JP2904565B2 - Method for analyzing stratum corneum components - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for analyzing a stratum corneum component, and more particularly, to a method for analyzing a surface layer component of a stratum corneum.

[Conventional technology]

Conventionally, as a method for collecting lipid components of the stratum corneum surface layer, an extraction solvent is impregnated into absorbent cotton or the like, a wet wiping method of wiping the stratum corneum surface, an organic solvent is put into a frame placed on the stratum corneum, and lipids are extracted. A cup method, a blotting paper method in which extra-stratum corneum extracellular lipids are absorbed by blotting paper, and the lipid content is extracted with a solvent are used.

As a method for collecting cells from the stratum corneum, a scrub method, a cyanoacrylate resin method, and the like are known.

The scrub method is a method in which a cylindrical cup with an open top and bottom is erected on the skin of a fixed arm or the like, and a solution such as a surfactant is dropped into the cup with a pipette or the like. This is a method in which horny cells are released into a solution such as a surfactant by stirring with a stick, and the released horny cells are transferred to a slide glass together with the solution.

On the other hand, the cyanoacrylate resin method is a method in which cells on the skin surface are peeled off and collected using a cyanoacrylate resin instant adhesive.

The analysis of the stratum corneum thus collected has an important meaning even in a compatibility test of, for example, cosmetics.

[Problems to be solved by the invention]

By the way, with the conventional wet wiping method, cup method, and blotting paper method, it is difficult to accurately narrow down the measurement range and perform accurate analysis on the range, and it is difficult to obtain highly reliable analysis results. There was a problem.

Certainly, the analysis of the components appearing on the surface of the stratum corneum
It can be said that the conventional method is also possible if some error is prepared. However, the component composition of the stratum corneum, for example, the surface layer portion, changes greatly even with a slight difference in depth. The conventional method has a problem that it is almost impossible to accurately know the component composition of a portion having such a small depth.

That is, the conventional scrubbing method and the cyanoacrylate-based resin method have a problem that any components appearing on the surface of the stratum corneum cannot be accurately analyzed.

In particular, the scrubbing method and the cup method require special instruments and containers for sampling, and the subject must be fixed. Therefore, there has been a problem that the subject suffers pain for a long time during the collection, and the place where the sample can be collected cannot be restricted.

Furthermore, a solution such as a surfactant or an organic solvent directly contacts the subject's skin (skin), which is not preferable in terms of safety.
Moreover, there is a problem that it is difficult to collect a large amount of stratum corneum details, and it is insufficient for analyzing constituent components.

Originally, when selling cosmetics and the like, dealers and the like should accurately grasp the condition of the user's skin and start makeup according to the condition. In addition, for patients with skin diseases, etc., it is important to know the information on the skin surface and the inside of the stratum corneum in order to accurately understand the condition of the skin, etc. Should.

In recent years, it has become an important issue to know skin quality, skin properties, skin condition, and the like based on such an idea.

Conventionally, various methods have been proposed as methods for knowing these skin conditions. The main method has been to collect cells in the stratum corneum on the skin surface and observe them with a microscope or the like. Was.

This method is mainly for observing the morphological aspects of the stratum corneum cells, and although it has a certain effect in knowing the condition of the skin, it has not been satisfactory for examining the skin in detail.

The present inventor has studied the morphological observation of the stratum corneum cells in combination with the biochemical components contained in the stratum corneum cells collected from the skin, such as proteins, amino acids, nucleic acids, sugars, lipids, enzymes, and living organisms. We thought that a more accurate skin test could be performed by qualitative and quantitative analysis of constituents such as elements.

However, the conventional method has a problem in that the location (position, spread, depth) of a component cannot be strictly controlled, and accurate analysis cannot be performed.

The present invention solves such a conventional problem,
An object of the present invention is to provide a method for analyzing a stratum corneum surface layer component, which allows the composition of the stratum corneum surface layer component to be easily and accurately known.

