WO2006033351A1 - Thioredoxin expression inducing composition - Google Patents

Thioredoxin expression inducing composition Download PDF

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Publication number
WO2006033351A1
WO2006033351A1 PCT/JP2005/017383 JP2005017383W WO2006033351A1 WO 2006033351 A1 WO2006033351 A1 WO 2006033351A1 JP 2005017383 W JP2005017383 W JP 2005017383W WO 2006033351 A1 WO2006033351 A1 WO 2006033351A1
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Prior art keywords
extract
thioredoxin
expression
blue
mugwort
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PCT/JP2005/017383
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French (fr)
Japanese (ja)
Inventor
Junji Yodoi
Hiroshi Masutani
Yuji Kunimoto
Koji Takatera
Nobue Kanou
Kazuo Murata
Shogo Tamasu
Original Assignee
Kyoto University
Rock Field Co., Ltd.
Redox Bioscience Inc.
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Application filed by Kyoto University, Rock Field Co., Ltd., Redox Bioscience Inc. filed Critical Kyoto University
Priority to JP2006536394A priority Critical patent/JPWO2006033351A1/en
Publication of WO2006033351A1 publication Critical patent/WO2006033351A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/535—Perilla (beefsteak plant)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to a composition that induces expression of thioredoxin. Specifically, the present invention relates to a composition for inducing thioredoxin expression that induces expression of thioredoxin and is useful for mitigating oxidative stress. Furthermore, the present invention relates to a method for inducing the expression of thioredoxin. Background art
  • Thioredoxin is a molecule that performs a reversible acid-acid reduction reaction (redox control) using two cysteine residues in its active site as a catalyst for the NADPH-dependent enzyme thioredoxin reductase.
  • redox control a reversible acid-acid reduction reaction
  • thioredoxin reductase a reversible acid-acid reduction reaction
  • NTF-a tumor necrosis factor ex
  • cheoredoxin transgenic mice exhibit increased life span resistance to ischemic injury, acute lung failure, diabetes, etc. (eg, Non-Patent Documents 4 to 8). Since these pathologies are closely related to oxidative stress, thioredoxin is considered to have an important protective effect against oxidative stress.
  • Non-Patent Document 1 Holmgren A. et al., Methods Enzymol 1995; 252: 199-208.
  • Non-Patent Document 2 Nakamura H. et al., Immunol Lett 1994; 42: 75-80.
  • Non-Patent Document 3 Matsuda M. et al, J Immunol 1991; 147: 3837-3841.
  • Non-Patent Document 4 Mitsui A. et al., Antioxid Redox Signal 2002; 4: 693-696.
  • Non-Patent Document 5 Takagi Y. et al., Proc Natl Acad Sci USA 1999; 96: 4131-4136.
  • Non-Patent Document 6 Hoshino T. et al., Am J Respir Crit Care Med 2003; 168: 1075-1083
  • Non-Patent Document 7 Hotta M. et al "J Exp Med 1998; 188: 1445-1451.
  • Non-Patent Document 8 Yoon BI. Et al., Arch Environ Contam Toxicol 2001; 41: 232-236. Disclosure of the invention
  • An object of the present invention is to provide a safe and useful composition that induces the expression of thioredoxin and alleviates the effects of oxidative stress. Furthermore, an object of the present invention is to provide a method for inducing the expression of thioredoxin in mammals including humans. Means for solving the problem
  • the present inventors have found that mugwort extract and blue diso extract have an effect of inducing expression of thioredoxin.
  • the present invention has been completed based on powerful knowledge.
  • the present invention provides the following aspects of the invention:
  • Item 1 A composition for inducing thioredoxin expression, comprising at least one selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
  • Item 2 A composition for inducing thioredoxin expression comprising mugwort extract and Z or blue diso extract.
  • Item 3 The composition for inducing thioredoxin expression according to Item 1 or 2, for reducing oxidative stress.
  • Item 4 Total capacity of mugwort extract and persimmon or blue diso extract 0.
  • Item 3 The composition for inducing thioredoxin expression according to Item 2, which is 1 to 25% by weight.
  • Item 5 The composition for inducing thioredoxin expression according to claim 1, which is a food.
