Screening method for papilla projection formation promoter and papilla projection formation promoter

Abstract

To provide a screening method for papilla projection formation promoter, and a novel papilla projection improvement agent.SOLUTION: The inventors have clarified that MMP-9 is involved in promoting papilla projection formation, and have invented a method for screening papilla projection formation promoters using at least one of an amount of MMP-9 gene expression in epidermal cells, an amount of MMP-9 protein in epidermal cells, and a degree of cell contraction in epidermal cells as indicators. This has solved the above-mentioned problem. Furthermore, the inventors have conducted screening using the screening method, and have confirmed that an extract of Moringa oleifera determined to have a papilla projection formation promoting effect by the indicator of the present invention, actually has a papilla projection formation promoting effect, thereby completing the present invention.SELECTED DRAWING: Figure 9

Description

Technical Field

[0001] The present invention relates to a method for screening a useful papilla formation promoter and a papilla formation promoter selected thereby.

Background Art

[0002] The skin is composed of three layers: the epidermis, the dermis, and the subcutaneous tissue, in order from the surface layer where the human body contacts the outside air. The epidermis consists of cells called keratinocytes (keratinized cells) and is classified into the basal layer, the spinous layer, the granular layer, and the stratum corneum from the deep part near the dermis. In the epidermis, keratinocytes are pushed from the basal layer toward the stratum corneum by division and are peeled off as so-called dirt from the surface layer in order.

[0003] The dermis is classified into the papillary layer near the epidermis and the reticular layer existing deeper than that. The papillary layer mainly contains collagen fibers composed of collagen, elastic fibers composed of elastin, other extracellular matrix components, and fibroblasts (fibroblasts). The reticular layer is thicker than the papillary layer and occupies most of the dermis. The reticular layer also contains collagen fibers composed of collagen and elastic fibers composed of elastin, but is thicker than the collagen fibers of the papillary layer and is more mature than the elastic fibers of the papillary layer.

[0004] Papillae protruding toward the epidermis are formed in the upper part of the papillary layer. The epidermal layer enters between these papillae, and an uneven structure in which the papillary layer of the dermal layer and the epidermal layer are interlocked with each other is formed between the epidermis and the dermis. The space between the epidermal layer and the dermal layer is separated by a membrane structure called the basement membrane, and it can be said that the papilla is a structure composed of the epidermal layer, the dermal layer, and the basement membrane.

[0005] The dermis and the epidermis are separated by the basement membrane. Between the dermis and the epidermis, signal transmission and substance transport such as the transport of waste products and nutrients are carried out through dermal papillae. It is considered that the engagement of the dermis and the epidermis has a buffering effect on physical stimuli from the outside. In addition, it has been reported that the shape of the dermal papillae is also related to skin conditions (stratum corneum water content, transepidermal water loss, stratum corneum cell area, skin color) (Patent Document 1). To maintain the skin condition normally, it is considered important to appropriately maintain the dermal papillae. However, it is known that the dermal papillae become flattened by aging and ultraviolet rays (Non-Patent Document 1), resulting in deformation, a decrease in number, or loss of the dermal papillae. Therefore, a method for appropriately maintaining the dermal papillae is required.

[0006] As described above, appropriately maintaining the dermal papillae is very effective as a method for maintaining the skin condition. However, the formation mechanism of the dermal papillae has not been clarified so far. When searching for a dermal papilla improving agent, it was necessary to actually apply it to human skin and observe and evaluate the state of the dermal papillae itself. However, since the dermal papillae are structures existing inside the skin, it is not easy to evaluate their state. Methods such as excising human skin to prepare sections for observation, excising human skin to peel off the epidermal layer and observing it with an electron microscope, and observing it using expensive equipment such as a confocal laser scanning microscope have been adopted. These methods require a physical and mental burden on the observation sample provider, as well as the cost and labor of the researcher. Therefore, it has been desired to elucidate the formation mechanism of the dermal papillae and develop a simple screening method for a dermal papilla formation promoter.

