Composition for inducing heme oxygenase-1 and its use

Abstract

To provide a composition for inducing heme oxygenase-1 capable of inducing expression of heme oxygenase-1, a cytoprotective composition capable of protecting a cell from cellular injury, a composition for protecting organs capable of protecting organs from organ damage and a composition for the treatment, amelioration or prevention of heat stroke capable of treating, ameliorating or preventing heat stroke.SOLUTION: There is provide a composition for inducing heme oxygenase-1 which comprises at least a soybean protein and an extract of fermented soybean and rice bran obtained by fermenting rice bran.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition for inducing heme oxygenase-1, and a composition for protecting cells, a composition for protecting organs, and a composition for treating, ameliorating, or preventing heat stroke, each of which contains the composition for inducing heme oxygenase-1. [Background technology]

[0002] Acute organ failure, such as liver failure, renal failure, and multiple organ failure due to sepsis, is an inflammatory disease that has a high mortality rate. However, a decisive drug for treating it has not yet been developed. Cell damage caused by reactive oxygen species generated by oxidative stress is involved in the tissue damage of these acute organ failures.

[0003] On the other hand, to avoid this invasion, heme oxygenase-1 (hereinafter sometimes referred to as "HO-1") is induced in cells. Heme oxygenase-1 breaks down heme into biliverdin, carbon monoxide, and iron, the former of which is reduced to bilirubin. Bilirubin has an antioxidant effect, carbon monoxide has signal transduction and anti-inflammatory effects, iron has a ferritin-inducing effect, and heme metabolites produced by heme oxygenase-1 have a variety of physiological effects, including organ protection.

[0004] It has been shown that symptoms worsen in models of liver injury caused by carbon tetrachloride or kidney injury caused by ischemia-reperfusion injury when treated with a heme oxygenase-1 inhibitor (see Non-Patent Documents 1 and 2). In a sepsis model caused by lipopolysaccharide (LPS) administration, it has been confirmed that damage caused by oxidative stress is alleviated in areas where heme oxygenase-1 is induced, compared to areas where it is not induced (see Non-Patent Document 3). The inhibitory effect of heme oxygenase-1 on gastric ulcer formation has also been shown, and the effectiveness of heme oxygenase-1 induction has been shown in models of gastric ulcer induced by NSAIDs, ethanol, hydrochloric acid, etc. (see Non-Patent Documents 4 and 5).

[0005] Furthermore, heme oxygenase-1 has been shown to have a preventive effect against heat stress injury, and it has been reported that heme oxygenase-1 plays an important role in protecting against heat-induced brain and nerve damage (see Non-Patent Document 6). For these reasons, induction of heme oxygenase-1 is expected to have a mitigating effect on the prevention of heatstroke.

[0006] As described above, heme oxygenase-1 plays an important role, and inducing the expression of this heme oxygenase-1 is highly effective in protecting cells and organs, and in treating, ameliorating, or preventing heatstroke, but no such composition has been obtained to date.

[0007] Therefore, there is a strong demand for the development of a composition that can induce the expression of heme oxygenase-1 and exert more effective effects in cell protection, organ protection, and the treatment, amelioration, or prevention of heat stroke. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Nakahira K et al., Biochem.Pharmacol.66(6):1091-1105, 2003 [Non-Patent Document 2] Toda N et al., Crit.Care.Med.30(7):1512-1522, 2002 [Non-Patent Document 3] Fujii H et al., Crit. Care. Med. 31(3):893-902, 2003 [Non-Patent Document 4] Aliye Uc et al., J.Pediatr.Gastroenterol.Nutr.54(4):471-476, 2012 [Non-Patent Document 5] Mohamed SA et al., PloS One 14(6):e0216737, 2019 [Non-Patent Document 6] Ya-Ting W et al., Int. J. Med. Sci. 12(9):737-741, 2015 Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the current situation, the present invention aims to achieve the following objectives: That is, the present invention aims to provide a composition for inducing heme oxygenase-1 capable of inducing expression of heme oxygenase-1, a composition for protecting cells from cell damage, a composition for protecting organs from organ damage, and a composition for treating, ameliorating, or preventing heat stroke capable of treating, ameliorating, or preventing heat stroke. [Means for solving the problem]

[0010] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they found that an extract of fermented soybean and rice bran obtained by fermenting at least soybean protein and rice bran (hereinafter, sometimes referred to as "OE-1") has activity to induce the expression of heme oxygenase-1. They also confirmed that the OE-1 suppresses the production of inflammatory cytokines from macrophage cell lines, improves cell damage caused by heat or oxidative stress, and further suppresses the formation of gastric ulcers caused by ethanol, leading to the completion of the present invention.