  • a food for inducing thioredoxin expression comprising at least one selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
  • Item 7 Food for inducing thioredoxin expression, including mugwort extract and persimmon or blue diso extract
  • Item 8 The food for inducing thioredoxin expression according to Item 6 or 7, which is for reducing oxidative stress
  • Item 9 For improvement of liver disorder, improvement of climacteric disorder, reduction of blood cholesterol, reduction of blood neutral fat, reduction of blood glucose level, reduction of blood pressure, prevention of arteriosclerosis, or prevention of aging
  • Item 10 A method for producing a food for inducing thioredoxin expression, comprising adding a mugwort extract and a Z or blue diso extract in an amount of 0.1 to 25% by weight in the food.
  • Item 11 The composition for inducing thioredoxin expression according to Item 1, which is a pharmaceutical composition.
  • the composition for inducing thioredoxin expression according to Item 1 which is a pharmaceutical composition for treatment, prevention or treatment of liver disease, or antiallergy.
  • Item 13 The composition for inducing thioredoxin expression according to Item 1, which is an animal feed.
  • Item 14 A method for inducing the expression of thioredoxin, comprising administering or ingesting a mammal with at least one selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
  • Item 15 Use for the production of a composition for inducing thioredoxin expression, selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
  • Item 16 Use of mugwort extract and Z or blue diso extract for production of thioredoxin expression-inducing composition.
  • Item 17 Use of mugwort extract and Z or blue diso extract for the production of thioredoxin expression-inducing food.
  • Item 18 Use of mugwort extract and Z or blue diso extract for the production of pharmaceutical thread and compound for induction of thioredoxin expression.
  • the mugwort extract or blue diso extract which is one of the active ingredients contained in the composition for inducing thioredoxin expression of the present invention, can induce the expression of thioredoxin more effectively. Therefore, the thioredoxin expression-inducing composition of the present invention can be used for the purpose of alleviating oxidative stress (free radical elimination).
  • Thioredoxin maintains a reduced state in the body by cooperating with choredoxin peroxidase to eliminate peroxyhydrogen in the cell, and this pathway plays an essential role in eliminating intracellular oxidative stress. It is fulfilling.
  • Antioxidant substances include polyphenols in addition to thioredoxin. And vitamin C.
  • blue diso contains rosmarinic acid, a kind of polyphenol, and is known to act to trap phenolic hydroxyl group S-hydroxy radicals and the like. However, these antioxidants do not exhibit antioxidant effects when they are oxidized.
  • thioredoxin exerts an antioxidant effect by oxidation and reduction of two cysteine residues. Even if it becomes oxidized, it is reduced again by reducing enzymes such as NADPH and thioredoxin reductase in the body. It is known to return. Therefore, administration of a substance that induces the expression of thioredoxin Z intake is thought to be effective because it can eliminate oxidative stress inside the cell and uses a physiological oxidative stress elimination system.
  • thioredoxin for example, it is possible to provide a food or a pharmaceutical composition effective for improving liver disorders including hangover, improving menopause, and nutritional tonic. Is possible.
  • the active ingredient used in the composition for inducing thioredoxin expression of the present invention is derived from edible plants, it is extremely safe.
  • FIG. 1A shows the activity of thioredoxin gene by an aqueous extract of mugwort in K562 cells.
  • FIG. 1B shows activation of thioredoxin gene by blue disoacetone extract in K562 cells.
  • FIG. 2A shows the activity of thioredoxin gene in K562 cells treated with mugwort extract.
  • FIG. 2B shows the activity of thioredoxin gene in K562 cells treated with blue diso extract.
  • FIG. 3A Activation of thioredoxin gene by mugwort extract in homozygous assay.
  • FIG. 3B Activation of the thioredoxin gene by blue diso extract in homozygous assay.
  • FIG. 4B shows the results of Real Time RT-PCR for induction of thioredoxin mRNA expression by mugwort extract.
  • FIG. 4C shows the result of RT-PCR of induction of thioredoxin mRNA expression by blue diso extract.
  • FIG. 4D shows the result of Real Time RT-PCR of induction of thioredoxin mRNA expression by blue diso extract.
  • FIG. 5A shows induction of thioredoxin protein expression by mugwort extracts.
  • FIG. 5B shows induction of thioredoxin protein expression by blue diso extract.
  • FIG. 6 shows inhibition of peroxyhydrogen-induced cytotoxicity by mugwort extract. 2% TritonX-10
  • the absorbance value at 0 is 100% cell death, and the absorbance value at medium + cells is 0% cell death.
  • composition for inducing thioredoxin expression of the present invention comprises one or more of mugwort, mugwort extract, blue diso extract and blue diso extract (hereinafter sometimes simply referred to as “active ingredient”). It is characterized by that.