[0007] In addition to maintaining the dermal papilla structure of the human body, promoting dermal papilla formation is also important in the production of artificial skin. Artificial skin is used for the replacement or regeneration of damaged skin such as burns and trauma. Methods for obtaining a skin model having dermal papillae that can be used for artificial skin are known (Japanese Patent Application No. 2020-101725). However, in order to produce a skin model having dermal papillae with stable quality that can be industrially utilized, a method for promoting dermal papilla formation has been required.

[0008] Matrix metalloproteinase (hereinafter referred to as "MMP") is a type of protease in which a metal is involved in the catalytic mechanism, and is also called neutrophil gelatinase, type IV collagenase, and gelatinase B. It is an enzyme that exhibits broad substrate specificity for denatured collagen (gelatin) and native collagen (type IV, V, and XI collagen), and is known to be involved in physiological phenomena such as the activation of bioactive substances in addition to protein degradation. For example, MMP-9 is known to generate the contractility of actomyosin and cause cell motility such as cell migration via the Rho / Rock signaling pathway (Non-Patent Document 2). However, until now, the contribution of MMP-9 to promoting papilla formation and cell contraction has not been known. In addition, as described above, MMP-9 has been reported to degrade type IV collagen, which is specifically expressed in the basement membrane. Although production inhibition has been studied for the improvement of skin diseases, skin inflammation, and aging (Non-Patent Document 3), there has been no idea of enhancing the function of MMP-9 with the intention of normalizing the skin condition. That is, it has never been known that MMP-9, which is known to degrade the basement membrane, one of the components of the papilla, contributes to promoting papilla formation.

[0009] Although the use of at least one protein fraction of the extract of Wasabia japonica in skin care (Patent Document 2) is known, the possibility that the extract of Wasabia japonica, rather than a specific protein fraction, enhances the function of MMP-9 has not been known at all. Further, a method for improving the skin condition (Patent Document 3) including one or more components selected from the group consisting of Moringa oleifera or an extract thereof has been disclosed, but the action of Moringa oleifera or an extract thereof is unclear, and since it is a method based on the idea that an increased MMP expression level by fibroblasts is associated with accelerated aging, it was not at all an intended use for promoting the expression of MMP. Furthermore, an extract of Wasabia japonica with 90% ethanol has an effect of inhibiting the production of the above-described MMP-9, and an effect of suppressing the degradation of the extracellular matrix by MMP-9 has been reported (Patent Document 4). As described above, although some uses of the extract of Wasabia japonica are known, they have been used exclusively with the intention of reducing the function of MMP or MMP-9, and the possibility that the extract of Wasabia japonica enhances the function of MMP-9 has not been known at all.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0011]

Non-Patent Document 1

[0012] The problem of the present invention is to provide a method for screening a papilla formation promoter and a novel papilla improver. [Means for Solving the Problems]

[0013] As a result of intensive studies, the present inventors have clarified that MMP-9 is involved in promoting papilla formation, and have invented a method for screening a papilla formation promoter using at least one of the MMP-9 gene expression level in epidermal cells, the MMP-9 protein level in epidermal cells, and the cell contractility of epidermal cells, thereby solving the above problems. Furthermore, screening was carried out using the screening method, and the promoting effect of papilla formation was actually confirmed in the extract of Wasabinooki, which was determined to have a promoting effect on papilla formation according to the index of the present invention, and the present invention was completed. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a method for screening a papilla formation promoter, an MMP-9 production promoter containing an extract of Wasabinooki, and a papilla formation promoter. [Brief Description of the Drawings]

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0016] The screening method for a papilla formation promoter in the present invention is as follows: (1) A step of adding a test substance to epidermal cells and culturing them. (2) A step of measuring at least one selected from the MMP-9 gene expression level in epidermal cells, the MMP-9 protein level in epidermal cells, and the cell contractility of epidermal cells. (3) Comparing at least one selected from the MMP-9 gene expression level in epidermal cells, the MMP-9 protein level in epidermal cells, and the cell contractility of epidermal cells obtained in step (2) with a group without the test substance, and determining a substance that improves at least one of the selected MMP-9 gene expression level in epidermal cells, the MMP-9 protein level in epidermal cells, and the cell contractility of epidermal cells as a substance having a papilla formation promoting effect. It includes.

