it is suitable for sampling from a living body (particularly a human).

The observation method of the present invention has the following step (4) or (5) after the steps (1) to (3).
(4) The step of observing the coating film on the collected stratum corneum, or (5) The step of observing the collected stratum corneum The observation method of the present invention is the observation sample obtained by the preparation method of the present invention. It is used for observation, and the coating on the stratum corneum, the stratum corneum (corneal cells and intercellular lipids), or both can be observed. Moreover, since it can observe including the stratum corneum which apply | coated the coating film and there are few quality changes and deformation | transformation of a coating film, the state very near at the time of actual use can be observed. In addition, since it is a method that can be applied to living bodies, the applied composition is affected by body temperature, moisture evaporation from the skin surface, etc., and how its properties change over time Can be observed. In addition, since the sample after peeling from the skin is used for observation, there are no restrictions on the observation method and equipment used in consideration of safety to the human body, and observation of the cross-sectional structure and observation of the fine structure using an electron microscope is possible. It is.

The evaluation method of the present invention evaluates the performance of the external composition for skin based on the observation results obtained by the observation method of the present invention. In the observation method of the present invention, an observation result reflecting the actual use state more accurately than in the conventional method can be obtained, so that evaluation with higher accuracy is possible.
Hereinafter, the present invention will be described in detail with reference to the drawings as appropriate and with examples.

1. 1. Preparation of observation sample 1-1. Substrate and support [support]
As illustrated in FIG. 1, the support body 1 includes a base material 3 provided with a through hole 2 and an adhesive portion 4 formed on the skin bonding surface (back surface) side of the base material 3.
[Base material]
There may be a plurality of through holes provided in the substrate as shown in FIG. 1 or a single hole. In addition, examples of the planar shape of the through hole include a circle, an ellipse, a quadrangle, and a hexagon, but other shapes may be used.

In terms of collection efficiency of coating properties and film or stratum corneum of the skin external composition, pore area of each pore is preferably 0.8 [mu] m ² or more, more preferably 70 [mu] m ² or more, more preferably 300 [mu] m ² or more, 700 .mu.m ² The above is more preferable, 636200 μm ² or less is preferable, 196400 μm ² or less is more preferable, 49100 μm ² or less is more preferable, and 7900 μm ² or less is even more preferable. From the same viewpoint, the pore diameter is preferably 1 μm or more, more preferably 10 μm or more, further preferably 20 μm or more, still more preferably 30 μm or more, preferably 900 μm or less, more preferably 500 μm or less, further preferably 250 μm or less, 100 μm. The following is even more preferable.
In the present invention, the hole diameter means the diameter when the through-hole is circular, the long diameter when it is elliptical, the long side when it is square, and the opposite side when it is hexagonal.

The opening ratio of the base material is preferably 10% or more, more preferably 20% or more, more preferably 50% from the viewpoint of the adherence of the support to the skin and the efficiency of collecting the coating film and stratum corneum. More preferably, it is 90% or less, more preferably 85% or less, and further preferably 80% or less. In addition, an aperture ratio means the ratio which the sum total of the hole area of a through-hole occupies for the area of a base-material plane in a base-material plane.
It is preferable that the plurality of through holes on the substrate are regularly arranged at regular intervals. In this case, the distance (pitch) between the holes is preferably 10 μm or more, more preferably 20 μm or more, further preferably 30 μm or more, preferably 1000 μm or less, more preferably 500 μm or less, and further preferably 100 μm or less.

Examples of the planar shape of the substrate include a circle, an ellipse, a quadrangle, and a hexagon, but other shapes may be used.
From the viewpoint of coatability of bonded wear resistance and external composition for skin to skin, the area of the substrate plane is preferably 0.7 mm ² or more, more preferably 4 mm ² or more, more preferably 7 mm ² or more, 1970Mm ² or less Preferably, it is 320 mm ² or less, more preferably 80 mm ² or less. From the same viewpoint, the diameter of the substrate is preferably 1 mm or more, more preferably 2 mm or more, further preferably 3 mm or more, preferably 50 mm or less, more preferably 20 mm or less, and further preferably 10 mm or less. Here, the diameter of the base material means the diameter when the base material is circular, the long diameter when it is elliptical, the long side when it is square, and the opposite side when it is hexagonal.
The thickness of the base material is preferably 1 μm or more, more preferably 3 μm or more, further preferably 5 μm or more, preferably 100 μm or less, and preferably 50 μm or less, from the viewpoint of adhesiveness to the skin and applicability of the composition for external use on the skin. More preferably, it is more preferably 20 μm or less.

