Inhibitors of the expression of cellular senescence-related secretory plasma factors, inhibitors of the expression of TGFBR1 and / or TGFBR2, and modulators of the expression levels of aging-related genes.

Abstract

To provide novel inhibitors of cellular senescence-related secretory phenotypes, inhibitors of TGFBR1 and / or TGFBR2 expression, and modifiers of the expression levels of aging-related genes. [Solution] An inhibitor of the expression of cellular senescence-related secretory phenotypes, an inhibitor of the expression of TGFBR1 and / or TGFBR2, and an expression level modifier of aging-related genes, all containing royal jelly as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an expression inhibitor of cellular senescence-associated secretory phenotype factors, an expression inhibitor of TGFBR1 and / or TGFBR2, and an expression level regulator of aging-related genes.

Background Art

[0002] Cells undergo senescence by receiving damage from inside and outside the living body such as oxidative stress and ultraviolet radiation. Senescent cells accumulate with aging. Senescent cells release senescence-associated secretory phenotype (SASP) factors. Some SASP factors have the effect of inducing cells surrounding the senescent cell to become senescent cells, thereby promoting aging of the entire tissue (Non-Patent Document 1).

[0003] Patent Document 1 describes that a component eluted by a specific method from enzyme-treated royal jelly exerts an anti-aging effect by regulating the expression level of genes involved in the insulin / IGF1 signaling pathway. In the examples of Patent Document 1, in a test using Caenorhabditis elegans, the life-extending effect of the eluted component of enzyme-treated royal jelly is demonstrated. As its mechanism of action, an increase in the gene expression levels of ins-20 and dod-3 and a decrease in the gene expression levels of dod-19, dao-4, fkb-4, and ins-9, which are involved in the insulin / IGF1 signaling pathway, are shown, but the involvement of SASP factors is not indicated.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] The present invention aims to provide a novel inhibitor of the expression of cellular senescence-related secretory phenotypes. Furthermore, the present invention aims to provide a novel inhibitor of TGFBR1 and / or TGFBR2 expression, as well as an agent for adjusting the expression levels of senescence-related genes. [Means for solving the problem]

[0007] The inventors of this invention conducted diligent research to achieve the above objectives and discovered that royal jelly has excellent inhibitory effects on the expression of cellular senescence-related secretory phenotypic factors, regulating effects on the expression levels of aging-related genes, and inhibitory effects on the expression of TGFBR1 and / or TGFBR2, thereby completing the present invention.