[0017] The cell type used in the screening method for the papilla formation promoter in the present invention is not particularly limited as long as it is an epidermal cell, but is preferably a cultured epidermal cell derived from a human, and it is more preferable to use a cell in which the MMP-9 gene expression level and / or the MMP-9 protein amount is decreased. Examples of cells in which the MMP-9 gene expression level and / or the MMP-9 protein amount of epidermal cells is decreased include cells from donors of old age, cells mimicking cells from donors of old age by known aging induction means, or cells in which the MMP-9 gene expression level and / or the MMP-9 protein amount is decreased by damaging with known drugs or treatments as compared with the case without damage.

[0018] As the medium for culturing epidermal cells, it is possible to use a known medium suitable for culturing the epidermal cells used in the present invention. For example, a medium for epidermal keratinocyte proliferation (Humedia KG2 (KURABO)), DMEM (Dulbecco's modified Eagle's medium), etc. can be mentioned. When cells are proliferating, it is preferable to add serum such as fetal bovine serum, and additives such as growth factors, antibacterial agents, and insulin.

[0019] The test substance is not particularly limited. An extract derived from animals or plants, a culture of fungi, or a processed product thereof such as an enzyme, a compound or its derivative, etc. can be used as the test substance, and it may be in a liquid state, or in a powder state, a gel state, etc.

[0020] As used in the present invention, "using the MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells as an indicator" means that, using any method, the MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells is used as a criterion for effect determination. The "MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells" used as an indicator in the present invention only needs to be able to quantitatively grasp the amount of MMP-9 produced in cells. In addition to directly quantifying the MMP-9 gene expression level and the MMP-9 protein level, for example, by quantifying the amount of degradation of a known protein as a substrate, the enzyme activity of MMP-9, etc., due to the variation of the "MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells", the object whose measured value varies can be quantified, and indirectly, it can also be grasped as the "MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells".

[0021] The quantification of the MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells can be performed by known methods. For example, in the case of measuring the gene expression level, real-time PCR, semi-quantitative PCR can be used, and in the case of measuring the protein level, immunohistochemistry, Western blotting, ELISA, liquid chromatography, gas chromatography, mass spectrometry, etc. can be used for measurement. When indirectly grasping the "MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells" by quantifying the object whose measured value varies due to the variation of the "MMP-9 gene expression level in epidermal cells and / or the MMP-9 protein level in epidermal cells", for example, the degradation of denatured collagen can be measured using MMP-9 enzyme activity measurement, zymography, etc.

[0022] As used in the present invention, "using the cell shrinkage degree as an indicator" means that, using any method, the cell shrinkage degree is used as a criterion for effect determination. The cell shrinkage degree used as an indicator in the present invention represents the force that, when a cell adheres to a deformable substrate such as a gel, the cell shrinks and at the same time applies a force to the adhered substrate to deform and shrink the substrate. The higher the force to shrink the substrate, the higher the cell shrinkage degree is judged.

[0023] The measurement of cell contractility can be performed by known methods. For example, it can be measured from the observation of cell size changes by microscope or visually, the degree of deformation of the culture substrate around the cells, the actomyosin activity that causes cell contraction, etc. When measuring the degree of deformation of the culture substrate around the cells, for example, a FLECS plate commercially available as a method for measuring cell contractile force can be used (https: / / www.funakoshi.co.jp / contents / 69303). The FLECS plate consists of a fluorescently labeled cell adhesion factor. When the cells bound to the cell adhesion factor contract, the adhesion factor also contracts, and the displacement amount of the size of the cell adhesion factor can be analyzed as the degree of cell contraction.

[0024] The non-additive group of the test substance in the present invention is not particularly limited as long as it can be used as a control for evaluating the improvement effect of the MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells of the test substance, or the cell contractility. For example, in addition to using the solvent used to dissolve the test substance, it also includes adding nothing.

[0025] In the present invention, for example, the MMP-9 gene expression level, the MMP-9 protein level in epidermal cells or the cell contractility used as an index is compared with the non-additive group of the test substance, and it is determined that the test substance that improves the MMP-9 gene expression level, the MMP-9 protein level in epidermal cells or the cell contractility can be used as a papilla formation promoter. A test substance in which the gene expression level or protein level increases by 10% or more compared to the non-additive group, or a test substance in which the cell contractility increases by 5% or more compared to the non-additive group is determined to be highly effective as a papilla formation promoter.

