JP5595164B2 - Evaluation model of dermal yellowing - Google Patents

Description

  The present invention relates to a method for evaluating a substance that causes skin discoloration, a substance that suppresses skin discoloration, or a method for screening a substance that suppresses skin yellowing.

  Skin color is one of the important cosmetic elements and is known to be determined by the optical properties of the tissues that make up the skin and the various pigment components in the skin tissues. The optical characteristics of the tissue constituting the skin include light absorption and scattering due to the skin being a translucent solid. Specifically, it has been reported that the stratum corneum and epidermis contribute mainly to light absorption, and the dermis contributes largely to light scattering (Rerson, RR et al., J. Invest. Dermatol. 77: 13-19, 1981). For this reason, in order to evaluate the dermis color, an evaluation in a solid state that can be evaluated including the contribution of scattering is desired. Until now, the evaluation of dermal color has been a method of solubilizing the protein constituting the dermis with acid or alkali and measuring the absorbance with a spectrophotometer or the like. It was not possible to say that the skin color of the skin was fully evaluated. Therefore, a color evaluation method in a solid state capable of evaluating an apparent color is desired.

  Major pigment components that affect skin color include melanin and hemoglobin. Melanin is a dark brown pigment produced by melanocytes, which are pigment cells present in the epidermis, and is classified into eumelanin that exhibits black color and pheomelanin that exhibits red color. Hemoglobin is present in the blood, and the state in the capillaries existing above the dermis affects the skin color. The color tone changes depending on whether or not it binds to oxygen. Oxyhemoglobin, which is oxidized hemoglobin bound to oxygen, exhibits a bright red color, and deoxyhemoglobin, which is a reduced hemoglobin that releases oxygen, exhibits a dark red color. For this reason, excessive production of melanin pigment is a cause of typical senile appearance as age spots, and a decrease in hemoglobin oxygen saturation associated with blood flow stagnation is one of the causes of bears that develop under the eyes. It is thought that.

  As pigments other than melanin and hemoglobin, as a factor affecting skin color, bilirubin, which is a degradation product of hemoglobin and presents in serum and exhibits yellow color, and bilirubidine present in tissue in the case of internal bleeding, exhibits yellow color. It is also known that carotenoids, which are food-derived pigments, migrate from the blood to the epidermis.

  The skin color is determined by the complex factors of these pigments, and changes in the skin color due to aging, stress, etc. are also a cause of cosmetic problems. As one of them, a change in the color of the skin called yellowing is known, and the colorimetric data indicates that the hue of the skin actually turns yellow with aging (Nishimori, Y. et al., Skin Res Technol. 4: 79-82, 1998).

  Yellowing of the skin is also known as yellowing that causes the skin to lose its transparency, which may include factors such as deterioration of the proteins that make up the skin, as well as quantitative or qualitative changes in pigments. Denaturation is possible. That is, it is speculated that the modification of the protein constituting the skin tissue causes a change in the absorption spectrum related to the skin tone, and is one of the factors in the yellowing of the skin. Glycation is known as a protein modification that causes such a change in absorption spectrum. Protein saccharification is well studied in the food field for coloring phenomena associated with food heating and fermentation, but the proteins that make up the epidermis and dermis, including the stratum corneum, are also actually glycated in the skin. (JP 2009-8460; 129th Annual Meeting of the Pharmaceutical Society of Japan. 2009, 27P-pm168-169; Mizutari K. et al., J. Invest Dermatol. 108: 797-802, 1997; Jeanmaire , C. et al., Br. J Dermatol. 145: 10-18, 2001), there is also a report that indirectly showed that glycation of the proteins that make up the skin contributes to yellowing of the skin (Ohshima H. et al., Skin Res Technol. 15: 496-502, 2009). However, there is no direct evidence that glycation of the proteins that make up skin tissue is involved in skin yellowing, and the relationship between protein modifications other than glycation and skin yellowing Little research has been done.

