JP7822610B2 - Inhibin βA (INHBA) expression inhibitor - Google Patents

Description

The present invention relates to an inhibin βA (INHBA) expression inhibitor.

Inhibin βA (INHBA) is highly conserved among vertebrates and, after extracellular secretion, forms a dimer to become activin A. Therefore, it can be said that quantitative changes in INHBA in the body are positively correlated with quantitative changes in activin A. Activin A was identified as a gonadal protein that induces the biosynthesis and secretion of follicle-stimulating hormone from the anterior pituitary gland (Non-Patent Document 1). Previous research has confirmed that INHBA/activin A is expressed in various tissues from embryonic development to adulthood, and has revealed that it is involved in various biological functions, including stem cell maintenance and differentiation, cell proliferation, apoptosis, immune responses, and inflammatory responses (Non-Patent Document 2).

Activin A transmits signals via two receptors, type I and type II. When activin A binds to the type II receptor, the type I receptor is recruited, forming a tetrameric receptor complex and becoming phosphorylated. The activated type I receptor phosphorylates SMAD2/3 within the cell, and the phosphorylated SMAD2/3 then forms a complex with SMAD4, translocates to the nucleus, and regulates the transcription of target genes (Non-Patent Documents 2 and 3). Signal transduction not mediated by SMADs also exists, and activated type I receptors are known to activate intracellular signaling pathways such as ERK 1/2 (extracellular signal-regulated kinases 1/2), MAPK (p38 mitogen-activated protein kinase), and JNK (c-Jun N-terminal kinases), thereby influencing cell migration and differentiation (Non-Patent Document 4). Activin A is also known to induce the Wnt signaling pathway by activating SMAD2 (Non-Patent Document 5).

As such, activin A is involved in multiple intracellular signaling mechanisms and regulates diverse cellular responses, making it essential for maintaining the function of cells that make up biological tissues. However, due to its various biological effects and involvement in intracellular signaling mechanisms, its abnormal expression is associated with a variety of diseases and pathological conditions. In fact, activin A is overexpressed in tumor cells of lung adenocarcinoma, esophageal cancer, and malignant pleural mesothelioma, promoting cell proliferation and tumor expansion. It has also been reported that activin A promotes tumor cell invasion and metastasis in several tumors, including prostate cancer, breast cancer, oral squamous cell carcinoma, and melanoma (Non-Patent Documents 6-8). In addition to cancer, increased levels of activin A have been reported to be involved in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, allergic diseases such as allergic asthma and atopic dermatitis, and bone metabolic disorders (Non-Patent Documents 9, 10).

For this reason, efforts are underway to develop treatments that control activin A signaling for such diseases. For example, intraperitoneal injection of follistatin, an activin A antagonist protein, into colitis model mice has been shown to suppress colon inflammation and increase survival rates by preventing activin A receptor binding and the initiation of the signaling pathway (Non-Patent Document 11). Additionally, methods using anti-activin A antibodies and activin antagonists are available to treat diseases and disorders by inhibiting activin A signaling (Patent Documents 1 to 4).

Appropriate control of activin A signaling is therefore useful not only for treating various diseases, but also for efficiently controlling stem cell differentiation and cell proliferation. However, the above-mentioned antagonistic proteins and antibody proteins are problematic in their use because they are easily degraded by enzymes present in the body and also bind to transforming growth factor-β (TGF-β) and myostatin (GDF-8), which are structurally similar to activin A. Therefore, there is a need to identify new factors that can replace them.

Japanese Patent Publication No. 2020-11965 Special Publication No. 2021-512919 Japanese Patent Application Publication No. 2018-162312 Japanese Patent Application Laid-Open No. 2018-145198

