A screening method for papillary tract formation promoters, and papillary tract formation promoters

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, 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 towards the stratum corneum by division, and are peeled off as so-called dirt in order from the surface layer.

[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. 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 more mature than the elastic fibers of the papillary layer.

[0004] Papillae protruding towards 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 dermis layer and the epidermal layer mesh with each other is formed between the epidermis and the dermis. The space between the epidermal layer and the dermis layer is separated by a membrane structure called the basement membrane, and it can be said that the papillae are structures composed of the epidermal layer, the dermis layer, and the basement membrane.

[0005] The dermis and epidermis are separated by a basement membrane, but signal transduction and transport of waste products and nutrients occur between the dermis and epidermis via papillary processes, and the interlocking of the dermis and epidermis is thought to mitigate physical stimuli from the outside. In addition, it has been reported that the shape of the papillary processes is related to skin condition (stratum corneum water content, transepidermal water loss, stratum corneum cell area, skin color) (Patent Document 1), and it is considered important to properly maintain the papillary processes in order to maintain a normal skin condition. However, it is known that papillary processes flatten with age and ultraviolet radiation (Non-Patent Document 1), and deformation, reduction in number, or loss of papillary processes can occur. Therefore, there is a need for methods to properly maintain papillary processes.

[0006] Thus, maintaining proper papillary processes is a very effective way to maintain skin condition. However, the mechanism of papillary process formation has not been clarified until now, and when searching for papillary process improvement agents, it has been necessary to actually apply them to human skin and observe and evaluate the state of the papillary processes themselves. However, since papillary processes are structures located inside the skin, evaluating their condition is not easy, and methods such as excising human skin and creating sections for observation, excising human skin, peeling the epidermal layer and observing with an electron microscope, or using expensive equipment such as a confocal laser biomicroscope have been employed, which have placed a physical and mental burden on the sample provider and require significant expense and effort from the researchers. Therefore, there has been a desire to elucidate the mechanism of papillary process formation and develop a simple screening method for papillary process formation promoters.

[0007] Furthermore, promoting papillary formation is important not only for maintaining the papillary structure of the human body but also in the manufacture of artificial skin. Artificial skin is used to replace or regenerate damaged skin such as burns and trauma, and methods for obtaining skin models with papillary formation that can be used for artificial skin are known (Japanese Patent Application No. 2020-101725). However, in order to manufacture skin models with papillary formation of stable quality that can be used industrially, a method for promoting papillary formation has been required.

[0008] Matrix metalloproteinases (MMPs) are a type of protease in which metals are involved in the catalytic mechanism. Also known as neutrophil gelatinase, type IV collagenase, or gelatinase B, MMPs are enzymes that exhibit broad substrate specificity for denatured collagen (gelatin) and native collagen (type IV, V, and XI collagen). They are known to be involved in physiological phenomena such as protein degradation and the activation of bioactive substances. For example, MMP-9 is known to generate actomyosin contractility via the Rho / Rock signaling pathway, which triggers cell movement such as cell migration (Non-Patent Literature 2). However, the contribution of MMP-9 and cell contraction to the promotion of papillary ridge formation had not been previously known. In addition, as mentioned above, MMP-9 has been reported to degrade type IV collagen, which is specifically expressed in the basement membrane. While inhibition of its production has been studied to improve skin diseases, skin inflammation, and aging (Non-Patent Literature 3), the idea of ​​enhancing the function of MMP-9 with the aim of normalizing skin conditions had not existed. In other words, it was completely unknown that MMP-9, which is conventionally known to degrade the basement membrane, one of the components of papillary ridges, could contribute to the promotion of papillary ridge formation.

