Promoter of new protein synthesis

Abstract

Traditional research focusing on protein synthesis has focused on the total amount of protein, and therefore requires components that either suppress the decrease in the amount of newly synthesized protein or promote its synthesis. [Solution] Focusing on the synthesis of new proteins, and utilizing a method for evaluating the amount of new protein synthesis in dermal fibroblasts and epidermal keratinocytes, neem leaf extract and kudzu extract are provided as inhibitors of the decrease in the amount of new protein synthesis or as promoters of new protein synthesis.

Description

Technical Field

[0001] The present invention relates to the technical field of skin activation. In particular, provided is a protein synthesis promoter that promotes the synthesis of new proteins in dermal fibroblasts and epidermal keratinocytes.

Background Art

[0002] The skin is an organ that constitutes the outermost layer of the living body and serves as a boundary with the outside world. The skin is constantly exposed to stimuli from the outside world, such as ultraviolet rays, low temperature, high temperature, dryness, allergens, and chemical substances. The epidermal keratinocytes in the epidermal layer and the dermal fibroblasts in the dermal layer, which are the main cells constituting the skin, change their physiological activities in response to the stimuli. As an example, when dermal fibroblasts and epidermal keratinocytes are irradiated with ultraviolet rays, reactive oxygen species increase, and as a result, the ability to synthesize new proteins decreases. It is also known that the protein translation function declines during aging and in a hypoxic state (Non-Patent Document 1: Aleksandra S. Anisimova et al., Aging 2018 Dec; 10(12):9425-94288, Non-Patent Document 2: Molecular Cancer volume 18, Article number: 49 (2019)). Dermal fibroblasts mainly produce extracellular matrices such as fibronectin, laminin, collagen, and elastin. Thinning of the dermal layer may occur due to a decrease in the ability to synthesize new proteins. Since epidermal keratinocytes produce filaggrin and keratin as structural proteins, a decrease in the ability to synthesize new proteins may cause a decline in the skin barrier function in the stratum corneum. In anticipation of the improvement effect of the skin, various components have been studied, and components having effects such as suppression of oxidative stress, suppression of DNA damage, and promotion of collagen synthesis have been identified (Patent Documents 1 to 7).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

[0004] [Non-Patent Document 1] Aleksandra S. Anisimova et al., Aging 2018 Dec; 10(129:42594288) [Non-Patent Document 2] Molecular Cancer (2019) 18:49 [Overview of the project] [Problems that the invention aims to solve]

[0005] It was known that UV exposure and age-related declines in protein synthesis affect the appearance of the skin. Previous studies focusing on protein synthesis have focused on the total amount of protein, and have not adequately investigated components that suppress the decline in new protein synthesis or promote it. [Means for solving the problem]

[0006] The inventors focused on the synthesis of newly synthesized proteins and, by performing a method to evaluate the amount of newly synthesized proteins synthesized in dermal fibroblasts and epidermal keratinocytes, identified components that suppress the decrease in the amount of newly synthesized proteins synthesized or promote their synthesis. Therefore, the present invention relates to the following: [1-1] A new protein synthesis promoter containing kudzu extract or neem leaf extract in skin cells. [1-2] Use of kudzu extract or neem leaf extract for the production of a nascent protein synthesis promoter in skin cells. [1-3] Use of kudzu root extract or neem leaf extract on the skin for the purpose of promoting the synthesis of new proteins. [1-4] A method for promoting the synthesis of new proteins in subjects suffering from reduced synthesis of new proteins, comprising applying kudzu extract or neem leaf extract. [2] Use or method of a nascent protein synthesis promoter as described in items 1-1 to 1-4, which suppresses the decrease in protein synthesis caused by a stimulus response. [3] The nascent protein synthesis promoter, use, or method described in item 1-1 to 1-4 or 2, wherein the stimulus is ultraviolet light stimulation. [4] The nascent protein synthesis promoter, use, or method described in any one of items 1-1 to 1-4, 2, or 3, wherein the promoter contains Pueraria lobata extract and suppresses UV-induced decrease in protein synthesis in dermal fibroblasts. [5-1] A composition for improving wrinkles and sagging, comprising a new protein synthesis promoter as described in item 4. [5-2] Use of the neoprotein synthesis promoter described in item 1-1, or Pueraria lobata extract or Neem leaf extract, for the manufacture of compositions for improving wrinkles and sagging. [5-3] Use of kudzu extract or neem leaf extract on the skin to improve wrinkles and sagging by promoting the synthesis of new proteins. [5-4] A method for improving wrinkles or sagging in subjects suffering from decreased protein synthesis by applying kudzu extract or neem leaf extract. [6] A nascent protein synthesis promoter according to any one of items 1 to 3, wherein the inhibitor contains neem leaf extract and suppresses UV-induced decrease in protein synthesis in epidermal keratinocytes. [7] A composition for improving blemishes or skin barrier function, comprising a new protein synthesis promoter as described in item 6. [8-1] A UV resistance enhancer in dermal fibroblasts, containing kudzu extract. [8-2] Use of Pueraria lobata extract for the production of UV resistance enhancers in dermal fibroblasts. [8-3] Use of kudzu extract on the skin for the purpose of promoting UV resistance in dermal fibroblasts. [8-4] A method for promoting UV resistance in dermal fibroblasts, comprising applying Pueraria lobata extract to subjects requiring enhanced UV resistance. [9] The UV resistance enhancer described in item 8, wherein the effect of ultraviolet light is DNA damage or decreased collagen expression. [10-1] An agent that enhances UV resistance in epidermal keratinocytes, containing neem leaf extract. [10-2] Use of neem leaf extract for the production of UV resistance enhancers in epidermal keratinocytes. [10-3] Use of neem leaf extract on the skin, for the purpose of promoting UV resistance in epidermal keratinocytes. [10-4] A method for promoting UV resistance in subjects requiring enhanced UV resistance in epidermal keratinocytes, comprising applying neem leaf extract.

