JP7161170B2 - Zinc transporter expression promoter - Google Patents

Description

The present invention relates to zinc transporter expression promoters, zinc uptake promoters into cells, and various compositions for promoting zinc uptake for the purpose of treating or preventing diseases or symptoms associated with zinc deficiency.

Zinc is an important essential trace element that is involved in various biological phenomena such as cell proliferation, differentiation, fertilization, and biological defense. It is thought to be involved in maintaining the conformation of molecules. Therefore, although zinc is only about 0.003% in the human body, its deficiency is wide-ranging, such as taste abnormality, anemia, stomatitis, male dysfunction, immunocompromise, osteoporosis, dermatitis, and hair loss. and cause serious illness and symptoms. Furthermore, it has been pointed out that many patients with liver cirrhosis, diabetes, chronic inflammatory bowel disease, and chronic kidney disease have low serum zinc levels and are zinc deficient (Non-Patent Document 1). In addition, it is believed that Japan is currently in a state of zinc deficiency, particularly among children, the elderly, and young women (Non-Patent Document 2). This is thought to be caused by the fact that food additives with a strong zinc chelating action are often used in processed foods that are often used in Japanese dining tables in recent years, and by excessive dieting. In addition, it has been suggested that the cause of zinc deficiency associated with aging is the decrease in the expression of genes necessary for zinc uptake into cells (Non-Patent Document 3). Against the backdrop of an increase in single-person and double-income households, the dependence on processed foods is expected to increase further in the future. It is estimated that there are 2 billion people with zinc deficiency, mainly in developing countries, on a global scale (Non-Patent Document 4), and large-scale countermeasures are desired as an important public health problem.

As described above, zinc exists in a very small amount in the human body, but its function is very important. Therefore, zinc homeostasis in living organisms is strictly controlled by more than 20 types of zinc transporters. Zinc transporters are broadly classified into two families. The ZIP family (Zrt-, Irt-like protein, SLC39A family) increases the zinc ion concentration in the cytoplasm, while the ZnT family (Zn transporter, SLC30A family) functions to decrease it. These zinc transporters are expressed in different tissues and have different intracellular localizations, and regulate the intracellular zinc concentration as necessary (Non-Patent Document 1). For example, there are transporters such as ZIP1 that are ubiquitously expressed in various tissues in vivo, and transporters that are expressed in a wide range of tissues such as ZIP3, while ZIP4 is expressed in the cell membrane of small intestinal epithelial cells. There are also localized ones. ZIP4 is thought to primarily regulate zinc uptake in food. ZIP2 has also been confirmed to be expressed in the cell membrane of epidermal cells, and is known to be involved in cell differentiation (Non-Patent Document 5). Others, such as ZIP7 and ZIP13, are localized not in the cell membrane but in the Golgi apparatus, which is one of the intracellular organelles (Non-Patent Document 1). Several previous studies have reported that enhanced expression of ZIP leads to zinc uptake into cells and an increase in intracellular zinc levels (Non-Patent Documents 5 to 11). For this reason, materials that enhance the expression of zinc transporters, especially ZIP, which is involved in the uptake of zinc into cells, are being explored for the fundamental treatment and prevention of zinc deficiency (Non-Patent Document 12). ). Furthermore, in recent years, it has become clear that zinc and its transporters are also involved in regulation of proliferation and differentiation of somatic stem cells and pluripotent stem cells (Non-Patent Documents 13 and 14). Therefore, from the viewpoint of application to regenerative medicine, which is expected as next-generation medicine, it is important to elucidate the mechanism that controls the expression of zinc transporters and to search for materials.

Keigai (scientific name: Schizonepeta tenuifolia) is an annual plant belonging to the genus Catnip of the Labiatae family. It is used in Chinese herbal medicine for its sweating, antipyretic, analgesic, and anti-inflammatory effects. A mussel extract, alone or in combination with other plant extracts, is an epidermal cell cathepsin D production inhibitor (Patent Document 1), an aquaporin 3 expression enhancer (Patent Document 2), and a neurite outgrowth inhibitor. (Patent Document 3) and the like have been reported. Mint (scientific name: Mentha arvensis L.var.piperascens) is a perennial herb belonging to the genus Mentha of the Labiatae family. It has a unique aroma and refreshing feeling, and is used in Chinese herbal medicine with antipyretic, sweating, and stomachic effects. Peppermint is also used as a raw material for mint potency (L-menthol crystals) and mint oil, which are also used in insect repellents, deodorants, toothpastes, and candies. Mint extract, alone or in combination with other plant extracts, is an adrenocortical hormone secretion inhibitor (Patent Document 4), a platelet aggregation inhibitor (Patent Document 5), and an in vivo antioxidant (Patent Document 6). , TGR5 activator (Patent Document 7) and the like. However, nothing has been known about the zinc uptake-enhancing effect of extracts of mussels and mint.

