Method for evaluating type-vi collagen

Abstract

To develop a method capable of evaluating aging and to develop an agent capable of improving or preventing photo-aging.SOLUTION: There is provided a method for evaluating type-VI collagen in the skin by finding out although type VI collagen was previously considered to show no difference in expression between exposed and unexposed skin, the amount of type-VI collagen in facial skin, particularly in the dermal papillary layer, changes, and that the level of photo-aging (solar elastosis) correlates with the amount of type VI collagen. In addition, there is provided a method for screening by using the expression of type-VI collagen as an index, and further provided is an inhibitor of type-VI collagen expression containing camellia seed extract.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention provides a method for evaluating type VI collagen in skin, a method for screening an inhibitor of type VI collagen expression, and an inhibitor of type VI collagen expression. [Background technology]

[0002] Wrinkles and sagging skin significantly affect the appearance of aging, making maintaining skin firmness and improving wrinkles and sagging a major cosmetic challenge. Loss of skin firmness not only changes the skin's texture, but also leads to sagging or sunken cheeks and wrinkles around the eyes, resulting in an aged appearance. Skin firmness is primarily due to the thickness and physical properties of the dermis, and firm skin is rich in extracellular matrix components such as collagen and elastin, which fill the dermis. Synthesis of extracellular matrix components such as collagen and elastin in the dermis by dermal fibroblasts decreases with age. Furthermore, in sun-exposed areas such as the face, in addition to intrinsic aging due to aging, photoaging due to exposure to ultraviolet light occurs. This activates matrix metalloproteinases, decomposing collagen, while denatured elastin deposits, resulting in solar elastosis (also known as actinic elastosis). This causes changes in the elasticity and stiffness of the dermis, leading to loss of firmness, wrinkles, and sagging. In skin with solar elastosis, collagen is lost deep in the dermis and large amounts of denatured elastin accumulate, but collagen remains in the papillary dermis just below the epidermis. The area where a certain width of normal dermal components is present between the epidermis and the lesions in the dermis is called the Grenz zone. Age-related changes in the dermal extracellular matrix are also affected by skin care, UV exposure, and diet.

[0003] Collagen accounts for approximately 30% of animal body protein and is the major protein constituting not only skin but also bones, teeth, tendons, etc. More than 10 types of collagen are known to exist in humans, and collagens present in skin include type I collagen, type III collagen, type IV collagen, type V collagen, and type VII collagen, which are distributed differently. In the field of dermatology, active research has been conducted on type I collagen (Patent Document 1: Japanese Patent No. 7265015, Patent Document 2: Japanese Patent Application Publication No. 2014-172849, Patent Document 3: Japanese Patent Application Publication No. 2014-055116) and type III collagen (Patent Document 4: Japanese Patent Application Publication No. 2013-203683) due to their abundance in skin, type IV collagen specifically distributed in basement membranes (Patent Document 5: Japanese Patent Application Publication No. 2004-18471), and type VII collagen (Patent Document 6: Japanese Patent Application Publication No. 2023-089539), among others. On the other hand, type VI collagen has been reported to be involved in the malignancy of gastric cancer (Patent Document 7: JP 2004-248502 A), fibrosis (Patent Document 8: JP 2012-522233 A), and muscular dystrophy (Patent Document 9: JP 2009-532404 A), and has been attracting attention in relation to disease rather than cosmetic aspects. In recent years, however, it has been reported to be present in the dermis and basement membrane, attracting attention (Patent Document 10: JP 2024-006969 A, Patent Document 11: JP 2024-006968 A). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7265015 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-172849 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-055116 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-203683 [Patent Document 5] Japanese Patent Application Laid-Open No. 2004-18471 [Patent Document 6] Japanese Patent Application Publication No. 2023-089539 [Patent Document 7] Japanese Patent Application Laid-Open No. 2004-248502 [Patent Document 8] Special Publication No. 2012-522233 [Patent Document 9] Special Publication No. 2009-532404 [Patent Document 10] Japanese Patent Application Laid-Open No. 2024-006969 [Patent Document 11] Japanese Patent Application Publication No. 2024-006968 Summary of the Invention [Problem to be solved by the invention]