  As a general modification of proteins other than glycation, carbonylation can be considered. Carbonylated proteins are known to be detected in various tissues in the body, and for example, it is reported that they generally increase with age in the liver and brain (Stadtman ER et al., EXS. 62: 64-72, 1992). In addition, it has been reported that carbonylated protein increases in progeria, etc. that appear with accelerated aging (Stadtman ER et al., J biol Chem. 262: 5488-5491, 1987). Has been studied. The presence of carbonylated protein is also known in the skin, and accumulation in the horny layer of the exposed area and in the dermis of the photoaging site has been reported (Fujita H. et al., Skin Res Tech. 13: 84- 90, 2007; Sander, CS et al., J Invest Dermatol. 118: 618-25, 2002). Specifically, it has been reported that the carbonylated protein in the exposed stratum corneum causes changes in physical properties such as a decrease in optical transmission (Iwai I. et al., Int J Cosmet Sci. 30: 41- 46, 2008), and dermal carbonylated proteins have been reported to accumulate depending on the severity of photoelastic fibrosis (Tanaka, N. et al., Arch Dermatol Res. 293: 363-367). , 2001; Sander, CS et al., J Invest Dermatol. 118: 618-25, 2002). However, the relationship between the carbonylation of the protein constituting the skin tissue and the yellowing of the skin has never been clarified so far.

JP2009-8460

Rerson, R.R. et al., J. Invest. Dermatol. 77: 13-19, 1981 Nishimori, Y. et al., Skin Res Technol. 4: 79-82, 1998 The 129th Annual Meeting of the Pharmaceutical Society of Japan. 2009, 27P-pm168-169 Mizutari K. et al., Invest Dermatol. 108: 797-802, 1997 Jeanmaire, C. et al., Br. J Dermatol. 145: 10-18, 2001 Ohshima H. et al., Skin Res Technol. 15: 496-502, 2009 Stadtman E.R. et al., EXS. 62: 64-72, 1992 Stadtman E.R. et al., J biol Chem. 262: 5488-5491, 1987 Fujita H. et al., Skin Res Tech. 13: 84-90, 2007 Sander, C.S. et al., J Invest Dermatol. 118: 618-25, 2002 Iwai I. et al., Int J Cosmet Sci. 30: 41-46, 2008 Tanaka, N. et al., Arch Dermatol Res. 293: 363-367, 2001 Thiele, J.J. et al., Curr Probl Dermatol. 29: 26-42, 2001

  Skin discoloration is known to occur with stress, aging, and the like. In particular, yellowing of the skin causes a cosmetic problem because it loses the transparency of the skin. Therefore, the search for substances having an action of suppressing skin discoloration, particularly yellowing, is extremely important in the beauty industry and the pharmaceutical industry. The subject of this invention is providing the method for evaluating and selecting the substance which suppresses discoloration of skin, especially yellowing simply and objectively.

  The present inventor created a high-density collagen gel-like dermis model that mimics the dermis of living skin based on the knowledge that the modification of the protein present in the dermis is closely related to the discoloration of the skin. By measuring the discoloration in a solid state, a method for evaluating discoloration of the skin that fully reflects the optical characteristics of the tissue constituting the living body skin was completed. Surprisingly, if the dermis model is treated with an aldehyde that induces carbonylation to experimentally carbonylate the protein in the dermis model, it is considered to be one of the major factors of skin yellowing so far. We found that the dermis model was significantly yellowed more than if it had been led by saccharification. Based on this finding, the present inventors have succeeded in producing an evaluation system that sufficiently reflects the yellowing state of the skin observed in a living body.

This application includes the following inventions:
[1] A method for evaluating a causative factor of skin discoloration, wherein a gel-like dermis model in which collagen is densely concentrated is exposed to a candidate factor, and a change in color tone in the gel-like dermis model is detected. The method is characterized by evaluating from the change in spectral reflectance using a color difference meter or the like.
[2] The method according to [1], wherein an L ^* a ^* b ^* color system or a Munsell color system is used for evaluating a color tone change.
[3] A method for evaluating a substance that suppresses skin yellowing, wherein a substance causing yellowing is brought into contact with a candidate substance in a high-density collagen gel dermis model, and the gel-like dermis model A method of evaluating a change in color tone from a change in spectral reflectance using a color difference meter or the like.
[4] For the evaluation of the color tone change, the b ^* value of the L ^* a ^* b ^* color system or the YR value or Y value in the hue (Hue) of the Munsell color system should be used as an index of skin yellowing. The method according to [3], characterized in that
[5] The method according to [3] or [4], wherein the substance that causes yellowing is an aldehyde that induces carbonylation.
[6] The method according to any one of [3] to [5], wherein the aldehyde that induces carbonylation is acrolein or 4-hydroxy-2-nonenal.
[7] A method for screening a substance that suppresses skin yellowing, comprising contacting a candidate substance with an aldehyde that induces carbonylation in a high-density collagen gel dermis model When the detection level of the carbonyl group present in the dermis model is lower than when the candidate substance is not contacted, the candidate substance is selected as a substance that suppresses skin yellowing And how to.
[8] The method according to [7], wherein the aldehyde for inducing carbonylation is acrolein or 4-hydroxy-2-nonenal.
[9] The method according to [7] or [8], wherein the detection of the carbonyl group is performed using a labeling reagent having a hydrazino group.
[10] A method for producing a high-density collagen gel-like dermis model that mimics discoloration of living skin, wherein a culture solution containing fibroblasts is added to an acid-soluble collagen solution to neutralize the collagen in the solution. Culturing the fibroblasts until the collagen gel is sufficiently densified, and immersing the densified collagen gel in sterile water to kill fibroblasts surviving in the gel A method characterized by.
[11] The high-density collagen gel-like dermis model that mimics the discoloration of living skin, produced by the method according to [10].