Japanese Patent Application Laid-Open No. 2001-322940 Japanese Patent Application Laid-Open No. 2011-32191 Japanese Patent Application Laid-Open No. 2016-204310 JP-A-9-227399 Japanese Patent Application Laid-Open No. 2012-153671 Japanese Patent Application Laid-Open No. 2011-236149 JP 2016-8197 A

Clinical Guidelines for Zinc Deficiency 2016, Japanese Society of Clinical Nutrition Michio Komai, Zinc Nutrition Treatment Vol.6 No.1, 2015 Wong CP et al., J Nutr Biochem., 2013, 24: 353-9. Prasad AS et al., BMJ., 2003, 22; 326: 409-410. Inoue Y et al., J Biol Chem., 2014, 289: 21451-62. Dufner-Beattie J et al., J Biol Chem., 2003, 278: 33474-33481. Wang F et al., Mol Genet., 2004, 13: 563-571. Kim BE et al., J Biol Chem., 2004, 279: 4523-4530. Mao X et al., J Biol Chem., 2007, 282: 6992-7000. Kambe T et al., Mol Cell Biol., 2009, 29: 129-139. Li M et al., Proc Natl Acad Sci., U S A. 2007, 104: 18636-18641. Hashimoto A et al., Biochem J., 2015, 472: 183-93. Ohashi W et al., PLoS Genet., 2016, e1006349. Stefanie Pfaender et al., Neural Plast., 2016, 3760702.

In view of the circumstances described above, the present invention finds a new factor that enhances the expression of the zinc transporter ZIP, which is involved in the uptake of zinc into cells and the improvement of the intracellular zinc level. The aim is to provide a formulation that is effective in treating or preventing symptoms.

As a result of intensive studies to solve the above problems, the present inventors have found that a mussel extract and/or a mint extract promote the expression of the zinc transporter ZIP and increase the intracellular zinc concentration. The present inventors have found that they are effective and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A zinc transporter ZIP expression-promoting agent containing a mussel extract and/or a mint extract as an active ingredient.
(2) An agent for promoting zinc uptake into cells, containing a mussel extract and/or a mint extract as an active ingredient.
(3) A composition for promoting zinc uptake into cells, containing a mussel extract and/or a mint extract.
(4) The composition for promoting zinc uptake into cells according to (3), wherein the composition is a drug, quasi-drug, or cosmetic.
(5) The composition for promoting zinc uptake into cells according to (3), wherein the composition is a food or drink.
(6) The composition for promoting zinc uptake into cells according to any one of (3) to (5), which is used for treating or preventing diseases or symptoms associated with zinc deficiency.

INDUSTRIAL APPLICABILITY According to the present invention, an agent for promoting the expression of the zinc transporter ZIP and an agent for promoting zinc uptake into cells, which contain an extract of mussels and/or an extract of mint as an active ingredient, are provided. The mussel extract and/or the mint extract can promote the expression of the zinc transporter ZIP in cells and promote the uptake of zinc into cells. As a result, the intracellular zinc concentration is increased, which is effective in treating or preventing various diseases or symptoms associated with zinc deficiency. In addition, since the mussel extract and/or the mint extract are natural edible plants, they have no side effects and are highly safe. Therefore, pharmaceuticals, quasi-drugs, cosmetics, and food and drink containing the extract of mussels and/or the extract of mint can be safely taken, ingested, or used.

The present invention will be described in detail below.
A zinc transporter ZIP expression promoter according to the present invention contains a mussel extract and/or a mint extract as an active ingredient.

