Screening method for papilla formation promoter and papilla formation promoter

Abstract

To provide a screening method for a papilla formation promoter, and a novel papilla improver.SOLUTION: The present invention discloses a screening method for papilla formation promoter that uses, as an index, at least one of a level of expression of MMP-9 gene in epidermal cells, an amount of MMP-9 protein in epidermal cells, and a degree of contraction in epidermal cells, and an MMP-9 production promoter and a papilla formation promoter containing extract from Moringa oleifera.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to a method for screening useful papillae formation promoters and to papillae formation promoters selected thereby. [Background technology]

[0002] The skin is made up of three layers, starting from the surface layer that comes into contact with the outside air: the epidermis, dermis, and subcutaneous tissue. The epidermis is made up of cells called keratinocytes, and from the deepest part close to the dermis, it is divided into the basal layer, spinous layer, granular layer, and stratum corneum. In the epidermis, keratinocytes are pushed up from the basal layer toward the stratum corneum by division, and are exfoliated from the surface layer as so-called dirt.

[0003] The dermis is divided into the papillary layer, which is close to the epidermis, and the reticular layer, which lies deeper. The papillary layer mainly contains collagen fibers and elastic fibers made up of elastin, as well as other extracellular matrix components and fibroblasts. The reticular layer is thicker than the papillary layer and makes up the majority of the dermis. The reticular layer also contains collagen fibers and elastic fibers made up of elastin, but they are thicker than the collagen fibers of the papillary layer and are more mature than the elastic fibers of the papillary layer.

[0004] The upper part of the papillary layer has papillae protruding toward the epidermis. The epidermis layer fits between these papillae, and an uneven structure is formed between the epidermis and dermis, where the papillary layer of the dermis and the epidermis layer interlock. The epidermis and dermis are separated by a membrane structure called the basement membrane, and the papillae can be said to be a structure composed of the epidermis, dermis, and basement membrane.

[0005] Although the dermis and epidermis are separated by a basement membrane, signal transmission and transport of substances such as waste products and nutrients are carried out between the dermis and epidermis via the papillae, and the interlocking of the dermis and epidermis is said to have the effect of mitigating physical stimuli from the outside. In addition, it has been reported that the shape of the papillae is also related to skin conditions (stratum corneum moisture content, transepidermal water loss, stratum corneum cell area, skin color) (Patent Document 1), and it is considered important to properly maintain the papillae in order to keep the skin in a normal condition. However, it is known that the papillae become flattened by aging and ultraviolet rays (Non-Patent Document 1), and deformation, a decrease in number, or loss of papillae occur. Therefore, a method for properly maintaining the papillae is required.

[0006] Thus, maintaining the papillae appropriately is a very effective method for maintaining the skin condition, but the mechanism of papillae formation has not been clarified so far, and when searching for a papillae improvement agent, it was necessary to actually apply it to human skin and observe and evaluate the papillae condition itself. However, since papillae are structures that exist inside the skin, it is not easy to evaluate their condition, and methods that require physical and mental burdens on the observation sample provider and cost and labor of researchers, such as removing human skin and making slices for observation, removing human skin and peeling off the epidermal layer and observing it with an electron microscope, or observing it using expensive equipment such as a confocal laser biomicroscope, have been used. Therefore, it has been desired to clarify the mechanism of papillae formation and develop a simple screening method for papillae formation promoters.

[0007] In addition, promoting papilla formation is important not only for maintaining the papilla structure of the human body, but also for producing artificial skin. Artificial skin is used to replace or regenerate skin damaged by burns, trauma, etc., and a method for obtaining a skin model with papillae that can be used for artificial skin is known (Patent Application No. 2020-101725). However, in order to produce a skin model with papillae of stable quality that can be used industrially, a method for promoting papilla formation was required.