[0008] In other words, the present invention relates to the following [1] to [7]. [1] An inhibitor of cellular senescence-associated secretory phenotype (SASP) expression, containing royal jelly as an active ingredient. [2] The expression inhibitor according to [1], wherein the above SASP factor is at least one selected from the group consisting of IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14 and PAI1. [3] An inhibitor of the expression of TGFBR1 and / or TGFBR2, which are receptors for TGF-β, containing royal jelly as an active ingredient. [4] An expression regulator of aging-related genes, containing royal jelly as an active ingredient. [5] ABCA7, ACTG1, ALDH4A1, AMIGO2, ANKRD50, AP2M1, ASB1, AT P11A、B3GNT2、B4GALT1、BAZ2B、BCAR3、C11orf24、C1QTNF1、C1RL、CALD1、CAM SAP2, CASP8, CCND2, CD274, CDH3, CDK6, CDYL, CEP170, COL1A1, COL4A1, COL 4A2、COL4A5、COL4A6、COL5A1、COLGALT1、CORO1C、CPA4、CRIM1、CRNN、CSF3、C SNK1E, CSRP1, CSTF3, CXCL1, CXCL10, CXCL14, CXCL2, CXCL3, CXCL5, CYP24A 1、DECR1、DHX16、DRAM1、DUSP4、DUSP5、DYNC1H1、EDN1、EMC1、EPAS1、EREG、FA P, FAT1, FBN2, FNBP1L, FSIP1, FUS, G0S2, GNB4, GPR157, GPR68, GPRC5A, HEG 1、HELZ、HIVEP2、HLA-B、HSPA4L、HSPG2、HUWE1、ID3、IL6、IRF6、ITGB6、IVNS1 ABP, JAG1, JPT2, KCNJ15, KCTD11, KIAA0895L, KIFC3, KRT7, KYNU, LGALS3BP 、LIMK2、LOXL2、LTBP1、LTBP2、MAT2A、MB21D2、MCAM、MDN1、MEST、MICAL2、MOB 3B、MOCOS、MPZL2、MRAS、MRGPRX3、MTAP、MT-CO1、MT-CO2、MTHFD2L、MT-ND2、 MYD88、MYH9、NCEH1、NEFL、NIPAL3、NIPSNAP3A、NOMO2、P4HA1、PALM2AKAP2、P DCD11, PDCD5, PFKFB3, PGAM1, PGK1, PHF20, PLAT, PLOD2, PMEPA1, POLR1A, P OMK, PPP1R3B, PPP1R3C, PRKAR1A, PRORP, PRR5L, PSME3, PTGES, PTGFRN, PTPN 14、PTPRK、RAI14、RGMB、RHOBTB2、RNF152、RRAS、SACS、SBNO2、SDC4、SEMA7A、 SERPINE1, SERPINH1, SGK1, SH2D3A, SHFL, SIPA1L1, SKILL, SLAMF9, SLC16A3An expression regulator described in [4], which is an expression level increase inhibitor, comprising at least one gene selected from the group consisting of SLC2A1, SLC35F6, SLC38A4, SMURF2, SOWAHC, SPARC, SPTAN1, STC1, SUSD6, TAGLN2, TDRP, TENM2, TFRC, TGFBI, TGFBR1, TGFBR2, TGM2, THBS1, THEM4, TNC, TNFAIP1, TNFRSF10B, TRAF4, TRIM21, TRIM65, TSEN34, TUBA1A, TUBA1C, TVP23B, TXNDC9, TYMP, UBE2Q2, UFM1, UGP2, USB1, VCAN, WNT5A, YWHAG, ZBED2, ZNF486, and ZNFX1. [6] F1、ADK、AIF1L、AKAP12、ALCAM、ALDH1A3、ALDH3A2、ALOXE3、ANK3、ANXA5、AP PL2、AQP3、ARAP1、ARHGEF40、ARNTL2、BEX4、BMP4、BTBD11、CASP1、CAT、CCNI 、CCZ1B、CD109、CD9、CDC14B、CDHR1、CEBPB、CELSR2、CFAP46、CLIP4、CLMN、CN TN1、COG5、CPNE1、CRYBG1、CSRP2、CTDSP2、DDHD2、DENND4C、DSC3、ECH1、ECM 1、EFL1、EIF4B、ELK3、ELOVL4、EML1、EPHB3、ERO1B、ERRFI1、ETV1、FAM110C、F AM117A, FCHSD2, FERMT1, FHL1, FOSL1, FOXQ1, GABRE, GCLC, GPR176, H1-0, H1-10, HADHB, HDAC5, HMGA1, HPGD, HPSE, HSDL2, IMPA2, INSYN2B, IRF2BPL, JA RID2、JPH2、KANK2、KCNG1、KCNK1、KLF11、KLF4、KMO、KRT14、KRT15、KRT5、LH FPL2、LMNA、LMO4、LRP8、LRRC8A、LYST、MAL2、MAN1A1、MAP4K4、MAPKAPK3、MB OAT2、ME1、MMRN2、MOSMO、MPP7、MYO1D、NAPRT、NAV3、NIBAN1、NKX1-2、NQO2、 NR1D1、NRROS、NTSR1、OPN3、OSBPL1A、P3H2、PABPC1、PAG1、PAK6、PCDHGC3、PD E7A、PER3、PFKFB2、PIR、PITPNC1、PKIB、PLA2G4A、PLEKHG6、PPFIBP2、PRNP、 PRR15、PRX、PRXL2A、PSMG3、PTGR1、PTGS1、RAPGEFL1、RNF180、RPS6KA3、SC5D SIX4, SLC1A3, SLC41A1, SLC48A1, SLC7A11, SLC7A8, SLC9A3R1, SMN1, SMO, SMOC2, SNX29, SOX2, SOX9, SRGAP3, STEAP1, SUN1, SYT7, TAB3, TACC2, TAOK3An expression regulator according to [4], which is at least one gene selected from the group consisting of TEF, TGFBR3, TIMP1, TKT, TNIK, TNNT1, TOP1MT, TRAPPC9, TRPV3, TSC22D1, TSHZ2, TTC39A, UBASH3B, VWA5A, WLS, XDH, and ZNF395, and is an expression reduction inhibitor. [7] A cosmetic, quasi-drug, pharmaceutical, or food / beverage agent as described in any one of [1] to [6]. [Effects of the Invention]

[0009] The present invention provides a novel inhibitor of the expression of cellular senescence-related secretory phenotypes. Furthermore, the present invention provides a novel inhibitor of TGFBR1 and / or TGFBR2 expression, as well as an agent for adjusting the expression levels of senescence-related genes. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram illustrating the estimated mechanism of aging in cells. [Figure 2] This graph shows the level of IL6 expression in response to changes in royal jelly concentration. [Figure 3] (a) is a graph showing the expression level of CXCL1 in response to changes in royal jelly concentration, and (b) is a graph showing the expression level of CXCL2 in response to changes in royal jelly concentration. [Figure 4] This graph shows the expression level of the IL6 protein. [Modes for carrying out the invention]

[0011] The following describes in detail embodiments for carrying out the present invention, but the present invention is not limited to the following embodiments.