[0026] The screening method of the papilla formation promoter using the MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells in the present invention can be performed as follows, for example, when measuring the MMP-9 gene expression level. (1) Culture epidermal cells in a medium in a petri dish. (2) Add the test substance dissolved in water, and simultaneously add water in the same volume as the test substance as the group without the test substance. (3) Incubate for a certain period to allow the test substance to act on the epidermal cells. (4) Extract total RNA from the epidermal cells. (5) Synthesize cDNA from the total RNA. (6) Quantify the expression level of the MMP-9 gene by real-time PCR, and calculate the expression level of the MMP-9 gene when the test substance is added relative to the group without the test substance. (7) If the expression level of the MMP-9 gene when the test substance is added increases by 10% or more compared to the group without the test substance, it is determined that the test substance can be used as a papilla formation promoter.

[0027] The wasabi tree (scientific name: Moringa oleifera Lam.) used in the present invention is a tree cultivated in India, the Philippines, Taiwan, etc., and is also called Moringa, which is the genus name. It is rich in nutrients such as polyphenols, amino acids, and vitamin E, and is also called "green milk" or "tree of life" in some regions due to its high nutritional value and efficacy.

[0028] The extraction site of the wasabi tree extract used in the present invention is not particularly limited. For example, seeds, stems, branches, leaves, flowers, roots, etc. can be used. Among them, it is preferable to use seeds, and more preferably to use ground seeds including the outer skin. Also, the extraction solvent is not particularly limited, and examples include lower alcohols such as water and propyl alcohol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, etc., but it is particularly preferable to select water.

[0029] The extraction method of the wasabi extract used in the present invention is not particularly limited. For example, a mixed solution of 1 to 100 parts by mass of water and 1,3-butylene glycol or ethanol is used per 1 part by mass of dry plant, and extraction is preferably carried out at a temperature of 5 to 70 °C, preferably 10 to 60 °C, for 1 to 7 days, particularly 3 to 4 days. After extraction, filtration is performed, and it can be used as it is, but if necessary, purification treatments such as deodorization and decolorization can also be carried out within a range that does not affect the effect. Furthermore, it can also be used in a powdered state by freeze-drying or the like.

[0030] The method of using the papilla formation promoter and MMP-9 production promoter of the present invention is not particularly limited. As the final form, there is no problem in any form such as liquid, emulsion, gel, solid, powder, granule, etc., and optional components can be appropriately blended as needed within a range that does not impair the effect. Examples of the optional components include oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, preservatives, fragrances, various medicinal components, pH adjusters, neutralizing agents, and the like. In addition, as an MMP-9 production promoter, it is also possible to control the promotion of tissue formation other than papillae through promoting cell movement. The cell type on which the papilla formation promoter and MMP-9 production promoter act is not particularly limited as long as it is an epidermal cell, and it can act not only on epidermal cells in human skin tissue but also on epidermal cells in excised skin tissue used in skin transplantation, epidermal cells in artificial skin using a culture substrate, and the like.

Example

[0031] Hereinafter, the examples of the present invention will be specifically described, but the present invention is not limited by these examples.

[0032] [Test 1] <Evaluation of the Relationship between MMP-9 Production and Aging> Normal human epidermal keratinocytes from young donors (19 years old) and old donors (51 years old) were seeded in Humedia KG2 (KURABO) at 8.5×10 4Dispersed to obtain cells / mL, and seeded 2 mL each on a 6-well plate. Cultured at 37 °C and 5% CO 2 for 72 hours. Collected 2 mL of the medium. According to the protocol of Human MMP Antibody Array Membrane (abcam), measured the amount of MMP-9 protein in the collected medium, and determined the relative amount of MMP-9 protein in the aged donor when the amount of MMP-9 protein in the young donor was set to 1.

[0033] As shown in Figure 1, in the aged donor, the protein amount was 2 / 3 or less compared to the young donor. From this, it was found that the amount of MMP-9 protein produced by epidermal cells decreased with aging.