[0009] While the use of at least one protein fraction of Moringa oleifera extract in skincare (Patent Document 2) is known, the possibility that Moringa oleifera extract other than a specific protein fraction could enhance MMP-9 function was completely unknown. Furthermore, a method for improving skin condition (Patent Document 3) has been disclosed that includes one or more components selected from a group of various materials including Moringa oleifera or its extract, but the action of Moringa oleifera or its extract is unknown, and the method is based on the idea that elevated MMP expression levels by fibroblasts are associated with accelerated aging, so it was not intended at all to promote MMP expression. In addition, it has been reported that an extract of Moringa oleifera in 90% ethanol has the effect of inhibiting the production of the aforementioned MMP-9 and suppressing the degradation of the extracellular matrix by MMP-9 (Patent Document 4). As described above, although several uses of Moringa oleifera extract are known, they have been used primarily with the intention of reducing the function of MMP or MMP-9, and the possibility that Moringa oleifera extract could enhance the function of MMP-9 was completely unknown. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2016-016277 [Patent Document 2] Special Publication No. 2002-507575 [Patent Document 3] Special Publication No. 2014-521698 [Patent Document 4] Special Publication No. 2012-530769 [Non-patent literature]

[0011] [Non-Patent Document 1] Kawasaki K, Yamanishi K, Yamada H., Int J Dermatol.2015 54(3):295-301. [Non-Patent Document 2] Jose L Orgaz et al.,Nat Commun.2014 jun 25;5:4255. [Non-Patent Document 3] Takashi Kobayashi, Ensho Saisei. 2004 24(5):578–583. [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide a screening method for papillary tract formation promoters and a novel papillary tract improvement agent. [Means for solving the problem]

[0013] As a result of diligent research, the inventors have revealed that MMP-9 is involved in promoting papillary ridge formation, and have invented a screening method for papillary ridge formation promoters using at least one of the following as indicators: 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, thereby solving the aforementioned problem. Furthermore, by conducting screening using the screening method described above, we confirmed that the extract of Moringa oleifera, which was judged to have a papillary protrusion formation promoting effect according to the indicators of the present invention, actually exhibited a papillary protrusion formation promoting effect, thus completing the present invention. [Effects of the Invention]

[0014] The present invention provides a screening method for papillary ridge formation promoters, as well as an MMP-9 production promoter and a papillary ridge formation promoter containing an extract of Moringa oleifera. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the amount of MMP-9 protein in epidermal cells from young donors and epidermal cells from aged donors. [Figure 2]It is a diagram showing the MMP-9 gene expression levels of epidermal cells from young donors and epidermal cells from old donors. [Figure 3] It is a diagram showing the results of immunostaining of skin tissues with MMP-9 antibody. [Figure 4] It is a diagram showing the basal contractility of epidermal cells under each condition. [Figure 5] It is a diagram showing a remarkable example of epidermal cells with decreased contractility due to aging. [Figure 6] It is a diagram showing the results of observing the structure of actin filaments when an MMP-9 pathway inhibitor is applied. [Figure 7] It is a diagram showing the ability of epidermal cells under each condition to form papilla-like structures. [Figure 8] It is a diagram showing the MMP-9 gene expression levels of epidermal cells when each plant extract is added. [Figure 9] It is a diagram showing the change rate of the number of papillae before and after using each plant extract-containing cream.

Mode for Carrying Out the Invention

[0016] The screening method for the 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 of epidermal cells, the MMP-9 protein amount of epidermal cells, and the cell contractility of epidermal cells. (3) Comparing at least one selected from the MMP-9 gene expression level of epidermal cells, the MMP-9 protein amount of epidermal cells, and the cell contractility of epidermal cells obtained in step (2) with the non-addition group of the test substance, and determining a substance that improves at least one of the selected MMP-9 gene expression level of epidermal cells, the MMP-9 protein amount of 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 papillary ridge formation promoters in the present invention is not particularly limited as long as it is an epidermal cell, but is preferably a human-derived cultured epidermal cell, and is even more preferably a cell in which the MMP-9 gene expression level and / or MMP-9 protein level has been reduced. Examples of cells in which the MMP-9 gene expression level and / or MMP-9 protein level of epidermal cells have been reduced include cells from older donors, cells that mimic older donor cells by known aging induction methods, or cells in which the MMP-9 gene expression level and / or MMP-9 protein level have been reduced compared to undamaged cells by being damaged by known drugs or treatments.

[0018] For culturing epidermal cells, any known culture medium suitable for culturing epidermal cells used in this invention can be used. Examples include epidermal keratinocyte proliferation medium (Humedia KG2 (KURABO)) and DMEM (Dulbecc's modified Eagle medium). During cell proliferation, it is preferable to add serum such as fetal bovine serum, growth factors, antibacterial agents, insulin, and other additives.