Ling N, Ying SY, Ueno N, et al. Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature. 1986;321(6072):779-782. Morianos I, Papadopoulou G, Semitekolou M, Xanthou G. Activin-A in the regulation of immunity in health and disease. J Autoimmun. 2019;104:102314. Namwanje M, Brown CW. Activins and inhibitors: roles in development, physiology, and disease. Cold Spring Harb Perspect Biol. 2016;8(7):a021881. de Guise C, Lacerte A, Rafiei S, et al. Activin inhibits the human Pit-1 gene promoter through the p38 kinase pathway in a Smad-independent manner. Endocrinology. 2006;147(9):4351-4362. Hirota M, Watanabe K, Hamada S, et al. Smad2 functions as a co-activator of canonical Wnt/beta-catenin signaling pathway independent of Smad4 through histone acetyltransferase activity of p300. Cell Signal. 2008;20(9):1632-1641. Ries A, Schelch K, Falch D, Pany L, Hoda MA, Grusch M. Activin A: an emerging target for improving cancer treatment? Expert Opin Ther Targets. 2020;24(10):985-996. Donovan P, Dubey OA, Kallioinen S, et al. Paracrine activin-a signaling promotes melanoma growth and metastasis through immune evasion. J Invest Dermatol. 2017;137(12):2578-2587. Chen L, De Menna M, Groenewoud A, Thalmann GN, Kruithof-de Julio M, Snaar-Jagalska BE. A NF-κB-Activin A signaling axis enhances prostate cancer metastasis. Oncogene. 2020;39(8):1634-1651. Hardy CL, Rolland JM, O’Hehir RE. The immunoregulatory and fibrotic roles of activin A in allergic asthma. Clin Exp Allergy. 2015;45(10):1510-1522. Mundy C, Yao L, Sinha S, et al. Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice. Sci Signal. 2021;14(669):eabd0536. Jones KL, Mansell A, Patella S, et al. Activin A is a critical component of the inflammatory response, and its binding protein, follistatin, reduces mortality in endotoxemia. Proc Natl Acad Sci U S A. 2007;104(41):16239-16244.

Therefore, an objective of the present invention is to discover a substance that can suppress the expression of activin A, which controls various functions such as cell proliferation and differentiation, apoptosis induction, immune responses, and inflammatory reactions, and to provide this substance as a drug for treating and/or preventing diseases or pathological conditions associated with increased expression (overexpression) of activin A.

As a result of intensive research to solve the above-mentioned problems, the inventors discovered that rice extract, or a mixture of rice extract and purple barley seed extract, significantly suppresses the expression of inhibin βA (INHBA), a subunit of activin A, and thus completed the present invention.

That is, the present invention includes the following inventions.
(1) An inhibin βA (INHBA) expression inhibitor containing a rice extract as an active ingredient.
(2) An inhibin βA (INHBA) expression inhibitor containing a mixture of rice extract and purple barley seed extract.
(3) An inhibin βA (INHBA) expression inhibitor described in (1) or (2), characterized in that the extract is extracted with one or more solvents selected from the group consisting of water, lower alcohols, and hydrocarbons.
(4) An inhibin βA (INHBA) expression inhibitor according to (1) or (2), which is used for the treatment and/or prevention of a disease or pathological condition associated with increased expression of activin A.

The INHBA expression inhibitor of the present invention can significantly suppress intracellular INHBA expression and is therefore effective in treating and/or preventing diseases and pathological conditions associated with increased expression of activin A, an INHBA homodimer.

The inhibin βA (INHBA; hereinafter referred to as "INHBA") expression inhibitor of the present invention contains, as an active ingredient, a rice extract or a mixture of a rice extract and a purple barley seed extract. In the present invention, "inhibition of INHBA expression" refers to the inhibition of INHBA expression at the living organism level or culture level. Furthermore, in the present invention, "inhibition of INHBA expression" refers to the inhibition of INHBA mRNA expression and protein expression.

INHBA and its homodimer, activin A, are proteins belonging to the transforming growth factor-β (TGF-β) family. By controlling the activin A signaling pathway, they regulate many physiological functions, including cell proliferation and differentiation, apoptosis induction, immune responses, and inflammatory reactions. For example, the sequence information for the INHBA gene and protein in humans is registered in GenBank under the numbers Nucleotide NM_002192.4 and Protein NP_002183.1.

The rice used in the present invention is the seed of Oryza sativa L., a species of grass family, and may be brown rice (rice from which the husk has been removed), germinated rice (rice from which brown rice has been germinated), germinated rice (rice from which the bran has been removed and the germ remains), polished rice (rice from which the bran and germ have been removed and only the endosperm remains), or rice bran, germ, or endosperm. There are no particular limitations on the variety, but examples include edible varieties such as Koshihikari, Sasanishiki, and Akitakomachi.