  The material of the base material is preferably such that the collected coating film and stratum corneum can be maintained in a state close to that of a living body, and has a certain degree of flexibility so as to be adaptable to the unevenness of the human skin surface. Moreover, in order to improve the applicability to the skin of the composition for external application to be applied later, those having good affinity for the composition for external application are preferable. For example, copper, gold, platinum, aluminum, nickel, stainless steel, titanium, molybdenum, a copper alloy, an aluminum alloy, and the like can be given. Among these, copper, platinum, stainless steel, titanium, copper alloy, and aluminum alloy are preferable, and copper, platinum, stainless steel, and titanium are more preferable.

The base material may be subjected to a surface treatment in order to enhance the affinity with the composition for external use on the skin. As an example of the surface treatment, a method of coating a metal such as rhodium, gold or platinum on the surface of the base material of the above material can be mentioned. The coating can be performed by a method usually used for metal surface coating, and for example, a vapor deposition method, a sputtering method, or an ion plating method can be used. The thickness of the metal to be coated on the surface can be appropriately selected according to the type of metal used, but it is preferable that the coating film is not peeled off when the support is peeled off. As an example, when coating with platinum, it is preferable to set the thickness to about 10 nm.
Further, the substrate may be subjected to a discharge treatment under an inert gas. As the inert gas, for example, a rare gas such as argon or krypton, nitrogen, or the like can be used. As for the conditions of the discharge treatment, the pressure during the treatment is preferably 20 Pa or less, and more preferably 10 Pa or less. The voltage is preferably 1 kV or less, and more preferably 500 V or less. The treatment time is preferably 30 seconds or shorter, and more preferably 20 seconds or shorter.

[Adhesive part]
The adhesive portion of the support is formed on the skin bonding surface side of the substrate. By the action of the adhesive part, the support can be attached to the skin, and at the time of peeling, the stratum corneum on the skin surface can be collected while being bonded to the support.
The thickness of the adhesive part is preferably 20 μm or less, and more preferably 5 μm or less.

  From the following viewpoint, the shape of the pressure-sensitive adhesive portion requires that at least a part of the through holes of the base material is not blocked by the pressure-sensitive adhesive portion, and it is preferable to form the pressure-sensitive adhesive portion so as not to block the through holes as much as possible. As shown in FIG. 2, the support 1 is attached to the skin 5 with the surface (skin joint surface) provided with the adhesive portion 4 facing down. At this time, the adhesive portion 4 is joined to the stratum corneum 6 present on the outermost surface of the skin 5. After sticking a support body, as shown to Fig.3 (a), the skin external composition 7 is apply | coated from the support body 1 (from the non-skin joint surface side). The cross-sectional state of the support after applying the composition for external skin 7 is as shown in FIG. In addition, in FIG.3 (b), the composition 7 for external skin applied to the upper part and peripheral part of the support body 1 is abbreviate | omitted. The applied composition for external skin 7 is applied to the skin 5 (keratin layer 6) under the through hole through the through hole 2 of the base material 3. At this time, in order to observe the behavior of the composition for external skin on the skin, it is necessary to apply the composition for external skin 7 directly to the skin located under the through-hole 2. If the skin joint surface side of the through hole 2 is blocked by the adhesive portion 4, the composition for external skin 7 cannot be applied onto the skin in the through hole.