[Means for solving the problem]

In order to solve the above-mentioned problems, the first method for analyzing the components of the stratum corneum of the present invention comprises a method of laminating an adhesive film having a pressure-sensitive adhesive surface by laminating a pressure-sensitive adhesive on the surface of a base material, and temporarily attaching the adhesive film to the stratum corneum. Peeling off, thereby transferring the stratum corneum to the adhesive surface, and then washing the adhesive film with an organic solvent, thereby extracting the lipid in the stratum corneum transferred to the adhesive surface into the organic solvent, Analyze lipids extracted in organic solvents.

The second method for analyzing the components of the stratum corneum of the present invention comprises the steps of: laminating a pressure-sensitive adhesive on the surface of a base material, pasting an adhesive film provided with an adhesive surface to the stratum corneum once, and peeling off the stratum corneum by the adhesive surface. Then, the adhesive film is washed with an organic solvent, whereby the cells of the stratum corneum transferred to the adhesive surface are released into the organic solvent, and then the components of the cells released into the organic solvent are analyzed. I do.

In the third method for analyzing a stratum corneum component of the present invention, each of a plurality of pressure-sensitive adhesive films having a pressure-sensitive adhesive surface provided by laminating a pressure-sensitive adhesive on a base material surface, once in a stratum corneum, by limiting the site, By sticking and peeling each time, the stratum corneum is sequentially transferred to each adhesive surface at each depth, while each adhesive film thus transferred the stratum corneum is washed with an organic solvent, thereby The lipid components at each depth of the stratum corneum are analyzed by extracting the lipid at each depth of the stratum corneum into an organic solvent and then analyzing the lipid extracted into the organic solvent.

In the fourth method for analyzing a stratum corneum component of the present invention, a plurality of pressure-sensitive adhesive films each having a pressure-sensitive adhesive surface provided by laminating a pressure-sensitive adhesive on a base material surface, once in the stratum corneum, by limiting the site, By sticking and peeling each time, the stratum corneum is sequentially transferred to each adhesive surface at each depth, while each adhesive film thus transferred the stratum corneum is washed with an organic solvent, thereby The cells at each depth of the stratum corneum are released into an organic solvent, and then the components of the cells released into the organic solvent are analyzed to analyze the cell components of the stratum corneum at each depth.

The fifth method for analyzing the surface layer components of the stratum corneum of the present invention comprises the steps of: laminating an adhesive film having a pressure-sensitive adhesive surface by laminating a pressure-sensitive adhesive on the surface of a base material; Is removed by adhering to the adhesive surface, a lipid absorber is applied to the newly formed outermost surface, and the lipid oozing on the new outermost surface is absorbed by the lipid absorber and collected. The body analyzes for collected lipids.

Cellophane tape is usually sufficient for the adhesive film.

 Hereinafter, the present invention will be described in more detail.

In the present invention, the adhesive film 1 is once attached to the stratum corneum and then peeled off.

The stratum corneum usually contains lipid, but in the present invention, the stratum corneum to which the adhesive film is adhered may be a part of the human body itself such as the epidermis of the arm A as shown in FIG. However, it may be a stratum corneum separated from the human body. In addition, the present invention is not limited to the human body, and may be a part of another living body or a separated body thereof. Either case can be preferably used.

The adhesive film 1 to be stretched on the stratum corneum is provided with an adhesive surface by laminating an adhesive on the surface of the substrate 1a.

The base material 1a on which the adhesive is laminated may be cellophane paper or cloth, or any other material that is generally used as the base material 1a of the adhesive film 1 represented by an adhesive tape or the like. Can be widely used. The thickness of the substrate does not directly matter. Also, the thickness of the adhesive surface does not directly matter.

In addition, the adhesive constituting the adhesive film 1 together with the base material 1a is preferably a substance which is harmless to the skin when it is used by directly attaching it to the skin of a human body. As such a substance, for example, specifically, natural rubber or a derivative thereof, SBR
Rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, gum arabic, tragacanth rubber, and the like.