  • Artemisia princeps is a perennial Asteraceae plant.
  • the mugwort extract can be obtained by extracting mugwort.
  • blue perilla (Perilla frutescens) is a perennial Lamiaceae plant.
  • a blue diso extract can be obtained by extracting blue diso.
  • mugwort and Z or blue disodium when mugwort and Z or blue disodium are used as active ingredients, those that have been dried can also be used in the form of a paste, chopped, or the like.
  • any part to be extracted is not particularly limited as long as it has a thioredoxin expression-inducing action, and the whole plant body (whole plant) or any part ( For example, those extracted from leaves, rhizomes, stems, etc.) can be used.
  • the mugwort extract or green disodium extract used in the present invention can be obtained by an extraction processing method commonly used by those skilled in the art.
  • the extraction treatment method is not particularly limited. For example, water, an organic solvent, or a mixture thereof is used as the extraction solvent. And a method of extracting mugwort or blue disodium.
  • the solvent used for the extraction is not particularly limited.
  • examples thereof include lower alkyl esters such as alcohol and ethyl acetate, hydrocarbons such as benzene and hexane, conventionally known solvents such as acetone and methylene chloride, or a mixed solution thereof.
  • These extraction solvents may be used alone or in combination of two or more.
  • preferred solvents are water, ethanol or a mixed solution of ethanol and water.
  • extraction may be performed with a supercritical solvent such as supercritical CO.
  • a single extraction may be performed a plurality of times.
  • the extract obtained in this manner can be used as a mugwort extract or a blue disodium extract as it is, but further, purification such as deodorization and decolorization is carried out within a range not losing the effect of the present invention. You may use for a process. Further, the extract may be subjected to separation and purification steps such as treatment with a synthetic adsorbent, filtration treatment, concentration treatment, etc., if necessary.
  • the treatment method using the synthetic adsorbent is not particularly limited, and a conventionally known method may be used. Specifically, the synthetic adsorbent is passed through a column filled with the synthetic adsorbent, and then with eluent such as water, ethanol, or a mixed solution thereof.
  • the synthetic adsorbent examples include aromatic (cross-linked styrene) synthetic adsorbents, substituted aromatic synthetic adsorbents, and acrylic synthetic adsorbents.
  • mugwort extract or green disodium extract is obtained in a liquid form, and the extract is prepared using water-soluble dietary fibers such as pectin and dextrin as an excipient, spray drying method, etc.
  • the extract may be prepared in a powder form by drying by a known method.
  • the powdered extract can be used by re-dissolving in a solvent such as water, ethanol, propylene glycol, 1,3-butylene glycol, glycerin and the like.
  • these extracts are preferably in a dry state.
  • the composition for inducing thioredoxin expression of the present invention may contain one or more of mugwort, mugwort extract, blue disodium and blue disodium extract. Among these components, mugwort extract and blue diso extract are preferably used in the present invention because they can induce thioredoxin expression more effectively.
  • the composition for inducing thioredoxin expression of the present invention is applied to a mammal in various forms such as internal use, ingestion, injection, infusion, etc., thereby inducing the expression of thioredoxin in the body of the mammal. Can do.
  • the applied amount (intake or dose) of the thioredoxin expression-inducing composition of the present invention may be an effective amount for inducing thioredoxin expression, as long as it is an effective form for the formation of tyredoxin. It is selected as appropriate according to the type of active ingredient, the age, sex, weight, health status, other conditions of the subject, the degree of symptoms, etc. As an example, if mugwort extract and Z or blue dissociation extract are applied to humans as ingredients, the amount applied is the total amount of mugwort extract and Z or blue dissociation extract in terms of dry weight.
  • the amount per person per day is about 2 to 400 mg / kg, preferably about 10 to 150 mg / kg, more preferably about 20 to 80 mg / kg.
  • the thioredoxin expression-inducing composition of the present invention may be applied in a single dose or in several divided doses.
  • the blending ratio of the active ingredient may be appropriately set according to the type of the composition, the type of active ingredient used, the amount applied per day, and the like. it can. If mugwort extract and Z or blue diso extract are used as the above active ingredients, as an example of the blending ratio, mugwort extract and Z with respect to the total amount of thioredoxin expression-inducing yarn and composition. Or the total amount of blue diso extract is 0
  • the composition for inducing thioredoxin expression of the present invention is used in the fields of food, medicine, feed and the like. That is, by preparing the composition for inducing expression of thioredoxin according to the present invention in the form of food, pharmaceuticals, feed, etc., it is possible to provide foods, pharmaceutical compositions, and feeds that enable induction of thioredoxin expression. Become. Hereinafter, specific embodiments in the food, pharmaceutical and feed fields will be described in detail.