  According to the present invention, it is possible to simply and objectively evaluate and select a substance effective for suppressing discoloration of the skin, in particular, yellowing of the skin.

The influence with respect to the yellowing of the dermis model by a control (phosphate buffer), D-ribose, glyoxal, 4-hydroxy-2-nonenal, and acrolein is shown. The action which suppresses yellowing of the dermis model by pyridoxine hydrochloride is shown. The action which suppresses yellowing of the dermis model by an olive leaf extract is shown.

  In one embodiment of the present invention, a method for assessing a causative factor of skin discoloration, wherein a high density collagen gel dermis model is exposed to a candidate factor and the color change in the gel dermis model Is evaluated from a change in spectral reflectance using a color difference meter or the like.

  In order to provide an evaluation system that sufficiently mimics the skin discoloration observed in the living body, it is preferable that the dermis model used reflects the composition of the living skin. More than 90% of the dermis is type I collagen and exists in a very dense state. For this reason, it is preferable that the dermis model used in this invention is comprised from the high density collagen. As a material for producing such a dermis model, for example, an acetic acid-solubilized type I collagen solution derived from bovine dermis can be used. A translucent collagen gel can be prepared by neutralizing and diluting type I collagen and refibrosis.

  Furthermore, in order to produce a collagen gel concentrated at high density, it is preferable to use mammalian dermis-derived fibroblasts. The fibroblast can be any mammalian fibroblast, but fibroblasts derived from human dermis are preferred. Fibroblasts are the most important cells present in the dermis. The culture medium for fibroblasts is not particularly limited, and Dulbecco's modified Eagle medium (DMEM) containing fetal bovine serum can be used. When fibroblasts are cultured in type I collagen gel, collagen fibers are attracted around the cells, so that the density of collagen is concentrated, and a high-density collagen gel as seen in the dermis of a living body can be produced. . In addition, when the collagen gel produced in this way is cultured for a long time, the extracellular matrix produced by fibroblasts is produced in the gel, so it is possible to create a dermis model with a composition closer to that of living dermis. become. Fibroblasts are cultured for at least 2 days or more, preferably 5 days or more, more preferably 1 week or more. In addition, in order to eliminate the secondary factors that cells in the collagen gel respond to and more clearly capture the discoloration of the protein, the collagen gel thus obtained is immersed in sterilized water and subjected to a low tone treatment. By doing so, it is preferable to kill the living cells in the gel. By this treatment, the medium and soluble components are sufficiently removed, and a dermis model formed only from the dermal extracellular matrix that does not contain living cells can be produced. Therefore, in another aspect of the present invention, there is provided a method for producing a high-density collagen gel dermis model that mimics discoloration of living skin, wherein a solution containing fibroblasts is added to an acid-soluble collagen solution, Neutralize the collagen therein, culture the fibroblasts until the collagen gel is sufficiently densified, and survive in the gel by immersing the densified collagen gel in sterile water There is provided a method characterized by killing fibroblasts, and a high density collagen gel dermis model that mimics the discoloration of living skin made by such a method.

  In this way, it is possible to produce a dermis model very similar to the living dermis constituted by the extracellular matrix having a composition and density close to that of the living dermis.