[0008] Matrix metalloproteinase (MMP) is a type of protease in which metals are involved in the catalytic mechanism. It is also known as neutrophil gelatinase, type IV collagenase, and gelatinase B. It is an enzyme that shows broad substrate specificity toward denatured collagen (gelatin) and native collagen (type IV, type V, and type XI collagen), and is known to be involved in physiological phenomena such as the activation of physiologically active substances in addition to the degradation of proteins. For example, it is known that MMP-9 generates actomyosin contractility via the Rho / Rock signaling pathway, and induces cell motility such as cell migration (Non-Patent Document 2). However, the contribution of MMP-9 or cell contraction to the promotion of papilla formation has not been known, and in addition, as mentioned above, it has been reported that MMP-9 degrades type IV collagen, which is specifically expressed in the basement membrane, and although inhibition of production has been studied to improve 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 skin condition. In other words, it was completely unknown that MMP-9, which has been known to degrade the basement membrane, one of the components of papilla formation, contributes to the promotion of papilla formation.

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

[0010] [Patent Document 1] JP 2016-016277 A [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. Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a method for screening a papilla formation promoter and a novel papilla improvement agent. [Means for solving the problem]

[0013] As a result of extensive research, the inventors have revealed that MMP-9 is involved in promoting papilla formation and have invented a screening method for papilla formation promoters using at least one of the following as an indicator: the level of MMP-9 gene expression in epidermal cells, the amount of MMP-9 protein in epidermal cells, or the degree of cell contraction in epidermal cells, thereby solving the above-mentioned problem. Furthermore, screening was carried out using the screening method, and it was confirmed that the extract of Wasabinoki, which was determined to have a papilla formation-promoting effect using the indicators of the present invention, actually does have the papilla formation-promoting effect, thereby completing the present invention. Effect of the Invention

[0014] INDUSTRIAL APPLICABILITY The present invention can provide a method for screening a papillary process formation promoter, and an MMP-9 production promoter and a papillary process formation promoter containing an extract of Moringa oleifera. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the amounts of MMP-9 protein in epidermal cells derived from young donors and epidermal cells derived from old donors. [Diagram 2]FIG. 1 shows the expression levels of MMP-9 gene in epidermal cells derived from young donors and epidermal cells derived from old donors. [Diagram 3] FIG. 1 shows the results of immunostaining of skin tissues with an MMP-9 antibody. [Figure 4] FIG. 13 shows the degree of basal contraction of epidermal cells under each condition. [Diagram 5] FIG. 1 shows a remarkable example of epidermal cells whose contraction rate has decreased due to aging. [Figure 6] FIG. 1 shows the results of observing the structure of actin fibers when an MMP-9 pathway inhibitor was applied. [Figure 7] FIG. 1 shows the ability of epidermal cells to form papilla-like structures under each condition. [Figure 8] FIG. 1 shows the expression level of MMP-9 gene in epidermal cells when each plant extract was added. [Figure 9] FIG. 1 shows the rate of change in the number of papillae before and after use of creams containing each plant extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The method for screening a papillary process formation promoter according to the present invention comprises the steps of: (1) adding a test substance to epidermal cells and culturing them; (2) measuring at least one selected from the expression level of MMP-9 gene in epidermal cells, the amount of MMP-9 protein in epidermal cells, and the degree of cell contraction in 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 contraction level of epidermal cells obtained in step (2) with a group to which the test substance was not added, and determining that 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 contraction level of epidermal cells is a substance having a papillary process formation promoting effect; Includes.

[0017] The type of cells used in the screening method for papillary formation promoters of the present invention is not particularly limited as long as they are epidermal cells, but preferably human-derived cultured epidermal cells are used, and it is more preferable to use cells in which the MMP-9 gene expression level and / or MMP-9 protein level are reduced. Examples of cells in which the MMP-9 gene expression level and / or MMP-9 protein level of epidermal cells is reduced include cells from older donors, cells that mimic older donor cells using known aging induction methods, and cells that have been damaged by known drugs or treatments and have reduced MMP-9 gene expression level and / or MMP-9 protein level compared to undamaged cells.

[0018] The medium for culturing epidermal cells may be any known medium suitable for culturing the epidermal cells used in the present invention. Examples include a medium for growing epidermal keratinocytes (Humedia KG2 (KURABO)) and DMEM (Dulbecco's modified Eagle's medium). During cell growth, it is preferable to add serum such as fetal bovine serum, and additives such as growth factors, antibacterial agents, and insulin.

[0019] There are no particular limitations on the test substance. Extracts derived from animals and plants, cultures of fungi, or products of these treated with enzymes or the like, compounds or derivatives thereof, etc. can be used as the test substance, and they can be in liquid, powder, gel, etc.