  • thioredoxin expression-inducing composition of the present invention By preparing the thioredoxin expression-inducing composition of the present invention as a food product, a food product capable of inducing thioredoxin expression is provided.
  • Examples of the food include foods and drinks such as nutritional supplements, non-nutrition nutritional foods, health foods, nutritional functional foods, foods for specified health use, and foods for the sick. Made of these foods The production method is not particularly limited as long as a retinal protective effect can be obtained. Suitable examples of the food include supplements having the form of powder, granules, capsules, tablets and the like. In such a form of food, a mugwort extract and / or a blue diso extract is preferably used as a component. In addition to the above forms, the food includes gum.
  • the oral intake form it may be in a tube intake form (liquid food, etc.).
  • the content ratio of the active ingredient in the food can be appropriately adjusted according to the daily application amount, the form of the food, the flavor of the food, and the like.
  • the amount of mugwort extract and Z or blue diso extract is generally 0.1 to Examples include a ratio of 25% by weight, preferably 0.5 to: LO% by weight, more preferably 1 to 5% by weight.
  • the food may contain a high content of mugwort extract and Z or blue diso extract. In this way, when the food contains a high content of mugwort extract and Z or blue diso extract, the total amount of mugwort extract and Z or blue diso extract is, for example, 20% by weight or more. A proportion of 30 to 70% by weight is preferred.
  • mugwort extract and Z or blue biloba extract are used as active ingredients
  • mugwort and / or blue biloba itself may further be included.
  • the food can be prepared according to a conventional method in accordance with the form of food by mixing the active ingredient with a carrier containing food raw materials or additives.
  • the additive include sweeteners, colorants, antioxidants, vitamins, and fragrances.
  • the food can induce the expression of thioredoxin in the human body ingested.
  • This expression of thioredoxin can improve liver damage including hangover, etc.
  • arteriosclerosis is caused by the accumulation of peroxylipid, which is a kind of active oxygen species.
  • peroxylipid which is a kind of active oxygen species.
  • thioredoxin By ingesting the food, the expression of thioredoxin is induced, and it is expected that atherosclerosis is improved by the reactive oxygen species scavenging action of thioredoxin itself or thioredoxin-dependent peroxidase.
  • the excessive increase in blood glucose level is caused by the involvement of injury caused by oxidative stress in insulin-producing cells in the spleen La Isle.
  • Ingestion of the food induces the expression of thioredoxin, which is expected to reduce blood glucose levels by inhibiting the injury of insulin-producing cells by thioredoxin itself or thioredoxin-dependent peroxidase scavenging action. Is done.
  • the ability to induce the expression of thioredoxin in the food and body and exert its antioxidant effect helps to relieve (oxidation) stress and maintain the balance of oxidation / reduction (redox) of the body (oxidation) ) It can be suitably applied to people who are concerned about stress.
  • the food can also be used for the purpose of nutrition tonic, fatigue recovery, and the like.
  • composition for inducing thioredoxin expression of the present invention is provided as a pharmaceutical composition for inducing thioredoxin expression by being prepared by blending a pharmaceutically acceptable carrier with the above active ingredient.
  • Carriers to be blended in the pharmaceutical composition for retinal protection include binders, disintegrants, surfactants, absorption enhancers, humectants, adsorbents, lubricants, fillers, extenders, and the like. Examples include wetting agents, preservatives, stabilizers, emulsifiers, solubilizers, salts or buffers for adjusting osmotic pressure, and diluents or excipients. used.
  • the reticuledoxin expression-inducing pharmaceutical composition may contain additives such as colorants, preservatives, fragrances, flavors, sweeteners, and other pharmacologically active ingredients as necessary.
  • the thioredoxin expression-inducing pharmaceutical composition is used as an internal preparation; an injection such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection;
  • the dosage form of the pharmaceutical composition for retinal protection is appropriately set according to the application form.
  • solid preparations such as tablets, powders, powders, granules, capsules, etc .
  • liquid preparations such as suspensions.
  • the pharmaceutical composition for inducing expression of thioredoxin is an injection such as a liquid, emulsion, suspension, etc.