When using brown rice or polished rice, it may be subjected to a pulverization process. For pulverization, for example, a ball mill, roll mill, impact mill, ultracentrifugal mill, airflow mill, or shear friction mill can be used.

The term "purple barley" used in the present invention refers to barley with purple glumes, caryopses, stems, or leaves, and its scientific name is Hordeum vulgare L. Varieties include, but are not limited to, OUC321, CI158, and CI244, which can be obtained from the Wild Barley Resources Research Center, Research Institute for Bioresources, Okayama University.

The extraction method for obtaining rice extracts and purple barley seed extracts is not particularly limited. For example, it may be a heated extraction method, or a room temperature or cold extraction method. Examples of solvents used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), hydrocarbons (hexane, heptane, liquid paraffin, squalane, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water, lower alcohols, and hydrocarbons are preferred, with lower alcohols and hydrocarbons being more preferred, and hydrocarbons being even more preferred. Particularly preferred extraction solvents include ethanol and hexane. These solvents may be used alone or in combination. Furthermore, among the above solvents, the water-soluble organic solvent may be used alone, or a mixed solvent of water and a water-soluble organic solvent (so-called aqueous organic solvent) may be used. The concentration of the organic solvent contained in the aqueous organic solvent is not limited, but for example, in the case of aqueous ethanol (ethanol aqueous solution), it is 10 to 90 v/v%, preferably 30 to 70 v/v%. Furthermore, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent may also be used.

There are no particular limitations on the amount of solvent used; for example, it may be at least 10 times, and preferably at least 20 times, the amount of rice or purple barley seeds (dry weight). However, for convenience in operations such as concentration and isolation after extraction, it is preferable that the amount be no more than 100 times. The extraction temperature and time will depend on the type of solvent used, but examples include temperatures of 10 to 100°C, preferably 30 to 90°C, and times of 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, a rice or purple barley seed extract can be obtained by adding water to dried rice or purple barley seeds and performing hot water extraction at 95 to 100°C. Alternatively, a rice or purple barley seed extract can be obtained by adding a lower alcohol (e.g., ethanol), a liquid polyhydric alcohol (e.g., propylene glycol, 1,3-butylene glycol), and/or a hydrocarbon (hexane, heptane, liquid paraffin, squalane, etc.) to dried rice or purple barley seeds and performing extraction at room temperature (e.g., 15 to 35°C). The extract may be used as is as an extracted solution, or, if necessary, may be subjected to treatments such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, and ethanol precipitation, to an extent that does not affect the extract's effectiveness. Furthermore, the extracted solution may be concentrated to dryness, spray-dried, freeze-dried, or other treatments, and then used as a dried product.

In the present invention, rice extract may be used alone, but is preferably used in combination with purple barley seed extract, as this enhances the inhibitory effect on INHBA expression. When rice extract and purple barley seed extract are used in combination, the mixing ratio is not limited, but is preferably 1:10 to 10:1, more preferably 1:5 to 5:1, even more preferably 1:2 to 2:1, and most preferably 1:1.

Cells targeted for INHBA expression inhibition are not particularly limited as long as they express INHBA and are present in biological tissues. Examples include cells from bone marrow, brain, head and neck, nasopharynx, bone, blood, skin, heart, lung, breast, esophagus, stomach, pancreas, liver, colon/rectum, small intestine, spleen, testes, ovaries, thyroid, bladder, salivary gland, duodenum, endometrium, placenta, prostate, gallbladder, kidney, and lymph node. Furthermore, the cells may be undifferentiated pluripotent stem cells, such as embryonic stem cells (ES cells); somatic stem cells present in bone marrow, blood, skin (epidermis, dermis, and subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidney, muscle, and other tissues; and artificially generated stem cells (induced pluripotent stem cells: iPS cells) by gene transfer or other methods. Cells of mammalian origin, such as human, mouse, rat, hamster, rabbit, dog, cat, pig, cow, horse, and monkey, are preferred. The cells may be in vivo cells or isolated cells. Isolated cells can be maintained and cultured using known techniques.