[0020] In the present invention, "using the MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells as an index" means that any method is used to use the MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells as a criterion for effect evaluation. The "MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells" used as an index in the present invention may be any amount that can quantitatively grasp the amount of MMP-9 produced in cells, and in addition to directly quantifying the MMP-9 gene expression level and MMP-9 protein amount, it can also be indirectly grasped as the "MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells" by quantifying the measurement value fluctuating due to the fluctuation of the "MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells", such as the amount of decomposition of a protein known as a substrate and the enzyme activity of MMP-9.

[0021] The quantification of the MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells can be performed by known methods. For example, the gene expression level can be measured by real-time PCR or semi-quantitative PCR, and the protein level can be measured by immunostaining, Western blotting, ELISA, liquid chromatography, gas chromatography, mass spectrometry, etc. When the measurement value fluctuates due to the fluctuation of the "MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells", the "MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells" can be indirectly grasped by quantifying the subject whose measurement value fluctuates due to the fluctuation of the "MMP-9 gene expression level and / or the MMP-9 protein level in epidermal cells", for example, the decomposition of denatured collagen can be measured by MMP-9 enzyme activity measurement, zymography, etc.

[0022] In the present invention, "using the cell contraction degree as an index" means that any method is used to use the cell contraction degree as a criterion for effect evaluation. The cell contraction degree used as an index in the present invention represents the force exerted on the adhered substrate by which the cells contract and deform the substrate when the cells adhere to the substrate and contract when the cells adhere to the substrate and contract, and the higher the force that causes the substrate to contract, the higher the cell contraction degree is determined to be.

[0023] The cell contraction degree can be measured by a known method. For example, it can be measured by observing the change in cell size with a microscope or by visual observation, the degree of deformation of the culture substrate around the cells, and the actomyosin activity that causes cell contraction. When measuring the degree of deformation of the culture substrate around 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). The FLECS plate is made of fluorescently labeled cell adhesion factors, and when cells bound to the cell adhesion factors contract, the adhesion factors also contract, and the amount of change in the size of the cell adhesion factors can be analyzed as the cell contraction degree.

[0024] In the present invention, the group to which the test substance is not added is not particularly limited as long as it serves as a control for evaluating the effect of the test substance on the MMP-9 gene expression level and / or the MMP-9 protein amount in epidermal cells, or the effect of improving cell contractility, but it also includes, for example, the addition of nothing other than the solvent used to dissolve the test substance.

[0025] In the present invention, for example, the MMP-9 gene expression level, MMP-9 protein amount in epidermal cells, or cell contractility as indicators are compared with a group to which the test substance is not added, and a test substance that improves the MMP-9 gene expression level, MMP-9 protein amount in epidermal cells, or cell contractility is judged to be usable as a papillary formation promoter. A test substance that increases the gene expression level or protein amount by 10% or more compared to the non-added group, or a test substance that increases the cell contractility by 5% or more compared to the non-added group, is judged to be highly effective as a papillary formation promoter.

[0026] In the present invention, a screening method for a papilla formation promoter using the expression level of the MMP-9 gene in epidermal cells and / or the amount of MMP-9 protein in epidermal cells as an indicator can be carried out as follows, for example, when measuring the expression level of the MMP-9 gene. (1) Epidermal cells are cultured in a culture medium in a petri dish. (2) The test substance dissolved in water is added, and at the same time, the same volume of water as the test substance is added to serve as a control group. (3) The cells are cultured for a certain period of time and the test substance is allowed to act on the epidermal cells. (4) Extract total RNA from epidermal cells. (5) Synthesize cDNA from total RNA. (6) The expression level of the MMP-9 gene is quantified by real-time PCR, and the expression level of the MMP-9 gene when the test substance is added is calculated relative to the level in a group without the test substance added. (7) If the expression level of MMP-9 gene is increased by 10% or more when the test substance is added compared to the non-addition group, the test substance is determined to be usable as a papillary formation promoter.

[0027] The horseradish 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 its genus name. It is rich in nutrients such as polyphenols, amino acids, and vitamin E, and is also called "green milk" and "tree of life" in some regions due to its high nutritional value and efficacy.