[0019] There are no particular restrictions on the test substance. It can be an extract derived from plants or animals, a culture of fungi, or an enzyme-treated product thereof, a compound, or a derivative thereof. It may also be in liquid, powder, or gel form.

[0020] In this invention, "using the MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells as indicators" means using any method to determine the effectiveness of the method. The "MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells" used as indicators in this invention only needs to be able to quantitatively determine the amount of MMP-9 produced in the cells. In addition to directly quantifying the MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells, it is also possible to indirectly determine the "MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells" by quantifying the amount of MMP-9 that changes in the measured value due to changes in the "MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells," such as the amount of degradation of known substrate proteins or the enzyme activity of MMP-9.

[0021] The quantification of MMP-9 gene expression levels and / or MMP-9 protein levels in epidermal cells can be performed using known methods. For example, gene expression levels can be measured using real-time PCR or semi-quantitative PCR, while protein levels can be measured using immunohistochemistry, Western blotting, ELISA, liquid chromatography, gas chromatography, or mass spectrometry. When indirectly understanding "MMP-9 gene expression levels and / or MMP-9 protein levels in epidermal cells" by quantifying the factors whose measured values ​​fluctuate due to these fluctuations, for example, the degradation of denatured collagen can be measured using MMP-9 enzyme activity measurement, zymography, etc.

[0022] In this invention, "using the degree of cell contraction as an indicator" means using the degree of cell contraction as a criterion for evaluating the effect using any method. The degree of cell contraction used as an indicator in this invention represents the force exerted on the substrate by which cells contract when they adhere to a deformable substrate such as a gel, causing the substrate to deform and contract simultaneously. The higher the force that causes the substrate to contract, the higher the degree of cell contraction is judged to be.

[0023] The degree of cell contraction can be measured using known methods. For example, it can be measured by observing changes in cell size using a microscope or visually, by measuring the degree of deformation of the culture substrate surrounding the cells, or by measuring actomyosin activity that causes cell contraction. When measuring the degree of deformation of the culture substrate surrounding the cells, for example, a commercially available FLECS plate can be used as a method for measuring cell contraction force (https: / / www.funakoshi.co.jp / contents / 69303). A FLECS plate consists of fluorescently labeled cell adhesion molecules, and when cells bound to these molecules contract, the adhesion molecules also contract, allowing the amount of size displacement of the cell adhesion molecules to be analyzed as the degree of cell contraction.

[0024] The group without the test substance in this invention is not particularly limited as long as it can serve as a control that can evaluate the effect of the test substance on improving the MMP-9 gene expression level and / or the amount of MMP-9 protein in epidermal cells, or the degree of cell contraction. For example, it includes using the solvent used to dissolve the test substance, and adding nothing else.

[0025] In this invention, for example, the MMP-9 gene expression level, the amount of MMP-9 protein in epidermal cells, or the degree of cell contraction are compared with a group without the test substance, and it is determined that a test substance that improves the MMP-9 gene expression level, the amount of MMP-9 protein in epidermal cells, or the degree of cell contraction can be used as a papillary ridge formation promoter. A test substance that increases the gene expression level or protein level by 10% or more compared to the group without the substance, or a test substance that increases the degree of cell contraction by 5% or more compared to the group without the substance, is determined to be highly effective as a papillary ridge formation promoter.

[0026] The present invention provides a screening method for papillary ridge formation promoters using the amount of MMP-9 gene expression and / or the amount of MMP-9 protein in epidermal cells as indicators. For example, when measuring the amount of MMP-9 gene expression, the method can be performed as follows. (1) Cultivate epidermal cells in a culture medium in a petri dish. (2) Add the test substance dissolved in water, and at the same time add the same volume of water as the test substance to represent the group without the test substance. (3) The cells are cultured for a certain period of time to allow the test substance to act on the epidermal cells. (4) Extract total RNA from epidermal cells. (5) Synthesize cDNA from total RNA. (6) The MMP-9 gene expression level is quantified by real-time PCR, and the MMP-9 gene expression level when the test substance is added is calculated compared to the group without the test substance. (7) If the MMP-9 gene expression level increased by 10% or more when the test substance was added compared to the control group, the test substance is deemed usable as a papillary ridge formation promoter.