  The causative factor of skin discoloration can be evaluated by exposing the dermis model prepared above to a candidate factor and measuring the color tone of the dermis model. Candidate factors may be any chemical or physical factor that has the potential to induce or suppress discoloration of the dermis model. For example, when it is desired to evaluate a factor that induces skin discoloration as a causal factor of skin discoloration, it is performed by directly evaluating the color tone of the dermis model after exposing the dermis model to a candidate factor. On the other hand, when it is desired to evaluate a factor that suppresses skin discoloration as a causative factor of skin discoloration, it is performed by exposing the dermis model to a protein modifier in addition to the candidate factors. When the dermis model is exposed to a protein modifier, a color change of the dermis model is induced by modifying the protein in the dermis model. Such a protein modifier means any factor that can induce skin discoloration by modifying the protein that constitutes the skin. For example, in addition to chemical or biological substances such as oxidants and enzymes, ultraviolet light, dryness, etc. It also includes physical factors such as The dermis model treated with the protein modifier can be used as an evaluation system that sufficiently reflects the skin discoloration seen in the living body. Specifically, a protein modifier and a candidate substance are applied to the dermis model, reacted under conditions sufficient to cause skin discoloration, and then the color of the dermis model is evaluated to evaluate the candidate substance. It becomes possible to evaluate the inhibitory effect on skin discoloration.

Further, when the aldehyde that induces carbonylation is treated as such a protein modifier, the protein constituting the dermis model is carbonylated to induce yellowing of the dermis model. Protein carbonylation means a general term for a phenomenon in which the amino acid side chain of a protein is modified with various highly reactive aldehydes to introduce a carbonyl group into the protein. The introduction of carbonylation involves reaction when NH ₂ groups of amino acid residues such as Lys, Arg, and Pro in proteins are directly oxidized to carbonyl groups, and when lipids are oxidized to lipid peroxides and further decomposed. It is considered that the aldehyde is a highly active aldehyde, which is generated by binding to a protein.

  Aldehydes are produced from lipid peroxidation substances generated in vivo by ultraviolet rays and stress, and are also known to be contained in tobacco smoke and environmental pollutants. When unsaturated fatty acids widely distributed in the living body undergo oxidative degradation, acrolein, 4-hydroxy-2-nonenal and the like are produced. In addition, it is known that these aldehydes are produced in the inflammatory reaction in the skin and the degradation reaction of sebum secreted to the skin surface (Thiele, JJ et al., Curr Probl Dermatol. 29: 26-42 , 2001; Stevens, JF et al., Mol Futr Food Res. 52: 7-25, 2008). Therefore, the aldehyde used in the present invention is not particularly limited as long as it is a substance that can modify the amino acid side chain of the protein in the dermis model and introduce a carbonyl group, but it is not limited to acrolein or 4-hydroxy-2- Nonenal is preferred.

  Thus, by treating the dermis model with aldehyde, the yellowing of the skin seen in the living body can be sufficiently reflected. Therefore, by applying aldehyde and a candidate substance to the above dermis model and reacting under conditions sufficient to cause yellowing of the skin, the color of the dermis model is evaluated, thereby inhibiting the candidate substance from yellowing. Can be evaluated.

  Further, as described above, in the measurement of the color tone of the dermis model, it is necessary to sufficiently reflect the optical characteristics in the composition of living skin. That is, the color cannot be accurately evaluated by a general method in which protein is dissolved with acid or alkali and the absorbance of the protein solution is measured with a photometer or the like. For this reason, it is necessary to directly analyze the color in the solid dermis model.

  As such a method, it is conceivable that the spectral reflectance in the dermis model is measured by a color difference meter (spectral colorimeter) or the like and the measured value is used as a color index. Spectral reflectance means the reflectance for each wavelength when a substance is irradiated with light. Reflection includes specular reflection with the same incident angle and reflection angle as a mirror, and diffuse reflection that is reflected in various directions from the surface irregularities and the inside of the object, but when measuring the spectral reflectance, Using an integrating sphere having a function of collecting light, specular reflection and diffuse reflection can be measured simultaneously. In the measurement of the spectral reflectance in the dermis model, an absorption spectrum in a wavelength range of about 400 nm to 750 nm is typically used. When yellowing is evaluated, an absorption spectrum on the short wavelength side of the visible light region is used. (For example, about 450 nm) can also be evaluated. Although the color difference meter etc. which can be used for a measurement are not restrict | limited in particular, The color difference meter etc. based on Japanese Industrial Standard (JIS) are preferable.

Various color systems can be used as the skin color notation method. As typical notation methods, L ^* a ^* b ^* color system having perceptually equal color difference, color Examples include Munsell color system using three attributes.