[0011] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> The composition for inducing heme oxygenase-1 is characterized by containing at least an extract of soybean and rice bran fermentation product obtained by fermenting soybean protein and rice bran. <2> The above <1> The present invention relates to a composition for protecting cells, the composition comprising the heme oxygenase-1 induction composition described above. <3> The above <1> The present invention relates to a composition for protecting organs, which comprises the composition for inducing heme oxygenase 1 described above. <4> The above <1> The present invention relates to a composition for treating, ameliorating, or preventing heatstroke, comprising the composition for inducing heme oxygenase-1 described above. <5> The above-mentioned food, beverage or pharmaceutical products <1> from <4> The composition according to any one of claims 1 to 5, Effect of the Invention

[0012] According to the present invention, it is possible to solve the above-mentioned problems in the conventional art and achieve the above-mentioned object, and to provide a composition for inducing heme oxygenase-1 capable of inducing expression of heme oxygenase-1, a composition for protecting cells from cell damage, a composition for protecting organs from organ damage, and a composition for treating, ameliorating, or preventing heat stroke capable of treating, ameliorating, or preventing heat stroke. [Brief description of the drawings]

[0013]

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[0014] (Composition for inducing heme oxygenase-1) The composition for inducing heme oxygenase-1 of the present invention contains at least an extract of fermented soybean and rice bran obtained by fermenting soybean protein and rice bran, and further contains other components as necessary. The composition for inducing heme oxygenase-1 has an activity of inducing the production of heme oxygenase-1 in cells.

[0015] <Extract of fermented soybean and rice bran> The extract of the fermented soybean and rice bran is an extract of the fermented soybean and rice bran obtained by fermenting at least soybean protein and rice bran.

[0016] <<Fermented soybeans and rice bran>> The soybean and rice bran fermented product is a fermented product obtained by fermenting at least soybean protein and rice bran. The soybean and rice bran fermented product may be a fermented product obtained by fermenting a composition containing soybean protein, rice bran, and other components as necessary, or a fermented product obtained by mixing fermented products obtained by separately fermenting soybean protein, rice bran, and other components as necessary. Also, as long as it contains at least fermented soybean protein and rice bran, it may contain unfermented components.

[0017] -Soy protein- The soybean protein is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include isolated soybean protein, concentrated soybean protein, defatted soy milk, defatted soybeans, etc. These may be used alone or in combination of two or more kinds. The soybean protein may be appropriately prepared or a commercially available product, such as New Fujipro SE, New Fujipro 1700, Fujipro E (all powdered soybean proteins, manufactured by Fuji Oil Co., Ltd.), New Fujinic P50, Apex 600 (all granular soybean proteins, manufactured by Fuji Oil Co., Ltd.), etc.

[0018] The soy protein may be a solid fraction of ground soybeans using soybeans or their related species as a raw material. The solid fraction of ground soybeans may be, for example, okara, which is a by-product in the process of producing tofu, and the type of soybeans as a raw material and the production conditions are not particularly limited. The solid fraction may be the one filtered in the production process or may be the one dried.

[0019] The composition of the soybean solid fraction is not particularly limited and can be appropriately selected depending on the purpose, but the dry matter contents are preferably 20% to 30% by mass of crude protein, 10% to 15% by mass of crude fat, 25% to 35% by mass of soluble nitrogen-free matter, and 10% to 20% by mass of crude fiber, and the moisture content in the solid fraction is preferably 75% to 80% by mass.

[0020] -Rice bran- The rice bran is not particularly limited as long as it contains at least one of rice bran, defatted rice bran, rice germ, and defatted rice germ, and can be appropriately selected depending on the purpose, and for example, ordinary rice bran containing rice pericarp, seed coat, aleurone layer, germ, etc., which are removed in the process of polishing brown rice to white rice, can be used as it is. These may be used alone or in combination of two or more types. The rice bran may be appropriately prepared or a commercially available product may be used. Examples of the commercially available product include Oryza Germ-DLP, Oryza Germ-DLS, Oryza Dorim, Defatted Rice Bran (all of which are defatted rice bran, manufactured by Oryza Oil & Fat Chemical Co., Ltd.), Defatted Bran (manufactured by Tsuno Foods Industry Co., Ltd.), etc.

[0021] The mass ratio of the soybean protein to the rice bran (soybean protein / rice bran) is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1 / 5 to 10 / 1, more preferably 1 / 2 to 5 / 1.

[0022] -Other ingredients- The other components in the soybean and rice bran fermented product are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include tea catechins, anthocyanins, rutin, hesperidin, quercetin, isoflavones, tannins, chlorogenic acid, ellagic acid, curcumin, lignans, saponins, dietary fiber, vitamins, etc. These may be used alone or in combination of two or more. The other ingredients may be added before or after fermentation.

[0023] The mass ratio of the other components to the soybean protein and rice bran (other components / soybean protein and rice bran) is not particularly limited and can be appropriately selected depending on the purpose.