Rice extract or a mixture of rice extract and purple barley seed extract is effective in treating and/or preventing various diseases or conditions associated with increased expression (overexpression) of activin A due to the inhibitory effect of INHBA expression. Diseases or conditions associated with increased expression of activin A include cancer (colon cancer, melanoma, gastric cancer, hepatocellular carcinoma, prostate cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, kidney cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, head and neck cancer, lymphoma, glioma, glioblastoma, etc.), allergic diseases (hay fever, atopic dermatitis, bronchial asthma, allergic rhinitis (runny nose, nasal congestion, sneezing), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, food allergies), and other conditions. , urticaria, collagen disease, serum sickness, viral hepatitis, contact dermatitis, etc.), autoimmune diseases (rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, type 1 diabetes, polymyositis, systemic sclerosis, dermatomyositis, pernicious anemia, etc.), inflammatory bowel diseases (ulcerative colitis, Crohn's disease, etc.), nervous system diseases accompanied by nerve degeneration or damage (memory loss, dementia, Alzheimer's disease, spinal cord injury, facial nerve paralysis, spinocerebellar degeneration, Huntington's disease (HD), amyotrophic lateral sclerosis, Amyotrophic lateral sclerosis (ALS), polyneuropathy, spinal muscular atrophy, Parkinson's disease, multiple sclerosis (MS), cerebrovascular disorders (cerebral infarction, stroke, cerebral aneurysm), movement disorders due to cerebrovascular disorders, progressive supranuclear palsy, tremors, epilepsy, brain trauma, depression, insomnia, learning disabilities, anxiety disorders (panic disorder, obsessive-compulsive disorder, etc.), muscle diseases (sarcopenia, muscular atrophy, muscular dystrophy, etc.), skin aging and damage (wrinkles, sagging, dullness, loss of firmness and luster, rough skin, dryness) These include, but are not limited to, skin, sensitive skin, hyperkeratosis, enlarged pores, cracks, chapped skin, asteatosis (xeroderma), asteatotic eczema, skin pruritus, hand eczema, psoriasis (accompanied by erythema, scaling, and desquamation), delayed healing of burns and wounds, bedsores, etc.), bone metabolic disorders (osteoporosis, osteomalacia, osteogenesis imperfecta, osteopetrosis, Paget's disease of bone, etc.), and infectious diseases (infections caused by influenza virus, norovirus, herpes virus, AIDS virus, etc.).

The INHBA expression inhibitor of the present invention can be used as is, or can be incorporated into various compositions, such as pharmaceuticals, quasi-drugs, cosmetics, foods and beverages, for inhibiting INHBA expression, together with appropriate additives within a range that does not impair the effects of the present invention, for the purpose of treating and/or preventing various diseases or pathologies associated with the increased expression of activin A. It should be noted that the pharmaceuticals of the present invention also include drugs for use on animals, i.e., veterinary drugs.

The pharmaceuticals of the present invention function as preventive drugs that suppress the onset of the above-mentioned diseases or pathological conditions and/or as therapeutic drugs that improve the condition to a normal state. Because the active ingredients of the pharmaceuticals of the present invention are derived from natural products, they are extremely safe and have no side effects. Therefore, when used as pharmaceuticals for the treatment and/or prevention of the above-mentioned diseases or pathological conditions, they can be administered orally or parenterally in a wide range of dosages to mammals such as humans, mice, rats, rabbits, dogs, and cats.

When the INHBA expression inhibitors of the present invention are incorporated into pharmaceuticals, they can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various dosage forms suitable for application to affected areas. Pharmacologically and pharmaceutically acceptable additives include formulation bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonicity agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, and flavoring agents, selected appropriately depending on the dosage form and intended use. Various dosage forms that can be administered orally or parenterally, systemically, or locally, can be prepared using various known methods. When providing the pharmaceuticals of the present invention in the above forms, they can be manufactured using methods commonly used by those skilled in the art, such as the methods set forth in the General Provisions for Preparations [2], each article of preparations, of the Japanese Pharmacopoeia.