The L ^* a ^* b ^* color system is a color system standardized by the International Commission on Illumination (CIE), and is widely known in the art as a notation for representing skin color. CIE 1976 (L ^* a ^* b ^* ) Also referred to as a color system. In the L ^* a ^* b ^* color system, lightness is represented by L ^* , and chromaticity indicating hue and saturation is represented by a ^* and b ^* . Each of a ^* and b ^* indicates a color direction, a ^* indicates a red direction, -a ^* indicates a green direction, b ^* indicates a yellow direction, and -b ^* indicates a blue direction. The values of L ^* , a ^* and b ^* can be obtained from the following equations from the tristimulus values X, Y, Z.
L ^* = 116 (Y / Y ₀ ) ¹ /3-16;
a ^* = 500 [(X / X ₀ ) ¹ /3-(Y / Y ₀ ) ^1/3 ]; and
b ^* = 200 [(Y / Y ₀ ) ¹ / ^3- (Z / Z ₀ ) ^1/3 ]
However, X / X _0, Y / Y ₀ and Z / Z ₀ are> 0.008856, where X ₀ , Y ₀ and Z ₀ represent the tristimulus values of the standard light source.
The color difference ΔE between two different colors, L ^* ₁ , a ^* _1, and b ^* _1, and L ^* ₂ , a ^* _2, and b ^* ₂ can be obtained from the following equation.
ΔE = √ (L ^* ₂ -L ^* ₁ ) ² + (a ^* ₂ -a ^* ₁ ) ² + (b ^* ₂ -b ^* ₁ ) ²

The color analysis using the L ^* a ^* b ^* color system can be easily performed using an integrating sphere type spectrocolorimeter, and for example, a spectrocolorimeter CM-2600d manufactured by Konica Minolta can be used.

  The Munsell color system is standardized by JIS, and the three attributes of color, hue (Hue), lightness (Value), and saturation (Chroma) are represented by H, V, and C, respectively, and scaled. It is a color system. The hue (Hue) is R (red), YR (yellow red), Y (yellow), GY (yellowish green), G (green), BG (blue green), B (blue), PB (purple blue), The hues are divided into 10 hues of P (purple) and RP (red purple), and these hues are further divided into 10 parts each, which are represented by a total of 100 hues. The value (Value) is expressed in 11 steps at equal intervals, with 0 being completely black and 10 being completely white, and Chroma is 0 with achromatic color being 0. According to the degree of vividness, it is represented by a larger number. For example, in the skin color, when the hue is 5YR, the lightness is 6, and the saturation is 3.8, it is expressed as 5YR 6 / 3.8.

  A color difference meter or the like used in color analysis by the Munsell color system is not particularly limited, but is preferably compliant with JIS.

  In addition, since the dermis model is a semi-transparent body like living body skin, in order to prevent the color under the object from being reflected in the measurement value, the object is not covered with a white calibration tile or the like. It is preferable to carry out the measurement on the top.

In addition, as described above, the b ^* value according to the L ^* a ^* b ^* color system and the YR value or Y value in the hue (Hue) of the Munsell color system directly represent yellowishness, and thus are obtained in the dermis model. By using these obtained values as an index of yellowing, the degree of yellowing of the dermis model can be easily and objectively evaluated. For example, when the L ^* a ^* b ^* color system is used, if the b ^* value decreases significantly, it can be evaluated that yellowing has been significantly suppressed, but the color is different in human eyes. There is a range and color identification area where the difference cannot be identified. The degree of discrimination varies depending on the hue, lightness, and saturation, but the density collagen gel-like dermis model can be recognized by a difference of about 3 or so.

Accordingly, in a more specific embodiment of the present invention, a method for evaluating a substance that suppresses skin yellowing, comprising contacting a high-density collagen gel dermis model with an aldehyde and a candidate substance, and The L ^* a ^* b ^* color system b ^* value in the gel-like dermis model or the YR value or Y value in the Munsell color system hue (Hue) is used as an index of skin yellowing. A method is provided.

  In another embodiment of the present invention, there is provided a method for screening a substance that suppresses skin yellowing, wherein an aldehyde that induces carbonylation is contacted with a candidate substance in a high-density collagen gel dermis model. And when the detection level of the carbonyl group present in the dermis model contacted with the candidate substance is significantly lower than when the candidate substance is not contacted, the candidate substance is A method is provided that is selected as a substance that suppresses crystallization.