The mussel used in the present invention refers to Schizonepeta tenuifolia of the Labiatae family, the genus Catnip, which is an annual plant native to China. In the present invention, the extract of the mussel is an extract of the whole plant (whole plant) of the mussel, or a part of the plant such as leaves, stems, flowers, buds, fruits, seeds, roots, etc., or a mixture thereof. However, the part used as the raw material for extraction in the present invention is particularly preferably the spike. For extraction, these plant bodies may be used as they are, or may be subjected to treatments such as drying, pulverization, and shredding.

The mint used in the present invention refers to Mentha arvensis L. var. piperascens, which is a perennial herb originating from China. In the present invention, the mint extract refers to the whole plant of mint (whole plant), or the above-ground parts (leaves, branches, bark, trunk, stem, fruit, seeds, flowers, etc.), plant bodies such as rhizomes. Although it refers to an extract of a part or a mixture thereof, the parts used as raw materials for extraction in the present invention are particularly preferably aerial parts (for example, stems and leaves). For extraction, these plant bodies may be used as they are, or may be subjected to treatments such as drying, pulverization, and shredding.

The method for extracting the mussel extract and/or the mint extract is not particularly limited, and may be, for example, a heating extraction method, a room temperature extraction method, or a cold temperature extraction method. Solvents used for extraction include, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 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.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran , propyl ether, etc.). Among these solvents, water, lower alcohols and liquid polyhydric alcohols are preferred, and water and ethanol are more preferred. These solvents may be used singly or in combination of two or more. For example, 30 to 70 v/v % ethanol aqueous solution may be used. Also, a solvent whose pH is adjusted by adding an acid or an alkali to the extraction solvent can be used.

The amount of the solvent to be used is not particularly limited. It is preferably 100 times or less for convenience of operation in the case of The extraction temperature and time depend on the type of solvent used, but can be exemplified, for example, at 10 to 100° C., preferably 30 to 90° C., for 30 minutes to 24 hours, preferably 1 to 10 hours. More specifically, water is added to flower spikes of mussels and aerial parts of mint, and hot water extraction at 95 to 100° C. is performed to obtain extracts of mussels and mint. Alternatively, a lower alcohol (eg, ethanol, etc.) or a liquid polyhydric alcohol (eg, propylene glycol, 1,3-butylene glycol, etc.) is added to flower spikes of mussels and the aerial part of mint, and the temperature is maintained at room temperature (eg, 5 to 35°C). Extraction of mussels and mint can be obtained.

The extract may be used as an extracted solution, but if necessary, concentration (concentration by organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, activated carbon, etc., within a range that does not affect the effect. It may be used after being subjected to treatments such as decolorization, deodorization, and ethanol precipitation. Furthermore, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

The zinc transporters are ZnT (Zn transporter, SLC30A family) that transports zinc from the cytoplasm to the extracellular and intracellular organelles, and Zip (Zip) that transports zinc from the extracellular and intracellular organelles into the cytoplasm. Zrt-, Irt-like protein, SLC39A family), all of which are transmembrane proteins with multiple transmembrane domains. The zinc transporter whose expression is to be regulated in the present invention refers to the latter Zip transporter (referred to as "zinc transporter ZIP" in the present invention). The zinc transporter ZIPs have so far been identified as ZIP1, ZIP2, ZIP3, ZIP4, ZIP5, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP12, ZIP13, ZIP14. Therefore, the ZIP on which the zinc transporter ZIP expression promoter of the present invention acts may be any one of ZIP1 to ZIP14, or two or more of them, and is not particularly limited. For example, abnormal expression of ZIP4, a zinc transporter that has been identified as a causative gene for acrodermatitis enteropathica, which is a congenital zinc deficiency, and plays a central role in the absorption of dietary zinc, and zinc-dependent diseases or symptoms. ZIP1 to ZIP3 (developmental abnormalities), ZIP10 (deficiency of acquired immunity, hypoplasia of hair follicles and epidermis), ZIP13 (abnormalities in tissues such as skin and bones), etc., which have been clarified to be related to zinc according to the present invention It is preferable as a target for expression control of transporter ZIP.