[0034] [Test 2] <Evaluation of the relationship between MMP-9 gene expression level and aging> Suspended normal human epidermal keratinocytes from young donors (19 years old) and aged donors (51 years old) in Humedia KG2 (KURABO), and prepared a cell suspension to obtain 8.5×10 4 cells / mL, and seeded 500 μL each in a 24-well culture plate. At 37 °C and 5% CO 2After culturing for 2 days below, total RNA was extracted using the Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using the PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of MMP-9 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) were measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using the following primers and enzymes. For the primers, a sense primer for MMP-9 (5’-GACGCAGACATCGTCATCCA-3’), an antisense primer (5’-AACTCGTCATCGTCGAAATGG-3’), a sense primer for GAPDH (5’-CCACATCGC TCAGACACCAT-3’), and an antisense primer (5’-TGACCAGGC GCCCAATA-3’) were used. Power SYBR Green Master Mix (Applied Biosystems) was used for the PCR reaction, and the analysis of gene expression was performed by the comparative Ct method. That is, the change in the gene expression level due to the addition of the test substance was determined as the relative amount with respect to a value obtained by correcting the Ct value of MMP-9 of young donors with the Ct value of GAPDH and setting it to 1.

[0035] As shown in Figure 2, it was found that the gene expression level of MMP-9 in elderly donors was lower compared to that in young donors, and the gene expression level of MMP-9 in epidermal cells decreased with aging. From this, it was found that the protein amount of MMP-9 and the gene expression level of MMP-9 could be grasped by measuring one of them.

[0036] [Test 3] <Evaluation of the Relationship between MMP-9 Secretion Site and Aging> Human skin tissues were obtained by purchasing abdominal skin from donors of young age (29 years old) and old age (72 years old) from BIOPREDIC. The human skin tissues were embedded in O.C.T compound (Sakura Finetek Japan), frozen, sectioned into 4-μm slices using a cryostat, and attached to glass slides. These were immersed in 95% ethanol for fixation and immunostained. As the primary antibody, Anti MMP-9 rabbit polyclonal antibody was used, and as the secondary antibody, Alexa Fluor 594 conjugate anti-rabbit IgG(H+L) chicken secondary antibody was used. Thereafter, the stained sections were subjected to microscopic observation (20× magnification) and photographed using a fluorescence microscope (BZ-X700, KEYENCE).

[0037] Figure 3 shows the results of immunostaining of skin tissues with the MMP-9 antibody. In the immunostaining using MMP-9, the antibody binds to MMP-9 in the skin, and a secondary antibody that emits fluorescence binds thereto, causing the MMP-9 presence part to emit red fluorescence. From Figure 3, as indicated by the white arrow, in the skin derived from a young human having a distinct papillary structure, there is a part that strongly emits red fluorescence, which is not seen in the skin derived from an old human where the papillary structure is not recognized, around the papilla. It was found that the MMP-9 presence part well coincides with the contour part on the papillary epidermal cell side. That is, the presence of MMP-9 on the papillary epidermal cell side correlates with the presence or absence of papillae.

[0038] [Test 4] <Evaluation of the effect of aging change in cell contractile force and MMP-9 pathway inhibition> Human epidermal keratinocytes derived from neonatal donors (hereinafter referred to as neonatal keratinocytes), human epidermal keratinocytes derived from 50-year-old adult donors (hereinafter referred to as 50-year-old adult keratinocytes), and human epidermal keratinocytes derived from 51-year-old adult donors (hereinafter referred to as 51-year-old adult keratinocytes) at 5.0×10 4 Cells / mL were dispersed in Humedia KG2 (KURABO) and seeded in 500-μL aliquots into a cell contractile force measurement kit FLECS Plate (Forcyte Biotechnologies). At 37°C, 5% CO2 After 6 hours of culture under conditioned conditions, the MMP-9 pathway inhibitor Y-27632 was added to neonatal donor-derived human epidermal keratinocytes at a concentration of 20 μM. Further, at 37 °C, 5% CO 2 After 20 hours of culture under conditioned conditions, the nuclei of the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI), and microscopic observation (20× magnification) and photography were performed using a fluorescence microscope (BZ-X700, KEYENCE). The lengths of the lines constituting the cross of the cruciform gel to which the cells adhered in the photographed images were measured using imageJ (OPEN SOURCE), and the degree of substrate contraction by the cells was calculated as follows. In addition, for neonatal donor-derived human epidermal keratinocytes treated with the cell actin staining reagent Alexa Fluor (registered trademark) 488 Phalloidin, cell structure observation (20× magnification) was performed using a fluorescence microscope (BZ-X700, KEYENCE), and photography was carried out.