[0028] The part of the extract of Moringa oleifera used in the present invention is not particularly limited. For example, seeds, trunks, branches, leaves, flowers, roots, etc. can be used. Among them, it is preferable to use seeds, and it is more preferable to crush seeds including the outer skin and use them. In addition, the extraction solvent is not particularly limited, and examples thereof include water, lower alcohols such as propyl alcohol, and polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, but it is particularly preferable to select water.

[0029] The extraction method of the wasabi tree extract used in the present invention is not particularly limited. For example, 1 to 100 parts by mass of a mixed solution of water and 1,3-butylene glycol or ethanol is used with respect to 1 part by mass of the dried plant, and extraction is preferably performed 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 performed 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 oil agents, surfactants, powders, coloring materials, 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 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 the skin tissue of the human body, but also on epidermal cells in excised skin tissue used in skin transplantation, etc., and epidermal cells in artificial skin using a culture substrate.

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 amount and aging> Normal human epidermal keratinocytes from young donors (19 years old) and old donors (51 years old) were seeded at 8.5×10 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 culture medium was collected. According to the protocol of the Human MMP Antibody Array Membrane (abcam), the amount of MMP-9 protein in the collected culture medium was measured, 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 a concentration of cells / mL and seeded at 500 μL per well in a 24-well culture plate. After culturing for 2 days at 37 °C and 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, 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 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 MMP-9 gene expression level in epidermal cells decreased with aging. From this, it was found that the protein amount of MMP-9 and the MMP-9 gene expression level can 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 by purchasing abdominal skin from young (29 years old) and old (72 years old) donors from BIOPREDIC. Human skin tissue was embedded in OCT compound (Sakura Finetech Japan) and frozen, and 4 μm sections were prepared using a cryostat and attached to slides. These were fixed by immersion in 95% ethanol and immunostained. Anti-MMP-9 rabbit polyclonal antibody was used as the primary antibody, and Alexa Fluor 594 conjugate anti-rabbit IgG(H+L) chicken secondary antibody was used as the secondary antibody. The stained sections were then observed (20x) and photographed using a fluorescent microscope (BZ-X700, KEYENCE).

[0037] FIG. 3 shows the results of immunostaining of skin tissue with MMP-9 antibody. In immunostaining using MMP-9, the antibody binds to MMP-9 in the skin, and a fluorescent secondary antibody binds to the MMP-9, causing the area where MMP-9 is present to emit red fluorescence. As shown by the white arrow in FIG. 3, in the skin derived from a young person having a clear papilla structure, there are areas around the papilla that emit strong red fluorescence, which are not seen in the skin derived from an old person in which the papilla structure is not observed, and it was found that the area where MMP-9 is present closely matches the outline of the papilla epidermal cell side. In other words, the presence of MMP-9 on the papilla epidermal cell side correlates with the presence or absence of papillae.

[0038] [Test 4] <Age-related changes in cell contractile force and evaluation of the effects of MMP-9 pathway inhibition> Human epidermal keratinocytes from newborn donors (hereafter referred to as newborn keratinocytes), human epidermal keratinocytes from 50-year-old adult donors (hereafter referred to as 50-year-old adult keratinocytes), and human epidermal keratinocytes from 51-year-old adult donors (hereafter referred to as 51-year-old adult keratinocytes) 5.0 × 10 4Cells / mL were dispersed in Humedia KG2 (KURABO) and 500μL was seeded on a cell contraction force measurement kit FLECS Plate (Forcyte Biotechnologies). After 6 hours of culture at 37℃ and 5% CO2, MMP-9 pathway inhibitor Y-27632 was added to human epidermal keratinocytes derived from newborn donors at 20μM. After further culture at 37℃ and 5% CO2 for 20 hours, the cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and observed (20x) using a fluorescent microscope (BZ-X700, KEYENCE) and photographed. The length of the lines constituting the cross of the cross-shaped gel to which the cells were attached in the photograph was measured using imageJ (OPEN SOURCE), and the degree of substrate contraction by the cells was calculated as follows. In addition, the cell structure of human epidermal keratinocytes derived from newborn donors treated with the cell actin staining reagent Alexa Fluor (registered trademark) 488 Phalloidin was observed (20x) using a fluorescence microscope (BZ-X700, KEYENCE) and photographed.