  The adhesive part can be formed using an adhesive. The pressure-sensitive adhesive to be used is not particularly limited as long as it can be peeled off after the support is attached to the skin. Specifically, anionic resins (for example, vinyl acetate-crotonic acid copolymer, crotonic acid-vinyl acetate-vinyl neodecanoate copolymer, crotonic acid-vinyl acetate-vinyl propionate copolymer, vinyl acetate-malein) Acid monobutyl-acrylic acid isobornyl copolymer, acrylic acid octylamide-acrylic acid ester copolymer, N-vinylpyrrolidone-acrylic acid ester- (meth) acrylic acid copolymer, (meth) acrylic acid- (meth) acrylic Acrylic resin alkanolamine liquid which is an acid ester copolymer, acrylic acid ester-methacrylic acid ester-diacetone acrylamide-methacrylic acid copolymer, methyl vinyl ether-monoalkyl maleic acid copolymer), cationic resin (for example, hydroxy Ethylcellulose dimethyldialy Ammonium chloride, O- [2-hydroxy-3- (trimethylammonio) propyl] hydroxyethylcellulose, N-vinylpyrrolidone-dimethylaminoethyl methacrylate copolymer, vinylimidazolium trichloride-N-vinylpyrrolidone copolymer N-vinylpyrrolidone-dimethylaminoethyl methacrylate copolymer, N-vinylpyrrolidone-aminoalkyl acrylate-N-vinylcaprolactam copolymer, N-vinylpyrrolidone-methacrylamidepropyltrimethylammonium chloride copolymer, N- Alkylacrylamide-acrylic acid-N- (alkylamino) alkylacrylamide-polyethylene glycol methacrylate copolymer, polydimethylmethylenepiperidinium chloride solution, dimethyldiallyl Ammonium chloride, acrylamide copolymer, cationized guar gum, etc.), nonionic resin (for example, polyvinyl alcohol, poly (N-vinylpyrrolidone), N-vinylpyrrolidone-vinyl acetate copolymer, N-vinylpyrrolidone-vinyl acetate) Vinyl propionate copolymer, N-vinylpyrrolidone-vinyl acetate-aminoalkyl acrylate copolymer, vinyl acetate-N-vinyl-5-methyl-2-oxazoline copolymer, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose , Methylcellulose, carboxymethylcellulose, carboxyvinyl polymer, polyvinylformamide, polyvinylacetamide, etc.), amphoteric resins (for example, octylamide acrylate-hydroxyacrylate) Propyl-butylaminoethyl methacrylate copolymer, N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine-methacrylic acid ester copolymer, acrylic acid-methacrylic acid-acrylic acid 2-hydroxy Propyl-dimethylaminoethyl methacrylate-ethyl methacrylate-diacetone acrylamide-N-vinylpyrrolidone copolymer and its alkanolamine solution). One or more of these pressure-sensitive adhesives can be used for the pressure-sensitive adhesive part.

  As shown in FIG.3 (b), since the adhesion part 4 contacts with the applied external composition 7 for skin, the adhesive has a little affinity with the external composition for skin. Moreover, the thing stable with respect to the composition for external use of a skin is preferable so that it may melt | dissolve in contact with the composition for external use of skin, or adhesive force may not fall. From this viewpoint, a nonionic resin or an amphoteric resin is preferable as the adhesive, and an amphoteric resin is more preferable.

As described above, the adhesive portion is required to be formed so as not to block the through-hole of the substrate as much as possible, but also requires an adhesive force that does not shift or peel off when the external composition for skin is applied.
In order to realize both, it is also preferable to change the position of the adhesive portion in addition to the selection of the adhesive. For example, as shown in FIG. 5, the base material 3 is divided into an outer edge area 8 and an observation area 9 inside thereof, and an adhesive portion is formed on the entire skin bonding surface side of the outer edge area 8. It is preferable to form an adhesive portion on the side so as not to block the through hole. That is, since the adhesive portion is uniformly formed so as to close the through hole in the outer edge region 8, the adhesive strength of the support can be enhanced. On the other hand, in the central observation region 9, the through hole is not blocked by the adhesive part, so that the composition can be directly applied onto the skin. By such separate application of the adhesive portion, it is possible to increase the adhesive strength of the support to the skin without affecting the observation of the coating film.

The area of the observation region is preferably 90% or less, more preferably 80% or less, and preferably 10% or more, and 20% or more with respect to the area of the substrate plane. Is more preferable.
Moreover, it is preferable that the hole opening ratio of the said observation area | region is 50% or more and 100% or less, and it is more preferable that it is 80% or more and 100% or less. In the present invention, the aperture ratio of the observation region is calculated by the following mathematical formula (I).

Formula (I)
Hole opening ratio (%) of observation region = (number of through holes not blocked by adhesive portion in observation region / total number of through holes in observation region) × 100

Whether or not the through hole is blocked by the adhesive portion can be observed and measured with an optical microscope.

1-2. Application of skin external composition [skin external composition]
The composition for external use of skin can be used as long as it is in a form that can be used by directly contacting the product with the skin. For example, skin external preparations including cosmetics and pharmaceuticals can be mentioned. The cosmetics include those belonging to both cosmetics and quasi-drugs under the Japanese Pharmaceutical Affairs Law.
The external composition for skin used in the present invention is preferably a cosmetic. As cosmetics, skin care cosmetics (for example, lotion, milky lotion, cosmetic liquid, cream, oil, pack, sunscreen), makeup cosmetics (for example, lipstick, lip gloss, foundation, concealer), cleaning cosmetics (For example, face wash, cleansing, body shampoo) and the like.