[0039] The cell contractility measurement kit FLECS Plate has a structure in which a cruciform gel is arranged on plastic, and the cruciform gel is observed as red fluorescence. Also, by staining the nuclei of the cells blue with DAPI, it is possible to determine the cruciform gel to which the cells adhere. The degree of substrate contraction (cell contractility) was expressed by the following formula from the lengths of the lines constituting the cross of the cruciform gel to which the cells did not adhere (Control gel) and the lengths of the lines constituting the cross of the cruciform gel to which the cells adhered.

[0040]

Equation

[0041] The degree of cell contraction by epidermal cells was measured for N=6, and the average was plotted. The results are shown in Figure 4. Compared to human epidermal cells derived from newborn donors, human epidermal cells derived from aged adult donors aged 50 and 51 years had lower cell contraction, indicating that cell contraction decreases with age. An example of a remarkable effect is shown in Figure 5. It was also found that the cell contraction decreases with age when the MMP-9 pathway inhibitor Y-27632 is applied to human epidermal cells derived from newborn donors. In other words, this test showed that MMP-9 plays an important role in cell contraction of epidermal cells, and at the same time, it was shown that the amount of MMP-9 gene expression and / or the amount of MMP-9 protein in epidermal cells can be understood by measuring the cell contraction.

[0042] Figure 6 shows the results of observing the structure of actin fibers when the MMP-9 pathway inhibitor Y-27632 was applied to human epidermal keratinocytes derived from newborn donors. In neonatal keratinocytes to which Y-27632 was not added, actin fibers (white arrows) were present in the cells, but in neonatal keratinocytes to which Y-27632 was added, actin fibers were not formed and the cells were observed to be enlarged (not contracted). In other words, cell contractility is controlled by MMP-9, and taking the results so far into consideration, it was hypothesized that MMP-9, which decreases with age, is the cause of the decrease in cell contractility, and that the decrease in cell contractility with age makes it difficult for epidermal morphological changes to occur, resulting in a decrease in the ability to form papillae. Test 5 proved this hypothesis.

[0043] [Test 5] <Confirmation of the effect of MMP-9 pathway inhibition on papilla structure-forming skin model> The skin model with papillae structure was created in accordance with Patent Application No. 2020-101725.

[0044] [Creation of a skin model with nipple structure formation] A culture substrate, which is a component of a papilla structure-forming skin model, was prepared by the following procedure. As the fibrous collagen, a 0.3 w / v% solution of commercially available type I collagen (manufactured by Kurabo Industries Ltd., Cellmatrix (registered trademark) Type-A) and 10×PBS (Phosphate-Buffered Saline: an aqueous solution containing 1370 mmol / L of NaCl, 81 mmol / L of Na 2 HPO 4 , 26.8 mmol / L of KCl, and 14.7 mmol / L of KH2PO4) were mixed at a ratio of 8:1. As the basement membrane matrix preparation, a commercially available Matrigel (manufactured by Corning, standard Matrigel (registered trademark) basement membrane matrix preparation: protein concentration 10 mg / mL) was thawed by leaving it standing for 2 hours under ice cooling, and the resulting solution was used. A 0.1 w / v% aqueous solution of glutaraldehyde was used as the cross-linking agent, and a 2 w / v% aqueous solution of glycine was used as the cross-linking terminator. The solution of fibrous collagen (type I collagen), the solution of basement membrane matrix (Matrigel), and the cross-linking agent solution were finally mixed so that the contents were 0.08 (w / v%) for collagen, 0.7 (w / v%) for Matrigel, and 0.01 (w / v%) for glutaraldehyde, and the total volume was adjusted to 100 w / v% with water. 150 μL of this mixture was injected into each well of an 8-well type chamber slide (manufactured by IWAKI, 10 mm×10 mm) and incubated at 4°C for 12 hours. Then, it was incubated at 37°C for 2 hours to obtain a gel-like substance. Next, 200 μL of a 2 w / v% aqueous glycine solution, which is the cross-linking terminator, was added per well and incubated at 37°C for 2 hours. Then, the liquid on the gel-like substance was removed to obtain a chamber slide holding the culture substrate. Neonatal human epidermal keratinocytes were dispersed in Humedia KG2 (KURABO), which is a culture medium, so as to be 1×10 5 Cells / mL, and 200 μL per well was seeded onto the culture substrate of the chamber slide prepared above. After culturing at 37°C in the presence of 5% CO 2 for 24 hours, a papilla structure-forming skin model was obtained.