In the present invention, for example, an adhesive film 1 such as a cellophane tape contained in a tape holder 2 or the like is attached to the surface of the stratum corneum on the surface of the skin such as the arm A as shown in the conceptual diagram of FIG. A certain amount of load is applied from the direction shown by the arrow in (2) of FIG. Then, it is peeled off, and the stratum corneum is transferred to the adhesive film 1, for example, in the form of a thin film.

There is no limitation on the number of the adhesive films 1 used by being attached to the surface of the stratum corneum. The number of adhesive films 1 used is 1
It is possible to collect and analyze the stratum corneum appearing on the outermost surface even on a single sheet.

If two or more adhesive films 1 are used,
When the site on the stratum corneum to be attached is specified, the stratum corneum can be transferred and collected at different depths. Specifically, one sheet of each adhesive film 1
When the sheets are sequentially attached at the same place and peeled off each time, the stratum corneum layers having different depths can be transferred and obtained by the number of the plurality of adhesive films 1. In this way, it is possible not only to extract and know a component at a specific depth, but also to easily analyze how the component continuously changes in the depth direction.

In the present invention, the components of the stratum corneum which are transferred and collected on the adhesive surface of the adhesive film 1 in this manner are analyzed. The component analysis is divided into a lipid component and a component other than the lipid, and preferably both components may be analyzed.

Analysis of lipid components was performed using a sticky film 1 with a transferred stratum corneum.
Is put into, for example, a test tube 3a with a cap as shown in (3) of FIG. 1, and an organic solvent is put therein with a pipette 4 as shown in (4) of FIG. May be extracted in the organic solvent, and the lipid component extracted in the organic solvent may be analyzed. The lipid component may be extracted by stirring the adhesive film 1 in an organic solvent.

Examples of the organic solvent used for washing the adhesive film 1 include:
For example, xylene, toluene, benzene, chloroform, methylene chloride, petroleum ether, n-hexane, ethyl ether, acetone, methyl ethyl ketone, tetrahydrofuran, ethanol, methanol and the like can be mentioned.

As a method for analyzing lipids in an organic solvent, for example, chromatography is specifically mentioned. Examples of the chromatography include gas chromatography, thin-layer chromatography, and mass spectroscopy (GC mass). In addition to the chromatography, a gravimetric method using an ultra-trace amount bottle, an osmius acid method using a reaction with a double-bonded lipid, and a chromic acid oxidation method in which a color change is caused by reacting with a lipid can be given. Further, a thiobarbituric acid (hereinafter, referred to as "TBA") may be added, and a TBA method for measuring the degree of red coloring may be mentioned.
An iodine titration method in which an iodine solution is dropped and the degree of discoloration associated with ionization of iodine is measured, and an oxygen absorption method in which the amount of oxygen absorbed in fats and oils is measured, and 233 n
The conjugated diene method for measuring the absorbance at m can be mentioned, and the POV method for quantifying iodine generated by reacting hydroperoxide with potassium iodide can also be mentioned.

Among these, the TBA method is suitable for analyzing lipid peroxide in the stratum corneum, and in this case, the amount of the sample to be measured is small, which is preferable. In addition, gas chromatography is suitable for analyzing simple lipids. In particular, even if the amount of a sample to be measured is small, it is preferable because multiple components can be quantified almost simultaneously by a single measurement.

Components other than lipids usually constitute the cells themselves in the stratum corneum cells. Therefore, for analysis of components other than lipids, the adhesive film 1 to which the stratum corneum cells are adhered is put into a test tube 3b, for example, as shown in (1) of FIG.
As shown in (2), the test tube 3b may be washed by adding an organic solvent with a pipette 4, for example. Washing with an organic solvent may be usually immersion for 5 to 20 minutes. Next, as shown in (3) of FIG. 2, the base portion 1a of the adhesive film 2 is removed from the test tube 3b.