[0012] In this specification, "comprise" encompasses both the meanings of "essentially consist of" and "consist of."

[0013] A first aspect of the present invention is an inhibitor of expression of senescence-associated secretory phenotype (SASP) factors, which contains royal jelly as an active ingredient.

[0014] Senescent cells release SASP factors to the extracellular space. Some SASP factors have the effect of inducing senescence in cells surrounding the senescent cell, thereby promoting aging of the entire tissue. The SASP factor expression inhibitor of the present invention, which contains royal jelly as an active ingredient, can suppress the expression of SASP factors in senescent cells.

[0015] By suppressing the expression of SASP factors in senescent cells, it is possible to prevent surrounding cells from being induced to become senescent cells, consequently prevent an increase in the number of senescent cells, and suppress aging of biological tissues. The SASP factor expression inhibitor of the present invention can also be referred to as a senescence propagation inhibitor.

[0016] Furthermore, by suppressing the expression of SASP factors, damage to biological tissues can be ameliorated or prevented. The biological tissue may be, for example, skin tissue. In addition, suppressing SASP factor expression can lead to reduction of wound healing delay caused by chronic inflammation and suppression of tumor formation (Non-Patent Documents 2 and 3).

[0017] SASP factors include, for example, inflammatory cytokines, chemokines, and the like. In the SASP factor expression inhibitor of the present invention, the SASP factors may be, for example, IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, PAI1 (SERPINE1), and the like.

[0018] The SASP factor expression inhibitor can suppress the expression of SASP factors by suppressing the expression of genes encoding each SASP factor protein. Therefore, the SASP factor expression inhibitor of the present invention can be used as an expression inhibitor for genes encoding SASP factors.

[0019] SASP factor expression inhibitors can suppress SASP factor expression depending on the concentration or amount of royal jelly used as the active ingredient. In other words, the more of the active ingredient used, the more effectively SASP factor expression can be suppressed.

[0020] A second aspect of the present invention is an inhibitor of TGFBR1 and / or TGFBR2 expression, which are receptors for TGF-β, containing royal jelly as an active ingredient. TGFBR1 and TGFBR2 are located in the cell membrane upstream of the SASP factor. TGF (Transforming growth factor)-β has the function of regulating the action of the SASP factor. Therefore, by suppressing the expression of TGFBR1 and / or TGFBR2, it is possible to suppress cell cycle arrest and the expression of the SASP factor downstream via SMAD2 / 3 phosphorylation or a SMAD-independent pathway (Non-Patent Literature 4).

[0021] TGFBR1 and / or TGFBR2 expression inhibitors can suppress the expression of TGFBR1 and / or TGFBR2 by suppressing the expression of the genes encoding these proteins. Therefore, the TGFBR1 and / or TGFBR2 expression inhibitor of the present invention can be used as an expression inhibitor for the genes encoding TGFBR1 and / or TGFBR2.

[0022] A third aspect of the present invention is an expression regulator for aging-related genes, containing royal jelly as an active ingredient. In this specification, aging-related genes are genes whose expression levels increase or decrease due to cellular aging.

[0023] The gene expression regulators for aging may specifically be agents that suppress the increase or decrease in the expression level of aging-related genes.

[0024] The inhibitors that suppress the increased expression of aging-related genes have the effect of suppressing the expression of genes selected from Table 1. These genes are expressed in greater amounts in senescent cells than in less senescent cells. By using aging-related gene expression inhibitors, the increased expression of these genes can be suppressed. Among these, CCND2 and RRAS are involved in cell cycle arrest.

[0025] [Table 1]

[0026] The gene expression inhibitors that reduce the expression of aging-related genes increase the expression of genes selected from Table 2. These genes are expressed at lower levels in senescent cells than in less senescent cells. By using gene expression inhibitors that reduce the expression of aging-related genes, the decrease in expression of these genes can be suppressed.

[0027] CAT is an antioxidant enzyme involved in the suppression of SASP factor expression. TIMP1 is a collagen-degrading enzyme inhibitor. CD109 has functions in epidermal formation and TGF-β signaling suppression. The gene expression regulator for aging-related genes according to the present invention can comprehensively adjust the expression levels of the genes shown in Tables 1 and 2, including these genes, and therefore has an overall anti-aging effect.

[0028] [Table 2]

[0029] The above-mentioned gene expression regulators can adjust the expression level of aging-related genes depending on the concentration or amount of royal jelly used as the active ingredient. In other words, in the case of gene expression increase inhibitors, the more you use, the more you can suppress the increase in expression, and in the case of gene expression decrease inhibitors, the more you use, the more you can suppress the decrease in expression.