Application of the composition for external use on skin can be performed in the same manner as the normal use of the composition, and may be applied by hand or a dedicated product (brush, sponge, etc.) may be used.
It is preferable that the application amount of the composition for external use of skin is a normal use amount of the composition to be used. For example, in the case of skin care cosmetics, the coating amount is preferably 1 mg / cm ² or more, more preferably 4 mg / cm ² or less. In the case of makeup cosmetics, the application amount is preferably 0.2 mg / cm ² or more, more preferably 0.6 mg / cm ² or less. In particular, in a liquid foundation or the like, the coating amount is preferably 0.4 mg / cm ² or more, and more preferably 0.6 mg / cm ² or less. Moreover, in powder foundation etc., the application amount is preferably 0.2 mg / cm ² or more, and more preferably 0.4 mg / cm ² or less.

1-3. Collection of coating film After applying the external composition for skin, the support is peeled off from the skin.
The time from application to peeling can be appropriately selected according to the purpose of observation. For example, the change of the coating film over time can be examined by applying the composition at the same time using a plurality of supports and sequentially performing peeling immediately after the application and after a lapse of a certain time. By performing such time-lapse observation, information on changes in the feeling of use of the cosmetics, sustainability of the effects, makeup collapse, and the like can be obtained.

As shown in FIGS. 3 and 4, when the support 1 is peeled from the skin 5, a part of the stratum corneum 6 that is the outermost part of the skin 5 is peeled from the skin 5 together with the support 1. At this time, along with the stratum corneum 6 bonded to the adhesive portion 4, the stratum corneum 6 located under the through-hole 2 where the adhesive portion is not formed is also peeled off. The stratum corneum of the skin is formed by laminating about 10 to 20 stratum corneum cells, and has a structure that is easily peeled off from the surface. For this reason, when the stratum corneum joined to the adhesion part 4 peels, the stratum corneum 6 located under the through-hole 2 surrounded by the adhesion part 4 can also be peeled together. On the stratum corneum 6 located under the through-hole 2, a coating film of the external composition 7 is formed.
Although there is no restriction | limiting in particular in the thickness of the stratum corneum peeled and extract | collected, 500 nm or more is preferable. The stratum corneum sampling rate of the support is preferably 10% or more and 100% or less, and more preferably 30% or more and 100% or less. The stratum corneum collection rate of the support is calculated by the following mathematical formula (II).

Formula (II)
Stratum corneum collection rate (%) = (total area of stratum corneum collected on support skin interface / area of support plane) × 100

2. Observation of coating film and stratum corneum The method for observing the coating film and stratum corneum sample of the collected external composition for skin is not particularly limited, and a technique used for observation and analysis of a normal biological sample can be used. In addition, the observation method of the present invention includes not only structure and form observation but also analysis of chemical properties and measurement of physical properties.
The observation sample collected by the method of the present invention is suitable for both surface observation and cross-sectional observation of the coating film and stratum corneum, and is suitable for both structural (morphological) observation and physical property measurement.
Specific observation methods include optical microscope, transmission electron microscope (TEM), scanning electron microscope (SEM), scanning probe microscope (SPM), laser confocal microscope, etc. Analysis of constituent elements and electronic structure by energy dispersive X-ray spectroscopy (EDX), wavelength dispersive X-ray spectroscopy (WDX), and electron energy-loss spectroscopy (EELS); X-ray diffraction method Analysis of fine periodic structures (for example, lamellar structure of intercellular lipid in stratum corneum, lamellar structure in composition for external use of skin) by electron beam diffraction method, etc. can be performed.
Among these, SEM observation (for example, Cryo SEM, low vacuum SEM) or TEM observation (for example, Cryo TEM) is preferable from the viewpoint that the fine structure of the coating film and the stratum corneum can be observed.