[11] A UV resistance enhancer as described in item 10, which suppresses the effects of UV radiation through antioxidant, anti-inflammatory, and differentiation-inducing mechanisms.

[12] A method for screening UV-resistant agents using the ability to synthesize new proteins in UV-irradiated cell cultures as an indicator.

[13] The screening method according to item 12, wherein the cell culture is a culture of epidermal keratinocytes or dermal fibroblasts.

[14] A step of culturing UV-irradiated epidermal keratinocytes or dermal fibroblasts in a culture medium to which candidate components have been added, A step of measuring the ability to synthesize new proteins in cell cultures cultured in a medium containing candidate components, and A step of determining the added candidate component as an ultraviolet resistance enhancer when the ability of de novo protein synthesis is enhanced by comparing the measured ability of de novo protein synthesis with a control. The screening method according to item 13, including this.

[15] The screening method according to item 14, wherein the ultraviolet resistance enhancer is a de novo protein synthesis promoter or a drug that promotes the expression of a gene selected from the group consisting of DNA repair genes, antioxidant genes, differentiation induction promoting genes, and anti-inflammatory genes.

[16] A screening method for an ultraviolet resistance enhancer using the expression of an ultraviolet resistance gene as an index in an ultraviolet-irradiated cell culture.

[17] The screening method according to item 16, wherein the cell culture is a culture of epidermal keratinocytes or dermal fibroblasts.

[18] A step of culturing ultraviolet-irradiated epidermal keratinocytes or dermal fibroblasts in a medium to which a candidate component is added. A step of measuring the expression level of an ultraviolet resistance gene in a cell culture cultured in a candidate component-added medium, and A step of determining the added candidate component as an ultraviolet resistance enhancer when the expression level of the ultraviolet resistance gene is enhanced by comparing the measured expression level of the ultraviolet resistance gene with a control. The screening method according to item 17, including this.

[19] The screening method according to item 18, wherein the ultraviolet resistance gene is at least one selected from the group consisting of GPX3, AKR1C1, EIF3L, DUSPI, involucrin, transglutaminase, and KDM48.

[20] The screening method according to item 18 or 19, wherein the ultraviolet resistance enhancer is a drug that promotes the expression of a gene selected from the group consisting of a de novo protein synthesis promoter, DNA repair genes, antioxidant genes, differentiation induction promoting genes, and anti-inflammatory genes.

Effects of the Invention

[0007] The nascent protein synthesis promoter according to the present invention promotes the synthesis of nascent proteins in skin cells, particularly epidermal keratinocytes and dermal fibroblasts. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a fluorescence image showing the decrease in the amount of newly synthesized protein after UV irradiation. Newly synthesized proteins are indicated by fluorescence using fluorescent labeling. [Figure 2] Figure 2 shows the decrease in nascent protein synthesis in cultured dermal fibroblasts due to UV irradiation (A). Nascent protein synthesis, which was reduced by UV irradiation, was increased by adding kudzu extract and culturing for 48 hours (B). [Figure 3] Figure 3 shows the decrease in nascent protein synthesis in cultured epidermal keratinocytes due to UV irradiation (A). Nascent protein synthesis, which was reduced by UV irradiation, was increased by adding neem leaf extract and culturing for 24 hours (B). [Figure 4] Figure 4 shows that in cultured dermal fibroblasts, the reduction in nascent protein synthesis caused by UV irradiation is not increased by scutellaria baicalensis extract, thyme extract, peony extract, and olive leaf extract, which have collagen production-promoting effects. [Figure 5] Figure 5 shows the results of RNA-seq analysis of changes in gene expression when neem leaf extract was added to cultured epidermal keratinocytes. Compared to gene expression after UV irradiation, gene expression was significantly increased (A: GPX3, B: AKR1C1, C: EIF3L, D: DUSP1, E: INV, F: TGM1). For DUSP1, the TPM value was low, so a follow-up test was conducted separately by quantitative PCR, and the results are shown in Figure 5G. Furthermore, (A) GPX3, (B) AKR1C1, and (D) DUSP1 also showed significantly increased gene expression after UV irradiation. [Figure 6]Figure 6 shows the results of quantitative PCR analysis of gene expression when kudzu extract was added to cultured dermal fibroblasts. KDM4B expression increased with UV irradiation, and further increased with the application of kudzu extract (A). COL5A1 expression decreased significantly with UV irradiation, while the application of kudzu extract suppressed the decrease in expression caused by UV (B). [Figure 7] Figure 7 shows a fluorescence microscope image (A) illustrating the change in rH2AX expression due to kudzu extract, and a graph (B) showing the fluorescence intensity of rH2AX. [Modes for carrying out the invention]