Formulations for oral administration may contain, but are not limited to, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, starch, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

Formulations for parenteral administration may contain, but are not limited to, solvents such as distilled water, saline, ethanol, glycerin, propylene glycol, macrogol, alum water, and vegetable oil; isotonicity agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, and organic bases.

Pharmaceutical forms include, but are not limited to, oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, suppositories, ointments, lotions, eye drops, sprays, transdermal absorption preparations, transmucosal absorption preparations, and patches. They may also be prepared as dried products that are reconstituted for use, and injectable preparations are provided in unit-dose ampoules or multi-dose containers. Suitable forms for use as pharmaceuticals for treating, ameliorating, or preventing skin-related diseases or symptoms associated with increased expression of activin A include topical preparations, such as ointments, creams, gels, liquids, patches (cataplasms and plasters), foams, sprays, and aerosols. Ointments are homogeneous, semi-solid topical preparations, and include oleaginous ointments, emulsion ointments, and water-soluble ointments. Gels are topical preparations in which a water-insoluble component, a hydrated compound, is suspended in an aqueous liquid. Solutions are liquid topical preparations, and include lotions, suspensions, emulsions, liniments, etc.

When the INHBA expression inhibitor of the present invention is incorporated into cosmetics or quasi-drugs, the formulation may be any of an aqueous solution, solubilized solution, emulsion, powder, powder dispersion, oil solution, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, such cosmetics or quasi-drugs can be manufactured according to methods known in the art by appropriately blending various ingredients, additives, bases, etc. commonly used in topical skin compositions together with the INHBA expression inhibitor of the present invention, selected according to their type. The formulation may be any of a liquid, emulsion, cream, gel, paste, spray, etc. Examples of the ingredients include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate ... These include: isopropyl, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

Examples of types of cosmetics and quasi-drugs include lotions, emulsions, gels, serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, powders, and body lotions.

When the extract is incorporated into the above-mentioned pharmaceuticals, quasi-drugs, and cosmetics, its content is not particularly limited, but is preferably 0.001 to 30% by weight (w/w), and more preferably 0.01 to 10% by weight (w/w), calculated as the dry solid content of the extract relative to the total weight of the formulation (composition). At less than 0.001% by weight (w/w), the effect is low, and at more than 30% by weight (w/w), a significant increase in effect is unlikely. Furthermore, the active ingredient may be added in advance or during production, and can be added as appropriate in consideration of workability.

The INHBA expression inhibitor of the present invention can also be incorporated into food and beverage products. In the present invention, the term "food and beverage products" refers not only to general food and beverage products but also to foods other than pharmaceuticals that can be consumed for the purpose of maintaining or improving health, such as health foods, functional foods, foods with health claims, and foods for special dietary uses. Health foods include foods sold under names such as dietary supplements, dietary supplements, and supplements. Health claims are defined by the Food Sanitation Act and the Health Promotion Act and include foods for specified health uses and foods with nutrient claims, which can display specific health benefits, nutritional functions, and disease risk reduction, as well as foods with functional claims, which can display scientifically based functionality reported to the Commissioner of the Consumer Affairs Agency. Special dietary claims also include foods for patients, foods for the elderly, foods for infants, and foods for pregnant and nursing mothers, which display information indicating that the foods are suitable for specific individuals or patients with specific diseases. The specific health benefits and nutritional functions of foods and beverage products can be displayed on product containers, packaging, instructions, package inserts, and other labels, as well as in product flyers, pamphlets, and product advertisements in newspapers and magazines.

The form of the food or drink may be any form suitable for consumption, such as solid, liquid, granules, powder, capsule, cream, or paste. In particular, for the health foods mentioned above, preferred forms include tablets, pills, capsules, powder, granules, fine granules, lozenges, and liquids (including syrup, milk, and suspension).

Types of food and beverages include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock foods, oils and fats and processed oil and fat foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated liquids and powders for adjusting such beverages. Food and beverages may contain commonly used additives as appropriate depending on the type. Any additive permitted under the Food Sanitation Act can be used, including, for example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorings, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

The present invention will be explained in more detail below using examples. However, the present invention is not limited to these examples.