  As described above, since the carbonylation of protein in the dermis is one of the main factors of skin yellowing, the detection level of the carbonyl group present in the dermis model contacted with the candidate substance decreased. In this case, it means that the candidate substance has a very high probability of having an inhibitory action on the skin. Therefore, after applying the aldehyde that induces carbonylation and a candidate substance to the dermis model prepared as described above, and reacting for a time sufficient for yellowing of the skin to occur, the carbonyl introduced into the protein present in the dermis model By detecting the group, the yellowing inhibitory action of the candidate substance can be easily evaluated.

Protein carbonyl groups can be detected with a fluorescent substance that specifically fluorescently labels the carbonyl group, and such fluorescent substances have a hydrazino group (-NHNH ₂ ) capable of binding to the carbonyl group of the protein. Is preferred. Examples of such fluorescent materials include fluorescein-5-thiosemicarbazide, Texas red hydrazide, lucifer yellow hydraside and the like.

  In addition, the specific fluorescent labeling of the carbonylated protein may be a combination of biotin hydrazide and fluorescently labeled avidin. Since biotin hydrazide also has a hydrazino group, it can bind to the carbonyl group of the protein. In this case, biotin hydrazide is first bound to the carbonylated protein, and then fluorescently labeled avidin is bound to biotin hydrazide via a biotin-avidin bond, and as a result, the carbonylated protein is fluorescently labeled. Biotin hydrazide is well known in the art, and for example, those manufactured and sold by Pierce can be used. As the fluorescent avidin, for example, fluorescein avidin can be used.

  Furthermore, specific fluorescent labeling of a carbonylated protein can also be performed by allowing dinitrophenylhydrazine to act and bind to the carbonyl group of the protein and labeling the dinitrophenyl moiety with a fluorescent dye. Therefore, in a further preferred embodiment of the present invention, the dinitrophenyl moiety of dinitrophenylhydrazine bonded to the carbonyl group of the protein can be detected by fluorescence immunoassay or the like.

  Moreover, the carbonyl group of the protein fluorescently labeled as described above can be easily detected by a fluorescence microscope, and may be arbitrarily photographed by a fluorescence microscope.

  The screening method of the present invention makes it possible to select a substance that suppresses skin yellowing very easily.

  The invention is illustrated in more detail by the following examples.

Example 1. Preparation of dermis model Dermal fibroblasts isolated from human dermis were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Dermal fibroblasts were collected by trypsin-EDTA treatment. As a final concentration, collagen solution (Koken) was diluted with a medium so as to be a 0.1% type I collagen solution containing 1.0 × 10 ⁵ cells / mL of fibroblasts to neutralize collagen. Gelled. For type I collagen, a solution extracted from bovine dermis with acetic acid was used. After neutralization gelation, the medium was changed once every 2-3 days and cultured for 7 days. Since dermal fibroblasts have the property of attracting surrounding collagen fibers, the collagen gel concentrated during the culture and contracted to about 1/3 of the original size. A sufficiently densified contracted collagen gel was immersed in sterilized water, and the fibroblasts surviving in the gel were killed by a hypotonic treatment. The sterilized water was exchanged 10 times or more to sufficiently remove the medium and soluble components, and a gel formed only with the dermal extracellular matrix component not containing living cells was prepared, and this was used as a dermis model.

Example 2. Examination of dermal yellowing factors The dermal model was used to compare the effects of glycation and carbonylation on dermal protein on dermal model yellowing. For glycation of the protein in the dermis model, D-ribose (Wako Pure Chemical Industries, Ltd.), which is a reducing sugar having a higher reactivity than glucose, and glyoxal (Alfa Aesar) produced by autooxidation of glucose were used. For carbonylation, acrolein (AccuStandard) and 4-hydroxy-2-nonenal (Oxis International), which are aldehydes derived from lipid peroxides, which are known to exist in the skin, were used. The dermis model prepared in Example 1 was immersed in 2.5 mL of a solution containing 200 mM D-ribose, 10 mM glyoxal, 10 mM acrolein, and 10 mM 4HNE prepared with a phosphate buffer, and the mixture was immersed at 37 ° C. for 5 days. Incubation was carried out to induce glycation and carbonylation experimentally. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out from the solution, washed thoroughly with a phosphate buffer, and then placed on a white calibration tile (CR-A43 (1849-701)) for measurement. CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum by b ^* was used as an index of yellowness. The change in yellowness (yellowing) was evaluated using a dermis model immersed in a phosphate buffer as a control.
The obtained results are shown in FIG. As can be seen from FIG. 1, when carbonylation of a protein in the dermis model is induced, the yellowing (yellowing) is more significant than glycation, which has been considered to be one of the major factors of skin yellowing so far. It was shown to induce