[0027] The Moringa oleifera Lam. used in this invention is a tree cultivated in India, the Philippines, Taiwan, and other regions, and is also known by its genus name, Moringa. It is rich in nutrients such as polyphenols, amino acids, and vitamin E, and due to its high nutritional value and efficacy, it is called "green milk" or "tree of life" in some regions.

[0028] The part of the Moringa oleifera plant used for extraction in the present invention is not particularly limited. For example, seeds, trunks, branches, leaves, flowers, roots, etc., can be used. Among these, seeds are preferred, and it is even more preferable to use seeds that have been crushed and include the outer shell. The extraction solvent is not particularly limited, and examples include water, lower alcohols such as propyl alcohol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, but water is particularly preferred.

[0029] The extraction method of the wasabi extract used in the present invention is not particularly limited. For example, 1 to 100 parts by mass of water and a mixed solution of 1,3-butylene glycol or ethanol are 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 freeze-dried and used in a powder state.

[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 oil agents, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizing agents, 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 papilla through the promotion of cell motility. The cell type to 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, etc.

Examples

[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 at 8.5×10 in Humedia KG2 (KURABO). 4They were dispersed to a concentration of Cells / mL and seeded at 2 mL per well on a 6-well plate. They were cultured at 37 °C under 5% CO2 for 72 hours. 2 mL of the medium was collected. The amount of MMP-9 protein in the collected medium was measured according to the protocol of the Human MMP Antibody Array Membrane (abcam), and the relative amount of MMP-9 protein in the elderly donor was determined with the amount of MMP-9 protein in the young donor set as 1.

[0033] As shown in Figure 1, in the elderly 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 decreases with aging.

[0034] [Test 2] [Evaluation of the Relationship between MMP-9 Gene Expression Level and Aging] Normal human epidermal keratinocytes from young donors (19 years old) and elderly donors (51 years old) were suspended in Humedia KG2 (KURABO), 8.5×10 4The cell suspension was prepared to be [[ID=]], and 500 μL of it was seeded into each well of a 24-well culture plate. After culturing for 2 days at 37 °C under 5% CO2, total RNA was extracted using the Total RNA Purification Kit (Jena Bioscience). Subsequently, 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, the sense primer for MMP-9 (5’-GACGCAGACATCGTCATCCA-3’), the antisense primer (5’-AACTCGTCATCGTCGAAATGG-3’), the sense primer for GAPDH (5’-CCACATCGC TCAGACACCAT-3’), and the 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 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 tissue was obtained from the abdomen of young (29 years old) and elderly (72 years old) donors, purchased from BIOPREDIC. The human skin tissue was embedded in OCT compound (Sakura FineTech Japan) and frozen. 4 μm sections were prepared using a cryostat and mounted on glass slides. These were fixed by immersion in 95% ethanol and then immunostained. The primary antibody used was Anti MMP-9 rabbit polyclonal antibody, and the secondary antibody was Alexa Fluor 594 conjugate anti-rabbit IgG(H+L) chicken secondary antibody. Subsequently, the stained sections were observed under a fluorescence microscope (BZ-X700, KEYENCE) at 20x magnification and photographed.

[0037] Figure 3 shows the results of immunostaining of skin tissue using an MMP-9 antibody. In immunostaining using MMP-9, the antibody binds to MMP-9 in the skin, and a fluorescent secondary antibody binds to it, causing the area where MMP-9 is present to fluoresce red. As indicated by the white arrows in Figure 3, in skin derived from young humans with a clear papillary structure, there is a region around the papillary structure that fluoresces strongly red, which is not observed in skin derived from aged humans without a papillary structure. It was found that the area where MMP-9 is present closely coincides with the contour of the papillary epidermal cell side. In other words, the presence of MMP-9 on the papillary epidermal cell side correlates with the presence or absence of papillary structures.