[0039] The cell contraction force measurement kit, FLECS Plate, has a structure in which a cross gel is placed on plastic, and the cross gel is observed as red fluorescence. In addition, cross gels to which cells have adhered can be identified by staining the cell nuclei blue with DAPI. The degree of substrate contraction (degree of cell contraction) is calculated using the following formula based on the length of the lines that make up the cross of the cross gel to which cells have not adhered (control gel) and the length of the lines that make up the cross of the cross gel to which cells have adhered.

[0040]

number

[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 the papillary structure-forming skin model, was prepared by the following procedure. As the fibrous collagen, a solution was used in which a commercially available 0.3 w / v% solution of type I collagen (Kurashiki Boseki Co., Ltd., Cellmatrix (registered trademark) Type-A) was mixed with 10xPBS (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 a ratio of 8:1. As the basement membrane matrix preparation, a commercially available Matrigel (Corning Co., Ltd., standard Matrigel (registered trademark) basement membrane matrix preparation: protein concentration 10 mg / mL) was used, which was left to stand for 2 hours under ice cooling and thawed. As the crosslinking agent, a 0.1 w / v% aqueous solution of glutaraldehyde was used, and as the crosslinking terminator, a 2 w / v% aqueous solution of glycine was used. A solution of fibrous collagen (type I collagen), a solution of basement membrane matrix (matrigel), and a crosslinking agent solution were mixed so that the final content was 0.08 (w / v%) collagen, 0.7 (w / v%) matrigel, and 0.01 (w / v%) glutaraldehyde, and the total amount was adjusted to 100 w / v% with water. 150 μL of this mixture was poured into each well of an 8-well type chamber slide (IWAKI, 10 mm x 10 mm) and incubated at 4 ° C for 12 hours. Then, the mixture was incubated at 37 ° C for 2 hours to obtain a gel-like substance. Next, 200 μL of a 2 w / v% glycine solution as a crosslinking stopper was added to each well, and the mixture was incubated at 37 ° C for 2 hours, after which the liquid on the gel-like substance was removed to obtain a chamber slide holding a culture substrate. Neonatal human epidermal keratinocytes were cultured in culture medium at 1 × 10 5 The cells were dispersed in Humedia KG2 (KURABO) culture medium at a concentration of 100 cells / mL, and 200 μL was seeded per well on the culture substrate of the chamber slide prepared above. After 24 hours at 37°C and in the presence of 5% CO2, a papillary structure-forming skin model was obtained.

[0045] [Confirmation of the effect of MMP-9 inhibition on papillary process formation model] The MMP-9 pathway inhibitor Y-27632 was added to the papillary structure-forming skin model at 20 μM. After that, the culture was continued for another 72 hours, and the resulting keratinocyte cell layer was observed. The medium was removed from the chamber slide, and the chamber slide was washed once with 100 μL of PBS(-) per well. Then, 100 μL of a solution of 0.5 v / v% Triton X-100 dissolved in PBS(-) was added to each well. After leaving it at room temperature for 15 minutes, washing with 100 μL of PBS(-) per well was repeated three times. 100 μL of a previously 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 well was left at room temperature for 20 minutes to stain actin. Then, 170μL of 3v / v% BSA in PBS(-) was added and reacted for 20 minutes to block. 70μL of a solution of the primary antibody (anti-laminin antibody rabbit host antibody (L9393 Sigma)) (100-fold dilution with PBS(-)) was added thereto and reacted at room temperature for 2 hours. Next, the sections were washed by repeatedly contacting with Wash buffer (T-PBS) for 5 minutes three times. 70μL of a solution of a fluorescently labeled secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermofisher)) (200-fold dilution with PBS(-)) was added and reacted at room temperature for 45 minutes. Again, the sections were washed by repeatedly contacting with Wash buffer (T-PBS) for 5 minutes three times. The sections were further washed three times with 100μL of PBS(-). Then, one drop of mounting fluid (Aqua poly / Mount, Diagnostic Biosystems) was added, and the keratinocyte cell layer and gel were observed under a fluorescence microscope (BZ-X700, KEYENCE) in sectioning mode (20x magnification). Images were analyzed using image analysis software (BZ-X Analyzer, KEYENCE).