Next, the cell phase and the liquid phase precipitated or suspended in a solid state in the organic solvent are separated by a centrifugal separator 5 preferably at 5,000 to 15,000 rpm as shown in FIG.
Separation takes about 30 minutes to about 30 minutes, and the organic phase is removed by a gradient method as shown in (5) to collect the cell phase B in the test tube 3b. Next, preferably, a volatile organic solvent such as ethanol is put into the test tube 3b in which the cell phase B remains by a pipette 4 as shown in (6), and the adhesive-capable solution adhering to the cell phase B is removed. It is better to wash and remove it more completely. further,
For example, as shown in FIG. 2 (7), the centrifugal separator 5 may be used again, and then most of the volatile organic solvent may be removed by a gradient method, and a small amount of the volatile organic solvent may be evaporated.

Next, various components may be extracted from the thus washed cell phase B, and the extracted various components may be quantitatively or qualitatively analyzed.

The quantitative or qualitative analysis may be performed as follows. For example, the cell phase from which the adhesive has been removed by washing is placed in pure water to extract water-soluble components, and the residue and the aqueous phase are separated. Then
The obtained aqueous phase is separated into components by liquid chromatography or the like, and each component according to the physical properties is confirmed.

Further, the residue obtained as above is boiled in a solvent of a reduction denaturing solution such as a mixed aqueous solution of sodium dodecyl sulfate and 2-mercaptoethanol, and various proteins are brought into a liquid phase by boiling. The protein is extracted and analyzed by SDS-polyacrylamide electrophoresis or the like. In the method using electrophoresis, the colloidal proteins collected on both electrodes are classified according to the rank according to mass, and the weight of the colloid allocated to each rank is measured.

Further, the components still remaining as residues after boiling as described above are hydrolyzed to amino acids by boiling in a strong acid such as hydrochloric acid. Subsequently, analysis is performed by liquid chromatography or the like.

On the other hand, in addition to analyzing the components of the stratum corneum obtained by transferring to the adhesive surface of the adhesive film 1, the component (1) in FIG.
As shown in (1), once the adhesive film 1 is peeled off on the surface of the stratum corneum and then peeled off as shown in (2), the outermost surface is removed, for example, in the form of a thin film, and the newly formed outermost surface has lipid absorption. The body may be applied and the new outermost surface absorbed and absorbed lipids may be collected and then analyzed for lipids contained in the lipid absorber.

Also in the case of this method using a lipid absorber, the number of the adhesive films 1 does not need to be limited to one. The site on the stratum corneum to be stuck is specified, each of the adhesive films 1 is stuck to the site, and each time it is peeled off, each time the adhesive film 1 is peeled off, an appropriate outermost surface is newly formed. If a lipid absorber is applied at the time, one or more lipids can be collected at the required depth.

Examples of the lipid absorber for absorbing lipid include blotting paper, filter paper, cigarette paper, absorbent cotton, and gauze.

The analysis of the lipid absorbed by the lipid absorber was carried out according to the above-described case in which the adhesive film 1 was washed with the organic solvent, and the lipid absorber was washed with the organic solvent, and the lipid extracted from the organic solvent was already described above. The analysis should be performed by a method.

In this way, for example, when trying to analyze simple lipids by, for example, gas chromatography, there is no room for the plasticizer or the like mixed in the adhesive of the adhesive tape to enter the sample to be measured, and there is little error. This is preferable because more accurate analysis is possible.

By the way, in the case of the invention in which each of the plurality of adhesive films 1 is adhered to the stratum corneum in a limited area, and the stratum corneum at each depth is collected and analyzed, for example, development of a cleaner more preferable for human skin Can provide guidance on cleaning methods, scientific proof of its effectiveness, and useful evaluation means.

Regular cleaning of lipids present on the skin surface is important for keeping the body clean. However, excessive cleaning results in rough skin. It is said to be caused by the shedding of lipids in the stratum corneum. Overwashing is caused by the number of washes and the detergency of the surfactant used. If the detergency is weak, it will not be possible to wash out lipids enough,
If it is too strong, it will wash away even the stratum corneum lipids.

Lipids present on the skin surface change to peroxide when exposed to sunlight or air. Peroxide is generated not only on the skin surface but also inside the stratum corneum. However, under what conditions the generation of lipid peroxide in the stratum corneum is generated to what depth in the stratum corneum has not been measured.

