JP4954908B2 - Evaluation method of skin quality using the amount of soluble protein in stratum corneum as an index - Google Patents

Description

  The present invention relates to a method for evaluating skin quality by measuring a quantitative ratio of soluble protein to insoluble protein of stratum corneum protein.

  Accurate grasping of skin quality (or skin condition) is important for accurate skin care for maintaining healthier skin. Therefore, when performing skin care with cosmetics, the skin quality of cosmetic users has been evaluated through, for example, an inquiry by a beauty engineer. In addition, for the purpose of objective evaluation of skin quality, the state or function of the skin is also evaluated using various types of measuring devices and the parameters observed or measured.

  Typical examples of such parameters are skin surface morphology by magnifying a replica of the skin surface and observing skin grooves and cuticles, stratum corneum water content by conductance measurement, and transepidermal water. Examples include a stratum corneum barrier function by measuring a transepidermal water loss (TEWL).

  In addition, natural moisturizing factor (NMF, specifically various free amino acids, etc.) is quantified as an index of the moisturizing ability of the stratum corneum, and specific proteins such as cytokines and calprotectin in the stratum corneum are quantified. Also, methods for evaluating skin quality are being applied (for example, JP-A-10-216106, JP-A-2007-33201, etc.). However, these have the problem that it is necessary to assay the protein by ELISA or the like, which is troublesome.

  The stratum corneum is composed of keratinocytes formed by terminal differentiation of epidermal keratinocytes and intercellular lipids surrounding them. It has become clear that intercellular lipids play an important role in the stratum corneum barrier function by forming a lamellar structure using ceramide, cholesterol, fatty acid and the like as components. This is supported by the fact that intercellular lipids are disordered both morphologically and compositionally in cases involving various skin diseases in which the stratum corneum barrier function decreases and skin troubles such as rough skin.

  On the other hand, keratinocytes are composed of a keratin fiber as a main component and a confined envelope (CE) enclosing it. CE is formed by cross-linking and insolubilizing a plurality of CE precursor proteins produced as epidermal keratinocytes differentiate with the enzyme transglutaminase. Furthermore, it is suggested that ceramide, etc. is covalently bonded to a part of it and forms a hydrophobic structure, thereby providing the foundation of the lamellar structure of the intercellular lipid described above and forming the basis of the stratum corneum barrier function. ing.

  CE boiled skin tissue or cultured skin cells in a solution containing a surfactant such as sodium dodecyl sulfate and a reducing agent such as mercaptoethanol, and an insoluble fraction from which soluble components have been removed by means such as centrifugation. It can be prepared by obtaining. By observing the form with a microscope, the property can be evaluated. Michel et al. Report that there are more CEs with a fragile structure deeper in the stratum corneum than in the outermost layer of the stratum corneum (J. Invest. Dermatol 91: 11-15, 1988). On the other hand, it is said that fragile CE is also observed in the outermost layer in psoriasis and phylloid ichthyosis (Br. J. Dermatol. 122: 15-21, 1990).

  Japanese Patent Application Laid-Open No. 2001-91514 discloses a skin quality evaluation method using the dyeability of CE. Specifically, it stains CE in a skin-derived stratum corneum sample with a dye that can selectively stain a hydrophobic region, and uses the staining property of the CE as an index for evaluation. Although the CE staining method is excellent as visual data, it is not quantitative and the method is somewhat complicated, and there is a difficulty in assaying a large amount of skin samples.

Japanese Patent Laid-Open No. 10-216106 JP 2007-33201 A JP 2001-91514 A J. Invest. Dermatol 91: 11-15, 1988 Br. J. Dermatol. 122: 15-21, 1990

  If it is possible to evaluate skin quality, specifically rough skin, quantitatively and in a simple manner, it is expected to be extremely beneficial in terms of dermatology and cosmetics. For example, the cosmetic industry in recent years It can be an effective means for providing counseling services for advice such as appropriate skin care methods.

  In view of the above facts, the present inventor examined the relationship between the amount ratio of soluble protein and insoluble protein in the stratum corneum and the rough skin state, and found that the ratio of the soluble protein increased as the rough skin state progressed. .