[0005] The goal is to develop a method to evaluate photoaging. [Means for solving the problem]

[0006] The present inventors conducted research focusing on the relationship between photoaging and collagen, and discovered that type VI collagen, which was thought to be expressed in the same way between sun-exposed and non-sun-exposed skin, changes in its abundance in the Grenz zone of the papillary dermis in facial skin, particularly in areas of solar elastosis caused by the progression of photoaging. They also found that the level of solar elastosis correlates with the amount of type VI collagen, leading to the present invention. The present invention therefore relates to: [1] measuring the level of dermal solar elastosis in a subject's skin; and determining the type VI collagen level based on the correspondence between the solar elastosis level and the type VI collagen level; A method for assessing type VI collagen in skin, comprising: [2] The method according to item 1, wherein the solar elastosis level is determined by irradiating the skin with excitation light and detecting the autofluorescence of elastin. [3] The method according to item 1, wherein the type VI collagen level is the amount of type VI collagen in the papillary dermis. [4] The method according to item 1, wherein the correspondence is a correspondence according to the skin area of ​​the exposed part. [5] The method according to item 1, wherein the correspondence is a correlation equation. [6] A method for determining a photoaging state based on a type VI collagen level determined by the method according to any one of items 1 to 5. [7] culturing a culture containing at least one cell selected from dermal fibroblasts, epidermal keratinocytes, immune cells such as macrophages, and the like, in a medium containing a candidate drug; measuring the expression of type VI collagen in the culture after culturing in a medium containing the candidate drug; and A step of comparing the measured expression level with a control to determine the inhibitory effect of the candidate drug on type VI collagen expression. A method for screening for an agent that inhibits the expression of type VI collagen, comprising: [8] The method according to Item 7, wherein the inhibitor of type VI collagen expression is a photoaging inhibitor. [9] The method according to Item 7 or 8, wherein the control is the expression level of type VI collagen in a culture containing at least one cell selected from dermal fibroblasts, epidermal keratinocytes, immune cells such as macrophages, and the like, cultured in a medium not containing the candidate drug, or a threshold value determined from the expression level. [10-1] A type VI collagen expression inhibitor containing camellia seed extract. [10-2] A method for inhibiting type VI collagen expression in a subject in need of prevention or improvement of photoaging, comprising applying camellia seed extract. [10-3] Camellia seed extract for use in improving or preventing photoaging through the inhibition of type VI collagen expression. [10-4] Use of camellia seed extract for the manufacture of an inhibitor of type VI collagen expression.

[11] A photoaging prevention or amelioration agent comprising the type VI collagen expression inhibitor according to Item 10-1. [Effects of the Invention]

[0007] The level of solar elastosis can be determined by utilizing the autofluorescence of elastin fibers, and the amount of type VI collagen can be evaluated based on the determined solar elastosis level. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the analysis results of skin samples from a 78-year-old woman's buttock (a non-exposed area) and the upper cheek and eye corner (exposed areas). Sections of the skin samples were subjected to Elastica van Gieson staining (A), picrosirius red staining (B), and immunofluorescence staining using anti-type VI collagen antibody (C). [Figure 2] Figure 2 shows the results of electron microscopy of collagen fibers in the upper dermis of skin samples taken from a non-exposed area (buttocks) and an exposed area (corner of the eye) (A). The results of measuring the thickness of the photographed collagen fibers are also shown (B). [Figure 3] Figure 3 shows the results of Elastica van Gieson staining of skin samples obtained from subjects with various levels of solar elastosis (A). Skin samples were obtained from the buttocks, cheeks, and corners of the eyes of subjects classified into various solar elastosis levels, and the correlation with type VI collagen levels is shown (B). [Figure 4] FIG. 4 shows that camellia seed extract suppressed the expression of COL6A1, COL6A2, and COL6A3, which are constituent genes of type VI collagen, in dermal fibroblast cultures. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for assessing type VI collagen levels in skin. Type VI collagen increases in the Grenz zone, a normal dermal region with a certain width between the dermal lesion and the epidermis, as solar elastosis progresses due to photoaging, contributing to skin fibrosis. Therefore, appropriate assessment is required. Furthermore, assessing type VI collagen levels can determine the progression of photoaging. More specifically, the method for assessing type VI collagen levels includes the following: measuring the level of dermal elastosis in the subject's skin; and A step of determining the type VI collagen level based on the correspondence relationship between the elastosis level and the amount of type VI collagen. Includes.