[0038] [Exam 4] <Evaluation of age-related changes in cell contractility and the effects of 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) 5.0 × 10 4Cells / mL were dispersed in Humedia KG2 (KURABO) and seeded in 500 μL portions on the FLECS Plate (Forcyte Biotechnologies) cell contraction force measurement kit. After culturing for 6 hours at 37°C under 5% CO2 conditions, 20 μM of the MMP-9 pathway inhibitor Y-27632 was added to the neonatal donor-derived human epidermal keratinocytes. After further culturing for 20 hours at 37°C under 5% CO2 conditions, the cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and microscopic observation (20x magnification) and photography were performed using a fluorescence microscope (BZ-X700, KEYENCE). The length of the lines forming the cross on the cross-shaped gel to which the cells were attached in the photographs was measured using imageJ (OPEN SOURCE), and the degree of substrate contraction by the cells was calculated as follows. Furthermore, the cell structure of human epidermal keratinocytes derived from neonatal donors, treated with the cell actin staining reagent Alexa Fluor® 488 Phalloidin, was observed (20x magnification) and photographed using a fluorescence microscope (BZ-X700, KEYENCE).

[0039] The FLECS Plate cell contraction force measurement kit has a structure in which a cross-shaped gel is placed on plastic, and the cross-shaped gel is observed as red fluorescence. Furthermore, by staining the cell nucleus blue with DAPI, it is possible to determine which cross-shaped gels cells are attached to. The substrate contraction degree (cell contraction degree) is expressed by the following formula based on the lengths of the lines forming the cross on the cross-shaped gel without cell attachment (control gel) and the lengths of the lines forming the cross-shaped gel with cell attachment.

[0040]

number

[0041] Figure 4 shows the results of plotting the average basal cell contraction of epidermal cells, measured with N=6. Compared to human epidermal cells derived from neonatal donors, human epidermal cells derived from aged 50-year-old and 51-year-old adult donors showed lower cell contraction, indicating that cell contraction decreases with age. A case of remarkable efficacy is shown in Figure 5. Furthermore, it was found that applying Y-27632, an MMP-9 pathway inhibitor, to human epidermal cells derived from neonatal donors reduced cell contraction, similar to age-related changes. In other words, this study demonstrated that MMP-9 plays an important role in epidermal cell contraction, and at the same time, that the amount of MMP-9 gene expression and / or MMP-9 protein in epidermal cells can be determined by measuring cell contraction.

[0042] Figure 6 shows the results of observing the structure of actin filaments in human epidermal keratinocytes derived from neonatal donors after treatment with the MMP-9 pathway inhibitor Y-27632. In neonatal keratinocytes without Y-27632 treatment, intracellular actin filaments (indicated by white arrows) were present, but in neonatal keratinocytes treated with Y-27632, actin filaments were not formed, and the cells appeared to be hypertrophied (not contracting). In other words, the degree of cell contraction is controlled by MMP-9, and taking all of these results together, we hypothesized that the decrease in MMP-9 with age is the cause of the decrease in the degree of cell contraction, and that this decrease in the degree of cell contraction with age makes it difficult for morphological changes in the epidermis to occur, thus reducing the ability to form papillary ridges. Experiment 5 validated this hypothesis.

[0043] [Exam 5] <Confirmation of the effect of MMP-9 pathway inhibition on papillary ridges using a skin model for papillary ridge formation> The creation of the skin model for papillary structure formation was carried out in accordance with Japanese Patent Application No. 2020-101725.

[0044] [Creation of a skin model with papillary ridge structure] The culture substrate, a component of the papillary structure-forming skin model, was prepared using the following procedure. As fibrous collagen, a solution was used which was an 8:1 mixture of a 0.3 w / v solution of commercially available type I collagen (Kurabo Industries Ltd., Cellmatrix® Type-A) and 10×PBS (Phosphate-Buffered Saline: an aqueous solution containing 1370 mmol / L NaCl, 81 mmol / L Na2HPO4, 26.8 mmol / L KCl, and 14.7 mmol / L KH2PO4) at pH 7.4. For the basement membrane matrix preparation, a commercially available Matrigel (Corning, Standard Matrigel® Basement Membrane Matrix Preparation: protein concentration 10 mg / mL) was used, which was thawed by leaving it on ice for 2 hours. A 0.1 w / v aqueous solution of glutaraldehyde was used as a crosslinking agent, and a 2 w / v aqueous solution of glycine was used as a crosslinking inhibitor. A solution of fibrous collagen (type I collagen), a solution of basement membrane matrix (Matrigel), and a crosslinking agent solution were mixed to a final content of 0.08 (w / v%) collagen, 0.7 (w / v%) Matrigel, and 0.01 (w / v%) glutaraldehyde, and the total volume was diluted to 100 w / v% with water. 150 μL of this mixture was injected into each well of an 8-well type chamber slide (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 2 w / v% glycine aqueous solution, a crosslinking inhibitor, was added to each well and incubated at 37°C for 2 hours. After removing the liquid from the gel-like substance, a chamber slide holding the culture substrate was obtained. 1 × 10⁶ neonatal human epidermal keratinocytes were cultured in the culture medium. 5 The cells were dispersed in Humedia KG2 (KURABO) culture medium to a concentration of Cells / mL, and 200 μL was seeded per well onto the culture substrate of the chamber slide prepared above. After 24 hours at 37°C in the presence of 5% CO2, a skin model with papillary structure formation was obtained.