[0045] [Confirmation of the effect of MMP-9 inhibition on the papilla formation model] The MMP-9 pathway inhibitor Y-27632 was added to the papillae-forming skin model to a concentration of 20 μM. Subsequently, the cells were cultured for an additional 72 hours, and the resulting keratinocyte cell layer was observed. The medium was removed from the chamber slides, and the chamber slides were washed once with 100 μL of PBS(-) per well. Next, 100 μL of a 0.5 v / v% Triton X-100 solution dissolved in PBS(-) was added to each well. After leaving it at room temperature for 15 minutes, washing was repeated three times with 100 μL of PBS(-) per well. 100 μL of a pre-prepared actin staining solution (2.5 w / v% Alexa Fluor® 488 Phalloidin solution (Thermo Fisher Scientific, in a 1 v / v% BSA in PBS(-) solution)) was added to each well, and the actin was stained by leaving it at room temperature for 20 minutes. Then, 170 μL of 3 v / v% BSA in PBS(-) was added and reacted for 20 minutes for blocking. To this, 70 μL of a solution of the primary antibody (rabbit host anti-laminin antibody (L9393 Sigma)), a 100-fold dilution with PBS(-), was added and reacted at room temperature for 2 hours. Next, washing was performed by repeating contact with Wash buffer (T-PBS) three times for 5 minutes each. 70 μL of a solution of the fluorescently labeled secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermofisher)), a 200-fold dilution with PBS(-), was added and reacted at room temperature for 45 minutes. Washing was performed again by repeating contact with Wash buffer (T-PBS) three times for 5 minutes each. Further washing was performed three times with 100 μL of PBS(-). Then, one drop of Mounting Fluid (Aqua poly / Mount, Diagnostic Biosystems) was added, and observation of the keratinocyte cell layer and the gel was performed in the sectioning mode of a fluorescence microscope (BZ-X700, KEYENCE) (20x magnification). Image analysis was performed using image analysis software (BZ-X Analyzer, KEYENCE).

[0046] 〔Observation Results〕 The observation results are shown in Fig. 7. Neonatal keratinocytes formed papilla-like convexities, and a cell layer was formed on the entire surface of the convexities (the shape of the cell layer is indicated by the white dotted line in the figure). However, in neonatal keratinocytes to which the MMP-9 pathway inhibitor Y-27632 was added, papilla-like convexities were not formed, and a cell layer was formed in a planar shape. From the above results, it became clear that MMP-9 regulates cell contraction and subsequent papilla formation, and if a certain point in that process is promoted, papilla formation can be promoted. That is, by using at least one selected from the amount of MMP-9 gene expression in epidermal cells, the amount of MMP-9 protein in epidermal cells, and the degree of cell contraction in epidermal cells as an index, it was found that a papilla formation promoter can be screened.

[0047] [Test 6] [Evaluation of the effect of Wasabinooki on promoting MMP-9 production] [Preparation of test substances] Preparation of an extract of Wasabinooki: Seeds of Wasabinooki containing dried outer skin were ground, and 10 times the mass of purified water was added, followed by heating extraction at 60 °C for 4 hours. To 1 part of the dry residue of the extract, 124 parts by volume of a 70% aqueous glycerol solution was added and diluted to obtain a test substance. Preparation of an extract of Physalis alkekengi: 10 times the mass of a 50% aqueous ethanol solution was added to dried fruits of Physalis alkekengi var. franchetii, and extraction was carried out at room temperature for 6 days. To 1 part of the dry residue of this extract, 49 parts by volume of distilled water and 50 parts by volume of 1,3-butylene glycol were added to obtain a sample solution. Comparison sample: As samples for comparison, Hikokoshi leaf / stem extract (Ichimaru Pharcos) and saffron root extract (Ichimaru Pharcos) were used.