[0046] [Observation results] The observation results are shown in Figure 7. In neonatal keratinocytes, papilla-like convexities were formed and a cell layer was formed over the entire surface of the convexities (the shape of the cell layer is indicated by the white dotted line in the figure), whereas in neonatal keratinocytes to which the MMP-9 pathway inhibitor Y-27632 had been added, papilla-like convexities were not formed and a flat cell layer was formed. These results demonstrated that MMP-9 regulates cell contraction and the subsequent papilla formation, and that the formation of papillae can be promoted by promoting any part of this process. In other words, it was found that papilla formation promoters can be screened by using at least one of the following as an index: 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.

[0047] [Test 6] <Evaluation of the MMP-9 production promoting effect of Moringa oleifera> [Preparation of test substance] Preparation of Moringa oleifera extract: Dried Moringa oleifera seeds including the outer skin were crushed, 10 times the mass of purified water was added, and the mixture was heated and extracted at 60°C for 4 hours. 1 part of the dried residue of the extract was diluted with 124 times the amount of 70% glycerin aqueous solution to prepare the test substance. Preparation of Physalis extract: Dried physalis fruits were added with 10 times the mass of 50% ethanol aqueous solution and extracted at room temperature for 6 days. To the dry residue of this extract, 49 volumes of distilled water and 50 volumes of 1,3-butylene glycol were added to prepare a sample solution. Comparative samples: The samples used for comparison were Scutellaria Baicalensis leaf / stem extract (Ichimaru Falcos) and Scutellaria Baicalensis root extract (Ichimaru Falcos).

[0048] [MMP-9 gene expression analysis] Normal human epidermal keratinocytes from a young donor (19 years old) and an old donor (51 years old) were suspended in Humedia KG2 (KURABO) at 5.0 × 10 4A cell suspension was prepared to give 1000 cells / mL, and 500 μL was seeded in a 24-well culture plate. After 24 hours of culture at 37°C and 5% CO2, the prepared test substance or the solvent for the test substance was added to a final concentration of 0.4% or 0.2%, respectively, and cultured at 37°C and 5% CO2 for 2 days. Total RNA was extracted using a Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using a 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 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'). Power SYBR Green Master Mix (Applied Biosystems) was used for PCR reactions, and gene expression analysis was performed using the comparative Ct method. In other words, the change in gene expression level due to the addition of the test substance was calculated as a relative amount to the Ct value of MMP-9 when the solvent for the test substance was added, which was corrected by the Ct value of GAPDH, which was set to 1.

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

[0050] [Test 7] <Evaluation of the effect of Moringa oleifera on promoting papilla formation> The following topical compositions containing Wasabinoki extract or Physalis orbiculatus extract were prepared, and their effects on improving papillae and skin condition were confirmed. 15 panelists (men aged 40-50) were asked to apply an appropriate amount of the composition to their faces once in the morning and once at night for two months. The papillae were observed on the cheeks using a confocal laser biomicroscope (Vivascope1500, Caliber ID).

[0051] JPEG0007665379000002.jpg75155

[0052] To analyze the papillae observation images, a 1.0 mm x 1.0 mm horizontal image was taken at a position approximately 25 μm from where the papillae began to be observed, and the number of papillae was counted visually. The number of papillae after use was calculated by setting the number of papillae before using the composition for two months as 100.

[0053] As shown in FIG. 9, the composition containing the Wasabinoki extract showed an increase in papillae of more than 50%, whereas no increase in papillae was observed in the comparative composition containing the Physalis alkekengi extract or the control. These results demonstrated that extracts from Wasabinoki, which have the effect of promoting MMP-9 production, also have the effect of promoting skin papilla formation, and that they can be used as a papilla formation promoter to improve the flattening of papillae that accompanies aging, etc.

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

Claims

[Claim 1] (1) adding a test substance to epidermal cells and culturing them; (2) measuring at least one selected from the expression level of MMP-9 gene in epidermal cells and the amount of MMP-9 protein in epidermal cells; (3) comparing at least one selected from the MMP-9 gene expression level in epidermal cells and the MMP-9 protein level in epidermal cells obtained in step (2) with a group to which the test substance was not added, and determining that a substance that increases at least one of the selected MMP-9 gene expression level in epidermal cells and the MMP-9 protein level in epidermal cells by 10% or more is a substance having a papillary process formation promoting effect; A method for screening for a papillary process formation promoter comprising the steps of:

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