JP2013227250A - Nrf2-ACTIVATING AGENT AND APPLICATION THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel Nrf2-activating agent that can replace Nrf2-activating agents known heretofore, and applications thereof.SOLUTION: There are provided: an Nrf2-activating agent including as an effective ingredient a culture solution obtained by cultivating Clostridium butyricum or a residue including the bacterium obtained by centrifuging the culture solution or a dried product of the residue; a prophylactic and/or therapeutic agent, including the Nrf2-activating agent, for tissue disorder or pathology for the treatment of which activation of Nrf2 is effective; and foods and drinks including the Nrf2-activating agent that have functions of preventing and/or ameliorating tissue disorder or pathology for the treatment of which activation of Nrf2 is effective and have the functions displayed thereon.

Description

  The present invention relates to an Nrf2 activator and use thereof.

  It is known that increasing free radicals and active oxygen (ROS; oxidative stress factor) in the body cause cancer, arteriosclerosis, myocardial infarction, cerebral infarction, liver dysfunction, diabetes and the like. Known oxidative stress factors include hydroxy radical, superoxide, hydrogen peroxide, hydroperoxyl radical, alkoxyl radical, alkyl peroxyl radical, nitric oxide, peroxynitrite, lipid peroxide, hypochlorous acid, ozone, etc. ing. Oxidative stress factors are likely to react with proteins, nucleic acids, lipids, etc., and when they react with them, protein denaturation, enzyme inactivation, DNA translational abnormalities, DNA degradation, lipid peroxide production, unsaturated fatty acid formation Causes oxidative degeneration, etc., thereby causing the above diseases.

  A living body naturally has a defense mechanism against such an oxidative stress factor, and recently, it has been reported that Nrf2 (NF-E2 related factor-2) as a transcription factor plays a major role (for example, , See Patent Document 1). Nrf2 is usually present in the cytoplasm in a state of being bound to actin via Keap1 (Kelch like ECH associated protein 1). On the other hand, in the nucleus, there are antioxidant genes encoding second-phase foreign substance metabolizing enzymes, antioxidant proteins, and the like. When cells are exposed to oxidative stress, Keap1 senses this and Nrf2 translocates into the nucleus. Nrf2 transferred into the nucleus forms a complex with small Maf and binds to the ARE region (antioxidant response element) of the antioxidant gene, and various second-phase foreign body metabolizing enzymes and antioxidant proteins Increases the expression of

  Antioxidant proteins whose expression is enhanced by Nrf2 include glutathione-S-transferase (GST), heme oxygenase (HO-1), NAD (P) H: quinone oxidoreductase-1 (NQO1), thioredoxin reductase 1 ( TXNRD1), glutamate-cysteine ligase (GCL-c; glutathione synthetase), superoxide dismutase (SOD), glutathione reductase, catalase and the like are known. These antioxidant proteins directly reduce or produce oxidative stress factors and protect cells from damage by oxidative stress factors.

  By the way, Clostridium butyricum called butyric acid bacteria is one type of intestinal bacteria and is a bacterium that comes out alive from the body in the stool like other intestinal bacteria. Clostridium butyricum is a useful bacterium that is also used in industrial production of butyric acid to produce a large amount of butyric acid from carbohydrates by butyric acid fermentation. Since it is known to be produced, it has been widely used as a viable bacterium having an intestinal regulating effect in medicines or feed ingredients for the purpose of improving abdominal symptoms. However, it is not known in the prior art that Clostridium butyricum exhibits Nrf2 activating action (and anti-stress action mediated thereby).

JP 2008-208038 A

  An object of the present invention is to provide a novel Nrf2 activator that can replace a conventionally known Nrf2 activator, and a use thereof.

  In view of the current state of the prior art as described above, the present inventors have intensively studied. In the process, it was surprisingly found that Clostridium butyricum exhibits an activating action on Nrf2, and thus exhibits an antioxidant stress action. And based on this knowledge, it came to complete this invention.

  That is, according to one aspect of the present invention, a culture solution obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution, or a dried product of the residue is contained as an active ingredient. , Nrf2 activators are provided. Here, the Clostridium butyricum is preferably Clostridium butyricum Miyairi 588 (Crostridium butyricum MIYAIRI 588, FERM BP-2789).

  According to another aspect of the present invention, there is provided a preventive and / or therapeutic agent for a tissue disorder or disease state in which activation of Nrf2 is effective for treatment, containing the Nrf2 activator.