[0024] -fermentation- The method for fermenting the soybean protein and the rice bran is not particularly limited and can be appropriately selected depending on the purpose. However, from the viewpoints of food and drink applications, nutrients contained in the bacteria themselves, fermentation aroma, and the like, it is preferable to ferment the soybean protein and the rice bran using at least one of Bacillus subtilis natto, Bacillus subtilis Tempeh, Lactic acid bacteria, and yeast. As the natto bacteria, tempeh bacteria, lactic acid bacteria, and yeast bacteria, mutant strains having altered bacteriological properties produced naturally or by artificial mutation means can also be used as long as their safety is guaranteed.

[0025] The above-mentioned natto bacteria ( Bacillus subtilis var. natto The natto bacteria are not particularly limited, and commercially available general natto bacteria can be used, for example, they can be obtained from Naruse Fermentation Chemistry Research Institute Co., Ltd. The natto bacteria may be used alone or in combination of two or more kinds.

[0026] The tempeh fungus is not particularly limited and can be appropriately selected depending on the purpose. For example, Rhizopus oligosporus , Rhizopus oryzae , Rhizopus stolonifer Among these, from the viewpoint of ease of fermentation, Rhizopus oligosporus These tempeh fungi are easily available as imported products from Indonesia or from Japanese koji manufacturers. The tempeh fungi may be used alone or in combination of two or more kinds.

[0027] The lactic acid bacteria is not particularly limited and can be appropriately selected depending on the purpose. For example, Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus bifidus ( Lactobacillus bifidus ), Streptococcus faecalis ( Streptococcus faecalis ), Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), Lactobacillus san francisco (Lactobacillus sanfrancisco ), Lactobacillus casei ( Lactobacillus casei ), Streptomyces lactis ( Streptomyces lactis These may be used alone or in combination of two or more. All of these lactic acid bacteria are known and easily available.

[0028] The yeast fungus is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include Saccharomyces, Schizosaccharomyces, Candida, and Kluyveromyces. Among these, yeasts of the Saccharomyces genus are preferred from the viewpoint of food and beverage applications, and sake yeast and brewer's yeast are particularly preferred. These yeast fungus can be obtained, for example, from the Brewing Society of Japan. The yeast fungus may be used alone or in combination of two or more kinds.

[0029] The amount of the bacteria to be inoculated is not particularly limited as long as the bacteria can grow, and can be appropriately selected depending on the type of bacteria. When the fermentation target is a liquid, the amount is usually 1×10 3 pieces / mL~1×10 8 When the fermentation target is a solid, the average is usually 1 × 10 3 pieces / g~1×10 8 The inoculation amount is 1×10 3 / mL or 1 x 10 3 If the number of bacteria is less than 1×10 / g, fermentation by bacteria may take a long time. 8 / mL or 1 x 10 8 If the number exceeds 100 / g, bacterial growth may be inhibited and fermentation may not proceed.

[0030] The fermentation conditions, such as fermentation temperature, fermentation time, fermentation type, pH, aeration conditions, etc., can be appropriately determined, but it is preferable to select conditions suitable for the characteristics of the growth, etc. of the bacteria used.

[0031] The fermentation temperature is not particularly limited as long as the fermentation by the bacteria proceeds, and can be appropriately selected depending on the type of bacteria used. The temperature is usually 10°C to 55°C, preferably 20°C to 50°C, and more preferably 25°C to 45°C.

[0032] The fermentation time is not particularly limited as long as the bacteria can grow, and can be appropriately selected depending on the type of bacteria. The fermentation time is usually 1 hour to 5 days, preferably 3 hours to 3 days, and more preferably 6 hours to 2 days.

[0033] The subject of the fermentation may be a solid or a liquid, and may be appropriately aerated. When the subject of the fermentation is a liquid, the fermentation may be performed while shaking or stirring, or may be performed while standing still, but shaking or stirring is preferable from the viewpoint of promoting the growth of the bacteria and promoting the fermentation. The shaking conditions are not particularly limited as long as the mixture is thoroughly stirred.

[0034] The pH during the fermentation is not particularly limited as long as the bacteria can grow, and can be appropriately selected depending on the purpose, but is usually 4.5 to 8.5, and preferably 5.5 to 7.5. The pH may be adjusted using hydrochloric acid or sodium hydroxide for pH adjustment, or a pH adjusting buffer may be used.

[0035] The fermented soybean and rice bran product is preferably a protease-treated product obtained by treating the fermented product with a protease after fermentation. The protease treatment is also referred to herein as "protease digestion" or "proteolysis", and is intended to degrade proteins or peptides derived from the fermentation product.

[0036] The protease is a general term for peptide bond hydrolases, and is also called protein decomposition enzymes. The protease is not particularly limited as long as it can decompose the protein or peptide contained in the fermentation product, and a proteinase that hydrolyzes peptide bonds in protein molecules or a peptidase that hydrolyzes peptide bonds at the amino or carboxy terminus of a peptide chain may be used. The proteases are classified into acidic proteases, neutral proteases, and alkaline proteases according to their optimum pH, and among these, neutral proteases are preferred because they have high enzyme processing efficiency and can produce products with high functionality. Specific examples of the proteases include metal proteases (such as thermolysin), serine proteases (such as trypsin, chymotrypsin, thrombin, plasmin, elastase, subtilisin), thiol proteases (such as papain, ficin, bromelain, cathepsin B), aspartic acid proteases (such as pepsin, chymosin, cathepsin D), aminopeptidases, and carboxypeptidases. The proteases may be used alone or in combination of two or more kinds. Among the above proteases, those containing serine protease and aminopeptidase are preferred, since they have high enzymatic processing efficiency and can give products with high functionality. These proteases may be purified or unpurified, and may be derived from any of microorganisms, plants, and animals.