  • these are preferably sterilized and are preferably isotonic with blood.
  • water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used as a diluent.
  • a sufficient amount of sodium chloride, glucose or glycerin for preparing an isotonic solution may be contained in the drug of the present invention.
  • Ordinary solubilizing agents, buffering agents, soothing agents, etc. may be added.
  • the pharmaceutical composition for induction of thioredoxin is a liquid preparation, it may be stored in a frozen or lyophilized state. If the product is freeze-dried, add distilled water for injection at the time of use and dissolve it again before use.
  • the blending ratio of the active ingredient in the pharmaceutical composition can be appropriately adjusted according to the amount of application per day, the form of the composition, and the like.
  • the pharmaceutical composition induces the expression of thioredoxin in the human body to which it is administered, and thereby exhibits various useful physiological activities.
  • the pharmaceutical composition can improve menopause, lower blood cholesterol, lower blood neutral lipid, lower blood glucose level, lower blood pressure, prevent aging, prevent or treat arteriosclerosis, diabetes It is useful for prevention or treatment of liver disease, prevention or treatment of liver disease, antiallergy and the like.
  • the pharmaceutical composition is suitably used as a pharmaceutical composition for preventing or treating arteriosclerosis, preventing or treating diabetes, or preventing or treating liver disease.
  • a feed capable of inducing the expression of thioredoxin is provided.
  • feeds include livestock and poultry feeds as well as pet food for mammals.
  • the feed is prepared by being prepared by blending the active ingredient together with feed raw materials and feed additives.
  • the feed is not particularly limited in its shape.
  • the intake of the feed can be appropriately selected with reference to the above-mentioned application amount for humans. Also, the mixing ratio of the active ingredient in the feed can be appropriately set according to the type of the target animal, the type of the active ingredient to be used, the intake amount of the active ingredient per day, etc.
  • the feed can reduce the effects of oxidative stress by inducing the expression of thioredoxin in the body of the ingested animal. Therefore, the feed is useful for promoting the healthy growth of animals and maintaining a healthy state.
  • At least one effective amount selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract is administered to a mammal in which induction of thioredoxin expression is desired
  • the present invention provides a method for inducing expression of thioredoxin, which is characterized by ingestion.
  • the mammal includes a human.
  • the types of active ingredients to be used, the effective dose or intake thereof, the number of doses or application per day, etc. are as described above.
  • the method can be used to improve menopause, lower blood cholesterol, lower blood neutral lipid, lower blood sugar, lower blood pressure, prevent aging, prevent or treat arteriosclerosis, prevent diabetes or It is useful for treatment, prevention or treatment of liver disease, prevention or treatment of allergic diseases, and the like.
  • Example 1
  • a filter cake of blue diso obtained by filtration was freeze-dried, dissolved in 60 volumes of acetone, filtered through a thin film filter, and dried by an evaporator at 30 ° C. The dried material was dissolved in 1 ml ethanol (acetone extract) and used for analysis.
  • the sulforan used throughout this example was purchased from LTK laboratories Inc. (product number: S 8046) and used at a concentration of 10M. The present inventors have confirmed that sulforafuan induces the expression of cheredoxin.
  • Human erythroblastic leukemia cell line K562 was inoculated with 10% urinary fetal serum (FC S) and antibiotics (100 IU / ml penicillin and 100 (g / ml streptomycin) inactivated by heat treatment. Cultured in RPMI 1640 medium (Life Technologies) under humid conditions at 37 ° C and 5% CO
  • the pTRXCAT plasmid was generated as described in Taniguchi Y. et al., Nucleic Acids Res 1996; 24: 2746-2752.
  • the Hindlll-BamHI insert from the pTRXCAT vector was subcloned into p Bluescriptll KS (+) (pTRXblue vector).
  • the pTRX-Luc vector used in this example is the method described in Kim YC. J Biol Chem 2001; 276, 18399-18406. And Kim YC. Et al., Oncogene 2003; 22: 1860-1865.
  • the pTRX (-1148) -Luc vector was prepared by ligating the Kpnl / BamHI fragment of the pTRXblue vector to the Kpnl / Bglll site of the pGL3 basic vector (Promega).
  • luciferase reporter expression vector or pGL3-basic vector was transfected into K562 cells according to the instructions for use. After 4 hours of incubation, K562 cells transfected with each vector were mixed with DMSO (final concentration 0.1%), 10 M sulforaphane, blue diso acetone extract (final concentration 1%) or mugwort aqueous extract (final concentration 3). %) And added 24 hours.