  Furthermore, according to still another aspect of the present invention, there is provided a food or drink containing the Nrf2 activator, wherein the Nrf2 activation has a function of preventing and / or improving a tissue disorder or a disease state effective for treatment. And the food / beverage products which the function display was attached are provided. The food or drink is preferably a health food, a functional food, a food for specified health use, a dietary supplement, a food with a disease risk reduction label, or a food for a sick person.

  ADVANTAGE OF THE INVENTION According to this invention, the novel Nrf2 activator which can substitute for the conventional Nrf2 activator, and its use can be provided.

In an Example, it is a microscope picture which shows the result of having examined the behavior of lipid peroxide by performing tissue staining of 4-HNE (4-hydroxy-2-nonenal) with respect to the tissue section derived from the liver tissue of a rat. . In the Example, it is a microscope picture which shows the result of having detected the production | generation of the superoxide which is 1 type of a reactive oxygen species (ROS) by performing the dihydroethidium (DHE) dyeing | staining using the tissue slice derived from the liver tissue of a rat. is there. In an Example, it is an electrophoresis photograph which shows the result of having confirmed the expression of various proteins in the liver tissue of a rat by the Western blot. In an Example, it is an electrophoresis photograph which shows the result of having confirmed the expression of various proteins in the large intestine tissue of a rat by Western blot.

  Hereinafter, specific embodiments for carrying out the present invention will be described in detail, but the technical scope of the present invention is not limited to the following specific embodiments.

  One embodiment of the present invention is an Nrf2 activator comprising, as an active ingredient, a culture solution obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution, or a dried product of the residue.

  The Nrf2 activator according to the embodiment contains, as an active ingredient, a culture solution obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution, or a dried product of the residue It is characterized in that

  Clostridium butyricum is a spore-forming and anaerobic Gram-positive gonococcus that repeats division and growth (vegetative cells) while nutritional balance is maintained, but when the balance is lost, spores are produced in the cells. Many bacteria, not only anaerobic bacteria, easily die when left in a dry state when they have the form of vegetative cells. However, since the spore is a resting cell, it has strong resistance to various external environments such as drying, heat and chemicals, and is convenient for storage.

  Further, as described above, Clostridium butyricum is spore-forming, and has resistance to various external environments when it is in the spore state. For this reason, when Clostridium butyricum is orally administered to humans and animals in the form of spores, even if it comes into contact with digestive fluids such as gastric acid, intestinal juice and bile acid, Clostridium butyricum does not completely die, It also reaches the fermentation site up to the large intestine and can proliferate.

  Furthermore, Clostridium butyricum is widely marketed as a viable agent, feed additive and food, and no side effects are observed even when administered to mammals such as humans and livestock for a long time, ensuring high safety. ing.

  Among the Clostridium butyricum, Clostridium butyricum Miyari, Clostridium butyricum NIP1020 (Clostridium butyricum NIP1020), Clostridium butyricum NIP1021 (Clostridium butyricum NIP1021), Crostridium butyricum NER1021 P-11868), Clostridium butyricum (FERM P-11869), Clostridium butyricum (FERM P-11870), Clostridium butyricum ATCC859 (Clostridium butyricum ATCC859), Clostridium butyricum N RC3315 (Clostridium butyricum NBRC3315), Clostridium butyricum · ATCC860 (Clostridium butyricum ATCC860) or Clostridium butyricum · ATCC19398 (Clostridium butyricum ATCC19398) are preferred. More preferably, Clostridium butyricum Miyairi 588 (Crostridium butyricum MIYAIRI 588, FERM BP-2789), Clostridium butyricum Miyairi 585 (FERM BP-06815), Clostridium butyricum Miyairi 595P ER One or more selected from the group consisting of Miyairi 630 (FERM BP-06817), and more preferably Clostridium butyricum Miyairi 588 (Clostridium butyricum MIYAIRI 588, FERM BP-2789).

  Clostridium butyricum or Miyairi is commercially available from Miyarisan Pharmaceutical Co., Ltd. as a viable bacterial agent, and is particularly suitable for use in the present invention because it has no side effects even when administered to humans and animals for a long time. In addition, as a Clostridium butyricum which is an active ingredient, only 1 type may be used independently and 2 or more types may be used together.