[0037] The protease may be a commercially available product. Examples of the commercially available product include Denatzyme AP ( Aspergillus oryzae Neutral protease (manufactured by Nagase ChemteX Corporation), Protease N "Amano" G, Protin SD-NY10, Protin SD-PC10F (all of the above, Bacillus subtilis Neutral protease, Amano Enzyme Co., Ltd.), Protease A "Amano" G ( Aspergillus oryzae Neutral protease, Amano Enzyme Co., Ltd.), Protease S "Amano" G ( Bacillus stearothermophilus Papain W-40 (neutral protease derived from papaya latex, manufactured by Amano Enzyme Co., Ltd.), Bromelain F ( Ananas comosus M Neutral protease derived from porcine pancreas, manufactured by Amano Enzyme Co., Ltd.), PTN (neutral protease derived from porcine pancreas, manufactured by Novozymes Japan Co., Ltd.), Orientase 90N, Nucleisin, Orientase 10NL (all of the above, Bacillus subtilis Neutral protease (manufactured by HIV I Co., Ltd.), orientase ONS ( Aspergillus oryzae Examples of such protease include neutral protease derived from yeast (manufactured by HIV I, Inc.).

[0038] The treatment conditions for the protease treatment, for example, the amount of protease relative to the fermentation product, treatment temperature, treatment time, pH, etc., are not particularly limited and can be appropriately selected depending on the purpose.

[0039] The mass ratio of the protease to the solid content of the fermentation product (protease / solid content of the fermentation product) is not particularly limited and can be appropriately selected depending on the purpose, but is usually 1 / 500 to 1 / 10, and preferably 1 / 200 to 1 / 20. If the mass ratio is less than 1 / 500, the protein decomposition reaction (protease digestion) may be insufficient or the reaction may require a long time, and if it exceeds 1 / 10, it is economically undesirable.

[0040] The temperature for the protease treatment should be determined taking into consideration the optimum temperature of the protease used, and is usually 15°C to 70°C, preferably 40°C to 65°C.

[0041] The time for the protease treatment is not particularly limited and may be appropriately determined depending on the purpose, but from the viewpoints of reactivity and prevention of contamination with foreign bacteria, it is usually 10 minutes to 2 days, and preferably 3 hours to 24 hours.

[0042] The pH of the protease treatment can be determined in consideration of the optimum pH of the protease used, and is usually 3 to 10. When the neutral protease is used, the pH is preferably 4.5 to 8.0, more preferably 5 to 7.5. The pH may be adjusted using hydrochloric acid or sodium hydroxide for pH adjustment, or a pH adjusting buffer may be used.

[0043] In the protease treatment, in addition to the fermentation product, other components may be included as necessary. The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include defatted rice bran, rice germ, tea catechin, etc. These may be used alone or in combination of two or more.

[0044] <<Extract>> The solvent used for the extraction is not particularly limited, and any known solvent can be used, such as water, alcohols such as methanol, ethanol, butanol, acetone, hexane, mixtures thereof (aqueous alcohol solution, etc.), etc. Among these, water, ethanol, or mixtures thereof are preferred in that they have no adverse effects on the human body, and hot water or an aqueous ethanol solution is more preferred in that active substances can be efficiently extracted, with hot water being particularly preferred. The hot water refers to water having a temperature of 70° C. or higher, and the water temperature is preferably 80° C. to 100° C., and more preferably 90° C. to 100° C., from the viewpoint of extraction efficiency. The concentration of the aqueous alcohol solution is preferably 80% by volume or more and less than 100% by volume, and more preferably 90% by volume or more and less than 100% by volume.

[0045] The extraction method is not particularly limited and may be any known method, for example, a method in which either hot water or ethanol is added to the fermented soybean and rice bran product, the mixture is stirred to extract, and then the mixture is centrifuged for solid-liquid separation. Examples of the centrifuge include a decanter continuous horizontal centrifuge and an automatic basket centrifuge, and these may be used in combination.

[0046] The extract of the fermented soybean and rice bran may be one which has been subjected to filtration treatment, if necessary, after extraction. The filtration method is not particularly limited, and a known filtration device can be used, such as a filter press, a line filter, etc. These may be used in combination.

[0047] The extract of the fermented soybean and rice bran may be concentrated or diluted as necessary, and may be subjected to a drying treatment such as freeze-drying or heat drying before use. The methods for the concentration, dilution, and drying treatment are not particularly limited, and any known method can be appropriately selected.