The sample can be appropriately processed and used so as to be suitable for observation and analysis. For example, a thin piece sample may be prepared using a microtome or the like so as to be suitable for microscopic observation. Moreover, when performing cross-sectional observation, a cross-sectional sample can be created using the freezing cutting method or the freezing cleaving method. In this case, it is preferable that the coating film and the stratum corneum sample are frozen together with liquid nitrogen or the like and cleaved.
Although the example of observation using Cryo-SEM and Cryo-TEM is shown below, it can also be observed by other methods.
When performing cross-sectional observation of the coating film and the stratum corneum, the sample collected in the step (3) is frozen, then cleaved with a knife, and Cryo-SEM observation is performed. When observing a finer structure, after freezing the sample, expose the cut section with a cryomicrotome (freeze cutting method) and perform Cryo-SEM observation. In order to observe a finer structure, after exposing the cut cross section with a cryomicrotome, an ultrathin section is prepared with a thickness of about 100 nm or less, and Cryo-TEM observation is performed.
In addition, the surface morphology of the coating film is observed in Cryo-SEM after freezing, or in low vacuum SEM mode without freezing if deformation due to freezing is large.
When performing elemental analysis, it is preferable to use a low vacuum SEM mode (for example, a degree of vacuum of about 5 Pa to 150 Pa). When there is a concern about damage to the sample due to heat, it is preferable to perform elemental analysis while cooling the sample, and it is also preferable to perform the low vacuum SEM mode under Cryo-SEM observation conditions. As the detector for observation, a detector for secondary electrons, a detector for secondary electrons for low vacuum, a backscattered electron detector, and the like can be appropriately selected.

3. Evaluation of composition for external skin Based on the observation and analysis data obtained, the performance of the composition for external skin can be evaluated. For example, the network structure, lamellar structure (plate-like lamellar structure, spherical lamellar structure, and ratio of these lamellae, etc.), sponge-like structure, particle size distribution of blended inorganic particles Dispersion state, aqueous phase, oil phase region discrimination, fluidity of external composition for skin, moisture retention state, etc. can be evaluated.
In addition, by observing the state of the stratum corneum, for example, the shape of the stratum corneum, the presence or absence of nuclei, the lamellar structure of intercellular lipids or the moisture retention state, the size of pores, etc., the effect of using the composition for external use on the skin Can be evaluated.

  In relation to the above-described embodiment, the present invention further discloses the following method.

<1> A coating film observation method having the following steps (1) to (4).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (4) The step of observing the coating film on the collected stratum corneum

<2> The method according to <1>, which is used for living skin.
<3> The opening ratio of the base material is 10% or more, preferably 20% or more, more preferably 50% or more, 90% or less, preferably 85% or less, more preferably 80% or less, <1 > Or <2>.
<4> open area of the through hole is 0.8 [mu] m ² or more, preferably 70 [mu] m ² or more, more preferably 300 [mu] m ² or more, more preferably 700 .mu.m ² or more, 636200Myuemu ² or less, preferably 196400Myuemu ² or less, more preferably 49100μm ² or less, more preferably 7900Myuemu ² or less, <1> to the method of any one of <3>.
<5> area of the substrate plane is 0.7 mm ² or more, preferably 4 mm ² or more, more preferably 7 mm ² or more, 1970Mm ² or less, preferably 320 mm ² or less, more preferably 80 mm ² or less, <1 The method according to any one of> to <4>.
<6> The thickness of the substrate is 1 μm or more, preferably 3 μm or more, more preferably 5 μm or more, 100 μm or less, preferably 50 μm or less, more preferably 20 μm or less, any one of <1> to <5> The method according to claim 1.
<7> The method according to any one of <1> to <6>, wherein the external composition for skin is a cosmetic.

<8> The method according to any one of <1> to <7>, wherein in the step (4), the stratum corneum and the coating film collected are frozen and cleaved together with the support, and a cross section of the coating film is observed. .
<9> The method according to any one of <1> to <8>, wherein the observation of the step (4) is performed by an X-ray diffraction method.
<10> The method according to any one of <1> to <8>, wherein the observation of the step (4) is performed with a scanning electron microscope (SEM).
<11> The method according to <10>, wherein the SEM is a low vacuum SEM.
<12> The method according to <10>, wherein the SEM is a cryo SEM.