[0009] This invention relates to a protein synthesis promoter comprising kudzu extract or neem leaf extract. The protein synthesis promoter according to the present invention can promote protein synthesis in skin cells, particularly dermal fibroblasts or epidermal keratinocytes. This can suppress the decline in protein synthesis caused by ultraviolet radiation, aging, and hypoxic conditions, thereby improving the function and condition of the epidermis and / or dermis.

[0010] Neem leaf extract particularly promotes the synthesis of new proteins in epidermal keratinocytes. Epidermal keratinocytes synthesize various proteins, but especially keratin, filaggrin, and loricrin. Among these, the synthesis of keratin, a structure-related protein, is exceptionally high, and the promotion of keratin synthesis improves skin barrier function and promotes proper epidermal turnover. As a result, neem leaf extract and other neem leaf extract-promoting agents have beneficial effects on blemishes and wrinkles, and can be used as blemish-improving agents, wrinkle-improving agents, skin barrier function-improving agents, or anti-aging agents. Furthermore, since neem leaf extract promotes the expression of UV resistance genes (Figure 5), neem leaf extract-promoting agents can also be called UV resistance enhancers. UV resistance genes whose expression is promoted by neem leaf extract include GPX3, AKR1C1, EIF3L, DUSPI, involucrin, and transglutaminase 1.

[0011] Pueraria lobata extract particularly promotes the synthesis of new proteins in dermal fibroblasts. Dermal fibroblasts synthesize various proteins, but they synthesize large amounts of collagen, elastin, fibronectin, and fibrillin. In particular, the synthesis of collagen and elastin, which are proteins that make up the extracellular matrix, is promoted, increasing the amount of the extracellular matrix and increasing the dermal layer. As a result, pueraria lobata extract and other agents that promote the synthesis of new proteins have an improving effect on sagging, firmness, and wrinkles, and can be used as an anti-aging agent for improving sagging, firmness, and wrinkles. Furthermore, KDM4B and COL5A1 are among the genes whose expression is promoted by pueraria lobata extract. In addition, since pueraria lobata extract also contributes to DNA repair (Figure 8), it can also be called a DNA repair promoter.

[0012] The term "nascent protein" is used to distinguish it from proteins synthesized in the past, and refers to proteins newly synthesized after a predetermined time. Experimentally, the synthesis of nascent proteins can be evaluated by detecting newly incorporated amino acids or their analogs into the protein. As an example, radiolabeled amino acids (for example) 35 By adding S-labeled methionine, the synthesis of newly synthesized proteins can be evaluated. Another example is supplying o-propalin-puromycin (OPP) to cultured cells to allow protein uptake, and then adding fluorescently labeled picolyl azide and a click reaction reagent to evaluate the synthesis of newly synthesized proteins. Tests using OPP have shown that the expression of newly synthesized proteins in epidermal keratinocytes and fibroblasts decreases upon UV irradiation (Figure 1). Furthermore, this experimental system allows for the screening of components that can promote the synthesis of newly synthesized proteins. For example, after irradiating a culture of epidermal keratinocytes or dermal fibroblasts with UV, the cells are cultured in a medium containing the candidate drug and OPP. After a predetermined period of culture, the synthesis of newly synthesized proteins can be measured by adding fluorescently labeled picolyl azide and a click reaction reagent. As a control, if the fluorescence intensity increases compared to a culture without the candidate component, the candidate component can be selected as a new protein synthesis promoter. Pueraria lobata extract and neem leaf extract were screened as promoters of nascent protein synthesis (Figures 2 and 3). In living organisms, nascent protein refers to protein synthesized after the start of extract application.