[0010] Type VI collagen is a non-fibrous collagen that exists as bead-like microfilaments outside cells. It exists in a meshwork that encases collagen fiber bundles and functions to thicken and organize collagen fibers. Its expression increases in fibrotic tissues, including the skin, liver, lungs, and joints. In normal skin, no difference in expression was observed between sun-exposed and non-sun-exposed areas, suggesting that, unlike type I and type III collagen present in the dermis, type VI collagen contributes little to photoaging. However, research by the present inventors has shown that type VI collagen is present in the Grenz zone (shown by the white dotted line in Figure 1(A)) in the papillary dermis of skin with solar elastosis, and its expression increases as photoaging (solar elastosis) progresses (Figures 1(A), (B), and (C)). In the dermis of photoaged skin, collagen disappears and abnormal elastin is deposited (Figure 1(A)). Meanwhile, the collagen in the Grenz zone is thick and dense (Figure 1(B)). In the figure, the arrows indicate type VI collagen ((C) white arrow) that has accumulated in the area corresponding to the Grenz zone ((A) black arrow, (B) white arrow). In Figure 1(A), the white arrow also indicates abnormal elastin deposition. Furthermore, thicker fibers are observed in sun-exposed skin (Figure 2(A), (B)).

[0011] Typically, the amount of collagen present in the dermis, such as type I collagen and type III collagen, decreases upon exposure to light. This decrease in collagen amount occurs due to the activation of matrix metalloproteinases associated with UV irradiation, and is also contributed to by a decrease in production due to decreased activity of dermal fibroblasts. Meanwhile, unlike other collagens present in the dermis, type VI collagen increases in abundance in the papillary dermis in photoaging skin upon UV exposure. Until now, no attention has been paid to the types of collagen present in skin, particularly type VI collagen. In the present invention, the type VI collagen level evaluated refers to the protein amount of type VI collagen from the perspective of measuring type VI collagen deposited in tissue. In particular, type VI collagen amount refers to the amount of collagen located in the Grenz zone generated in the papillary dermis in solar elastosis skin.

[0012] The level of type VI collagen correlates with photoaging, particularly with the level of solar elastosis (Figures 3(A) and (B)). This makes it possible to measure the solar elastosis level in skin and determine the type VI collagen level from the correlation between the solar elastosis level and the type VI collagen level. The solar elastosis level can be determined by detecting elastin in a skin sample or by a non-invasive method. Elastin can be detected in a skin sample by Elastica van Gieson staining or an immunological method using an anti-elastin antibody. Alternatively, elastin has autofluorescence, which can be quantified by externally irradiating the skin with light, such as excitation light. The elastin autofluorescence can be quantified using a multiphoton microscope. The elastosis level can also be determined based on the intensity of elastin autofluorescence (emission wavelength: ideally 410 nm to 490 nm, or even 300 nm to 600 nm) when the skin is irradiated with excitation light. For example, the excitation light may be light in the near-infrared region, for example, 700 nm to 1550 nm, particularly 700 nm to 1300 nm.The solar elastosis level may be measured using the autofluorescence of elastin detected under specified conditions.

[0013] The correspondence relationship between solar elastosis levels and type VI collagen levels can be determined in advance. The correspondence relationship can be created by classifying solar elastosis levels and measuring the type VI collagen level according to the classification. A correspondence table can be used to create such a correspondence relationship. In another embodiment, when autofluorescence measured under predetermined conditions is used as the elastosis level, a correlation equation or one or more thresholds indicating the type VI collagen level relative to the measured value of autofluorescence can be used as the correspondence relationship. Such a correspondence relationship can be created for a specific site, such as facial skin, or more specifically, for a limited area such as the corners of the eyes or cheeks.