[0030] Figure 1 shows the estimated mechanism of behavior in cellular aging. In Figure 1, the underlined genes are those whose expression increases with aging and decreases with the agent of the present invention, or decreases with aging and increases with the agent of the present invention. When cells age due to DNA damage, etc., SASP factors are secreted. When TGF-β binds to TGFBR1 and TGFBR2, which are receptors for TGF-β that regulate the function of SASP factors in other cells, it causes damage such as DNA damage in those cells and leads to cellular aging. As shown in the examples described later, due to the replication aging of cells, the expression of SASP factors secreted by senescent cells, TGF-β receptors located on the cell membrane upstream of SASP factors, CCND2 and RRAS which are involved in cell cycle arrest, etc. increases. Adding royal jelly to senescent cells alters various signals, such as suppressing the activity of TGF-β receptors, suppressing inflammatory signals including chemokines, and enhancing regulatory signals for keratinocyte differentiation. By adjusting the expression levels of the aging-related genes mentioned above, the increase in SASP factor expression can be suppressed.

[0031] The SASP factor expression inhibitor, TGFBR1 and / or TGFBR2 expression inhibitor, and aging gene expression level modifier of the present invention (hereinafter collectively referred to as "the agents of the present invention") contain royal jelly as an active ingredient. Royal jelly is a milky white jelly-like substance produced by mixing secretions secreted from the hypopharyngeal gland and mandibular gland by worker bees aged 3 to 12 days. The main physiologically active components in royal jelly include, for example, organic acids such as saturated fatty acids and unsaturated fatty acids unique to royal jelly, as well as proteins, amino acids, peptides, lipids, sugars, vitamins such as B vitamins, folic acid, nicotinic acid, and pantothenic acid, and various minerals.

[0032] The royal jelly used in this invention includes raw royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, unenzyme-treated royal jelly, royal jelly extract, and fermented royal jelly. The royal jelly may be sourced from any of the following countries: European countries, Oceania countries, the United States, Brazil, Japan, China, or other Asian countries.

[0033] Dried royal jelly powder is made by drying and pulverizing fresh royal jelly. Any known drying method used in general food processing can be used, including natural drying such as air drying or sun drying, forced drying by heating with electricity, or freeze-drying. Freeze-drying is preferred.

[0034] Enzyme-treated royal jelly is royal jelly treated with a protease (protein-degrading enzyme). Preferably, it is a low-allergen enzyme-treated royal jelly in which allergic reactions caused by proteins contained in royal jelly are suppressed by protease treatment. Therefore, in addition to the protease hydrolysates of proteins contained in royal jelly, enzyme-treated royal jelly may contain organic acids such as saturated and unsaturated fatty acids, lipids, sugars, vitamins, and various minerals.

[0035] The royal jelly used in the production of enzyme-treated royal jelly is not particularly limited and may include, for example, raw royal jelly, royal jelly powder obtained by drying and pulverizing raw royal jelly, or raw royal jelly extracted with water or aqueous ethanol.

[0036] Enzyme-treated royal jelly can be produced by treating royal jelly raw material with an enzyme having at least endopeptidase activity, an enzyme having at least exopeptidase activity, and / or an enzyme having both endopeptidase and exopeptidase activity.

[0037] Examples of proteolytic enzymes that possess at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

[0038] Examples of proteolytic enzymes that possess at least exopeptidase activity include carboxypeptidases, aminopeptidases, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus species, Rhizopus species, etc.), and pancreatin and pepsin, which also possess endopeptidase activity.

[0039] Among these various enzymes, preferred examples of enzymes possessing both exopeptidase and endopeptidase activity include Streptomyces griseus-produced peptidase (product name: Actinase AS), Aspergillus oryzae-produced peptidase (product names: Protease A, Flavorzyme, Proteax), and Aspergillus melleus-produced peptidase (product name: Protease P). Preferred examples of enzymes possessing exoprotease activity include Aspergillus oryzae-produced peptidase (product names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), and Aspergillus sojae-produced peptidase (product name: Sternzyme). Examples include peptidases produced by Aspergillus species (product name: Cocrase P) and peptidases produced by Rhizopus oryzae (product name: Peptidase R). Furthermore, preferred examples of enzymes possessing endoprotease activity include peptidase produced by Bacillus subtilis (trade names: Orientase 22BF, Nucleicin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutralase), and peptidase produced by Bacillus species (trade name: Protamex).

[0040] Enzymatic treatment to reduce the allergenicity of royal jelly can be carried out, for example, in accordance with the descriptions in Japanese Patent Publication No. 2007-295919 and Japanese Patent Publication No. 2007-295920.

[0041] Royal jelly extract is obtained by extracting royal jelly (including raw, dried, and pulverized forms) with water or aqueous ethanol, etc.

[0042] Royal jelly fermented products can be produced by conventional methods using microorganisms such as yeast and lactic acid bacteria.