In the present invention, a culture of Clostridium butyricum, that is, “a culture solution obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution and a dried product of the residue” is a culture of a known microorganism. It can be obtained by a method, for example, the method disclosed in Japanese Patent Application No. 08-252088. One embodiment is shown below: Clostridium butyricum is 1.0 (w / v)% peptone, 1.0 (w / v)% yeast extract, 1.0 (w / v)% corn starch and 0.2 (W / v)% Inoculated into a medium consisting of precipitated calcium carbonate at 10 ⁵ to 10 ⁶ cells / mL, and allowed to stand at 37 ° C. for 48 hours to obtain a “culture solution of Clostridium butyricum” obtain. Next, the obtained culture solution is centrifuged (2,000 to 6,000 g × 10 to 30 minutes) to separate “residue containing bacteria obtained by centrifuging the culture solution”. 0 to 80 ° C., preferably 10 to 20 ° C., 1 to 24 hours, preferably 5 to 18 hours by air drying, or 0 to 80 ° C., preferably 10 to 20 ° C., 0.05 to 500 Torr, preferably Is obtained by subjecting to a reduced-pressure drying treatment at 1 to 100 Torr for 1 to 24 hours, preferably 2 to 15 hours.

  The medium used for culturing Clostridium butyricum of the present invention varies depending on the type of strain used, but other sources such as carbon sources, suitable amounts of nitrogen sources, inorganic salts and vitamins that can be used by Clostridium butyricum. As long as the medium contains the nutrients, either a synthetic medium or a natural medium may be used.

  For example, the carbon source used in the medium according to the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the strain used. The carbon source is not necessarily limited to sugar, but considering the growth of bacterial cells, a sugar or sugar containing sugar that can be used by the bacterium to be used is preferably used. Specific examples of carbon sources that can be used include cellobiose, glucose, fructose, galactose, lactose, maltose, mannose, melibiose, raffinose, salicin, starch, sucrose, trehalose, xylose, dextrin, and molasses in view of utilization. Etc. Of these carbon sources, starch, glucose, fructose, sucrose and molasses are preferably used. In consideration of the Clostridium butyricum used, one or more carbon sources may be selected and used. At this time, the added concentration of the carbon source varies depending on the type of clostridium butyricum to be used and the type of the carbon source and the medium composition other than the carbon source of the medium to be used, but usually 0.5 to 5 (w / v)%, Preferably it is 2-4 (w / v)%.

  Examples of nitrogen sources and vitamins include meat extract, peptone, yeast extract, hydrolyzate of soybeans and wheat such as taste liquid, soybean powder, milk casein, casamino acid, various amino acids, corn steep liquor, and others. Examples thereof include organic nitrogen compounds such as hydrolysates of animals, plants, and microorganisms, and ammonium salts such as ammonium sulfate. Of these nitrogen sources, peptone, yeast extract, meat extract, corn steep liquor and taste liquid are preferably used. In order to improve the growth of Clostridium butyricum to be used, one or more of the above nitrogen sources and vitamins may be selected and used. At this time, the concentration of the nitrogen source added varies depending on the strain used, the type of nitrogen source, the medium composition other than the nitrogen source of the medium used, and the like, but when using peptone containing a large amount of nitrogen source, 0.5 to 4 (w / v)%, preferably 1 to 3 (w / v)%, and when using a taste liquid or corn steep liquor containing a large amount of nitrogen source and vitamins, usually 0. 5 to 5 (w / v)%, preferably 1 to 4 (w / v)%. Further, when using a yeast extract or meat extract containing a large amount of vitamins, usually 0.5 to 4 ( w / v)%, preferably 1 to 3 (w / v)%.

  In addition, inorganic salts include phosphates, hydrochlorides, sulfates, butyrate, propionate and acetic acid such as magnesium, manganese, calcium, sodium, potassium, molybdenum, strontium, boron, copper, iron, tin and zinc. One or more selected from salts and the like can be used. In addition, an antifoaming agent, vegetable oil, surfactant, blood and blood components, drugs such as antibiotics, physiologically active substances such as plant or animal hormones, and the like may be appropriately added to the medium as necessary.

  The culture conditions performed in the present invention vary depending on physiological properties such as the growth range (pH, temperature, etc.) of Clostridium butyricum used in the present invention. It is necessary to cultivate under anaerobic conditions, for example, by reducing the redox potential by aeration with nitrogen or carbon dioxide gas or by adding a reducing agent to the medium. The culture conditions at that time are appropriately selected depending on the growth range of the strain to be used, the composition of the medium and the culture method, and are not particularly limited as long as the strain can be grown. Specifically, the culture temperature is usually 20 to 42 ° C, preferably 35 to 40 ° C.