[0013] A nascent protein synthesis accelerator is a drug that exerts the effect of promoting the synthesis of new proteins in the target tissue after a predetermined timing, and has such an effect. In one example, a nascent protein synthesis accelerator is a drug that exerts the effect of restoring the protein synthesis ability that has decreased due to ultraviolet irradiation or the like in the target tissue, and has such an effect. From the viewpoint of exerting the effect of restoring the decreased protein synthesis ability, it is preferable to apply it to subjects in which the synthesis of nascent proteins is decreased. Such subjects include subjects affected by ultraviolet radiation and aging, and the drug is applied to the skin of such subjects that has been affected by ultraviolet radiation. In such subjects, the synthesis of nascent proteins is decreased. If the protein synthesis ability itself is decreased, the efficacy of drugs that promote the production of specific proteins, such as collagen production accelerators, will also decrease (Figure 4). The type of protein that is increased by a nascent protein synthesis accelerator differs depending on the cell type. The nascent protein synthesis accelerator according to the present invention may increase only the amount of synthesis while keeping the profile of proteins expressed under normal conditions unchanged, or it may change the profile to increase the amount of synthesis of specific proteins. Protein synthesis promoters can also be simply called cell activators or UV resistance gene expression promoters.

[0014] In epidermal keratinocytes, UV irradiation increases the gene expression of GPX3, AKR1C1, and DUSP1 (Figure 5). While not intended to be purely theoretical, these genes are involved in antioxidant and anti-inflammatory processes, respectively, and their expression is thought to be promoted as resistance genes to UV irradiation. Neem leaf extract can further increase the expression of GPX3, AKR1C1, and DUSP1, which are increased by UV irradiation. Therefore, neem leaf extract can be considered an anti-inflammatory or antioxidant agent. Furthermore, neem leaf extract can promote the expression of genes selected from the group consisting of involucrin, transglutaminase 1, and EIF3L (Figure 5). EIF3L is a translation-related factor, and increased expression can contribute to increased expression of newly synthesized proteins. Involucrin and transglutaminase contribute to the differentiation of epidermal keratinocytes and can contribute to the normalization of epidermal differentiation and turnover.

[0015] KDM4B expression increased in dermal fibroblasts upon UV irradiation, and further increased upon application of kudzu extract (Figure 6A). COL5A1 expression significantly decreased upon UV irradiation, while application of kudzu extract suppressed this UV-induced decrease (Figure 6B). Furthermore, kudzu extract can suppress the increased H2AX phosphorylation caused by UV irradiation, thereby improving or restoring DNA damage and conferring resistance (Figure 7).

[0016] A further aspect of the present invention relates to a screening method for UV resistance enhancers. The screening method of the present invention may use the ability to synthesize new proteins in UV-irradiated cell cultures as an indicator, or the expression of UV resistance genes as an indicator. Examples of cell cultures include those containing dermal fibroblasts or epidermal keratinocytes. When using the ability to synthesize new proteins as an indicator, more specifically, the following: A step of culturing epidermal keratinocytes or dermal fibroblasts irradiated with ultraviolet light in a culture medium to which candidate components have been added. A step of measuring the ability to synthesize new proteins in cell cultures cultured in a medium containing candidate components, and The measured nascent protein synthesis ability is compared with a control, and if the nascent protein synthesis ability is enhanced, the added candidate component is determined to be an ultraviolet resistance enhancer. Includes. When using the expression of UV resistance genes as an indicator, more specifically, the following: A step of culturing epidermal keratinocytes or dermal fibroblasts irradiated with ultraviolet light in a culture medium to which candidate components have been added. A step of measuring the expression level of UV resistance genes in cell cultures cultured in a medium containing candidate components, and The process involves comparing the measured expression level of the UV resistance gene with a control, and determining the added candidate component as a UV resistance enhancer if the expression level of the UV resistance gene is increased. This includes. UV resistance enhancers may be agents that promote the synthesis of new proteins, or agents that promote the expression of genes selected from the group consisting of DNA repair genes, antioxidant genes, differentiation induction promoting genes, and anti-inflammatory genes.

[0017] Candidate components may be added to the culture medium at a predetermined time, or the medium may be replaced with a medium containing the candidate drug. Candidate cells may be subjected to starvation before the addition of the candidate drug. As a control, the expression of nascent protein synthesis ability or UV resistance genes obtained by culturing a culture containing dermal fibroblasts or epidermal keratinocytes in a medium that differs only in that it does not contain the candidate drug can be used. The control group may have been experimentally prepared in advance to set a threshold based on the expression of nascent protein synthesis ability or UV resistance genes, or the culture and measurement steps may be performed in parallel. If the expression of nascent protein synthesis ability or UV resistance genes is increased compared to the expression of nascent protein synthesis ability or UV resistance genes in the control, the candidate drug can be screened as a nascent protein synthesis promoter or an expression promoter of UV resistance genes. Such drugs can be used as skin elasticity improvers and as agents for improving sagging, wrinkles, and firmness.