[0045] [Confirmation of the effect of MMP-9 inhibition on a papillary ridge formation model] The MMP-9 pathway inhibitor Y-27632 was added to a papillary ridge formation skin model to a concentration of 20 μM. The cells were then cultured for a further 72 hours, and the resulting keratinocyte cell layer was observed. The culture medium was removed from the chamber slide, and each well was washed once with 100 μL of PBS(-). Next, 100 μL of 0.5 v / v% Triton X-100 solution dissolved in PBS(-) was added to each well. After standing at room temperature for 15 minutes, the washing with 100 μL of PBS(-) per well was repeated three times. 100 μL of pre-prepared actin staining solution (2.5 w / v% Alexa Fluor® 488 Phalloidin solution (Thermo Fisher Scientific, with 1 v / v% BSA in PBS(-) solution)) was added to each well, and the cells were left at room temperature for 20 minutes to stain the actin. Next, 170 μL of 3v / v% BSA in PBS(-) was added and the mixture was blocked after 20 minutes of reaction. Then, 70 μL of a solution of the primary antibody (anti-laminin antibody, rabbit host antibody (L9393 Sigma)) (100-fold dilution in PBS(-)) was added and the mixture was reacted at room temperature for 2 hours. Subsequently, the mixture was washed by contacting it with Wash buffer (T-PBS) for 5 minutes three times. 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)) (200-fold dilution in PBS(-)) was added and the mixture was reacted at room temperature for 45 minutes. The mixture was washed again by contacting it with Wash buffer (T-PBS) for 5 minutes three times. Finally, it was washed three times with 100 μL of PBS(-). Then, one drop of Mounting Fluid (Aqua poly / Mount, Diagnostic Biosystems) was added, and the keratinocyte cell layer and gel were observed using a fluorescence microscope (BZ-X700, KEYENCE) in sectioning mode (20x magnification). Image analysis was performed using image analysis software (BZ-X Analyzer, KEYENCE).

[0046] [Observation results] The observation results are shown in Figure 7. In neonatal keratinocytes, papillary projections were formed and a cell layer was formed over the entire surface of the projections (the shape of the cell layer is shown by the white dotted line in the figure). However, in neonatal keratinocytes treated with the MMP-9 pathway inhibitor Y-27632, papillary projections were not formed, and a cell layer was formed in a planar manner. From these results, it became clear that MMP-9 regulates cell contraction and the subsequent papillary projection formation, and that promoting any part of this process can promote papillary projection formation. In other words, it was found that papillary projection promoters can be screened using at least one indicator selected from the MMP-9 gene expression level in epidermal cells, the amount of MMP-9 protein in epidermal cells, or the degree of cell contraction in epidermal cells.

[0047] [Exam 6] <Evaluation of the MMP-9 production-promoting effect of Moringa oleifera> [Preparation of the test substance] Preparation of Moringa extract: Moringa seeds, including the dried outer bark, were crushed, and extracted by heating at 60°C for 4 hours with 10 times the mass of purified water. The test substance was prepared by diluting the dried residue of the extract with 124 times the volume of 70% glycerin aqueous solution. Preparation of Physalis extract: Dried Physalis rupestris fruits were added to a 50% ethanol aqueous solution 10 times by mass and extracted at room temperature for 6 days. To the dry residue of this extract, 49 parts distilled water and 50 parts 1,3-butylene glycol were added to prepare the sample solution. Comparative samples: For comparison, we used Isodon japonicus leaf / stem extract (Ichimaru Pharcos) and Scutellaria baicalensis root extract (Ichimaru Pharcos).