[0043] Commercially available royal jelly may be used. Specific examples of commercially available royal jelly include, for example, Enzyme-hydrolyzed Royal Jelly King (manufactured by Yamada Bee Farm Co., Ltd.), Royal Jelly FD Powder (manufactured by Nakahara Co., Ltd.), Royal Jelly Extract SF (manufactured by Matsuura Pharmaceutical Co., Ltd.), Deproteinized Royal Jelly Powder F (manufactured by Maruzen Pharmaceutical Co., Ltd.), and Deproteinized Royal Jelly Extract (manufactured by Api Co., Ltd.).

[0044] Since the agent of the present invention uses royal jelly, which has been conventionally used as a food ingredient, it is highly safe.

[0045] The content of the above-mentioned active ingredient in the agent of the present invention may be, for example, 0.1% by mass or more, 1% by mass or more, 3% by mass or more, 3.5% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 93% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass relative to the total amount of the agent. The content of the above active ingredient in the agent is solid content based on the total amount of the agent, for example, 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 93% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 6 It may be 0 mass% or less, 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 8 mass% or less, 5 mass% or less, 3 mass% or less, or 1 mass% or less.

[0046] The agents of the present invention may also be administered to humans for use, for example, to suppress SASP factor expression, TGFBR1 and / or TGFBR2 expression, and to modulate the expression levels of aging-related genes. The agents of the present invention may be administered orally or parenterally. Oral administration includes enteral administration. Parenteral administration includes topical administration, and in particular includes transdermal administration.

[0047] The dosage of the agent of the present invention when administered orally may vary depending on the form of the agent and the method and amount of application. For example, for an adult weighing 60 kg, the daily dose should be 10 mg to 30,000 mg in terms of the dry solid content of the active ingredient, preferably 100 mg to 20,000 mg, 150 mg to 15,000 mg, 600 mg to 12,000 mg, 1,200 mg to 10,000 mg, or 2,400 mg to 8,000 mg. This dosage can be appropriately increased or decreased depending on factors such as the health condition of the person taking the drug, the method of administration, and combinations with other agents.

[0048] The agent of the present invention may be administered once a day, or divided into multiple doses, such as twice a day or three times a day, as long as the effective daily dose is within the range described above. The agent of the present invention provides immediate effects after administration, but continuous administration for 1 to 4 weeks, 1 month or more, 6 months or more, or 1 year or more is preferable because it allows for a longer-lasting effect.

[0049] The dosage of the agent of the present invention when administered parenterally may vary depending on the site and scope of application. For example, the amount applied to the skin may be 0.01 mg to 50 mg of the active ingredient in terms of dry solid content, preferably 0.02 mg to 40 mg, 0.02 mg to 35 mg, or 0.025 mg to 30 mg of the active ingredient.

[0050] The amount of the active ingredient in cosmetics, food and beverages, quasi-drugs, or pharmaceuticals containing the agent of the present invention is not particularly limited, and any effective amount that can achieve the oral or parenteral dosage of the agent is acceptable.

[0051] The agent of the present invention can be used in cosmetics, food and beverages (especially food and beverages intended for health, health maintenance, and promotion (e.g., health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, nutritional function foods, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. Furthermore, the agent of the present invention also encompasses the meaning of additives that impart SASP factor expression inhibitory activity, TGFBR1 and / or TGFBR2 expression inhibitory activity, and aging gene expression level regulating activity.

[0052] Cosmetics, food and beverages, quasi-drugs, or pharmaceuticals containing the agent of the present invention as one component can be manufactured, for example, by adding the agent of the present invention to an intermediate product in the manufacturing process of these products.

[0053] In addition to the royal jelly mentioned above, the above-mentioned cosmetics may contain, as needed, other ingredients commonly used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavoring agents, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), whitening agents, moisturizers, antioxidants, UV absorbers, powder components, colorants, various skin nutrients, etc.

[0054] Cosmetic ingredients may be in any form, such as solid, liquid, or paste. Cosmetics may be medicated cosmetics (i.e., quasi-drugs). Cosmetics include all cosmetics that can be applied to parts of animals (including humans), such as skin, mucous membranes, body hair, hair, scalp, nails, teeth, facial skin, and lips.

[0055] Cosmetic formulations can take a wide range of forms, including aqueous solutions, solubilized solutions, emulsified solutions, powders, oil-based solutions, gels, ointments, aerosols, two-layer water-oil systems, and three-layer water-oil-powder systems.

[0056] The uses of cosmetics are also arbitrary. For example, basic cosmetics include facial cleansers, toners, lotions, creams, gels, essences, serums, packs, masks, mists, and UV protection cosmetics. Makeup cosmetics include foundations, lipsticks, blushes, eyeshadows, eyeliners, and mascaras. Nail cosmetics include nail polish, base coats, top coats, and nail polish removers. Other products include facial cleansers, (paste or liquid) toothpaste, mouthwash, oral cosmetics, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, conditioners, hair treatments, hair styling products, hair tonics, hair mists, hair foams, hair liquids, hair gels, hair sprays, hand creams, hand soaps, hair growth products, antiperspirants, and bath additives.