[0018] The candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or synthesized compounds and prepared extracts may be used. Ultraviolet irradiation can be performed using an ultraviolet generator such as a solar simulator. An appropriate intensity can be selected to cause changes in cell activity without killing the cells. For example, the intensity of the ultraviolet generator can be set to 5mJ to 50mJ. Any range selected from 5mJ to 10mJ, 10mJ to 20mJ, 20mJ to 30mJ, 30mJ to 40mJ, and 40mJ to 50mJ, and any consecutive ranges thereof, can be selected and applied.

[0019] The cell culture may be a culture of established cell lines, a cell culture of cells obtained from living organisms, or an organ culture. A three-dimensional skin model created by layering cells and inducing differentiation may also be used. Culturing can be carried out using media and culture conditions commonly used in this art, for example, under a 37°C CO2 atmosphere. As a control, an index for UV resistance genes or nascent protein synthesis ability obtained by conducting experiments under conditions that differ only in that candidate components are not added can be used. Such control values ​​may have been determined in advance or in parallel with the experiments. If the experiments are performed in advance, a threshold value set from the control index can also be used.

[0020] The ability to synthesize new proteins may be measured using methods known in this art. For example, 35 The newly synthesized protein may be measured using S-methionine, or it may be measured using o-propalin-puromycin (OPP).

[0021] As UV resistance-constituting genes, at least one gene selected from the group consisting of GPX3, AKR1C1, EIF3L, DUSPI, involucrin, transglutaminase 1, and KDM48 may be selected. The expression of these genes can be determined by methods well known in the art, such as RT-PCR or RNA-seq. As described below, these genes are thought to be involved in reducing reactive oxygen species caused by UV radiation, repairing DNA damage, repairing or removing denatured proteins and lipids, and structural proteins, and thus contributing to reducing the effects of UV radiation.

[0022] The GPX3 gene encodes glutathione peroxidase 3, an enzyme with antioxidant properties. GPX3 is involved in protecting cells and tissues from oxidative stress caused by reactive oxygen species (ROS) (Biochemical Pharmacology Volume 184, February 2021, 114365).

[0023] The AKR1C1 gene encodes the aldoketo reductase (AKR1C1), which belongs to the aldoketo reductase family. AKR1C1 reduces aldose sugars to produce polyols and is also involved in the metabolism of steroid hormones and drugs (AKR1C1: Invest Ophthalmol Vis Sci. 2022 Sep 6;63(10):3).

[0024] The EIF3L gene encodes subunit L of EIF3, a translation initiation factor complex in eukaryotes. EIF3L functions in the initiation stage of protein synthesis and works in conjunction with other translation factors to contribute to ribosome construction and mRNA binding. It can assist in the translation of mRNAs that require it, thereby regulating protein expression (Br J Dermatol. 2013 Feb;168(2):339-45).

[0025] The DUSP1 gene encodes dual-specific phosphatase 1 (DUSP1). DUSP1 is involved in intracellular signaling pathways. Specifically, it suppresses the activation of MAP kinase family members such as ERK, JNK, and p38, thereby contributing to stress response, immune response, and regulation of cell proliferation and differentiation.

[0026] The IVL gene is a gene that codes for involucrin. It is a protein component of the skin and, by binding with loricrin, forms a cornifidobelli that protects keratinocytes. Ultraviolet light oxidizes the sebum film and reduces skin barrier function, while increased expression of the IVL gene contributes to the restoration of skin barrier function.

[0027] The TGM1 gene is a gene that encodes transglutaminase. Transglutaminase forms isopeptide bonds by cross-linking glutamine and lysine residues. Among the TGM family, TGM1 is present in the skin, is involved in keratinization, and contributes to the restoration of skin barrier function.

[0028] The KDM4B gene is the gene that encodes histone lysine demethylase 4B. Histone lysine demethylase 4B is mainly involved in histone modification and plays a role in epigenetic gene expression regulation. In particular, it is known to remove methylation of histone 3 lysine 9 (H3K9) and lysine 36 (H3K36), contributing to the activation of gene expression.

[0029] The plant extracts described herein can be obtained by conventional methods, for example, by immersing or refluxing the source plant with an extraction solvent at room temperature or under heating, followed by filtration and concentration. They can also be obtained by distillation, such as steam distillation. Any solvent commonly used for extraction can be used as the extraction solvent, for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols including ethanol, propylene glycol, 1,3-butylene glycol, glycerin, aqueous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., each can be used alone or in combination. Preferably, a mixed solvent of water and alcohol, such as 1,3-butylene glycol, is used as the solvent. The extract obtained by extraction with the above solvent can be used as is, or concentrated by methods such as freeze-drying. If necessary, an extract obtained by removing impurities using an adsorption method, such as an ion exchange resin, or an extract obtained by adsorption using a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration can also be used, and it can be formulated at a concentration equivalent to the raw material or raw powder. For plant extracts, commercially available extracts for cosmetic use can also be used, and commercially available extracts can be formulated at a predetermined concentration.