In addition, the cells that promote the expression of the zinc transporter ZIP are not particularly limited as long as they are cells that express the zinc transporter ZIP on the cell membrane or intracellular organelle membrane and are present in living tissue. For example, epidermal cells, hair matrix cells, epithelial cells of the gastrointestinal tract (esophagus, stomach, small intestine, large intestine), epithelial cells of the respiratory tract (nasal cavity, pharynx, respiratory tract, lung), corneal epithelial cells, lymphocytes, muscle cells, fibroblasts cells and the like. The cells may also be undifferentiated/pluripotent stem cells, embryonic stem cells (ES cells); Somatic stem cells present in liver, pancreas, kidney, muscle, and other tissues; stem cells artificially produced by gene transfer or the like (induced pluripotent stem cells: iPS cells). Cells are preferably derived from mammals such as humans, mice, rats, hamsters, rabbits, dogs, cats, pigs, cows, horses and monkeys. The cells may be in vivo cells or isolated cells. Isolated cells can be maintained and cultured by known techniques.

The mussel extract and/or the mint extract can promote zinc uptake into cells by promoting the expression of the zinc transporter ZIP, and thus can be used as an agent for promoting zinc uptake into cells. . Here, the term “intracellular” refers to the entire inside of the cell and the inside of the cytoplasm (excluding intracellular organelles).

Either one of the mussel extract and the mint extract may be used, but it is preferable to use the two together because the effect of promoting the expression of the zinc transporter ZIP and the effect of promoting zinc uptake into cells are enhanced. When the mussel extract and mint 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, and still more preferably 1 :2 to 2:1, most preferably 1:1.

In addition, the zinc transporter ZIP expression promoter or zinc uptake promoter of the present invention will be used to elucidate the mechanism of zinc transport by zinc transporters and the effects of zinc and zinc transporters on proliferation and differentiation of stem cells. available as a research reagent for

The mussel extract and/or the mint extract treat, ameliorate, or prevent various diseases or symptoms associated with zinc deficiency by promoting the expression of the zinc transporter ZIP and promoting zinc uptake into cells. is effective for Zinc deficiency-related diseases or symptoms are known to include sensory organ disorders, skin disorders, reproductive dysfunction, immune dysfunction, metabolic disorders, and developmental disorders. Specifically, dysgeusia, olfactory abnormality, night blindness (dark adaptation disorder), acral dermatitis enteropathica, around extremities and orifices (eyes, mouth, nostrils, ear canals, vulva, etc.), and around nails dermatitis, delayed wound healing (pressure ulcers, etc.), stomatitis, alopecia, developmental disorders (short stature, poor weight gain, etc.), gonadal dysfunction (underdevelopment of secondary sexual characteristics, oligospermia and ED, infertility, irregular menstruation, etc.), insulin metabolism abnormalities (diabetes), anemia, loss of appetite, diarrhea, osteoporosis, susceptibility to infections due to decreased immunity, and mental disorders (depression), etc., but are not limited to these.

The mussel extract and/or the mint extract can be used as they are, but they can be used together with suitable additives to the extent that they do not impair the effects of the present invention for the above-mentioned diseases or symptoms related to zinc deficiency. It can be provided by blending it in various compositions such as pharmaceuticals, quasi-drugs, cosmetics, food and drink, etc. for promoting zinc uptake into cells for the purpose of treatment or prevention. The drug of the present invention also includes drugs used for animals, that is, veterinary drugs.

When the mussel extract and/or the mint extract are incorporated into pharmaceuticals, they should be mixed with pharmacologically and pharmaceutically acceptable additives and prepared into various formulations suitable for application to the affected area. Can be formulated. Pharmacologically and pharmaceutically acceptable additives include formulation bases and carriers, excipients, diluents, binders, lubricants, and coatings that are appropriately selected according to the dosage form and application. agent, disintegrant or disintegration aid, stabilizer, preservative, preservative, bulking agent, dispersant, wetting agent, buffer, solubilizer or dissolution aid, tonicity agent, pH adjuster, propellant , colorants, sweeteners, corrigents, flavoring agents, etc., may be added as appropriate, and various formulations that can be administered systemically or locally via oral or parenteral administration may be prepared by various known methods. When the drug of the present invention is provided in each of the forms described above, it can be produced by a method commonly used by those skilled in the art, for example, the method shown in the Japanese Pharmacopoeia General Rules for Formulations [2] Each article for formulations.

Formulations for oral administration include, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, microcrystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; carboxymethylcellulose, carboxymethylcellulose calcium, Disintegrants or disintegration aids such as 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 hydroxypropyl methylcellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat; It is not limited to these.