  In the present invention, the cultivation of Clostridium butyricum is preferably promoted by neutralizing the acid produced during the cultivation with an alkali, so that calcium carbonate is preferably added to the medium in advance. At this time, the amount of calcium carbonate added is usually 0.1 to 4 (w / v)%, preferably 0.2 to 2.5 (w / v)%. Or it is also preferable to perform the said neutralization process, suppressing pH of a culture medium in the range of setting pH with alkaline aqueous solutions, such as sodium hydroxide, sodium hydrogencarbonate, sodium carbonate, potassium hydroxide, and potassium carbonate. When an alkaline aqueous solution is used, the “set pH” means the pH of the medium set in advance during the culture period, and the “set pH range” is allowed during the culture period. This is a pH range, and is generally expressed as a set pH ± tolerance. According to the present invention, the set pH is usually set within the range of 5.0 to 7.5, preferably 5.5 to 6.5, and the set pH range is set to pH ± 0.5, preferably set. pH ± 0.2.

  In the present invention, the pH of the medium during culture is about neutral at the time of inoculation with the bacteria, more preferably 6.5 to 7.5. In addition, when using alkaline aqueous solution, it is preferable to maintain within the range of setting pH, stirring gently so that oxygen may not mix. Thus, by controlling the pH at the time of bacterial inoculation and bacterial growth, the bacterial density can be dramatically increased.

In the culture according to the present invention, the initial culture concentration of Clostridium butyricum is not particularly limited as long as Clostridium butyricum can grow, and is usually the same as that performed in the culture of Clostridium butyricum. Specifically, it is usually 10 ⁴ to 10 ⁷ pieces / mL, preferably 10 ⁵ to 10 ⁶ pieces / mL.

  The culture of Clostridium butyricum thus obtained, in particular, the culture of Clostridium butyricum Miyairi 588 (Clostridium butyricum MIYAAIRI 588, FERM BP-2789) has a strong Nrf2 activation action (Nrf2 activation) Agent).

  As described in Examples below, Clostridium butyricum, which is an active ingredient of the Nrf2 activator according to this embodiment, promotes the expression of Nrf2 that is a transcription factor and the genes that are regulated by Nrf2. The Such Nrf2 activation action and Nrf2 regulatory gene group expression promoting action enhance the in vivo antioxidant capacity and detoxification action. These effects are clearly distinguishable from the conventionally known effects of Clostridium butyricum. That is, Clostridium butyricum has an action of enhancing the defense mechanism in vivo against xenobiotics such as oxidative stress and chemical substances, and these actions are unrelated to the action of conventionally known Clostridium butyricum. .

  According to the present invention, tissue damage or pathological conditions for which Nrf2 activation is effective for treatment are prevented or improved. That is, according to another aspect of the present invention, there is provided a preventive and / or therapeutic agent for a tissue disorder or disease state in which activation of Nrf2 is effective for treatment, containing the Nrf2 activator. The tissue damaged by active oxygen or xenobiotics can be any tissue in the living body, such as the eye, nerve, muscle, epithelium, blood, lymph, digestive tract, blood vessel, brain, bone, joint, skin, heart, Examples include kidney, liver, pancreas, spleen and thyroid. Examples of tissue disorders and conditions include chronic inflammation, cancer, arteriosclerosis, hypertension, cranial neurodegenerative disease, skin disease, asthma, liver dysfunction, keratoconjunctival disorder, glaucomatous retinopathy, optic neuropathy, diabetic retinopathy, Macular degeneration, cataract, photoretinopathy, diabetes, UV-responsive skin damage, neutropenia, cellular immunodeficiency, Alzheimer's disease, Parkinson's disease, amyotrophic side sclerosis, pulmonary bronchitis, rheumatoid arthritis, Examples include hepatitis, pancreatitis, vasculitis, esophagitis, ulcerative colitis.

  Furthermore, according to another aspect of the present invention, an effective amount of “a culture solution obtained by culturing Clostridium butyricum or a residue containing bacteria obtained by centrifuging the culture solution or a dried product of the residue” is provided to a mammal. There is provided a method for preventing and / or treating “a tissue disorder or condition in which activation of Nrf2 is therapeutically effective” in the mammal, comprising administering or ingesting the mammal. The “effective amount” means an amount of an active ingredient that is at least required for activating Nrf2 and exerting a desired effect such as prevention and / or treatment of the above-mentioned tissue damage / pathology.

  The Nrf2 activator according to the present invention can be used alone, but can be provided in the form of various compositions such as pharmaceuticals and foods and drinks.