[0014] The type VI collagen level may refer to either the expression level or protein amount of type VI collagen, but may also refer to the protein amount from the perspective of measuring the degree of deposition. The protein amount determined by immunological techniques can be used to create a correspondence relationship. In this case, it is preferable to use an antibody specific to type VI collagen. Apart from the antibody, a probe specific to type VI collagen can also be used.

[0015] The evaluated type VI collagen level may be further used to determine the photoaging state, which can be determined by comprehensively assessing the decrease in collagen in the dermis layer, the denaturation of elastin in the dermis layer, and the fibrosis caused by the increase in type VI collagen in the papillary dermis layer.

[0016] Another aspect of the present invention may relate to a method for screening for an inhibitor of type VI collagen expression. Screening for a compound having an inhibitory effect on type VI collagen expression can lead to the development of a product effective against photoaging. More specifically, the screening method for an inhibitor of type VI collagen expression comprises the following steps: Cultivating a culture containing at least one cell selected from dermal fibroblasts, epidermal keratinocytes, immune cells such as macrophages, and the like in a medium containing a candidate drug; measuring the expression of type VI collagen in the skin cultures after culturing in a medium containing a candidate drug; and A step of comparing the measured expression level with a control to determine the inhibitory effect of the candidate drug on type VI collagen expression. The compounds thus screened for having an inhibitory effect on type VI collagen expression can also be used as agents for preventing or ameliorating photoaging. Therefore, the method for screening for inhibitors of type VI collagen expression can also be said to be a method for screening for agents for preventing or ameliorating photoaging.

[0017] The cells used in the screening can be any cell found in the skin, such as dermal fibroblasts, epidermal keratinocytes, or immune cells, or a combination thereof. However, dermal fibroblasts expressing type VI collagen are preferred. Examples of immune cells found in the skin include macrophages and Langerhans cells. Melanocytes may also be included. The culture containing dermal fibroblasts may be a culture of isolated dermal fibroblasts or established dermal fibroblast cells, a harvested skin organ, or a three-dimensional skin model. The cells used may be derived from any animal, but mammalian, and especially human, cells are preferred. A candidate drug may be added to the culture at a predetermined time, or the medium may be replaced with a candidate drug-containing medium. Type VI collagen expression can be determined by measuring the protein or mRNA levels, each of which can be measured using immunological techniques or quantitative PCR, or other techniques well known in the art.

[0018] As a control, a culture containing dermal fibroblasts can be cultured in a medium that differs only in that it does not contain a candidate drug, and the expression of type VI collagen measured can be used. A control group may be prepared by conducting a preliminary experiment and setting a threshold based on the expression of type VI collagen in the control group, or the culture and measurement steps may be carried out in parallel. If the expression of type VI collagen is suppressed compared to the expression of type VI collagen in the control, the candidate drug can be screened as a type VI collagen expression inhibitor, and such a drug can be used as a drug for preventing or improving photoaging. It can be used.

[0019] The candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0020] The plant extracts described herein can be obtained by conventional methods, for example, by immersing or refluxing the plant with an extraction solvent at room temperature or elevated temperatures, followed by filtration and concentration. Any solvent commonly used for extraction can be used as the extraction solvent. For example, aqueous solvents such as water, saline, phosphate buffer, borate buffer, or organic solvents such as alcohols (e.g., ethanol, propylene glycol, 1,3-butylene glycol, glycerin), hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., can be used alone or in combination. A mixed solvent of water and an alcohol (e.g., 1,3-butylene glycol) is preferably used. The extract obtained by extraction with the above solvents can be used directly or after concentration by, for example, freeze-drying. If necessary, the extract can be used after removing impurities using adsorption techniques, such as ion exchange resins, or after adsorption on a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration. The plant extract may be an extract commercially available as a cosmetic raw material, and the commercially available extract may be blended at a predetermined concentration.