Detergent performance is sufficient if it reaches the site where lipid peroxides are formed. For proper washing, it is important to measure how much lipid peroxide is present in the stratum corneum and how much is removed by washing. The present invention is a useful evaluation tool for developing a preferable cleaning agent for the skin, providing guidance on the cleaning method, and scientifically proving its effect.

〔Example〕

 Hereinafter, examples of the present invention will be described.

<Example 1> Quantitative analysis of lipids in the stratum corneum was performed as follows using the cheeks of 10 male panelists who had washed their face with soap for 24 hours.

Hand five pieces of cellophane tape, 52mm x 24mm in size, to each person, spread it on the cheeks of each paneler, rub it with your thumb twice, and then put it in the same place.
The third and third sheets were stretched in the same manner and peeled off each time. Each operation was performed with a stripping frequency of 1 to a stripping frequency of 5, and five stratum corneum surface components having different depths were collected from each of ten persons in this way.

Collect the cellophane tapes of all members from the number of times of stripping 1 to the number of times of stripping 5 and divide them by number of times.
With respect to the lipid peroxide in the collected stratum corneum surface layer component, the average value of all the members was calculated for each time.

Specifically, each peeled tape was put in chloroform, and lipid peroxide was extracted with chloroform. Next, a TBA reagent was dropped into chloroform from which lipid peroxide had been extracted to react with malondialdehyde, and the fluorescence intensity of the formed complex at 553 nm was measured to determine the amount of lipid peroxide mixed in the extract. Next, the total value of all the quantitative values for each count was divided by the number of people to obtain an average value.

The average distribution of lipid peroxide in the stratum corneum is plotted on the horizontal axis with the number of times of stripping, and the reacted malondialdehyde (MD
A) The amount is shown on the vertical axis in FIG.

As abundance of lipid peroxide, it was possible to accurately measure a numerical distribution almost as theoretically estimated.

<Example 2> Immediately after the end of Example 1, the same 10 panelists were washed with water-based face wash, and then, on the cheek opposite to Example 1, five cellophane tapes were used one by one. In the same manner as in Example 1, five stratum corneum surface components having different depths were collected.

Based on this, the average value of the amount of lipid peroxide collected by each cellophane tape was determined for each of the stripping times 1 to 5.

In order to examine how much lipid peroxide remains even after the face washes, the average value of each stripping count before and after the face wash was compared, and the remaining amount was determined as a percentage.

FIG. 5 shows the number of times of stripping on the horizontal axis and the percentage of the residual amount of lipid peroxide on the vertical axis.

The washing removes 80% of the lipid peroxide in the first layer of the stratum corneum having a stripping frequency of 1. The second and subsequent layers of the stripping number 2 also remove 50 to 60% of the lipid peroxide.

Thus, the removal effect of the detergent on the lipid peroxide in the stratum corneum could be accurately measured.

<Example 3> One cheek stratum corneum was peeled off for another 10 panelists using the same method as in Example 1 at a stripping frequency of 1 to 5. At the same time, immediately after each time that the cellophane tape was peeled off 1 to 5 times, the blotting paper was pressed to the peeling position for 60 seconds each time to adsorb lipid to the blotting paper.

Next, each blotting paper was placed in chloroform to perform lipid extraction, and the squalene component and triglyceride component in the extracted lipid were quantified by gas chromatography, and the average value of all stripping times was determined.

In addition, the same 10 panelists were used to wash their faces with aqueous face wash potassium lauryl sulfate, and then the cellophane tape was similarly peeled off on the opposite cheek, and the lipid was adsorbed on the blotting paper each time.

Before and after face washing, to determine how much lipid remains at different depths, the squalene and triglyceride components in the lipid collected by blotting paper were determined by gas chromatography at each stripping cycle, and after washing the face. Was determined.

 The results are shown in FIG.

It was also experimentally revealed that the lipid on the upper part of the stratum corneum was removed by the detergent, but the part deeper than the third layer corresponding to stripping 3 was not washed.

<Example 4> It carried out similarly to Example 3 except having used potassium laurate instead of potassium lauryl sulfate.

 The results are shown in FIG.