  The pharmaceutical composition that can be used as “a preventive and / or therapeutic agent for a tissue disorder or disease state in which Nrf2 activation is therapeutically effective” according to the present invention includes the Nrf2 activator according to the present invention as described above. It is characterized by that. Here, it is preferable that the pharmaceutical composition contains the activator in an appropriate amount so that the effective amount of the active ingredient of the Nrf2 activator is contained in the pharmaceutical composition.

  Therefore, the pharmaceutical composition according to the present invention is a sufficient amount (ie, an effective amount) of a culture solution in which Clostridium butyricum is cultured or a fungus obtained by centrifuging the culture solution. Containing a residue or a dried product of the residue ”, which is prepared as an oral preparation or a parenteral preparation in accordance with a conventional method using additives that are acceptable for formulation. Additives that are acceptable for such formulations include, for example, excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavors, buffers, antioxidants. Agents, pH adjusters, binders, thickeners, dispersants, suspending agents, disintegrating agents and the like. In the present invention, when the pharmaceutical composition is an oral preparation, it is a solid preparation such as a food, a food additive, a tablet, a powder, a fine granule, a granule, a capsule, a pill, or a sustained release agent, a solution, a suspension. It can take the form of liquid preparations such as liquids and emulsions. In the case of parenteral preparations, it can take the form of injections, drops, external preparations, and suppositories. From the viewpoint of simplicity, an oral preparation is preferable.

  The pharmaceutical composition according to the present invention may further contain other auxiliary components as necessary. Examples of other auxiliary components that can be used in combination include vitamin components (for example, vitamin C and vitamin E), antibiotics, amino acids, peptides, and minerals (for example, zinc, iron, copper, manganese, etc.) , Nucleic acids, polysaccharides, fatty acids, herbal medicines and the like.

  Furthermore, in formulating, one or more medically effective active ingredients (active ingredients) other than the active ingredient according to the present invention may be further added and blended. In administration of the active ingredient according to the present invention, one or more kinds of medically effective active ingredients other than the active ingredient according to the present invention may be administered in combination.

  According to this invention, the food / beverage products containing the Nrf2 activator mentioned above are also provided. Here, it is preferable that the said food / beverage products contain the said activator in a suitable quantity so that the effective component of the Nrf2 activator may be included. Here, “including an effective amount of an active ingredient” means that the active ingredient is contained in such an amount that an effect as an active ingredient can be exhibited as a result of ingesting an amount of each food or drink normally consumed. You may mix | blend the active ingredient which concerns on this invention with the food / beverage products which concern on this invention as it is or in the form of the activator as mentioned above. In addition, the food and drink according to the present invention are prepared as food and drink by adding conventional additives such as stabilizers to the active ingredient according to the present invention, various proteins, sugars, fats, trace elements, vitamins, etc. It may be prepared by further blending them, liquid, semi-liquid or solid, paste, or a general food or drink with active ingredients added.

  In the present invention, the “food or drink” is not a drug and is not particularly limited as long as it is in a form that can be taken orally by mammals, and the form is also a liquid (solution, suspension, emulsion). Liquid, etc.), semi-liquid, powder, or solid molded product. Therefore, the food and drink may be in the form of a beverage, for example, or may be in the form of a dietary supplement tablet such as a supplement.

  Specific examples of food and drink include instant noodles, retort foods, canned foods, microwave foods, instant soups and miso soups, freeze-dried foods, etc .; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee Beverages such as beverages, tea beverages, powdered beverages, concentrated beverages, nutritional beverages, alcoholic beverages; flour products such as bread, pasta, noodles, cake mixes, fried flour, bread crumbs; rice cakes, caramels, chewing gums, chocolates, cookies, Confectionery such as biscuits, cakes, pies, snacks, crackers, Japanese confectionery, dessert confectionery; sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew Seasonings; processed fats and oils, butter, margarine, mayonnaise, etc .; milk drinks, yogurts, lactic acid bacteria drinks Dairy products such as ice cream and cream; processed fishery products such as fish ham and sausages and fish paste products; processed livestock products such as livestock ham and sausages; canned agricultural products, jams and marmalades, pickles, boiled beans, cereals, etc. Processed agricultural products; frozen foods; nutritional foods.

  The food or drink according to the present invention is affected by or suspected of having a tissue disorder or pathological condition in which the activation of Nrf2 is effective for treatment, or suffering from such a tissue disorder or pathological condition. It can be suitably used for those who are at high risk. Here, as a person with a high risk of morbidity, for example, it was determined that the risk was high in consideration of various indicators including body composition and eating habits, or from diagnosis and medical examination such as a health checkup. And those who have been perceived by the person or others as having such a high risk.