[0021] Camellia seed extract is an extract obtained from the seeds of plants belonging to the genus Camellia in the family Theaceae (Theaceae). It is particularly extracted from the seeds of Camellia japonica (Camellia japonica). Camellias are native to Japan but are widely distributed throughout the Japanese archipelago, as well as the Korean Peninsula, China, and Taiwan. It can be extracted from camellia seeds themselves, or it can be produced by further solvent extraction of the residue after squeezing camellia oil. It can also be an extract extracted after fermentation of camellia seeds. Camellia seed extract is designated by the Japan Cosmetic Industry Association (JCI) as the label name for all cosmetic ingredients and the international label name according to the INCI (International Nomenclature of Cosmetic Ingredients) name: Camellia japonica seed extract. Solvent extraction can be performed using water, alcohol, or a mixture of these. Ethanol, propylene glycol, or butylene glycol are used as the alcohol. More preferably, it can be extracted with a mixture of water and 1,3-butylene glycol in any ratio, for example, a mixture of 10:90 to 90:10, preferably a mixture of 30:70 to 70:30, and even more preferably a mixture of 50:50. Camellia seed extract is blended at a concentration of 0.0001% to 1%, more preferably 0.01% to 0.3%.

[0022] The inhibitor of type VI collagen expression of the present invention may be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, or may be incorporated into foods, such as dietary supplements or functional foods. These drugs may be administered orally or parenterally, for example, transdermally. When administered transdermally, they may be formulated into topical preparations for skin.

[0023] The topical skin preparation is not particularly limited as long as it is applicable to the skin, and any dosage form can be used, for example, a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a topical skin preparation, bases and excipients typically used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0024] When incorporated into cosmetics, the composition can be incorporated into facial or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into foods, the composition can be incorporated into capsules, tablets, beverages, and the like. The use of cosmetics, foods, pharmaceuticals, and quasi-drugs containing the components of the present invention results in the inhibitory effect of type VI collagen expression. The inhibitor of type VI collagen expression can be administered over a long period of time as the food or cosmetic product of the present invention. From the viewpoint of inhibiting type VI collagen expression, the inhibitor may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. The upper limit is not particularly limited, but may be several years or less, for example, one year or less.

[0025] The concentration of the type VI collagen expression inhibitor of the present invention can be selected arbitrarily from the viewpoint of achieving the desired effect, for example, improving or preventing photoaging. When formulated as an external skin preparation, the extract of the present invention can be formulated at 0.0005% to 0.5%. To fully exert its effect, it can be formulated at preferably 0.001% or more, more preferably 0.005% or more. To avoid a strong odor, it can be formulated at preferably 0.1% or less, more preferably 0.05% or less.

[0026] All documents mentioned herein are incorporated by reference in their entirety.

[0027] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0028] Example 1. Histological analysis of facial and buttock skin tissues Skin tissue samples taken from the buttocks or face (eye corners and cheeks) of individuals in their 20s to 80s were fixed using the AMeX fixation method, embedded in paraffin, and then sectioned at 4 μm. They were then stained with Elastica van Gieson and observed under a light microscope (A). Similar sections were also stained with Picro-Sirius Red using the Picro-Sirius Red Stain Kit (For Collagen) (PSR-1, ScyTek Laboratories, Inc.) and observed under a cross-polarized light microscope (B). Immunostaining for type VI collagen was performed using a mouse monoclonal anti-type VI collagen antibody (Merk, MAB1944) as the primary antibody and visualized using a fluorescently labeled secondary antibody, Alexa 488 anti-mouse IgG (Lifetechnologies). The samples were mounted using Vectasheeld with DAPI (VECTOR) and then observed under a fluorescence microscope (BX51, Evident) (C). Figure 1 shows the results of observation of a skin sample from a person in their 70s who showed photoaging in exposed areas.

[0029] Example 2. Analysis of collagen microstructure in facial and buttock skin Unfixed samples from the buttocks and face (corners of the eyes) of individuals in their 50s to 70s were cut into 2 mm cubes with a skin cutting blade and fixed by immersion in 1.5% paraformaldehyde and 0.5% glutaraldehyde-phosphate buffer. After post-fixation with 1% osmium tetroxide, the specimens were embedded in epoxy resin and then cut into blocks. Ultrathin sections were prepared, stained with uranyl acetate and lead, and photographed using a TEM electron microscope (Figure 2(A)). Images of collagen fibers were obtained within a range of approximately 50 μm from the basement membrane, and collagen fiber diameter was measured using the image analysis software ImageJ. The results are shown in Figure 2(B).