[0057] The above-mentioned food and beverages may use the royal jelly as is, or, if necessary, may be combined with minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solvents, humectants, etc.

[0058] Food and beverages include all foods and beverages that can be consumed by animals (including humans). The types of food and beverages are not particularly limited and include, for example, dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea, milk drinks, lactic acid bacteria drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, sake, Western liquors, fruit wines, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, gummies, jelly, candy, cookies, cakes, puddings, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice pops, sherbet, etc.); food products (curry, gyudon, zosui, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0059] The manufacturing method for food and beverages is not particularly limited and may be carried out according to publicly known methods as appropriate.

[0060] There are no particular restrictions on the dosage unit form when used as a supplement; it can be selected as appropriate. Examples include chewable tablets, lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0061] The above-mentioned pharmaceuticals may use only the royal jelly, or they may be used in combination with other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

[0062] When preparing the agent of the present invention as a pharmaceutical product, the royal jelly can be prepared together with pharmaceutical-permissible components in the form of tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, and injections (including cases where it is prepared as a liquid by mixing it with distilled water or an infusion solution such as an amino acid solution or an electrolyte solution at the time of use) to create a pharmaceutical preparation.

[0063] The administration of pharmaceuticals may be local or systemic. There are no particular restrictions on the method of administration, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intra-arterial administration, and transdermal administration.

[0064] In addition to royal jelly, the pharmaceutical product of the present invention may contain, as needed, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

[0065] Furthermore, the cosmetics and pharmaceuticals of this invention also include quasi-drugs.

[0066] The agent of the present invention described above is applicable to mammals, including humans (preferably humans). [Examples]

[0067] [Preparation of Royal Jelly] Keratinocyte Basic Medium 2 (without phenol red; PromoCell, C-20216) was supplemented with Keratinocyte Proliferation Medium 2 Supplement Mix (PromoCell, C-39016) and a 1% penicillin-streptomycin mixture (penicillin 10,000 u / mL, streptomycin 10,000 μg / mL; Nakarai, 09367-34) to prepare a medium (hereinafter referred to as KGM medium). Unenzyme-hydrolyzed royal jelly was dissolved in KGM medium to a final concentration of 1 mg / mL, ultrasonically ground for 30 minutes, and then sterilized by filtration. This was used as royal jelly in the following tests.

[0068] [Cell culture] Infant-derived normal human epidermal keratinocytes (NHEK, PromoCell, Lot.470Z031) were maintained in KGM medium. NHEK cells that reached subconfluence were passaged for 3 × 10⁶ cells. 4 cells / cm 2 Seeds were seeded in a 35mm dish (Thermo Scientific BioLite 130180) to achieve the desired result. P5 (5th subculture) was used as the young cell model, and P20 (20th subculture) was used as the aged cell model. The day after seeding, a portion of the P20 cells were mixed with prepared royal jelly at a concentration of 1 mg / ml, and the cells were used for measurement 24 hours after mixing. The following measurements were performed on P5, P20, and the royal jelly-added P20 cells, respectively.

[0069] [Gene expression level measurement] Total RNA was extracted using the Total RNA Purification Plus Kit (Norgen Biotek, 48300) according to the manufacturer's protocol. The amount of RNA was quantified using a NanoDropOne micro-spectrophotometer (ThermoScientific, ND-ONE-W). 2 μL of 5×RTMaster Mix (Toyobo, FSQ201) was added to 8 μL of 50 ng of prepared total RNA, and reverse transcription was performed using a CFX Opus96 real-time PCR system (Bio-rad) at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes to produce cDNA. SsoAdvanced Universal SYBR Green Supermix (Biorad, 172-5271B02) was used to prepare the PCR reaction mixture. The primers used and their sequences are shown in Table 3. The RPLP0 gene was used as a reference. Real-time PCR was performed using the CFX Opus96 real-time PCR system (Bio-rad) under the following conditions: 95°C for 30 seconds (1 cycle), 95°C for 15 seconds, and 60°C for 30 seconds (40 cycles).

[0070] [Table 3]

[0071] [Investigation of Royal Jelly Concentration] For the gene IL6, the concentration of added royal jelly was adjusted to 0 μg / ml, 125 μg / ml, 250 μg / ml, 500 μg / ml, or 1000 μg / ml, and the gene expression level was measured using the primers shown in Table 3 in the same manner as above. The results are shown in Figure 2. Similarly, for the genes CXCL1 and CXCL2, the concentration of added royal jelly was adjusted to 0 μg / ml, 500 μg / ml, or 1000 μg / ml, and the gene expression levels were measured using the primers shown in Table 3 in the same manner as above (n=3). The results for CXCL1 are shown in Figure 3(a), and the results for CXCL2 are shown in Figure 3(b). It was confirmed that the expression levels of IL6, CXCL1, and CXCL2 could be suppressed depending on the concentration of added royal jelly.