Example 3 Inhibition of yellowing of the dermis model by pyridoxine hydrochloride We investigated whether suppression of carbonylation would inhibit protein yellowing in the dermis model. The dermis model produced in Example 1 was immersed in a solution (control) containing only 1 mM acrolein prepared in a phosphate buffer and 2.5 mL of a solution containing 1 mM acrolein and a candidate substance. As a candidate substance, 10 mM pyridoxine hydrochloride, which is known to have a carbonylation-inhibiting effect, was used. Thereafter, the dermis model treated as described above was incubated at 37 ° C. for 5 days. The solution was changed to a freshly adjusted solution every day. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out of the solution and placed on a white calibration tile (CR-A43 (1849-701)). B ^* (yellowishness) based on the CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum was used as an index of yellowing. The obtained results are shown in FIG.

  As can be seen from FIG. 2, it is shown that suppression of dermal protein carbonylation suppresses yellowing of the skin, and this model can be used as a model for evaluating suppression of yellowing. It was shown that there is.

Example 4 Evaluation of Skin Yellowing Inhibition Action by Olive Leaf Extract As an example of evaluation, the yellowing inhibition of olive leaf extract (Maruzen Pharmaceutical Co., Ltd.) was evaluated. Olive (Olea europaea Linne) belongs to the magnoliaceae olive genus, and is an evergreen tree that is cultivated mainly in Shodoshima in Japan, where temperatures are relatively high throughout the world. Olive oil obtained by squeezing ripe fruits is widely used for edible, medicinal, and cosmetic applications, and olive leaves are known for their antiseptic, antipyretic and antihypertensive effects, and in recent years have been used as raw materials for tea . However, it has not been known so far that olive leaf extract has a carbonylation-inhibiting action. Since the olive leaf extract is colored, the same experiment as in Example 3 was carried out using activated carbon from which the pigment component was removed so as not to interfere with the color evaluation. Specifically, the dermis model produced in Example 1 was immersed in a solution (control) containing only 1 mM acrolein prepared in a phosphate buffer and 2.5 mL of a solution containing 1 mM acrolein and 10% olive leaf extract. did. Thereafter, the dermis model treated as described above was incubated at 37 ° C. for 6 days. The solution was changed to a freshly adjusted solution every day. The color change of the dermis model was measured with Konica Minolta spectrocolorimeter CM-2600d diffuse illumination and 8 ° direction light reception φ3 mm. At the time of measurement, the dermis model was taken out of the solution and placed on a white calibration tile (CR-A43 (1849-701)). B ^* (yellowishness) based on the CIE L ^* a ^* b ^* color system calculated from the obtained spectral reflection spectrum was used as an index of yellowing.

As can be seen from FIG. 3, yellowing of the dermis model was suppressed, and a significant suppression of b ^* increase was observed. This indicates that the olive leaf extract is very likely to have an action of suppressing skin yellowing.

Claims (6)

A method for evaluating a substance that suppresses skin yellowing, comprising contacting a aldehyde that induces carbonylation with a candidate substance in a high-density collagen gel-like dermis model, and changing a color tone in the gel-like dermis model Is evaluated from a change in spectral reflectance using a color difference meter. The evaluation of color change is characterized by using the b ^* value of the L ^* a ^* b ^* color system or the YR value or Y value in the hue (Hue) of the Munsell color system as an index of skin yellowing. The method of claim 1 . Aldehyde induces the carbonylation, characterized in that acrolein or 4-hydroxy-2-nonenal A method according to claim 1 or 2.   A method for screening a substance that suppresses skin yellowing, comprising contacting a candidate substance with an aldehyde that induces carbonylation in a high-density collagen gel dermis model, and contacting the candidate substance When the level of detection of the carbonyl group present in the product is lower than when the candidate substance is not contacted, the candidate substance is selected as a substance that suppresses skin yellowing . The method according to claim 4 , wherein the aldehyde that induces carbonylation is acrolein or 4-hydroxy-2-nonenal. The method according to claim 4 or 5 , wherein the carbonyl group is detected using a labeling reagent having a hydrazino group.

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