It was shown that the depth at which the cleaning effect on the removal of lipids in the stratum corneum depends on the type of cleaning agent.

From this example, it was found that it was possible to evaluate how much lipid or lipid peroxide was generated from the surface of the stratum corneum toward the inside by the evaluation method described in the present invention and how much it could be washed away by washing.

<Example 5> From the skin surface of a 36-year-old male body, cell layers of the stratum corneum were collected with separate cellophane tapes (1.8 cm x 2 cm), and then the cellophane tape with the cells attached thereto was mixed with 1.5 ml of xylene. The adhesive was immersed in the solution and left at room temperature for 10 minutes to wash and remove the adhesive.

Next, the basement cellophane was removed from the xylene solution, and the xylene solution was centrifuged to collect the stratum corneum cells as a precipitate. Next, the precipitate was washed with ethanol to remove the adhered xylene solution, and the cells were again centrifuged to isolate stratum corneum cells.

Next, the stratum corneum cells thus isolated and obtained are boiled in a mixed aqueous solution containing 2% by weight of sodium dodecyl sulfate (hereinafter, referred to as SDS) and 1% by weight of 2-mercaptoethanol, and the soluble protein After extracting the SD
Soluble proteins were analyzed by S-polyacrylamide electrophoresis. The electrophoresis was performed according to Laemli's method (Nature, Vol. 227, 1970).

From the above results, the mass of the molecule of each component in the stratum corneum was decoded in dalton units.

The forearm is 1, the upper arm is 2, the abdomen is 3, the thigh is 4, the back is 5, the hoho is 6, the nose is 7, the jaw is 8, the palm is 9, the sole is 10, and the horizontal axis is Dalton. The vertical axis represents the molecular weight of each component indicated by, and the components are sorted according to the difference in molecular weight for each part, and the total mass of each component is represented by the thickness of the line, and is shown in FIG.

4 to 5 for stratum corneum proteins on body surface other than 1 to 5
Keratin-specific bands of the book appeared uniformly.

The stratum corneum of the face of 6-8 has 55 of the keratin bands.
K-dalton keratin was strongly revealed. A strong band also appeared around 45k.

Keratins of different molecular weights could be detected in the palms and soles of 9-10.

It was found to be consistent with the theory that exposed skin, such as the face, which is less susceptible to external stimuli, shows a different pattern from unexposed skin.

<Example 6> The stratum corneum surface components were collected from the forearms of 11 male and female panelists aged 20 to 60 years according to Example 5, and the mass of each component molecule in the stratum corneum was decoded in daltons. The results of the change in the amount of keratin by age according to age are shown according to Example 5.

The results are shown in FIG. The horizontal axis shows individual male and female panelists, where m represents a male and f represents a female.

It was found that the amount of extracted protein was different depending on the age, and the amount was particularly large in the elderly.

<Example 7> The stratum corneum was collected from the forearm of a 36-year-old male panelist using cellophane, and quantitative analysis of amino acids was performed.

That is, according to Example 5, the stratum corneum cells were isolated from the forearm skin, and then dried by air drying.

The total weight of the dried stratum corneum cells was 120 ± 35 μg. Then, distilled water was added to a part of the dried stratum corneum cells to extract the water-soluble amino acid, and then the water-soluble amino acid and the residue were separated, and the obtained residue was poured into a sufficient amount of 6N-hydrochloric acid, It was hydrolyzed by heating at 110 ° C. for 24 hours.

Separately, the rest of the dried keratinocytes was replaced with 2 SDSs.
The soluble protein is extracted by boiling in a mixed aqueous solution containing 1% by weight of 2-mercaptoethanol and 1% by weight of 2-mercaptoethanol. Then, the soluble protein and the residue are separated, and the obtained residue is dissolved in a sufficient amount of 6N-hydrochloric acid. And heated at 110 ° C. for 24 hours to hydrolyze.

Each of the water-soluble amino acid group, the constituent amino acid group of the stratum corneum cell constituent protein, and the constituent amino acid group of the stratum corneum cell insoluble protein thus obtained was subjected to component analysis using liquid chromatography.