[0030] Neem leaf extract is an extract from the leaves of plants belonging to the genus Azadirachta of the family Meliaceae, particularly the Indian lily tree (Azadirachta indica). The Indian lily tree is native to India. In addition, the display name used in the full ingredient list of cosmetics as defined by the Japan Cosmetic Industry Association, and the international display name according to the INCI (International Nomenclature of Cosmetic Ingredients) are expressed as: Display name / INCI name: MELIA AZADIRACHTA LEAF EXTRACT. Neem leaf extract is obtained by pressing the leaves of the Indian lily tree, either as is, or after drying or fermentation, and then extracting with a solvent. Solvent extraction can be prepared by using water, alcohol, or a mixture thereof. As alcohol, ethanol, glycerol, propylene glycol, or butylene glycol can be used. More preferably, the extract can be obtained using a mixture of water and alcohol, such as 1,3-butylene glycol, in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. The neem leaf extract can be formulated at a concentration of 0.0001% to 5% by mass. More specifically, any range selected from 0.0001 to 0.0005% by mass, 0.0005% to 0.001% by mass, 0.001% to 0.005% by mass, 0.005% to 0.01% by mass, 0.01% to 0.05% by mass, 0.05% to 0.1% by mass, 0.1% to 0.5% by mass, 0.5% to 1.0% by mass, and 1.0% to 5.0% by mass, and any ranges thereafter, can be selected.

[0031] Pueraria lobata extract is an extract obtained from the roots of plants belonging to the genus Pueraria in the legume family, particularly Pueraria lobata subsp. lobata. Pueraria lobata is distributed in Southeast Asia, including Japan. It has been used as a herbal medicine since ancient times, known as kakkotou. The display name used in the full ingredient list of cosmetics as defined by the Japan Cosmetic Industry Association, and the international display name according to the INCI (International Nomenclature for Cosmetic Ingredients), are expressed as: Display name / INCI name: Pueraria lobata Root Extract. Pueraria lobata extract is obtained by pressing the roots of Pueraria lobata, either as is, or after drying or fermentation, and then extracting with a solvent. Solvent extraction can be prepared by using water, alcohol, or a mixture thereof. As alcohol, ethanol, glycerol, propylene glycol, or butylene glycol can be used. More preferably, the extract can be obtained with a mixture of water and alcohol, such as 1,3-butylene glycol, in any proportion, for example, a mixture of 10:90 to 90:10, preferably 30:70 to 70:30, and even more preferably 50:50. The kudzu extract can be formulated at a concentration of 0.0001% to 5% by mass. More specifically, any range selected from 0.0001 to 0.0005% by mass, 0.0005% to 0.001% by mass, 0.001% to 0.005% by mass, 0.005% to 0.01% by mass, 0.01% to 0.05% by mass, 0.05% to 0.1% by mass, 0.1% to 0.5% by mass, 0.5% to 1.0% by mass, and 1.0% to 5.0% by mass, and any ranges thereafter, can be selected.

[0032] The various formulations of the present invention, including the nascent protein synthesis promoter, allow for the selection of concentrations and dosage forms from the viewpoint of achieving desired effects resulting from the promotion of nascent protein synthesis, namely, improvements in blemishes, wrinkles, sagging, and firmness. The various formulations, including the nascent protein synthesis promoter, can be incorporated into foods, cosmetics, pharmaceuticals, or quasi-drugs. When incorporated into foods, they may be incorporated into nutritional supplements such as supplements and energy drinks, or into foods with functional claims. When incorporated into cosmetics, they can be incorporated into facial or body cosmetics such as lotions, emulsions, serums, creams, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into pharmaceuticals, they may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into topical skin preparations. By using cosmetics, foods, pharmaceuticals, and quasi-drugs containing various formulations, including nascent protein synthesis promoters, it is possible to prevent and / or improve skin blemishes, wrinkles, firmness, and sagging by promoting the synthesis of proteins in the skin, particularly proteins that decrease due to UV exposure and aging, by promoting the production of structural proteins such as collagen, or by promoting skin turnover. Nascent protein synthesis promoters, or UV resistance gene expression promoters, can be administered over a long period of time as foods, cosmetics, or pharmaceuticals of the present invention. From the viewpoint of improving blemishes, wrinkles, and sagging, they may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. There is no particular upper limit, but it may be several years or less, for example, one year or less.