[0030] Example 3. Analysis of the relationship between the progression of solar elastosis and the amount of type VI collagen in the papillary dermis Skin tissue samples from the buttocks and face of subjects in their 20s to 80s were subjected to Elastica van Gieson staining and anti-type VI collagen immunofluorescence staining using the method of Example 1. The progression of solar elastosis was assessed using Elastica van Gieson stained images and scored from 0 to 4 as shown in Figure 3(A). The solar elastosis levels are shown in Table 1. [Table 1] Next, the amount of type VI collagen in the papillary dermis was determined by calculating the average brightness of the type VI collagen stained image within a 15 μm range from the basement membrane using the image analysis software ImageJ. The staining intensity of type VI collagen is shown in Table 2 below. [Table 2] The solar elastosis level and the type VI collagen level in the papillary dermis were plotted on a scatter plot to examine the correlation. A correlation was found between the type VI elastosis level and the type VI collagen level in the sun-exposed areas (Fig. 3(B)). The correlation coefficient was 0.287 for the sun-exposed areas (cheeks and eye corners), 0.576 for the cheeks only, and 0.218 for the eye corners only.

[0031] Example 4. Selection of anti-aging drugs using type VI collagen as an indicator 7x10 dermal fibroblasts 4Cells were seeded at 1 mL / well in a 12-well plate and cultured in Dulbecco's Modified Eagle's medium containing fetal bovine serum at 37°C under a 5% CO2 atmosphere for 48 hours. 0.07% Camellia Seed Extract (Yuka Sangyo) was added as a candidate drug, and the cells were cultured at 37°C under a 5% CO2 atmosphere for 48 hours before harvesting. Control cells were cultured under the same conditions except for the absence of extract. After washing with PBS, RNA was extracted from the harvested cells using an RNeasy Mini Kit (Qiagen) and reverse-transcribed using SuperScript VILO (Thermo Fisher Scientific). The reverse-transcribed nucleic acids were subjected to quantitative PCR using the following primer set, Platinum® SYBR® green qRCR SuperMix-UDG with ROX (Thermo Fisher Scientific), and StepOne® plus (Applied Biosystems). The expression levels of COL6A1, COL6A2, and COL6A3 were quantified relative to the expression level of G3PDH as an internal standard gene, and changes due to the extract drug were analyzed. The results are shown in Figure 4. [Table 3]

Claims

1. measuring the level of dermal solar elastosis in the subject's skin; and determining the type VI collagen level based on the correspondence between the solar elastosis level and the type VI collagen level; A method for assessing type VI collagen in skin, comprising:

2. 2. The method of claim 1, wherein the solar elastosis level is determined by irradiating the skin with an excitation light and detecting the autofluorescence of elastin.

3. 2. The method of claim 1, wherein the type VI collagen level is the amount of type VI collagen in the papillary dermis.

4. The method of claim 1 , wherein the correspondence is a correspondence according to the skin area of ​​the exposed part.

5. The method of claim 1 , wherein the correspondence is a correlation equation.

6. A method for determining a photoaging state based on a type VI collagen level determined by the method according to any one of claims 1 to 5.

7. Cultivating a culture containing skin cells selected from dermal fibroblasts, epidermal keratinocytes, immune cells such as macrophages, etc. in a medium containing a candidate drug; measuring the expression of type VI collagen in the skin cultures after culturing in a medium containing a candidate drug; and A step of comparing the measured expression level with a control to determine the inhibitory effect of the candidate drug on type VI collagen expression. A method for screening for an agent that inhibits the expression of type VI collagen, comprising:

8. The method according to claim 7, wherein the inhibitor of type VI collagen expression is a photoaging inhibitor.

9. The method according to claim 7 or 8, wherein the control is the expression level of type VI collagen in a culture containing dermal fibroblasts cultured in a medium containing no candidate drug, or a threshold value determined from the expression level.

10. A type VI collagen expression inhibitor containing camellia seed extract.

11. A photoaging prevention or amelioration agent comprising the type VI collagen expression inhibitor according to claim 10.

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