The constituent amino acid group of the stratum corneum cell constituent protein is the stratum corneum total amino acid (a), the constituent amino acid group of the stratum corneum cell insoluble protein is the amino acid (b) of the insoluble stratum corneum protein, and the water-soluble amino acid group is the free amino acid (c);
The results of the analysis are shown in Table 1.

In addition, the total weight in Table 1 represents the total measurement of all the amino acids quantified by the amino acid quantification in each liquid. Proline, cysteine and tryptophan were excluded from the measurement.

<Reference Experiment 1> Stratum corneum cells were collected from 11 forearms of different ages between 20 and 65 years old using cellophane tape. In the harvested stratum corneum cells, the cornified envelope
velope keratinized skin or marginal b
The ratio of ε- (γ-glutamyl) lysine cross-linking existing in the peripheral zone) was examined.
Changes in age were tracked in two groups of names.

Pisano et al (1969) Bioc
Based on hemistry Vol. 18, the amount of lysine detected by cyanoethylation of the free lysine residue in the conified envelope with acrylonitrile and acid hydrolysis is expressed as% of the total amount of crosslinked and uncrosslinked lysine.

 The results are shown in Table 2.

<Reference Experiment 2> Using cellophane tape, the number of times of tape stripping was set to 10, and stratum corneum layers having different depths were collected in layers from the human forearm to 10 layers.

Elemental analysis of the first, third, fifth and tenth layers of the stratum corneum thus obtained was performed on calcium, sulfur and phosphorus.

 The results are shown in FIG.

〔The invention's effect〕

Since the present invention is configured as described above, the composition of the stratum corneum surface layer component can be easily and accurately known.

[Brief description of the drawings]

(1) to (4) of FIG. 1 are diagrams illustrating a method of analyzing a lipid component, (1) of FIG. 1 is a diagram showing a state in which an adhesive film is stuck to a stratum corneum, and (2) of FIG. ) Is a diagram showing how a load is applied to the adhesive film, (3) in FIG. 1 is a diagram showing a state where the adhesive film is placed in a test tube with a cap, and (4) is a pipette containing an organic solvent. FIGS. 2A to 2D illustrate a method of analyzing components other than lipids, and FIG. 2A illustrates a state in which an adhesive film is placed in a test tube with a cap. FIG. 2 (2) is a diagram showing a state where an organic solvent is injected into a test tube with a pipette using a pipette, and FIG. 2 (3) is a diagram showing a state where a base portion of an adhesive film is removed. FIG. 2 (4) of FIG.
Is a diagram showing a state in which components are centrifuged, (5) in FIG. 2 is a diagram showing a state in which an organic solvent is excluded by a gradient method, and (6) in FIG. 2 is a pipette for volatilization of ethanol and the like. Figure 2 shows a state in which a volatile organic solvent is placed in a test tube.
FIG. 7 (7) shows a state of centrifugal separation, FIG. 2 (8) shows a state of evaporating a volatile organic solvent, and FIG. 3 shows removal of the outermost surface as a thin film. A diagram showing a method of analyzing by a new outer surface bleeding lipid generated by
FIG. 3 (1) is a view showing a state in which an adhesive film is peeled off on the surface of the stratum corneum, FIG. 3 (2) is a view showing a state in which the adhesive film is peeled off, and FIG. FIG. 5 is a diagram showing the average distribution of lipid peroxide before, FIG. 5 is a diagram showing the residual ratio of lipid peroxide after face washing when a sample was collected on the tape adhesive surface,
FIG. 6 is a diagram showing the percentage of the residual amount of lipid peroxide after face washing when a sample is collected with a blotting paper, FIG. 7 is a diagram showing the total mass of each component, and FIG. 8 is a change in the amount of keratin by age. FIG. 9 is a graph showing the results of elemental analysis. 1 ... Adhesive film

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masahiro Sato 27-1, Takashimadai, Kanagawa-ku, Yokohama-shi, Kanagawa Prefecture Inside of the Yokohama Research Laboratory, POLA CHEMICAL INDUSTRIES, LTD. (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/92 G01N 33/50 BIOSIS