  • Renilla luciferase gene expression was monitored using pRL-TK (Promega) to normalize the effect of transfection. Luciferase gene expression standardized by the Renilla luciferase activity was analyzed 24 hours later using the Atsy kit (Promega). Atsey went twice.
  • the aqueous mugwort extract showed approximately 7.61 times the activity of thioredoxin gene expression compared to the control (Fig. 1A).
  • the acetone extract of blue diso showed about 6.14 times the activity of thioredoxin gene expression compared to the control (Fig. 1B).
  • ARE antioxidant responsive element
  • the pTRX-3xARE-Luc vector is obtained by inserting a 3xARE oligonucleotide into the Kpnl-Nhel site of the pGL3 promoter vector.
  • oligonucleotides used for the construction of the vector are as follows.
  • Mugwort or blue disodium was homogenized with lOOOOrpm for 5 minutes after adding 2 volumes of sterile water.
  • the homogenate was passed through a PTFE (Teflon®) mesh filter and centrifuged at 45000 rpm.
  • the supernatant was passed through a Diaion HP20 (Mitsubishi Chemical Co., Ltd.) adsorption column and eluted with 99.5% ethanol.
  • the eluate was dried under vacuum to prepare an extract.
  • the dried material (0.1% yield) was resuspended in ethanol to a final concentration of 5% (weight / volume%; g / lOOmL).
  • mugwort extract or blue diso extract prepared by this method was used.
  • the mugwort extract (final concentration 0.2%) activated the luciferase reporter gene approximately 82 times as shown in FIG. 2A. Ethanol (final concentration 0.5%) was used as a control.
  • K 562 cells expressing the luciferase gene under the control of the thioredoxin promoter can be transfected with pTRX (-1148) -Luc and pMAM2-BSD, followed by selection and limiting dilution with blasticidin S. Prepared.
  • Cells are plated in 96 uel plates at a concentration of 5 x 10 4 cell Z wels and DMSO (final concentration 0.1%), sulfaurauan (10 mM), ethanol (0.2%), mugwort extract or blue diso extract Product (the extract obtained from [Momogi extract Z blue diso extract preparation ⁇ ]) at final concentrations of 0.05, 0.1 or Was added at 0.2%), and after 24 hours of incubation, luciferase gene expression was analyzed using the Bright-Gloluciferase assay kit (Promega). Atsey went twice.
  • Oligonucleotide forward (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) for human cheredoxin were designed according to the instructions for use. PCR was performed with Klen Taq DNA polymerase (Sigma) using a thermal cycler. PCR conditions were 22 cycles of 95 ° C for 30 seconds, 48 ° C for 30 seconds and 72 ° C for 1 minute. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) amplification was used as an internal control.
  • RNA isolation The same method as RT-PCR was used for cell treatment and total RNA isolation.
  • mugwort extract or blue diso extract (final concentration 0.1% each) and sulfaurafuan (10 mM) were used, and DMSO (final concentration 0.1%) and ethanol (final concentration 0.1%) were used as controls.
  • the oligonucleotide forward (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6) and TaqMan probe (SEQ ID NO: 7) for human thioredoxin were designed according to the instructions for use.
  • TaqMan One-Step RT-PCR Master Mix Reagents quantitatively in 96 well plates using ABI PRISM7000 Real Time RT-PCR was performed. Each reaction mixture contained 50 ng of total RNA in a final volume of 1.
  • RT-PCR conditions RT step for 30 minutes at 48 ° C, PCR step for 15 seconds at 95 ° C and 1 minute at 60 ° C for 40 cycles.
  • 18S rRNA was amplified as an internal control assay, and the amount of thioredoxin gene was normalized.
  • Cells were treated with mugwort extract or blue diso extract (final concentration 1% each), ethanol (final concentration 1%). After 48 hours, cells were collected, washed with ice-cold phosphate buffered saline (PBS), and then on ice for 30 minutes, solubilizing solution (10 mM Tris-HCI (pH 7.4), 150 mM) The cell lysate was lysed with NaCl, 1% NP-40, 1 mM EDTA, 0.1 mM PMSF, 8 (g / ml cap tune and 2 (g / ml leupeptin).