[0033] The various formulations according to the present invention, including the nascent protein synthesis promoter, are preferably applied to the skin and can be incorporated into topical skin preparations. From the viewpoint of incorporation into topical skin preparations, neem leaf extract and / or kudzu extract can each be incorporated in amounts from 0.001% to 10% by mass. Neem leaf extract and / or kudzu extract may be incorporated individually or in combination. They can also be combined with other cosmetic ingredients. From the viewpoint of fully exhibiting the effect, they can preferably be incorporated in a topical skin preparation at a concentration of 0.01% by mass or more, and more preferably at a concentration of 0.05% by mass or more. From the viewpoint of economic efficiency, they can preferably be incorporated at a concentration of 1% by mass or less, and more preferably at a concentration of 0.5% by mass or less.

[0034] Topical skin preparations are not particularly limited as long as they can be applied to the skin, and any dosage form can be used, such as solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated as a topical skin preparation, bases and excipients commonly used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0035] Another aspect of the present invention may relate to a cosmetic method comprising applying a cosmetic composition containing neem leaf extract and / or kudzu extract to the skin, particularly the skin of the face. Such a cosmetic method may be used on the skin of a person who is troubled by sagging skin or wrinkles, or who wishes to prevent sagging skin or wrinkles. Mechanical stimulation, such as vibration, or electrical stimulation, such as iontophoresis, may be applied with the intention that the components of the extract reach the skin, particularly the dermis. The cosmetic method according to the present invention may be offered as a service in a beauty salon, a beauty clinic, etc., or may be offered together with the sale of cosmetics at a cosmetics store. The cosmetic method is a non-therapeutic or non-medical method that can be distinguished from treatment or preventive methods performed by doctors or medical professionals, and is provided by a practitioner who is not a doctor or other medical professional.

[0036] All references made herein are incorporated herein by citation in their entirety.

[0037] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0038] Example 1: Synthesis of nascent proteins in cultured cells Epidermal keratinocytes (KURABO) and dermal fibroblasts (KURABO) were cultured at 37°C in a 5% CO2 humidified atmosphere. Epidermal keratinocytes were cultured in HuMedia-KG2 (KURABO) medium, and dermal fibroblasts in DMEM, low glucose (Thermo Fisher Scientific) medium. After culturing, UVB was irradiated at 30 mJ using a constant-temperature ultraviolet irradiation device (Nippon Ika Kikai Seisakusho). Subsequently, the Click-iT OPP reagent was added to the culture medium using the Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit (Thermo Fisher Scientific), and the cells were cultured for 30 minutes. After culturing, the reaction was carried out according to the kit instructions, and observation was performed using a confocal microscope, STELLARIS 5 (LEICA). The results are shown in Figure 1. It was shown that UV irradiation suppressed the synthesis of new proteins in both epidermal keratinocytes and dermal fibroblasts.

[0039] Example 2: Screening of nascent protein synthesis promoters In Example 1, after UV irradiation, candidate drugs were added from the cosmetic candidate library, and the mixture was cultured for one or two days to screen for drugs that enhance nascent protein synthesis. The candidate drugs included splendor extract, thyme extract, peony extract, olive leaf extract, neem leaf extract (Ichimaru Pharcos), and kudzu extract (Ichimaru Pharcos), each used at a concentration of 0.1%. Kudzu extract and neem leaf extract were selected as candidate components that significantly increased nascent protein synthesis, which had decreased after UV irradiation (Figures 2-4).

[0040] Example 3: Changes in gene expression due to UV irradiation and addition of neem leaf extract or kudzu extract Epidermal keratinocytes (KURABO) and dermal fibroblasts (KURABO) were cultured in HuMedia-KG2 (KURABO) medium at 37°C under a 5% CO2 humidified atmosphere. After culturing, they were irradiated with 30 mJ of UVB using a constant-temperature ultraviolet irradiation device (Nippon Ika Kikai Seisakusho), and cultured for 1 day after irradiation to form the UV-irradiated group. Next, in addition to UV irradiation, the culture medium for epidermal keratinocyte cultures was replaced with a medium containing 0.1% by mass neem leaf extract (Ichimaru Pharcos), and the culture medium for dermal fibroblast cultures was replaced with a medium containing 0.1% by mass kudzu extract (Ichimaru Pharcos). These were cultured for 1 day to determine the UV irradiation + extract addition group. Cells were harvested from each group, RNA was extracted using the Qiagen Rneasy mini kit (Qiagen), and RNA sequencing analysis was performed by TAKARA BIO. Gene expression data was analyzed using iDEP2.0 software, and genes showing changes in gene expression in the UV irradiation group and the UV irradiation + extract addition group were graphed (Figure 5). In the epidermal keratinocyte cultures, changes in gene expression for GPX3, AKR1C1, EiF3I, DUSP1, involucrin, and TGM1 are graphed and shown (Figures 5A-F). The AKR1C1 result showed a low TPM value, so as an additional experiment, q-rtPCR was performed using the following primers, and the same trend was observed (Figure 5G). [Table 1] In dermal fibroblast cultures, changes in gene expression for KDM4B and collagen 5A1 are graphed and shown (Figure 6A, B).