Claims (12)

(57) [Claims] 1. An adhesive film having an adhesive surface provided by laminating an adhesive on the surface of a base material is once adhered to the stratum corneum, and then peeled off, whereby the stratum corneum is transferred to the adhesive surface. A method for analyzing the components of the stratum corneum, in which the functional film is washed with an organic solvent, whereby the lipid in the stratum corneum transferred to the adhesive surface is extracted into the organic solvent, and then the lipid extracted in the organic solvent is analyzed. 2. An adhesive film having a pressure-sensitive adhesive surface provided by laminating a pressure-sensitive adhesive on the surface of a base material and then peeling off the pressure-sensitive adhesive layer once to the stratum corneum, thereby transferring the stratum corneum to the pressure-sensitive adhesive surface. A method for analyzing the components of the stratum corneum, in which the film is washed with an organic solvent, thereby releasing the cells of the stratum corneum transferred to the adhesive surface into the organic solvent, and then analyzing the components of the cells liberated in the organic solvent. 3. A plurality of adhesive films each having an adhesive surface provided by laminating an adhesive on a substrate surface,
By limiting the site, once sticking and peeling off each time, the stratum corneum is sequentially transferred to each adhesive surface at each depth, while each adhesive film to which the stratum corneum is transferred in this way is organic solvent. By extracting the lipid at each depth of the stratum corneum into an organic solvent, and then analyzing the lipid extracted into the organic solvent,
A method for analyzing a stratum corneum component that analyzes a lipid component of a stratum corneum at each depth. 4. A plurality of adhesive films each having an adhesive surface provided by laminating an adhesive on a substrate surface,
By limiting the site, once sticking and peeling off each time, the stratum corneum is sequentially transferred to each adhesive surface at each depth, while each adhesive film to which the stratum corneum is transferred in this way is organic solvent. The cells at each depth of the stratum corneum are released into the organic solvent, and then the components of the cells released into the organic solvent are analyzed to analyze the cell components of the stratum corneum at each depth. Method for analyzing stratum corneum components. 5. An adhesive film having an adhesive surface provided by laminating an adhesive on the surface of a base material, is once adhered to the surface of the stratum corneum, and thereby the outermost surface is adhered to the adhesive surface and removed. A lipid absorber is applied to the newly formed outermost surface, lipids oozing on the new outermost surface are absorbed by the lipid absorber and collected, and then the stratum corneum is analyzed by the lipid absorber for the collected lipid. How to analyze the components. 6. The method according to claim 1, wherein the analysis is a quantitative or qualitative analysis.
5. The method for analyzing a stratum corneum component according to any one of 5. 7. The method for analyzing a stratum corneum component according to claim 1, wherein the analysis of lipids is an analysis of lipid peroxides performed using thiobarbituric acid. 8. The method for analyzing a stratum corneum component according to claim 1, wherein the lipid analysis is a simple lipid analysis performed by gas chromatography. 9. The method for analyzing the components of the stratum corneum according to claim 2, wherein the analysis of the components of the released cells is carried out by hydrolyzing intracellular protein components to amino acids and quantitatively analyzing the components by liquid chromatography. . 10. The analysis of the components of the released cells is carried out by converting the cell components into colloids, electrophoresing the colloids, classifying each component particle according to the rank by mass, and measuring the amount of colloids allocated to each rank. The method for analyzing a stratum corneum component according to claim 2 or 4, which is performed by the following method. (11) the pressure-sensitive adhesive is natural rubber or a derivative thereof, SB
One or more rubbers selected from the group consisting of R-based rubber, NBR-based rubber, butadiene-vinylpyridine-based rubber, butyl rubber, chloroprene-based rubber, recycled rubber, cyanoacrylate-based rubber, arabic rubber, and tragacanth rubber The method for analyzing a stratum corneum component according to any one of claims 1 to 5, which is a mixture. 12. The method for analyzing a stratum corneum component according to claim 1, wherein the adhesive film is a cellophane tape.