  • PBS ice-cold phosphate buffered saline
  • solubilizing solution 10 mM Tris-HCI (pH 7.4), 150 mM
  • the cell lysate was lysed with NaCl, 1% NP-40, 1 mM EDTA, 0.1 mM PMSF, 8 (g / ml cap tune and 2 (g
  • the cell lysate was allowed to stand at 95 ° C for 5 minutes, and then separated by 15% SDS-polyacrylamide gel electrophoresis (cell lysate: 2.5 g).
  • the membrane was transferred to a ureaidene difluoride membrane (PVDF membrane: Millipore), which was treated with 10% (weight / volume) skim milk dissolved in T-PBS (PBS containing 0.05% Tween20), Incubated with human thioredoxin monoclonal antibody (Redox Biosciences) for 1 hour, then peroxida Anti-mouse Ig G conjugated with Ze.: Were incubated for 1 hour at (1 5000 dilution) (Amersham Pharmacia Biotech) after which the Epitopu were visualized ELC Western blot detection kit (Amersham Pharmacia Biotech).
  • thioredoxin is a protein that plays an important role against oxidative stress
  • the ability of mugwort extract to induce thioredoxin expression was observed to be able to suppress oxidative stress damage in K562 cells.
  • the effect of pretreatment with mugwort extract on LDH release from injured cells was analyzed according to the LDH accessory method described below.
  • K562 cells were plated on 96-well plates at a concentration of 1 ⁇ 10 4 cells Z-well. After that, the cells were treated with 2% TritonX-100 (this value is converted to 100% cytotoxicity), DMSO (final concentration 0.1%), sulforan (10 M), ethanol (0.2%) or mugwort extract (final concentration). 0.05, 0.1 or 0.2%). After 48 hours of incubation at 37 ° C, the cells were treated with 200 ⁇ 0 for 48 hours.
  • TritonX-100 this value is converted to 100% cytotoxicity
  • DMSO final concentration 0.1%)
  • sulforan (10 M sulforan
  • ethanol ethanol
  • mugwort extract final concentration
  • SEQ ID NO: 1 is an ARE forward primer.
  • SEQ ID NO: 2 is an ARE reverse primer.
  • SEQ ID NO: 3 is an oligonucleotide forward primer for human thioredoxin.
  • SEQ ID NO: 4 is an oligonucleotide reverse primer for human thioredoxin.
  • SEQ ID NO: 5 is the oligonucleotide forward primer used in Real Time RT-PCR. It is one.
  • SEQ ID NO: 6 is an oligonucleotide reverse primer used in Real Time RT-PCR.
  • SEQ ID NO: 7 is a TaqMan probe for human thioredoxin used in Real Time RT-PCR.

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Abstract

A composition that induces thioredoxin expression, being useful in relaxing of influences from oxidation stress, etc., and that is safe. There is provided a composition for use in the fields of food, pharmaceutical, feed, etc., comprising at least one ingredient selected from the group consisting of tansy, a tansy extract, green perilla and a green perilla extract.

Description

明 細 書  Specification
チォレドキシン発現誘導用組成物  Thioredoxin expression inducing composition
技術分野  Technical field
[0001] 本発明は、チォレドキシンの発現を誘導する組成物に関する。具体的には、チォレ ドキシンの発現を誘導し、酸化ストレスの緩和等に有用であるチォレドキシン発現誘 導用組成物に関する。更に、本発明は、チォレドキシンの発現誘導方法に関する。 背景技術  [0001] The present invention relates to a composition that induces expression of thioredoxin. Specifically, the present invention relates to a composition for inducing thioredoxin expression that induces expression of thioredoxin and is useful for mitigating oxidative stress. Furthermore, the present invention relates to a method for inducing the expression of thioredoxin. Background art
[0002] チォレドキシンは、その活性部位にある 2つのシスティン残基によって、 NADPH依 存性酵素であるチォレドキシンリダクターゼを触媒として可逆的な酸ィ匕還元反応（レド ックス制御）を行う分子である（例えば非特許文献 1)。また、チォレドキシンは、細胞 内還元環境を維持し、酸化ストレス (フリーラジカル)や腫瘍壊死因子 ex (TNF- a )が 引き起こす障害力 細胞を保護する役割を持つことが知られている (例えば非特許文 献 2及び 3)。  [0002] Thioredoxin is a molecule that performs a reversible acid-acid reduction reaction (redox control) using two cysteine residues in its active site as a catalyst for the NADPH-dependent enzyme thioredoxin reductase. (For example, Non-Patent Document 1). Thioredoxin is also known to have a role in maintaining the intracellular reducing environment and protecting cells that are damaged by oxidative stress (free radicals) and tumor necrosis factor ex (TNF-a). References 2 and 3).