[0048] The content of the extract of the fermented soybean and rice bran in the composition for inducing heme oxygenase-1 is not particularly limited and can be appropriately selected depending on the purpose. The composition for inducing heme oxygenase-1 may consist only of the extract of the fermented soybean and rice bran.

[0049] <Other ingredients> Other components in the composition for inducing heme oxygenase-1 are not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include catechin, anthocyanin, rutin, hesperidin, quercetin, isoflavone, tannin, chlorogenic acid, ellagic acid, curcumin, lignan, saponin, dietary fiber, vitamins, and excipients, bulking agents, disintegrants, lubricants, binders, antioxidants, colorants, aggregation inhibitors, absorption promoters, solvents, solubilizers, isotonicity agents, stabilizers, flavorings, preservatives, pH adjusters, etc. These may be used alone or in combination of two or more.

[0050] The contents of other components in the composition for inducing heme oxygenase-1 are not particularly limited and may be appropriately selected depending on the purpose.

[0051] The form of the composition for inducing heme oxygenase-1 is not particularly limited and can be appropriately selected depending on the purpose. Examples of the form include a solution, a suspension, a semi-solid (e.g., a paste), a solid (e.g., a powder, a granule, etc.), etc.

[0052] As shown in the test examples described below, the composition for inducing heme oxygenase-1 of the present invention can induce the expression of heme oxygenase-1, thereby exhibiting an anti-inflammatory effect. Therefore, the composition for inducing heme oxygenase-1 of the present invention can be suitably used as a composition for protecting cells, organs, and for treating, ameliorating, or preventing heat stroke.

[0053] (Composition for cell protection, composition for organ protection, composition for treating, improving, or preventing heat stroke) The cell protection composition of the present invention contains at least the heme oxygenase-1 inducing composition of the present invention, and further contains other components as necessary. The cell protection composition is preferably for cell damage caused by at least one of heat and oxidative stress. In this specification, cell protection refers to protecting cells from cell damage, and more specifically, for example, refers to improving cell viability.

[0054] The organ-protecting composition of the present invention (sometimes referred to as "organ-protecting composition") contains at least the heme oxygenase-1-inducing composition of the present invention, and may further contain other components as necessary. The composition for organ protection is preferably for organ damage caused by acute inflammation. In this specification, organ protection refers to protecting an organ from organ damage, and more specifically, it refers to treating, improving, or preventing damage such as gastric ulcers that occur in an organ such as the gastric mucosa.

[0055] The composition for treating, ameliorating, or preventing heatstroke of the present invention contains at least the composition for inducing heme oxygenase-1 of the present invention, and further contains other ingredients as necessary. In this specification, heat stroke refers to a general term for health disorders including heat syncope, heat cramps, heat exhaustion, heat stroke, as well as fainting (heat syncope), shock, or impaired consciousness (heat stroke) caused by an increase in body temperature due to a hot bath (Guidelines for the Diagnosis and Treatment of Syncope, pp. 37-39 (2012 revised edition, Japanese Circulation Society)).

[0056] As used herein, treatment refers to alleviating or ameliorating a disease or condition, preventing or slowing the worsening of a disease or condition, or preventing, slowing, or reversing the progression of a disease or condition. As used herein, amelioration refers to alleviating or improving a disturbed condition, preventing or slowing the worsening of a disturbed condition, or preventing, slowing, or reversing the progression of a disturbed condition. As used herein, prevention refers to preventing or delaying the onset of a disorder, or reducing the risk of onset of a disorder.

[0057] <Composition for inducing heme oxygenase-1> The composition for inducing heme oxygenase-1 in the composition for protecting cells, the composition for protecting organs, and the composition for treating, ameliorating, or preventing heatstroke is the composition for inducing heme oxygenase-1 of the present invention described above.

[0058] The content of the heme oxygenase-1 inducing composition in the composition for protecting cells, the composition for protecting organs, and the composition for treating, ameliorating, or preventing heat stroke is not particularly limited and can be appropriately selected depending on the purpose. The composition for protecting cells, the composition for protecting organs, and the composition for treating, ameliorating, or preventing heat stroke may consist of only the heme oxygenase-1 inducing composition.

[0059] <Other ingredients> The other components in the cell protection composition, organ protection composition, and composition for treating, ameliorating, or preventing heatstroke are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include the same as the other components in the above-mentioned heme oxygenase-1 induction composition.

[0060] The content of other components in the cell protection composition, organ protection composition, and composition for treating, ameliorating, or preventing heatstroke in the cell protection composition, organ protection composition, and composition for treating, ameliorating, or preventing heatstroke is not particularly limited and can be selected appropriately depending on the purpose.

[0061] The form of the cell protection composition, organ protection composition, and composition for treating, ameliorating, or preventing heatstroke is not particularly limited and can be appropriately selected depending on the purpose, and examples include a solution, suspension, semi-solid (e.g., paste, etc.), solid (e.g., powder, granules, etc.), etc.