Based on the elucidation of the above facts, the present application includes the following inventions.
(1) A skin quality evaluation method using an amount ratio of soluble protein of stratum corneum as an index,
(A) The adhesive tape sample with the stratum corneum obtained by tape stripping is immersed in an aqueous buffer solution to elute soluble protein in the aqueous buffer agent,
(B) separating the adhesive tape sample from the aqueous buffer and quantifying the soluble protein eluted in the buffer.
(C) The adhesive tape sample separated in step (b) is then immersed in a reducing agent-containing buffer to elute insoluble protein,
(D) separating the adhesive tape sample from the reducing agent-containing aqueous buffer, quantifying the insoluble protein eluted in the buffer,
(E) determining the ratio of the quantitative value of the soluble protein to the quantitative value of the insoluble protein;
A method comprising that.
(2) The method according to (1), wherein the adhesive tape sample having a stratum corneum obtained by tape stripping is freeze-ground.
(3) The method of (1) or (2), wherein the reducing agent is 2-mercaptoethanol.

  According to the present invention, the higher the ratio of soluble protein in the stratum corneum, the more skin is judged to be, and the skin quality can be evaluated easily and quantitatively.

  The stratum corneum to be measured in the present invention is collected from the skin or the like by a tape stripping method. Tape stripping is performed by attaching and peeling an adhesive tape piece on the skin or the like. The preferred method of tape stripping is to first clean the surface layer of the skin with ethanol, etc. to remove sebum, dirt, etc., and lightly place an adhesive tape piece cut to an appropriate size (eg 2 x 5 cm) on the skin surface. This is done by applying a uniform force to the entire tape and pressing it flat, and then peeling off the adhesive tape with a uniform force. The adhesive tape may be a commercially available cellophane tape or the like, and for example, cello tape (registered trademark) (manufactured by Nichiban), Scotch Superstrength Mailing Tape (manufactured by 3M) or the like can be used. The skin part may be any part of the body, and may include, for example, the facial cheek, forehead, back and trunk.

  Preferably, the adhesive tape with the stratum corneum is frozen in, for example, liquid nitrogen (about −180 ° C.) and finely pulverized with a freeze press pulverizer or the like to facilitate protein elution. As the freeze press crushing apparatus, for example, Microtech Nichion's Cryopress (registered trademark) can be used. The cryopress treatment is performed by pre-cooling the stainless steel inner cell and lid with liquid nitrogen, putting a tape sample cut to about 5 mm square inside, placing it in the cryopress, and applying the pulsed pressure applied from the top. Can be carried out by grinding.

  As the aqueous buffer for eluting soluble protein from the adhesive tape with the stratum corneum attached, any buffer generally used in the biochemical field can be used, and is not particularly limited. For example, water, physiological saline , Phosphate buffer, borate buffer, TRIS buffer, HEPES buffer, CHAPS buffer, and the like. If necessary, a surfactant such as Tween-20 or Triton X-100 may be added to the buffer. The soluble protein herein means a water-soluble precursor protein that constitutes CE and is not cross-linked or insolubilized. Specific proteins include, for example, involucrin, loricrin, small proline rich, Examples include protein (SPR, cornifine), cystatin A, elafin, filaggrin, keratin, emboplatin, desmosome-constituting protein, annexin I, PAI-2, and the like. Extraction of soluble protein can be performed at room temperature or low temperature (for example, around 0 ° C.), but if necessary, boil or sonicate for several minutes, for example, 1 to 30 minutes, preferably about 10 minutes. You can go.

  The reducing agent-containing buffer for eluting insoluble protein from the adhesive tape to which the stratum corneum is attached is based on the above aqueous buffer, and preferably 2-mercaptoethanol, preferably 0.1 to 5% by mass as the reducing agent. More preferably, it can be used at a concentration of 1 to 3% by mass. Other reducing agents such as dithiothreitol, which are widely used in the biochemical field, can also be used. The insoluble protein referred to here is, for example, that the above-mentioned precursor protein constituting CE forms an isopeptide bond between its lysine residue and glutamic acid residue, or the glutamine residues are pseudo-mediated by polyamines. This means that CE is formed by cross-linking and insolubilization by forming an isopeptide bond. Extraction of insoluble protein can be performed at room temperature or the like, but is preferably performed under high temperature conditions such as boiling. Preferably, the extraction operation is performed by boiling for 2 to 10 minutes.

  Any method commonly used in the field of biochemistry can be used as the protein quantification method, and it is not particularly limited. All methods such as law are listed. Protein quantification can be easily performed using a commercially available kit. For example, BioRad's DC protein assay, RC DC protein assay, or the like can be used.

  In the present invention, no specific standard is specified, but the standard for determining that the skin quality is rough is that the mass ratio of soluble protein in the total amount of protein in the stratum corneum is, for example, 3% or more, particularly 5 % Or more.

  Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Experiment 1: Quantification of soluble protein and insoluble protein Tape stripping was performed on the cheeks of 39 subjects to prepare an adhesive tape sample with a stratum corneum attached. Cello tape (registered trademark) (Nichiban) was used as the adhesive tape. This adhesive tape sample was cut into 15 cm ² , frozen with liquid nitrogen (−180 ° C.), and freeze-ground with Cryopress (registered trademark) (Microtech Nithion). This frozen and ground sample was dispensed into a 1.5 ml tube containing 300 ml of 0.1M Tris-HCl (pH 8.0), 0.14M NaCl, 0.1% Tween-20, and left for 30 minutes to dissolve soluble protein. Extracted.
The tube was centrifuged (15,000 g, 10 minutes), the supernatant was removed, and the protein was quantified with a DC protein assay kit (BioRad).

Next, 300 ml of 1 × Laemmli Sample Buffer solution containing 2.5% 2-mercaptoethanol was added to the adhesive tape sample settled in the tube and boiled for 2 minutes to extract insoluble protein. The tube was centrifuged (15,000 g, 10 minutes), and the protein was quantified with a DC protein assay kit (BioRad).
FIG. 1 shows the relationship between the amount of soluble protein and the amount of insoluble protein in each subject. In general, the more rough the skin is, the more stratum corneum is peeled off, and the amount of stratum corneum collected by tape stripping increases, and the resulting protein (both soluble and insoluble proteins). The total amount of is expected to increase. As the graph curve in FIG. 1 shows, the sample in which both the soluble protein and the insoluble protein have higher values, that is, the sample of the subject who is in a rough skin state, the slope of the graph curve is slanted. It can be seen that the ratio is high.

  FIG. 2 is a graph showing the results of Experiment 1, with the horizontal axis representing soluble protein and the vertical axis representing the ratio of soluble protein to insoluble protein. From this figure, it can be seen that the rougher skin sample with a larger amount of protein has a higher ratio of soluble protein. Therefore, it can be seen that the stratum corneum protein in a rough skin state is not only a large amount of peel but also a qualitatively soluble protein.

Experiment 2: Relationship between soluble protein ratio and TEWL In addition to the soluble protein ratio, TEWL as a parameter of each subject's skin barrier function was also examined, and the correlation between the soluble protein ratio and TEWL was examined. The result is shown in FIG. (A) shows TEWL measured values by Vapometer (Delfin Technologies Ltd., Finland), and (b) shows TEWL measured values by TEWAmeter (Tewameter TM210; Courage + Khazaka., Germany). The correlation coefficient between the ratio of soluble protein and TEWL was 0.3629 for Vapometer and 0.372 for TEWAmeter, both of which were significantly correlated. Therefore, it was found that the TEWL is high and the ratio of the rough protein and the soluble protein correlate.

Experiment 3: Relationship between the effect of skin care and the ratio of soluble protein Nine subjects were subjected to skin care treatment on their cheeks, and the change in the soluble protein ratio before and after the skin care treatment was examined. DP lotion (d-program products (lotion (lotion 2), milky lotion (emulsion 2), cream (cream AD))) was used for skin care and continued for 1 month. The result is shown in FIG. It can be seen that all soluble protein ratios are significantly reduced in subjects before and after skin care. Moreover, FIG. 5 shows the average value of the soluble protein ratio before and after DP lotion continuous use of all the subjects. It is clear from these results that the soluble protein ratio is reduced by improving the rough skin by skin care.

Relationship between the amount of soluble protein and the amount of insoluble protein. The graph which showed the ratio of the soluble protein with respect to the insoluble protein for the result of Experiment 1 on the horizontal axis | shaft on the horizontal axis | shaft, and the vertical axis | shaft. Relationship between soluble protein ratio and TEWL. Relationship between skin care effect and soluble protein ratio. Relationship between skin care effect and soluble protein ratio.

Claims (3)

An evaluation method of skin quality using an amount ratio of soluble protein of stratum corneum as an index,
(A) The adhesive tape sample with the stratum corneum obtained by tape stripping is immersed in an aqueous buffer solution to elute soluble protein in the aqueous buffer agent,
(B) separating the adhesive tape sample from the aqueous buffer and quantifying the soluble protein eluted in the buffer.
(C) The adhesive tape sample separated in step (b) is then immersed in a reducing agent-containing buffer to elute insoluble protein,
(D) separating the adhesive tape sample from the reducing agent-containing aqueous buffer, quantifying the insoluble protein eluted in the buffer,
(E) determining the ratio of the quantitative value of the soluble protein to the quantitative value of the insoluble protein;
A method comprising that.   The method according to claim 1, wherein the pressure-sensitive adhesive tape sample with the stratum corneum obtained by the tape stripping is freeze-pulverized in advance.   The method according to claim 1 or 2, wherein the reducing agent is 2-mercaptoethanol.

Images