[0072] [RNA sequencing] The isolated total RNA was adjusted to 50-100 ng / μL, and gene expression quantification and differential gene (DEG) analysis of the samples were commissioned to Rhelixa Corporation. The RNA samples were confirmed not to be degraded using a bioanalyzer. RNA seq libraries were prepared using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and the NEBNext® Ultra™II Directional RNA Library Prep Kit (NEB, E7760), respectively, and subjected to a next-generation sequencer, NovaSeq6000 (Illumina).

[0073] Subsequently, informatics analysis was performed according to the following procedure. Sequence reads were trimmed using the software Trimmomatic (Version 0.38) based on the following settings: ILLUMINACLIP2:30:10, LEADING20, TRAILING20, SLIDINGWINDOW4:15, MINLEN36. Mapping to the reference genome was performed using the software HISAT2 (Version 2.1.0). RLE normalization was performed based on the number of reads calculated using the software featureCounts (Version 1.6.3), and differentially expressed genes were extracted using the software DESeq2 (Version 1.24.0).

[0074] Increases or decreases in gene expression levels were detected when the P value was less than 0.05. In P20, 2939 mRNAs showed increased expression compared to P5, i.e., increased expression due to replication senescence. In royal jelly-added P20, 740 mRNAs showed decreased expression compared to P20, i.e., decreased expression in replication senescent cells after royal jelly treatment. Of these, 197 mRNAs showed increased expression due to replication senescence and decreased expression in replication senescent cells after royal jelly treatment. Table 4 shows the genes whose expression increased due to replication senescence and decreased expression in replication senescent cells after royal jelly treatment.

[0075] In particular, the genes encoding the SASP factors IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, and PAI1, as well as the genes encoding TGFBR1 and TGFBR2, were found to increase in expression due to replication senescence and decrease in expression with the addition of royal jelly. Similarly, the genes encoding RRAS and CCND2 were also found to increase in expression due to replication senescence and decrease in expression with the addition of royal jelly.

[0076] On the other hand, 3097 mRNAs showed decreased expression levels in P20 compared to P5, meaning their expression was reduced due to replication senescence. 731 mRNAs showed increased expression levels in royal jelly-treated P20 compared to P20, meaning their expression increased in replication senescent cells after royal jelly treatment. Of these, 178 mRNAs showed decreased expression due to replication senescence and increased expression in replication senescent cells after royal jelly treatment. Table 5 shows the genes whose expression decreased due to replication senescence and increased expression in replication senescent cells after royal jelly treatment.

[0077] [Table 4]

[0078] [Table 5]

[0079] Table 6 shows the gene expression levels (multiplier changes) of IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, and PAI1, with the gene expression level in the young model P5 set to 1.

[0080] [Table 6]

[0081] [Protein expression level] The IL-6 levels in the collected culture supernatant were evaluated using the Human IL-6 Quantikine ELISA Kit (R&D Systems, D6050) according to the manufacturer's protocol.

[0082] The results are shown in Figure 4. At P20, the amount of IL6 protein in the culture supernatant was significantly reduced in the royal jelly-supplemented group compared to the group without royal jelly. This indicates that the expression of IL6 protein, which increases due to replication senescence, is suppressed by the addition of royal jelly.

Claims

1. An expression inhibitor of cellular senescence-related secretory traits (SASPs), comprising royal jelly as an active ingredient, wherein the SASP factor is at least one selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, and PAI1.

2. An inhibitor of TGFBR1 and / or TGFBR2 expression, which are receptors for TGF-β, containing royal jelly as an active ingredient.