[0062] The above-mentioned compositions for inducing heme oxygenase-1, compositions for protecting cells, compositions for protecting organs, and compositions for treating, ameliorating, or preventing heatstroke of the present invention are highly safe and can therefore be suitably used as foods, beverages, or pharmaceuticals.

[0063] <Food and drink> The food and drink are not particularly limited and can be appropriately selected depending on the purpose. For example, the composition for inducing heme oxygenase-1, the composition for protecting cells, the composition for protecting organs, or the composition for treating, ameliorating, or preventing heat stroke of the present invention may be used as it is, or in the form of pellets, powder, granules, or the like, or may be used as a food additive, seasoning, or sprinkle. The composition for inducing heme oxygenase-1, the composition for protecting cells, the composition for protecting organs, or the composition for treating, ameliorating, or preventing heat stroke of the present invention may be used by being contained in food ingredients. The food and drink can also be used as health foods, functional foods, foods with nutritional claims, foods for specified health uses, foods for the sick, and the like.

[0064] <Drugs> The pharmaceutical agent may be administered orally or parenterally. The dosage form of the pharmaceutical is not particularly limited and can be appropriately selected depending on the purpose. For example, for oral administration, tablets (including sugar-coated tablets), pills, capsules, powders, fine granules, granules, troches, chewable tablets, drops, emulsions, solutions, suspensions, syrups, etc. can be mentioned, and for parenteral administration, emulsion forms for intravenous administration, suppositories for rectal administration, etc. can be mentioned. Preparations in these dosage forms can be produced by known methods.

[0065] The subjects for which the heme oxygenase-1 inducing composition, cell protecting composition, organ protecting composition, and heat stroke treatment, amelioration, or prevention composition are used are not particularly limited and can be appropriately selected depending on the purpose, and examples include humans, cows, pigs, chickens, dogs, cats, rabbits, squirrels, and hamsters.

[0066] The amount, duration, and timing of use of the composition for inducing heme oxygenase-1, the composition for protecting cells, the composition for protecting organs, and the composition for treating, ameliorating, or preventing heatstroke are not particularly limited and can be selected appropriately depending on the subject and the condition of the subject. EXAMPLES

[0067] The present invention will be specifically explained below by giving Production Examples and Test Examples, but the present invention is not limited to these Production Examples and Test Examples.

[0068] (Production Example 1: Production of extract of fermented soybean and rice bran) <Step 1: Preparation of fermented soybean solid fraction> Soybeans were soaked in water for 17 hours to allow them to fully absorb water, and then ground together with water using a bean grinder (Supermass Colloider, manufactured by Masuko Sangyo Co., Ltd.). The suspension of ground soybeans (hereinafter sometimes referred to as "ground soybean suspension") was heated at 100°C for 20 minutes with thorough stirring. After heating, the liquid phase was removed from the ground soybean suspension using a squeezer (KM-1000, manufactured by Minami Sangyo Co., Ltd.) and the solid phase (soybean solid fraction) was collected. At this time, the liquid phase was removed by gravity. 100 kg of the obtained soybean solid fraction was moderately heated and thoroughly stirred, and then 0.6 L (cell count 1.0 x 10) of natto bacteria (obtained from Naruse Fermentation Chemistry Research Institute) was added. 10 The soybean solid fraction inoculated with Bacillus subtilis natto was transferred to a stainless steel container or a polyethylene bag, and fermented for 18 hours in a constant temperature incubator or room at 40°C while ensuring ventilation. The obtained fermented soybean solid fraction was stored frozen until use.

[0069] <Step 2: Preparation of soybean and rice bran fermented liquid> The raw materials used were soy protein (New Fujipro SE, manufactured by Fuji Oil Co., Ltd.), defatted rice bran, and rice germ (all manufactured by Oryza Oil & Fat Chemical Co., Ltd.). 3,000 L of water was placed in a stainless steel tank, followed by 200 kg of soy protein, 50 kg of defatted rice bran, and 50 kg of rice germ. The mixture was then heated to 90°C and stirred for 1 hour after the temperature increase. After stirring, the mixture was cooled to 42°C, and 1 L of the same natto bacteria used in step 1 was added to the resulting soybean and rice bran liquid (bacterial count: 1.0 x 10 11 After inoculation with Bacillus subtilis natto, the mixture was stirred at 42°C for 28 hours to obtain a soybean and rice bran fermentation liquid.

[0070] <Step 3: Preparation of protease-treated product> To the soybean and rice bran fermented liquid obtained in step 2, 200 kg of the soybean solid fraction fermented product obtained in step 1, 50 kg of defatted rice bran, 50 kg of rice germ, and 260 g of tea catechins were added. This was mixed with stirring for 3 hours, and then heated to 50°C. Aspergillus oryzae 10 kg of neutral protease (Denatym AP, Nagase Chemtex Corporation) was added, and the mixture was stirred at 50° C. for 15 hours to carry out protease digestion. Thereafter, the protease was inactivated by heating at 90° C. for 10 minutes to obtain a protease-treated product.