[Example 1] Example of production of rice and purple barley seed extracts Rice (form: rice bran, brown rice, polished rice, variety: "Koshihikari") and purple barley (variety: "OUC321") seeds were used as extraction materials, and extracts thereof were produced by the following method.

(Production Example 1) Production of hot water extract of each extraction material 200 mL of purified water was added to 10 g of dried extract of each extraction material, and the mixture was extracted at 95 to 100°C for 2 hours, followed by filtration. The filtrate was concentrated and freeze-dried to obtain a hot water extract.

(Production Example 2) Production of ethanol extract of each extraction material 200 mL of ethanol was added to 10 g of dried material of each extraction material, and extraction was carried out at room temperature for 7 days, followed by filtration. The filtrate was concentrated and dried to obtain an ethanol extract.

(Production Example 3) Production of hexane extract of each extraction material 200 mL of hexane was added to 10 g of dried material of each extraction material, and extraction was carried out at room temperature for 7 days, followed by filtration. The filtrate was concentrated to dryness to obtain a hexane extract.

The extraction materials, extraction solvents, and amounts of extract used for the extracts obtained in Production Examples 1 to 3 (Extracts 1 to 12) are shown in Table 1 below.

Example 2: INHBA expression inhibitory effect Experiments on the INHBA expression inhibitory effect of rice and purple barley seed extracts were carried out as follows.

In this example, to verify the INHBA-inhibitory effect of each extract, a test was conducted using normal human epidermal keratinocytes (HEK, manufactured by Kurabo Industries, Ltd.).

First, ultraviolet (UVB) irradiation was investigated to generate a model in which INHBA was overexpressed in HEK. Specifically, HEK cells were seeded at 3 x ¹⁰ cells per well in a 96-well plate. After confirming engraftment, the cells were irradiated with ultraviolet (UVB) light at 100 mJ/cm using a Toshiba FL20S-E lamp. ^Two days after UVB irradiation, the cells were fixed with PFA and immunostained with an antibody against INHBA (Proteintech) and a fluorescently labeled secondary antibody. Individual cell nuclei were labeled with DAPI staining. The INHBA expression level per cell (INHBA fluorescence intensity/cell number) was then calculated using a fluorescence microscope and image analysis software (ImageJ). The INHBA expression level in cells cultured without UVB irradiation was defined as 100%, and the relative INHBA expression level in cells cultured under UVB irradiation was calculated. The results are shown in Table 2.

As shown in Table 2, it was found that UVB irradiation increases the expression of INHBA.

Next, an addition test of each extract was conducted. Specifically, as described above, HEK cells were seeded at 3 x ¹⁰ cells per well on a 96-well plate. After confirming engraftment, the cells were irradiated with ultraviolet light (UVB) at 100 mJ/ ^cm2 . Each extract obtained in Example 1 was then added to the cells at a concentration of 100 μg/mL. When a mixture of two extracts was used, the ratio was 1:1 (by weight). Two days after the addition of the extracts, the cells were fixed with PFA, and the INHBA expression level (INHBA fluorescence intensity/cell count) was calculated as described above. The INHBA expression level in cells cultured without the extract (control) was set to 100%, and the relative INHBA expression level in cells cultured with the extract was calculated and evaluated. The results are shown in Table 3.

As shown in Table 3, cells cultured in medium supplemented with extracts (extracts 1-9) of rice (rice bran, brown rice, polished rice) showed reduced INHBA expression compared to the control (no addition), demonstrating the effect of suppressing INHBA expression. It was also confirmed that the combined use of rice extract (extract 6) and purple barley seed extract (extract 12) significantly enhanced the effect of suppressing INHBA expression.

The present invention can be used in the field of manufacturing pharmaceuticals and cosmetics for the purpose of treating and/or preventing diseases associated with increased expression of activin A.

Claims (2)

An inhibin βA (INHBA) expression inhibitor containing a mixture of rice extract and purple barley seed extract, characterized in that the rice extract and purple barley seed extract are extracted with water or hot water, an aqueous ethanol solution, ethanol, or hexane. The inhibin βA (INHBA) expression inhibitor according to claim 1 , which is used for the treatment and/or prevention of a disease or pathological condition associated with increased expression of activin A.