Preparations for parenteral administration include solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum water and vegetable oil; isotonic agents such as glucose, sodium chloride and D-mannitol; , organic acids, inorganic bases or organic bases, and the like may be used, but are not limited to these.

The form of the pharmaceutical of the present invention is not particularly limited, but examples include tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs. oral agents such as agents, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, suppositories, ointments, lotions, eye drops, sprays, oral Examples include parenteral agents such as skin absorbers, transmucosal absorbers, and patches. It may also be a dry product to be reconstituted for use, and in the case of an injectable formulation, provided in unit dose ampules or multi-dose containers. Forms suitable for use as pharmaceuticals for treating, ameliorating, or preventing skin-related diseases or symptoms related to zinc deficiency are external preparations, such as ointments and creams. agents, gels, liquids, patches (plasts, plasters), foams, sprays, aerosols and the like. An ointment refers to a homogeneous semi-solid preparation for external use, including oleaginous ointment, emulsion ointment, and water-soluble ointment. A gel is an external preparation in which a water-insoluble hydrate compound is suspended in an aqueous solution. Liquid preparations refer to liquid preparations for external use, including lotions, suspensions, emulsions, liniments and the like.

The dose of the drug of the present invention can be appropriately determined according to the type of disease, age, sex, body weight, severity of symptoms, etc. of the subject. For example, in the case of oral administration to adults, the daily dose is 0.1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 300 mg as the extract of mussels and/or mint extract. .

When the extract of mussels and/or the extract of mint is blended in cosmetics or quasi-drugs, the dosage form is aqueous solution system, solubilization system, emulsification system, powder system, powder dispersion system, oil liquid system, Any of a gel system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system may be used. In addition, for the cosmetics and quasi-drugs, various ingredients, additives, bases, etc. that are usually used in external skin compositions are selected according to the type, along with the mussel extract and/or mint extract. and can be appropriately blended and manufactured according to techniques known in the art. The form may be liquid, emulsion, cream, gel, paste, spray, or the like. Examples of components of the external composition for skin include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated 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, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid) acids, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant/animal extracts, various surfactants agents, moisturizing agents, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances and the like.

Types of cosmetics and quasi-drugs include lotions, milky lotions, gels, serums, creams, packs, masks, facial cleansers, toilet soaps, foundations, powders, bath agents, body lotions, body shampoos, and hair shampoos. , hair conditioners, hair restorers, and the like.

The content of the mussel extract and/or mint extract in the pharmaceuticals, quasi-drugs, and cosmetics of the present invention is not particularly limited, but the amount of the mussel extract and/or mint extract relative to the total weight of the formulation (composition) It is preferably 0.001 to 30% by weight, more preferably 0.01 to 10% by weight, in terms of the dried mint extract. The above amounts are only examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, the general usage amount, efficacy and effects, and the like. In addition, the method of adding the active ingredient in formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

The mussel extract and/or mint extract can also be added to food and drink. In the present invention, food and drink include general food and drink, as well as foods other than pharmaceuticals that can be ingested for the purpose of maintaining and improving health, such as health foods, functional foods, health functional foods, and special purpose foods. Used in the sense of including. Health foods include foods provided under the names of nutritional supplements, health supplements, supplements, and the like. Foods with health claims are defined by the Food Sanitation Law or the Health Promotion Law. This includes foods with function claims that can display the content notified to the Commissioner of the Consumer Affairs Agency regarding the functionality of the food. Foods for special dietary uses include foods for the sick, foods for the elderly, foods for infants, foods for pregnant women, etc., which are labeled as being suitable for specific subjects or patients with specific diseases. Here, indications such as specific health effects and functions of nutritional ingredients attached to food and drink are indicated on product containers, packaging, instructions, and attached documents, product flyers and pamphlets, newspapers and magazines, etc. can be advertisements for products, etc.

The form of food and drink may be any form suitable for eating, such as solid, liquid, granule, grain, powder, capsule, cream, or paste. In particular, the shape of the above-mentioned health food and the like is, for example, tablet, round, capsule, powder, granule, fine grain, troche, liquid (including syrup, milk, and suspension). etc. are preferred.