[0048] [Analysis of MMP-9 gene expression level] Normal human epidermal keratinocytes from young donors (19 years old) and old donors (51 years old) were suspended in Humedia KG2 (KURABO), and a cell suspension was prepared to a concentration of 5.0 × 10 4 cells / mL. Then, 500 μL of the cell suspension was seeded into each well of a 24-well culture plate. Incubation was carried out at 37 °C in 5% CO 2After culturing for 24 hours below, the prepared test substance or the solvent of the test substance was added so that the final concentration became 0.4% and 0.2% respectively, and cultured at 37 °C and 5% CO 2 for 2 days below. Total RNA was extracted using the Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using the PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of MMP-9 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) were measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using the following primers and enzymes. As primers, a sense primer for MMP-9 (5’-GACGCAGACATCGTCATCCA-3’), an antisense primer (5’-AACTCGTCATCGTCGAAATGG-3’), a sense primer for GAPDH (5’-CCACATCGC TCAGACACCAT-3’), and an antisense primer (5’-TGACCAGGC GCCCAATA-3’) were used. Power SYBR Green Master Mix (Applied Biosystems) was used for the PCR reaction, and the analysis of gene expression was performed by the comparative Ct method. That is, the change in the gene expression level due to the addition of the test substance was determined as the relative amount with respect to the value obtained by correcting the Ct value of MMP-9 at the time of adding the solvent of the test substance with the Ct value of GAPDH as 1.

[0049] As shown in Fig. 8, it was confirmed that the extract of Japanese horseradish has an effect of increasing the gene expression level of MMP-9 in the epidermis.

[0050] [Test 7] <Evaluation of the promoting effect of Japanese horseradish on papilla formation> The following topical composition containing wasabi tree extract or Chinese lantern extract was prepared, and the effects of improving papillae and skin condition were confirmed. An appropriate amount of the composition was applied to the faces of 15 panelists (40- to 50-year-old men) once in the morning and once in the evening for 2 months. Observation of papillae was performed on the cheeks using a confocal laser scanning microscope (Vivascope 1500, Caliber I.D.).

[0051] [Table 1]

[0052] For the analysis of the papillae observation images, a horizontal image at a position approximately 25 μm from the start of papillae observation was obtained as a 1.0 mm × 1.0 mm image, and the number of papillae was counted visually. When the number of papillae before using the composition for 2 months was set to 100, the number of papillae after use was calculated.

[0053] As shown in Fig. 9, in the composition containing wasabi tree extract, an increase of 50% or more in papillae was confirmed, while no increase in papillae was observed in the composition containing Chinese lantern extract or the control, which was the comparison target. From these results, it became clear that the extract of wasabi tree, which has an effect of promoting MMP-9 production, has an effect of promoting papillae formation in the skin, and it was found that it can be used as an agent for promoting papillae formation to improve the flattening of papillae associated with aging and the like.

[0054] [Test 8] [Evaluation of the effect of wasabi tree on promoting papillae formation in artificial skin] When the 4.00% aqueous solution of wasabi tree extract described in Test 6 was added to artificial skin prepared according to the production method of Japanese Patent Application No. 2020-101725, the formation of papillae in the artificial skin was promoted. It was confirmed that the wasabi tree extract can be used not only for application to the human body but also as an agent for promoting papillae formation in artificial skin.

Claims

1. A method for screening papilla formation promoters using the degree of cell contraction of epidermal cells as an index.

2. (1) adding a test substance to epidermal cells and culturing them; (2) measuring the degree of cell contraction of epidermal cells; (3) A step of comparing the degree of cell contraction of the epidermal cells obtained in step (2) with a group to which the test substance was not added, and determining that a substance that improves the degree of cell contraction of the epidermal cells is a substance having a papillary process formation promoting effect. The method for screening a papillary process promoter according to claim 1, comprising:

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