[0071] <Step 4: Extraction> The protease-treated product obtained in step 3 was stirred for 2 hours and extracted with hot water (90°C). After extraction, solid-liquid separation was performed using a centrifuge (decanter continuous horizontal centrifuge, Tanabe Wiltec Co., Ltd.; 3,000 rpm, 30 minutes). Only the liquid phase was collected, and the pH was adjusted to 3.8 using sodium citrate. After the pH adjustment, the mixture was heat-sterilized at 90°C for 10 minutes and allowed to stand at 10°C for 12 hours.

[0072] <Step 5: Filtration> The extract obtained in step 4 was filtered using a filter press and a 0.5 μm line filter to recover a clear solution. The resulting clear solution was designated as an extract of fermented soybeans and rice bran (OE-1).

[0073] The extract of the fermented soybean and rice bran thus obtained was used to carry out the following Test Examples 1 to 5.

[0074] (Test Example 1: Induction of HO-1) RAW264 cells (available from the RIKEN BioResource Center) were subcultured in flasks until they reached confluence, and then collected and suspended in RPMI medium (GIBCO) containing 10% fetal bovine serum at 1 × 10 6 The cell concentration was adjusted to 100 cells / mL. The adjusted cell suspension was seeded in a 48-well plate at 450 μL / well, and cultured at 37° C. in a 5% CO2 incubator for 2 hours. After incubation, 50 μL of OE-1 sample adjusted to 12.5 mg / mL, 25 mg / mL, 50 mg / mL, or 100 mg / mL was added (final concentration: 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL), and incubation was continued for an additional 18 hours. After incubation, the supernatant was discarded, and 200 μL of the extract provided with the HO-1 quantification kit was added to dissolve the cells and recover the extract. The recovered cell extract was diluted two-fold with the sample dilution buffer provided with the kit, and the HO-1 content was quantified using the HO-1 quantification kit (Mouse Heme Oxygenase ELA Kit; Takara Bio Inc.) according to the protocol. The results are shown in Figure 1. As a control, a control in which pure water was added instead of the OE-1 was also evaluated in the same manner.

[0075] Figure 1 shows that cells cultured with 1.25-10mg / mL OE-1 showed a dose-dependent HO-1 induction effect in cells. When cultured with the highest dose of 10mg / mL OE-1, a 2.6-fold increase in HO-1 was observed compared to the pure water control group.

[0076] (Test Example 2: Inhibitory effect on TNF-α production from LPS-stimulated macrophage cell line (Raw264)) Regarding the anti-inflammatory effect, we investigated the inhibitory effect on the production of TNF-α, an inflammatory cytokine. 5 × 10 Raw264 cells were plated in a 24-well microplate. 5 The cells were seeded at 800 μL per well, and 100 μL of OE-1 sample adjusted to a concentration of 12.5 mg / mL, 25 mg / mL, 50 mg / mL, or 100 mg / mL was added (final concentration: 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL), and the cells were cultured for 18 hours in a 37°C, 5% CO2 incubator. 100 μL of LPS (Lipopolysaccharide; Sigma) adjusted to 0.1 μg / mL was added (final concentration: 0.01 μg / mL), and the cells were further cultured for 18 hours. After the incubation, the culture supernatant was collected from each well, and the amount of TNF-α contained therein was quantified using a TNF-α measurement kit (Levis TNFα-Mouse; manufactured by Shibayagi Co., Ltd.). At the end of the incubation, the viability of the cells was confirmed by staining with trypan blue (manufactured by Wako Pure Chemical Industries, Ltd.), and an evaluation was performed at a concentration where no cytotoxicity was observed. The results are shown in Figure 2. As controls, a control in which pure water was added instead of the OE-1 (LPS stimulation+) and a control in which no LPS was added (LPS stimulation-) were also evaluated in the same manner.

[0077] As shown in Figure 2, stimulation of Raw264 cells with LPS induced the production of TNF-α, while the addition of OE-1 dose-dependently suppressed the production of TNF-α.

[0078] (Test Example 3: Protective effect against cell damage caused by heat treatment) Confluent NIH-3T3 cells (available from the RIKEN BioResource Center) were collected by subculture in a flask and suspended in RPMI medium containing 10% fetal bovine serum at 1 × 10 5 The cell suspension thus prepared was seeded in a 24-well plate at 900 μL / well, and 100 μL of OE-1 sample adjusted to 12.5 mg / mL, 25 mg / mL, 50 mg / mL, or 100 mg / mL was added (final concentration: 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL), followed by culturing for 18 hours in a 37°C, 5% CO2 incubator. The culture temperature was raised to 43°C for 2 hours of heat treatment, and then returned to 37°C and cultured for an additional 18 hours. After the culture was completed, the cell viability was determined by the MTT method using a cell proliferation kit (Cell Proliferation Kit I; Roche). The results are shown in Figure 3. As a control, a control in which pure water was added instead of the OE-1 was also evaluated in the same manner.