Types of food and drink include bread, noodles, confectionery, dairy products, marine and livestock processed foods, oils and fats processed foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks , dairy beverages, etc.), and concentrated stock solutions and powders for preparation of such beverages, but are not limited thereto.

The food or drink of the present invention may contain additives that are commonly used depending on the type thereof. Any additive can be used as long as it is acceptable under the Food Sanitation Act. Examples include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; citric acid and malic acid. , acidulants such as tartaric acid; excipients such as dextrin and starch; binders, diluents, flavoring agents, coloring agents, buffering agents, thickening agents, gelling agents, A turbidity agent, a preservative, etc. are mentioned.

When the food or drink of the present invention is a general food or drink, it can be produced by including a step of adding an extract of mussels and/or an extract of mint in the normal production process of the food or drink. In the case of health foods, the manufacturing method for pharmaceuticals may be used as described above. It can be produced by mixing and molding under pressure with a tableting machine or the like. In addition, other materials (for example, vitamins such as vitamin C, vitamin B ₂ and vitamin B ₆ , minerals such as calcium, dietary fiber, etc.) can be added as necessary.

The amount of the mussel extract and/or the mint extract in the food or drink of the present invention may be any amount as long as it exhibits the effect of promoting zinc uptake into cells. It can be set as appropriate in consideration of the form, efficacy, taste, palatability, cost, etc. of food and drink.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these.

[Example 1]
A mussel, mint extract was prepared as follows.
(Production Example 1) Preparation of hot water extract of mussels 200 g of water was added to 10 g of dried spikes of mussels, and extracted at 90 to 100°C for 2 hours. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 1.5 g of a hot water extract of mussels.

(Production Example 2) Preparation of 50% ethanol extract of mussels 200 g of 50% (v/v) ethanol was added to 20 g of dried spikes of mussels, and the mixture was allowed to stand at normal temperature for one week for extraction. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 0.87 g of a 50% ethanol extract of mussels.

(Production Example 3) Preparation of ethanol extract of mussels 200 g of ethanol was added to 20 g of dried spikes of mussels, and the mixture was allowed to stand at normal temperature for one week for extraction. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 0.14 g of an ethanol extract of mussels.

(Production Example 4) Preparation of hot water extract of mint 200 g of water was added to 10 g of dried aerial part of mint, and extracted at 90 to 100° C. for 2 hours. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 1.3 g of hot water extract of mint.

(Production Example 5) Preparation of 50% Ethanol Extract of Mint 200 g of 50% (v/v) ethanol was added to 20 g of the dried aerial part of mint, allowed to stand at normal temperature, and extracted for 1 week. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 0.65 g of a 50% ethanol extract of mint.

(Production Example 6) Preparation of Ethanol Extract of Mint 200 g of ethanol was added to 20 g of the dried above-ground part of mint, and the mixture was allowed to stand at room temperature and extracted for one week. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 0.33 g of an ethanol extract of mint.

[Example 2]
Using one of the extracts of mussels and mint prepared in Example 1, or a mixture of two extracts, the effect of promoting ZIP gene expression and the effect of promoting zinc uptake into cells was tested as follows. In addition, when using the mixture of 2 types of extracts, the ratio was made into 1:1 (weight ratio).

(Experimental example 1) Evaluation of various ZIP gene expression promoting effects Commercially available normal human epidermal keratinocytes (manufactured by Kurabo Industries) were seeded in 12-well plates at 1 x 10 ⁴ cells each, and Keratinocyte-SFM (manufactured by Thermo Fisher Scientific) was used. Cultured for 4 days. At that time, the various extracts prepared in Example 1 (the extracts of Production Examples 1 to 6 and an equal mixture of the extracts of Production Examples 1 and 4) were added to a final concentration of 0.01%. After culturing, the cells were collected, washed twice with PBS(-), and RNA was extracted from the cells with Trizol Reagent (Invitrogen). After reverse transcription of the extracted RNA into cDNA using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles), and confirmed gene expression of ZIP1, ZIP2, and ZIP3. Other operations followed the prescribed method.