[0048] [MMP-9 gene expression level analysis] Normal human epidermal keratinocytes from young donors (19 years old) and elderly donors (51 years old) were suspended in Humedia KG2 (KURABO) and 5.0 × 10⁻⁶ 4Cell suspensions were prepared to a concentration of cells / mL and seeded in 500 μL portions in 24-well culture plates. After incubation at 37°C under 5% CO2 for 24 hours, the prepared test substance or its solvent was added to final concentrations of 0.4% and 0.2%, respectively, and the cells were incubated at 37°C under 5% CO2 for 2 days. Total RNA was extracted using the Total RNA Purification Kit (Jena Bioscience). Subsequently, 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. The primers used were the MMP-9 sense primer (5'-GACGCAGACATCGTCATCCA-3'), antisense primer (5'-AACTCGTCATCGTCGAAATGG-3'), GAPDH sense primer (5'-CCACATCGC TCAGACACCAT-3'), and antisense primer (5'-TGACCAGGC GCCCAATA-3'). Power SYBR Green Master Mix (Applied Biosystems) was used for the PCR reaction, and gene expression analysis was performed using the comparative Ct method. In other words, the change in gene expression levels due to the addition of the test substance was determined as a relative amount to a value of 1, which was obtained by correcting the MMP-9 Ct value at the time of solvent addition of the test substance by the GAPDH Ct value.

[0049] As shown in Figure 8, it was confirmed that the extract from Moringa oleifera has the effect of increasing the gene expression level of MMP-9 in the epidermis.

[0050] [Exam 7] <Evaluation of the papillary projection formation promoting effect of Moringa oleifera> The following topical compositions containing Moringa oleifera extract or Physalis alkekengi extract were prepared, and their effects on improving papillary ridges and skin condition were confirmed. Fifteen panelists (men aged 40-50) applied an appropriate amount of the composition to their faces once in the morning and once in the evening for two months. Papillary ridges were observed on the cheek area using a confocal laser biomicroscope (Vivascope1500, Caliber ID).

[0051] [Table 1]

[0052] For the analysis of the papillary projection observation images, a horizontal image of 1.0 mm × 1.0 mm was obtained at a position approximately 25 μm from the point where the papillary projections first became visible. The number of papillary projections was visually counted, and the number of papillary projections after use was calculated, with the number of papillary projections before use of the composition set to 100 over a two-month period.

[0053] As shown in Figure 9, a 50% or greater increase in papillae was observed in the composition containing Moringa oleifera extract, while no increase in papillae was observed in the comparative composition containing Physalis alkekengi extract or in the control group. These results revealed that the extract of Moringa oleifera, which has an MMP-9 production-promoting effect, also has an effect of promoting the formation of papillary ridges in the skin, and that it can be used as a papillary ridge formation promoter to improve the flattening of papillary ridges associated with aging, etc.

[0054] [Test 8] <Evaluation of the effect of Moringa oleifera on promoting papillary ridge formation on artificial skin> When a 4.00% aqueous solution of Moringa oleifera extract described in Test 6 was added to artificial skin prepared according to the method of Japanese Patent Application No. 2020-101725, the formation of papillary projections on the artificial skin was promoted. This confirmed that Moringa oleifera extract can be used not only for topical application to the human body but also as a papillary projection promoting agent for artificial skin.

Claims

1. (1) A step of adding the test substance to cultured epidermal cells and culturing them, (2) A step of measuring the degree of cell contraction of cultured epidermal cells, (3) The degree of cell contraction of the cultured epidermal cells obtained in step (2) is compared with that of the group without the test substance. A step in determining whether a substance that improves the degree of cell contraction in cultured epidermal cells is a substance that promotes papillary ridge formation. A screening method for a papillary ridge formation promoting agent comprising the above.

2. A substance that increases the degree of cell contraction of cultured epidermal cells by 5% or more compared to the group without the substance. It is determined that the substance has the effect of promoting the formation of papillary protrusions. The screening method according to claim 1.

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