[0079] As shown in Figure 3, the heat-treated control group showed a decrease in the number of viable cells of approximately 25% compared to the untreated group, while the addition of OE-1 showed a dose-dependent improvement effect against cell damage.

[0080] (Test Example 4: Cytoprotective effect against oxidative stress damage) NIH-3T3 cells were plated in a 96-well microplate at 1 × 10 5 The cells were seeded at 80 μL per well, and 10 μL of OE-1 sample adjusted to a concentration of 12.5 mg / mL, 25 mg / mL, 50 mg / mL, or 100 mg / mL was added (final concentration: 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL), and the cells were cultured for 18 hours in a 37°C, 5% CO2 incubator. 10 μL of 20 mM 2,2'-Azobis-2-amidinopropane, Dihydrochloride (AAPH, Funakoshi) was added as an oxidative stress inducer and the cells were further cultured for 3 hours, after which the cell viability was determined by the MTT method. The results are shown in Figure 4. As a control, a control in which pure water was added instead of the OE-1 was also evaluated in the same manner.

[0081] As shown in Figure 4, a decrease in cell viability was observed due to oxidative stress caused by AAPH, a radical generator. On the other hand, the addition of OE-1 improved cell viability in a dose-dependent manner, demonstrating its effect of protecting cells from cell damage caused by reactive oxygen species.

[0082] (Test Example 5: Protective effect against gastric mucosal injury) Groups of eight 6-week-old male Wistar rats (available from Japan SLC Co., Ltd.) were fasted overnight and then orally administered 1 mL of a hydrochloric acid-ethanol mixture (35% hydrochloric acid:95% ethanol = 1:50) per rat. One hour after oral administration, the rats were anesthetized with ether to kill them, and their stomachs were immediately removed. 10 mL of 3% formalin solution was injected into the removed stomach and fixed for 10 minutes. After fixation, the stomach was cut along the greater curvature and the length (mm) of damage caused to the body of the stomach was measured, and the sum of these values ​​was used as the ulcer index. The ulcer inhibition rate was calculated using the following formula based on the measured ulcer index. Ulcer inhibition rate (%) = {(ulcer index of control group - ulcer index of test sample administration group) / ulcer index of control group} x 100

[0083] The test sample (OE-1) was orally administered at a dose of 300mg / kg or 1,000mg / kg 30 minutes before administration of the hydrochloric acid-ethanol mixed solution. The control group was orally administered purified water 30 minutes before administration of the hydrochloric acid-ethanol mixed solution. The administration volume was 10mL / kg for both groups. The results are shown in Figures 5 and 6. The obtained values ​​are expressed as mean ± standard error, and the statistical test of the difference between the control group and the OE-1 administration group was performed using Dunnett's multiple comparison test. The significance level in the test was set to less than 5%.

[0084] As shown in Figures 5 and 6, when a mixed solution of hydrochloric acid and ethanol was orally administered to fasted rats, the control group showed significant hemorrhagic gastric mucosal damage, with an average ulcer index of 112.0 mm. On the other hand, the occurrence of gastric mucosal damage was significantly suppressed in rats administered OE-1, and in the high-dose group, no damage was observed in some rats.

[0085] The results of the above test examples show that the extract of fermented soybean and rice bran used in the present invention can induce HO-1 and can effectively and safely treat, ameliorate, or prevent organ damage caused by acute inflammation as well as heat stroke. [Industrial Applicability]

[0086] The extract of the fermented soybean and rice bran product, which is an active ingredient of the present invention, exhibits an anti-inflammatory effect by inducing HO-1, and is therefore useful in treating, improving, or preventing organ damage caused by acute inflammation, and can be suitably used as an active ingredient in foods, beverages, medicines, etc. for preventing, improving, or treating acute hepatitis, nephritis, sepsis, and heat stroke. In addition, since the extract of the fermented soybean and rice bran product is derived from a natural product, it has no side effects and is highly safe.

Claims

1. A composition for inducing heme oxygenase-1, comprising an extract of fermented soybean and rice bran, The extract of the fermented soybean and rice bran is a hot water extract of the fermented soybean and rice bran, The composition for inducing heme oxygenase-1 is characterized in that the soybean and rice bran fermentation product is a protease-treated product obtained by fermenting at least soybean protein and rice bran with Bacillus subtilis natto and treating the fermentation product with a protease.

2. A composition for cell protection, comprising the composition for inducing heme oxygenase-1 according to claim 1.

3. A composition for protecting organs, comprising the composition for inducing heme oxygenase-1 according to claim 1.

4. A composition for treating, ameliorating, or preventing heatstroke, comprising the composition for inducing heme oxygenase-1 according to claim 1.

5. The composition according to any one of claims 1 to 4, which is a food, drink or pharmaceutical product.

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