[Primer set for ZIP1 gene]
Forward primer: 5'- GCGCCTACCCTCATACCTAT-3' (SEQ ID NO: 1)
Reverse primer: 5'- GAGGCCAGAGAAGATACCAAA-3' (SEQ ID NO: 2)
[Primer set for ZIP2 gene]
Forward primer: 5'- GGTCATCACCGGCGAGTC-3' (SEQ ID NO: 3)
Reverse primer: 5'-TCCAGGGCTTCAGCAGTCATA-3' (SEQ ID NO: 4)
[Primer set for ZIP3 gene]
Forward primer: 5'-CGGGAGTTGCTGGACTGAGA-3' (SEQ ID NO: 5)
Reverse primer: 5'-CCAAGTCCCACGATGTGCTAC-3' (SEQ ID NO: 6)
[Internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene primer set]
Forward primer: 5'-CCGTGTTCCTACCCCCAAT-3' (SEQ ID NO: 7)
Reverse primer: 5'-TGCCTGCTTCACCACCTTCT-3' (SEQ ID NO: 8)

The effect of promoting ZIP gene expression was calculated as the ratio of the ZIP gene expression level to the internal standard GAPDH gene expression level (ZIP gene expression level/GAPDH gene expression level) in epidermal keratinocytes of the sample-free group. ) was defined as 1, and the same relative expression level of each ZIP gene in the epidermal keratinocytes of the sample addition group was calculated and evaluated. The results of these tests are shown in Table 1 below.

As shown in Table 1, all of the mussel extract, the mint extract, and the mixture of the mussel and mint extract were found to have a remarkable ZIP gene expression promoting effect. In particular, the effect was more remarkable when the extracts of mussels and mint were mixed.

(Experimental Example 2) Evaluation of Effect of Accelerating Zinc Uptake Commercially available normal human epidermal keratinocytes (manufactured by Kurabo Industries) were seeded in 5×10 ⁴ cells each in a 6 cm dish, and treated with Keratinocyte-SFM (manufactured by Thermo Fisher Scientific) for 4 days. cultured. At that time, the various extracts prepared in Example 1 (the extracts of Production Examples 1 to 6 and an equal mixture of the extracts of Production Examples 1 and 4) were added to a final concentration of 0.01%. After culturing, the cells were collected by trypsinization and reacted in PBS(-) with a fluorescent indicator for zinc detection, FluoZin-3 (maximum excitation wavelength λex: 494 nm, maximum fluorescence wavelength λem: 516 nm, manufactured by Molecular Probes) for 1 hour. (125 nM concentration). After the reaction, the cells were washed with PBS(-) to remove unreacted indicator, and finally suspended in PBS(-). Next, the cell suspension was analyzed using a flow cytometer (FACS Aria: manufactured by BD) to determine the percentage of FluoZin-3 positive cells. Here, "FluoZin-3-positive cells" means that the result of staining cells with a fluorescent indicator FluoZin-3 is that the intensity of autofluorescence exhibited by cells unstained with the indicator is set as an indicator, and the fluorescence intensity is higher than the reference value. refers to cells that exhibit The results of these tests are shown in Table 2 below.

As shown in Table 2, all of the mussel extract, the mint extract, and the mixture of the mussel and mint extract were found to have a remarkable effect of promoting zinc uptake into cells. In particular, the effect was more remarkable when the extracts of mussels and mint were mixed.

INDUSTRIAL APPLICABILITY The present invention can be used in the field of manufacturing pharmaceuticals, quasi-drugs, cosmetics, and foods and beverages for the treatment or prevention of diseases or symptoms associated with zinc deficiency.

Claims (6)

A zinc transporter ZIP expression promoter containing, as active ingredients, a mixture of a hot water extract of flower spikes of mussels and a hot water extract of aerial parts of mint . An agent for promoting zinc uptake into cells containing, as active ingredients, a mixture of a hot water extract of flower spikes of mussels and a hot water extract of aerial parts of mint . A composition for promoting zinc uptake into cells, containing a mixture of a hot water extract of flower spikes of mussels and a hot water extract of aerial parts of mint . 4. The composition for promoting zinc uptake into cells according to claim 3, wherein the composition is a drug, a quasi-drug, or a cosmetic. The composition for promoting zinc uptake into cells according to claim 3, wherein the composition is a food or drink. The composition for promoting zinc uptake into cells according to any one of claims 3 to 5, which is used for treatment or prevention of diseases or symptoms associated with zinc deficiency.