  In the present invention, “food and beverage” includes foods classified as health foods, functional foods, foods for specified health use, dietary supplements, foods with a disease risk reduction label, or foods for the sick. Is done. Furthermore, the term “food or drink” can be used to include feed when used for mammals other than humans. The food for specified health here refers to any legal restrictions in each country (for example, Japan) from the viewpoint of health when manufacturing or selling food for the purpose of prevention and / or improvement of hypertension. It means food that may be received. Such a food may be a food that indicates that the food may reduce the risk of disease, that is, a food with a disease risk reduction label. Here, the disease risk reduction label is a label for foods that may reduce the disease risk, and is based on or based on the standard established by the FAO / WHO Joint Food Standards Committee (Codex Committee). The display may be a prescribed display or an approved display with reference to FIG.

  In the food / beverage products of this invention, in addition to the active ingredient mentioned above, you may further add the component which has another function. Further, for example, the active ingredient of the present invention is added to foods, health foods, functional foods and supplements (for example, foods containing one or more vitamins such as minerals such as calcium and magnesium and vitamin K) consumed in daily life. By mix | blending, in addition to the effect by this invention, the food / beverage products which have the function based on another component can be provided.

  According to another aspect of the present invention, “the culture solution obtained by culturing Clostridium butyricum or the residue containing bacteria obtained by centrifuging the culture solution, which is an active ingredient of the Nrf2 activator described above, or drying the residue Food / beverage products containing an effective amount of the product, wherein Nrf2 activation has a function of preventing and / or improving a tissue disorder or a disease state effective for treatment, and the function indication is provided. The Here, the function display attached to the food or drink can be attached to, for example, any of the main body of the product, the container, the packaging, the instruction, the attached document, or the promotional material.

  In the production of foods and drinks according to the present invention, sugars, fragrances, fruit juices, food additives, stabilizers and the like that are used in the usual food and beverage product design can be added as appropriate. Manufacture of food and drink products can be carried out with reference to manufacturing techniques known in the art. The food / beverage products according to the present invention can take various forms, and the food / beverage products according to the present invention may be manufactured according to known pharmaceutical manufacturing techniques. In that case, it can be produced using a carrier or additive as described in the item of production of the inhibitor or pharmaceutical composition according to the present invention. Moreover, in a manufacturing stage, it is good also as multifunctional food / beverage products by combining with the other component which exhibits functions other than the function in this invention, or another functional food.

  When administering or ingesting the pharmaceutical composition and food or drink according to the present invention, the dose or intake of the active ingredient according to the present invention is the recipient, the age and weight of the recipient, symptoms, administration time, dosage form, administration It can be determined depending on the method, the combination of drugs and the like. In the present invention, the dose or intake of the composition or food / drink per day is taken into consideration so that at least the amount of the active ingredient per day necessary for obtaining the Nrf2 activation effect can be administered or taken. It is preferable to appropriately set the content in the composition or food or drink.

  Therefore, the pharmaceutical composition or food or drink according to the present invention is preferably an active ingredient “a culture solution obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution, or a dried product of the residue. Is preferably provided in the range of 20 to 360 mg, more preferably 40 to 200 mg, and even more preferably 60 to 120 mg per day per adult in terms of solid content of the active ingredient.

  Hereinafter, preferred embodiments of the present invention will be described in more detail by way of examples. However, the technical scope of the present invention should not be construed as being limited to the following examples.

<Experimental animals>
For the purpose of exposing Fischer rats (F344, Japan Claire Co., Ltd.) to oxidative stress, they can freely take choline-deficient L-amino acid defined diet (CDAA, manufactured by Dyets) for 8 weeks. I let you.

In addition, as an experimental group, choline-deficient L-amino acid defined diet (CDAA, manufactured by Dyets) 100% by mass is 10% by mass (bacterial mass conversion) of Clostridium butyricum Miyari 588 A diet to which fungal powder (bacterial count: 10 ⁹ to 10 ¹⁰ / g) was added was similarly fed.

  On the other hand, as a control group, choline-supplemented, L-amino-acid-defined diet (CSAA, manufactured by Dyets) 100% by mass of 10% by mass of excipient (corn starch, lactose, etc.) ) Was added in the same manner.

  Next, liver tissue and large intestine tissue were excised from the three groups of rats described above and used in the following experiments.