<13> A method for observing the stratum corneum, comprising the following steps (1) to (3) and (5).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (5) A step of observing the collected stratum corneum

<14> A method for preparing a sample for coating film observation, comprising the following steps (1) to (3).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. Step <15> for collecting a coating film of the external composition for skin preparation The method for preparing a stratum corneum observation sample, comprising the following steps (1) to (3).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. A step of collecting a coating film of the external composition for skin <16> A skin for evaluating the performance of the external composition for skin based on the observation result obtained by the observation method according to claim 1. Evaluation method of composition for external use.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Example Preparation and Observation of Sample for Coating Film Observation Production of Supports Transmission electron microscope (TEM) observation grids G600HH, G600TT, and H400 (product name, manufactured by Nisshin EM Co., Ltd.) were used as a base material. The shape of each grid is shown in Table 1, and the shape of the through holes is shown in FIG.

An adhesive was applied to one side of the substrate to provide an adhesive part. As the pressure-sensitive adhesive, an adhesive stock solution (acrylic ester resin, trade name: Polysic 5423A, manufactured by Sanyo Chemical Co., Ltd.) diluted 10 times with ethyl acetate (manufactured by Kanto Chemical Co., Ltd.) was used.
The substrate having a diameter of 3 mm was divided into an outer edge region and an observation region (diameter 2 mm) inside thereof, and the adhesive was separately applied as shown below.
A substrate having a diameter of 3 mm was placed on a silicon plate having a diameter of 4 mm. A silicon plate having a diameter of 2 mm was placed on the center of the substrate to cover the observation area of the substrate. The pressure-sensitive adhesive was uniformly applied to the entire outer edge region of the base material that was not concealed by the silicon plate having a diameter of 2 mm. After volatilizing the ethyl acetate, the substrate was removed.
Next, a pressure-sensitive adhesive was applied to the observation region of the substrate in the procedure shown in FIG. The base material 3 having an adhesive applied to the outer edge region was sandwiched between two silicon plates 10. The silicon plate 10 has a diameter of 4 mm and a hole with a diameter of 2 mm in the center, and the outer edge region of the substrate 3 is covered. Both ends of the silicon plate 10 were further sandwiched between two nylon tubes 11 and 12, and the silicon plate 10 and the nylon tubes 11 and 12 were fixed so as not to shift. The nylon tube 12 was connected to a syringe. The tube was erected vertically so that the nylon tube 11 not connected to the syringe would be on top (FIG. 7 (a)). From the upper part of the hole of the nylon tube 11, 2 microliters of adhesives 13 were dripped, and the adhesive was apply | coated to the observation area | region of the base material 3 (FIG.7 (c)). Next, by pulling the syringe and sucking 1 mL of air from below the base material 3, the pressure-sensitive adhesive 13 attached to the through-hole portion in the observation region was removed (FIG. 7D). When measured with an optical microscope (trade name: ECLIPS600POL (manufactured by NIKON), objective lens: 20 times, eyepiece lens: 10 times), the aperture ratio of the base material observation region was 99%.

2. Preparation of Sample for Film Coating Observation A sample for film coating observation was prepared using the support prepared above.
The subject's forearm was washed with hand soap (2 pumps, 30 seconds washing time), then rinsed with running water for 1 minute, wiped with Kim Towel, and rested for 15 minutes in the sampling room.
The adhesive part forming surface of the support was affixed to the inner side of the subject's forearm, and then pressed from the opposite surface at a pressure of 500 g / cm ² for 10 seconds. Next, cosmetic A (Sophina Genne; manufactured by Kao Corporation) was applied to the skin from above the pasted support so as to be ² mg / cm ² (FIG. 3A). Thereafter, the support was peeled from the skin with tweezers.

3. X-ray diffraction In the method of the present invention, since the composition for external use on the skin is applied from above the uneven support, the coating film is affected by the structure of the support as compared to the normal use mode (applying directly to the skin). There is concern about the deformation. In addition, there is a concern that the coating film may be deformed due to stress received when the support is peeled off. Therefore, this point was verified as follows.
2. The coating film observation sample prepared in (1) was observed by X-ray diffraction.
The support peeled off from the skin was placed on an X-ray sample table (aluminum substrate), and X-ray diffraction was performed under the following conditions (Sample A). For comparison, cosmetic A was directly applied to an X-ray sample stage (application amount: 2 mg / cm ² ), and X-ray diffraction was performed under the following conditions (Comparative Sample B).