3. A gene expression regulator containing royal jelly as an active ingredient.

4. Said aging-related gene is ABCA7, ACTG1, ALDH4A1, AMIGO2, ANKRD50, AP2M1, ASB1, ATP11A, B3GNT2, B4GALT1, BAZ2B, BCAR3, C11orf24, C1QTNF1, C1RL, CALD1, CAMSAP2, CASP8, CCND2, CD274, CDH3, CDK6, CDYL, CEP170, COL1A1, COL4A1, COL4A2, COL4A5, COL4A6, COL5A1, COLGALT1, CORO1C, CPA4, CRIM1, CRNN, CSF3, CSNK1E, CSRP1, CSTF3, CXCL1, CXCL10, CXCL14, CXCL2, CXCL3, CXCL5, CYP24A1, DECR1, DHX16, DRAM1, DUSP4, DUSP5, DYNC1H1, EDN1, EMC1, EPAS1, EREG, FAP, FAT1, FBN2, FNBP1L, FSIP1, FUS, G0S2, GNB4, GPR157, GPR68, GPRC5A, HEG1, HELZ, HIVEP2, HLA-B, HSPA4L, HSPG2, HUWE1, ID3, IRF6, ITGB6, IVNS1ABP, JAG1, JPT2, KCNJ15, KCTD11, KIAA0895L, KIFC3, KRT7, KYNU, LGALS3BP, LIMK2, LOXL2, LTBP1, LTBP2, MAT2A, MB21D2, MCAM, MDN1, MEST, MICAL2, MOB3B, MOCOS, MPZL2, MRAS, MRGPRX3, MTAP, MT-CO1, MT-CO2, MTHFD2L, MT-ND2, MYD88, MYH9, NCEH1, NEFL, NIPAL3, NIPSNAP3A, NOMO2, P4HA1, PALM2AKAP2, PDCD11, PDCD5, PFKFB3, PGAM1, PGK1, PHF20, PLAT, PLOD2, PMEPA1, POLR1A, POMK, PPP1R3B, PPP1R3C, PRKAR1A, PRORP, PRR5L, PSME3, PTGES, PTGFRN, PTPN14, PTPRK, RAI14, RGMB, RHOBTB2, RNF152, RRAS, SACS, SBNO2, SDC4, SEMA7A, SERPINE1, SERPINH1, SGK1, SH2D3A, SHFL, SIPA1L1, SKIL, SLAMF9, SLC16A3, SLC2A1,SLC35F6, SLC38A4, SMURF2, SOWAHC, SPARC, SPTAN1, STC1, SUSD6, TAGLN2, TDRP, TENM2, TFRC , TGFBI, TGFBR1, TGFBR2, TGM2, THBS1, THEM4, TNC, TNFAIP1, TNFRSF10B, TRAF4, TRIM21, TRIM The gene expression regulator according to claim 3, which is an expression expression increase inhibitor, comprising at least one gene selected from the group consisting of 65, TSEN34, TUBA1A, TUBA1C, TVP23B, TXNDC9, TYMP, UBE2Q2, UFM1, UGP2, USB1, VCAN, WNT5A, YWHAG, ZBED2, ZNF486, and ZNFX1.

5. Said aging-associated gene is ABCC2, ABCC3, ABHD5, ABLIM1, ACSL1, ACSS2, ADGRA3, ADGRF1, ADK, AIF1L, AKAP12, ALCAM, ALDH1A3, ALDH3A2, ALOXE3, ANK3, ANXA5, APPL2, AQP3, ARAP1, ARHGEF40, ARNTL2, BEX4, BMP4, BTBD11, CASP1, CAT, CCNI, CCZ1B, CD109, CD9, CDC14B, CDHR1, CEBPB, CELSR2, CFAP46, CLIP4, CLMN, CNTN1, COG5, CPNE1, CRYBG1, CSRP2, CTDSP2, DDHD2, DENND4C, DSC3, ECH1, ECM1, EFL1, EIF4B, ELK3, ELOVL4, EML1, EPHB3, ERO1B, ERRFI1, ETV1, FAM110C, FAM117A, FCHSD2, FERMT1, FHL1, FOSL1, FOXQ1, GABRE, GCLC, GPR176, H1-0, H1-10, HADHB, HDAC5, HMGA1, HPGD, HPSE, HSDL2, IMPA2, INSYN2B, IRF2BPL, JARID2, JPH2, KANK2, KCNG1, KCNK1, KLF11, KLF4, KMO, KRT14, KRT15, KRT5, LHFPL2, LMNA, LMO4, LRP8, LRRC8A, LYST, MAL2, MAN1A1, MAP4K4, MAPKAPK3, MBOAT2, ME1, MMRN2, MOSMO, MPP7, MYO1D, NAPRT, NAV3, NIBAN1, NKX1-2, NQO2, NR1D1, NRROS, NTSR1, OPN3, OSBPL1A, P3H2, PABPC1, PAG1, PAK6, PCDHGC3, PDE7A, PER3, PFKFB2, PIR, PITPNC1, PKIB, PLA2G4A, PLEKHG6, PPFIBP2, PRNP, PRR15, PRX, PRXL2A, PSMG3, PTGR1, PTGS1, RAPGEFL1, RNF180, RPS6KA3, SC5D, SIX4, SLC1A3, SLC41A1, SLC48A1, SLC7A11, SLC7A8, SLC9A3R1, SMN1, SMO, SMOC2, SNX29, SOX2, SOX9, SRGAP3, STEAP1, SUN1, SYT7, TAB3, TACC2, TAOK3, TEF,The gene expression regulator according to claim 3, which is an expression reduction inhibitor, comprising at least one gene selected from the group consisting of TGFBR3, TIMP1, TKT, TNIK, TNNT1, TOP1MT, TRAPPC9, TRPV3, TSC22D1, TSHZ2, TTC39A, UBASH3B, VWA5A, WLS, XDH, and ZNF395.

6. The agent according to any one of claims 1 to 5, which is a cosmetic, quasi-drug, pharmaceutical, or food or beverage.

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