<4-HNE staining>
A tissue section was prepared from the liver tissue extracted above according to a conventional method, and the behavior of lipid peroxide was examined by performing tissue staining of 4-HNE (4-hydroxy-2-nonenal) on the tissue section. . Here, 4-HNE is an oxidized secondary product (aldehyde) generated by ω6 polyunsaturated fatty acid such as arachidonic acid among biological lipids under oxidative stress. A wide variety of lipid peroxide degradation products are produced in living organisms, and 4-HNE is known as the most representative oxidative stress product because of its production amount, reactivity, physiological action, and the like.

  Specifically, a section of a fixed liver tissue is prepared, an anti-4-HNE monoclonal antibody (manufactured by Nikken Zeil) is used as a primary antibody against 4-HNE-modified protein, and biotin-labeled anti-mouse IgG is used as a secondary antibody. Using the VECTASTAIN ABC-AP Kit manufactured by VECTOR as the staining kit and the VECTOR Red Alkaline Phosphatase Substrate kit I manufactured by the same as the coloring kit, the 4-HNE modified protein in the liver tissue was immunostained. References (T. Tanaka, Y. Nishiyama, K. Okada, K. Hirota, M. Matsui, J. Yodoi, H. Hiai, S. Toyokuni: Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse Experiments were performed with reference to kidney: independence of tubular necrosis and sulfhydryl depletion. Lab. Invest. 77 (2), p145-155 (1997)).

  The results are shown in FIG. In FIG. 1, (a) shows the results of the control group (CSAA intake group), (b) shows the results of the CDAA intake group, and (c) shows the experimental group (CDAA containing 10% by mass of Clostridium butyricum Miyairi 588). (Ingestion group) results.

  As shown in FIG. 1, in the rat liver tissue of (b) (CDAA intake group), the positive site of lipid peroxidase 4-HNE increased, and in (c) (experimental group) the positive site was marked. There was a significant decrease. This is probably because the administration of Clostridium butyricum or Miyairi 588 suppressed lipid peroxidation by reducing oxidative stress such as free radicals generated in the liver tissue.

<DHE staining>
Subsequently, reactive oxygen species (ROS) were detected by performing dihydroethidium (DHE) staining using the tissue section derived from the liver tissue extracted above. Here, DHE fluorescence staining was performed for the purpose of detecting the production of superoxide (O ₂ ⁻ ), which is one of the active oxygen species. DHE is oxidized to ethidium bromide by the presence of O ₂ ⁻ and binds to DNA. For this reason, DHE is known as an oxidative stress marker.

  Specifically, an unfixed fresh frozen section of liver tissue was prepared, and the liver tissue section was incubated for 30 minutes in the presence of 5 to 20 μmol / L dihydroethidium (DHE, manufactured by Molecular Probe) and stained. . Here, DHE can freely penetrate the cell membrane and is oxidized to ethidium in the presence of oxygen, but is trapped in the nucleus by being inserted into DNA. Ethidium is excited with an excitation wavelength of 488 nm and has an emission spectrum with an emission wavelength of 610 nm. In this experiment, images obtained with a confocal laser microscope were visualized using a spectro analysis system. References (Rhian M. Touyz, Chantel Mercure, Ying He, Danesh Javeshghani, Guoying Yao, Glaucia E. Callera, Alvaro Yogi, Nadheige Lochard and Timothy L. Reudelhuber: Angiotensin II-Dependent Chronic Hypertension and Cardiac Hypertrophy Are Unaffected by gp91phox-Containing NADPH Oxidase. Hypertension 2005; 45; 530-537).

  The results are shown in FIG. 2, (a) shows the results of the control group (CSAA intake group), (b) shows the results of the CDAA intake group, (c) shows the experimental group (CDAA containing 10% by mass of Clostridium butyricum Miyai 588). (Ingestion group) results.

As shown in FIG. 2, in the rat liver tissue of (b) (CDAA intake group), the number of DHE positive cells increased, and in (c) (experimental group), the number of DHE positive cells significantly decreased. It was. This is presumably because the administration of Clostridium butyricum or Miyairi 588 exerted anti-oxidant ability and reduced oxidative stress (especially superoxide (O ₂ ⁻ )) such as free radicals generated in the liver tissue.

<Confirmation of expression of various proteins in rat liver tissue by Western blot>
The liver tissue excised above was added with 0.05% Tween 20-containing Tris buffer (TBS-T) (20 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.6) supplemented with protease inhibitor cocktail (Sigma). After lysis and centrifugation at 15,000 × g, 4 ° C., 30 min, the supernatant was used as a tissue lysis protein sample.