X-ray diffractometer / conditions / device: MXP21 (currently Bruker Ax)
X-ray source: counter rotating cathode Cu (wavelength 1.54 mm), voltage 40-50 kV, current 50-400 mA
-Measurement conditions: X-ray irradiation area (25 mm x 25 mm)
Sample A: An X-ray output (20 kW), scanning speed (0.007 deg / min), and an aluminum substrate was used to hold the film during film measurement.
Sample B: X-ray output (2 kW), scanning speed (0.1 deg / min)

The results of X-ray diffraction of each sample are shown in FIG. 8 in the range of 2θ (0.5 degrees to 3.5 degrees).
In FIG. 8, two diffraction peaks are observed in the comparative sample B, and these indicate the distance between the lamellar layers of the cosmetic A coating film.
On the other hand, in sample A, two diffraction peaks were observed at the same position as in comparative sample B. That is, it was confirmed that even when the cosmetic was applied via the support, the same lamellar spacing was maintained as in the case where the cosmetic was applied on a flat substrate, and the lamella structure was maintained. This supports that the method of the present invention does not cause deformation of the coating film so as to affect the observation result.

4). Observation of Cryo-SEM (Cryo-SEM) The cross-sectional observation of the sample for coating film observation prepared in (1) was performed using a cryo SEM. The cryo SEM apparatus and observation conditions are shown below.
The support peeled from the skin was immediately immersed in liquid nitrogen and quickly frozen. After cooling the single-edged razor in liquid nitrogen, the single-edged razor was applied from above the support to apply impact force, and the support was cleaved. The sample was fixed to the sample stage in a direction in which the cross section of the support could be observed in liquid nitrogen. After confirming that the temperature of the sample stage of the cryo SEM was −120 ° C. or less, the sample stage was inserted into the cryo SEM, and ice was sublimated. Thereafter, the temperature was lowered and the temperature of the sample stage was confirmed to be −100 ° C. or less and observed.
The observation images of the cryo SEM are shown in FIGS. In addition, FIG. 10 is the photograph which expanded the coating-film part of FIG.

Cryo SEM equipment / conditions / cryo SEM: Cryo system (Hitachi) is mounted on scanning electron microscope S-4300SE / N (Hitachi) Observation conditions: acceleration voltage 1 kV, Gun brightness 3, sublimation conditions: temperature -80 degrees 5 Minutes Detector: Secondary electron detector

  As shown in FIG. 9, the support, the exfoliated stratum corneum, and the coating film of the cosmetic A were observed on the sample cross section. Furthermore, as shown in FIG. 10 which expanded the coating-film part of FIG. 9, the structure of the spherulite lamella and oil droplet which are the characteristic structures of the cosmetics A were confirmed by the coating film.

DESCRIPTION OF SYMBOLS 1 Support body 2 Through-hole 3 Base material 4 Adhesive part 5 Skin 6 Corneum layer 7 Skin external composition 8 Outer edge area 9 Observation area 10 Silicon plates 11, 12 Nylon tube 13 Adhesive

Claims (13)

The coating-film observation method which has the process of following (1)-(4).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (4) The step of observing the coating film on the collected stratum corneum   The method according to claim 1, which is used for living skin.   The method according to claim 1 or 2, wherein the opening ratio of the substrate is 10% or more. The pore area of the hole is 0.8 [mu] m ² or more and 636200Myuemu ² or less, any one method according to claims 1-3. Area of the substrate plane is 0.7 mm ² or more and 80 mm ² or less, any one method according to claims 1-4.   The method according to any one of claims 1 to 5, wherein the external composition for skin is a cosmetic.   The method according to any one of claims 1 to 6, wherein, in the step (4), the stratum corneum and the coating film collected are frozen and cut together with the support, and a cross section of the coating film is observed.   The method according to claim 1, wherein the observation of the step (4) is performed by an X-ray diffraction method.   The method of any one of Claims 1-7 which observes the said process (4) with a scanning electron microscope (SEM). A method for observing the stratum corneum, comprising the following steps (1) to (3) and (5).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. (5) A step of observing the collected stratum corneum The preparation method of the sample for coating-film observation which has the process of following (1)-(3).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. For collecting a coating film of the skin external composition A method for preparing a sample for stratum corneum observation, which comprises the following steps (1) to (3).
(1) The process of sticking the support body which has the base material which provided the through-hole, and the adhesion part formed in the skin joint surface side of this base material on the skin surface (2) The through-hole of the stuck support body (3) The support is peeled from the skin and applied to the stratum corneum bonded to the support and the stratum corneum. For collecting a coating film of the skin external composition   The evaluation method of the external composition for skin which evaluates the performance of the external composition for skin based on the observation result obtained by the observation method of any one of Claims 1-10.