[0041] Example 5: Recovery of DNA damage by Kudzu extract after UV irradiation Dermal fibroblasts (KURABO) were cultured in DMEM, low glucose (Thermo Fisher Scientific) medium at 37°C under a 5% CO2 humidified atmosphere. After culturing, the cells were irradiated with 30 mJ of UVB using a constant-temperature ultraviolet irradiation device (Nippon Ika Kikai Seisakusho), and cultured for 2 days to form the UV irradiation group. Subsequently, in addition to UV irradiation, the culture medium was replaced with one containing 0.1% by mass kudzu extract (Ichimaru Falcos), and cultured for 2 days to form the UV irradiation + extract supplement group. In each group, cell samples were fixed with 4% by mass PFA, and anti-rH2AX antibody (Abcam) was used as the primary antibody, reacted at a 200-fold dilution for 1 hour, followed by donkey anti-rabbit IgG (Invitrogen) as the secondary antibody, reacted at a 200-fold dilution for 1 hour. Nuclear staining was performed using DAPI. The presence of rH2AX was confirmed using a confocal microscope STELLARIS 5 (LEICA). Fluorescence microscope images are shown in Figure 7A, and the fluorescence intensity of rH2AX is graphed and shown in Figure 7B.

Claims

1. A nascent protein synthesis promoter containing kudzu extract or neem leaf extract in skin cells.

2. The nascent protein synthesis promoter according to claim 1, which suppresses the decrease in protein synthesis caused by a stimulus response.

3. The nascent protein synthesis promoter according to claim 2, wherein the stimulus is ultraviolet light stimulation.

4. The nascent protein synthesis promoter according to claim 1, wherein the promoter contains Pueraria lobata extract and suppresses UV-induced decrease in protein synthesis in dermal fibroblasts.

5. A composition for improving wrinkles and sagging, comprising the new protein synthesis promoter described in claim 4.

6. The nascent protein synthesis promoter according to claim 1, wherein the inhibitor contains neem leaf extract and suppresses UV-induced decrease in protein synthesis in epidermal keratinocytes.

7. A composition for improving blemishes or skin barrier function, comprising the nascent protein synthesis promoter described in claim 5.

8. A UV resistance enhancer for dermal fibroblasts, containing kudzu extract.

9. The UV resistance enhancer according to claim 8, wherein the effect of the ultraviolet light is DNA damage or decreased collagen expression.

10. An agent containing neem leaf extract that enhances UV resistance in epidermal keratinocytes.

11. The UV resistance enhancer according to claim 10, which suppresses the effects of ultraviolet radiation through antioxidant, anti-inflammatory, and differentiation induction mechanisms.

12. A screening method for UV-resistant agents in UV-irradiated cell cultures, using the ability to synthesize new proteins as an indicator.

13. The screening method according to claim 12, wherein the cell culture is a culture of epidermal keratinocytes or dermal fibroblasts.

14. A step of culturing epidermal keratinocytes or dermal fibroblasts irradiated with ultraviolet light in a culture medium to which candidate components have been added. A step of measuring the ability to synthesize new proteins in cell cultures cultured in a medium containing candidate components, and The measured nascent protein synthesis ability is compared with a control, and if the nascent protein synthesis ability is enhanced, the added candidate component is determined to be an ultraviolet resistance enhancer. The screening method according to claim 13, including the method described in claim 13.

15. The screening method according to claim 14, wherein the UV resistance enhancer is a nascent protein synthesis promoter, or a drug that promotes the expression of a gene selected from the group consisting of DNA repair genes, antioxidant genes, differentiation induction promoting genes, and anti-inflammatory genes.

16. A method for screening UV resistance enhancers in UV-irradiated cell cultures, using the expression of UV resistance genes as an indicator.

17. The screening method according to claim 16, wherein the cell culture is a culture of epidermal keratinocytes or dermal fibroblasts.

18. A step of culturing epidermal keratinocytes or dermal fibroblasts irradiated with ultraviolet light in a culture medium to which candidate components have been added. A step of measuring the expression level of UV resistance genes in cell cultures cultured in a medium containing candidate components, and The process involves comparing the measured expression level of the UV resistance gene with a control, and determining the added candidate component as a UV resistance enhancer if the expression level of the UV resistance gene is increased. The screening method according to claim 17, including the method described in claim 17.

19. The screening method according to claim 18, wherein the ultraviolet resistance gene is at least one selected from the group consisting of GPX3, AKR1C1, EIF3L, DUSPI, involucrin, transglutaminase, and KDM48.

20. The screening method according to claim 18, wherein the UV resistance enhancer is a drug that promotes the expression of a gene selected from the group consisting of a nascent protein synthesis enhancer, a DNA repair gene, an antioxidant gene, a differentiation induction enhancer gene, and an anti-inflammatory gene.

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