  Each 60 μg of the tissue lysed protein sample was developed by SDS polyacrylamide gel electrophoresis according to a conventional method. The acrylamide concentration of the gel was 7.5%. From the developed gel, the protein was transferred to a PVDF membrane using a semi-dry type transfer apparatus, and Western blotting was performed according to a conventional method. As the washing solution, 0.05% Tween 20-containing Tris buffer (TBS-T) was used, and as the blocking solution, a 5% skimmed milk TBS-T solution was used. For detection of Nrf2 expression, a 300-fold diluted rabbit-derived anti-Nrf2 polyclonal antibody (Santa Cruz) was used as the primary antibody, and an anti-rabbit IgG antibody (HRP labeled, Dako) was used as the secondary antibody. What was diluted 2000 times was used. For detection of HO-1 expression, a rabbit-derived anti-HO-1 polyclonal antibody (manufactured by Enzo Life Sciences) diluted 800-fold was used as a primary antibody, and an anti-rabbit IgG antibody (HRP) was used as a secondary antibody. Labeled, manufactured by Dako) was diluted 2000 times. Furthermore, for detection of NQO1 expression, a primary anti-NQO1 polyclonal antibody (manufactured by ProteinTech Group) diluted 500-fold was used as the primary antibody, and an anti-rabbit IgG antibody (HRP labeled, manufactured by Dako) was used as the secondary antibody. ) Was diluted 2000 times. For detection of β-actin expression, the primary antibody used was a mouse-derived anti-β-actin monoclonal antibody (manufactured by Sigma) diluted 8000 times, and the secondary antibody was anti-mouse IgG antibody (HRP labeled, Dako) was diluted 8000 times. Detection was performed by a chemiluminescence method using ECL Plus Western Blotting Detection System (GE HealthCare).

  The results are shown in FIG. As shown in FIG. 3, in the experimental group (“CDAA + C.Butyricum” shown in FIG. 3), it is not only the Nrf2 protein but also the target gene of Nrf2, and plays an important role in the antioxidant process in the oxidative stress response. It has been shown that the protein expression of the responsible HO-1 and NQO1 is dramatically induced. Β-actin protein is known as a housekeeping gene, and was used as an internal standard in this experiment.

  From the above results, it was shown that administration of Clostridium butyricum Miyari 588 contributes to the suppression of oxidative stress by inducing Nrf2 in liver tissue and simultaneously inducing the target protein of Nrf2.

<Confirmation of expression of various proteins in rat colon tissue by Western blot>
The expression of Nrf2 protein and β-actin protein in the rat large intestine tissue was confirmed by Western blotting in the same manner except that the large intestine tissue extracted above was used instead of the liver tissue.

  The results are shown in FIG. As shown in FIG. 4, it can be seen that Nrf2 is significantly induced in the experimental group (“CDAA + C.Butyricum” shown in FIG. 4). That is, it was suggested that administration of Clostridium butyricum or Miyairi 588 not only shows the effect of suppressing oxidative stress in the liver, but also activates the mechanism of suppressing oxidative stress in the large intestine tissue.

  From the results shown above, it was demonstrated that Clostridium butyricum has an activating effect on Nrf2, and thus exhibits an antioxidant stress action. Based on this fact, in one embodiment of the present invention, a culture solution obtained by culturing Clostridium butyricum or a residue containing bacteria obtained by centrifuging the culture solution or a dried product of the residue is contained as an active ingredient. An Nrf2 activator is provided. This Nrf2 activator is considered to be very useful for the prevention and / or treatment of tissue damage or pathological conditions in which Nrf2 activation is effective for treatment, and may greatly contribute to the development of medical treatment in the future. It is a highly superior technology with hidden properties.

Claims (5)

  A Nrf2 activator comprising, as an active ingredient, a residue obtained by culturing Clostridium butyricum, a residue containing bacteria obtained by centrifuging the culture solution, or a dried product of the residue.   The Nrf2 activator according to claim 1, wherein the Clostridium butyricum is Clostridium butyricum Miyairi 588 (FERM BP-2789).   A preventive and / or therapeutic agent for a tissue disorder or pathological condition wherein Nrf2 activation is therapeutically effective, comprising the Nrf2 activator according to claim 1 or 2. A food or drink containing the Nrf2 activator according to claim 1 or 2,
A food / beverage product having a function of preventing and / or improving a tissue disorder or pathological condition in which activation of Nrf2 is effective for treatment, and a function indication thereof.   The food or drink according to claim 4, which is a health food, a functional food, a food for specified health use, a dietary supplement, a food with a disease risk reduction label, or a food for a sick person.