[0003] さらに、チォレドキシントランスジエニックマウスは、寿命の延長ゃ虚血障害、急性肺 不全、糖尿病等に対して抵抗性を示す (例えば、非特許文献 4〜8)。これらの病態は 酸化ストレスに密接な関係があることから、チォレドキシンは酸化ストレスに対して重 要な保護作用を示すと考えられている。  [0003] Further, cheoredoxin transgenic mice exhibit increased life span resistance to ischemic injury, acute lung failure, diabetes, etc. (eg, Non-Patent Documents 4 to 8). Since these pathologies are closely related to oxidative stress, thioredoxin is considered to have an important protective effect against oxidative stress.
[0004] この様なチォレドキシンの特徴を利用し、チォレドキシンの発現を誘導することで、 酸化ストレスの影響を緩和できると考えられる。 [0004] It is considered that the influence of oxidative stress can be alleviated by inducing the expression of thioredoxin using such characteristics of thioredoxin.
非特許文献 1: Holmgren A. et al.， Methods Enzymol 1995; 252:199-208.  Non-Patent Document 1: Holmgren A. et al., Methods Enzymol 1995; 252: 199-208.
非特許文献 2:Nakamura H. et al.， Immunol Lett 1994; 42:75-80.  Non-Patent Document 2: Nakamura H. et al., Immunol Lett 1994; 42: 75-80.
非特許文献 3:Matsuda M. et al, J Immunol 1991; 147:3837-3841.  Non-Patent Document 3: Matsuda M. et al, J Immunol 1991; 147: 3837-3841.
非特許文献 4: Mitsui A. et al., Antioxid Redox Signal 2002; 4:693-696.  Non-Patent Document 4: Mitsui A. et al., Antioxid Redox Signal 2002; 4: 693-696.
非特許文献 5:Takagi Y. et al., Proc Natl Acad Sci U S A 1999; 96:4131-4136. 非特許文献 6:Hoshino T. et al., Am J Respir Crit Care Med 2003; 168:1075-1083. 非特許文献 7:Hotta M. et al" J Exp Med 1998; 188:1445—1451.  Non-Patent Document 5: Takagi Y. et al., Proc Natl Acad Sci USA 1999; 96: 4131-4136. Non-Patent Document 6: Hoshino T. et al., Am J Respir Crit Care Med 2003; 168: 1075-1083 Non-Patent Document 7: Hotta M. et al "J Exp Med 1998; 188: 1445-1451.
非特許文献 8:Yoon BI. et al., Arch Environ Contam Toxicol 2001; 41:232-236. 発明の開示 Non-Patent Document 8: Yoon BI. Et al., Arch Environ Contam Toxicol 2001; 41: 232-236. Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] 本発明は、チォレドキシンの発現を誘導し、酸化ストレスによる影響の緩和等の有 用で、安全性な組成物を提供することを目的とする。更に、本発明は、ヒトを含むほ乳 動物において、チォレドキシンの発現を誘導する方法を提供することを目的とする。 課題を解決するための手段  [0005] An object of the present invention is to provide a safe and useful composition that induces the expression of thioredoxin and alleviates the effects of oxidative stress. Furthermore, an object of the present invention is to provide a method for inducing the expression of thioredoxin in mammals including humans. Means for solving the problem
[0006] 本発明者らは、ョモギ抽出物及び青ジソ抽出物には、チォレドキシンの発現を誘導 する作用があることを見出した。本発明は、力かる知見に基づいて完成するに至った [0006] The present inventors have found that mugwort extract and blue diso extract have an effect of inducing expression of thioredoxin. The present invention has been completed based on powerful knowledge.
[0007] 即ち、本発明は、下記に掲げる態様の発明を提供する： That is, the present invention provides the following aspects of the invention:
項 1. ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物力 なる群より選択される少な くとも 1種を含むことを特徴とする、チォレドキシン発現誘導用組成物。  Item 1. A composition for inducing thioredoxin expression, comprising at least one selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
である飲食品。  Food and drink that is.
項 2. ョモギ抽出物及び Z又は青ジソ抽出物を含む、チォレドキシン発現誘導用組 成物。  Item 2. A composition for inducing thioredoxin expression comprising mugwort extract and Z or blue diso extract.
項 3. 酸化ストレス緩和用である、項 1又は 2に記載のチォレドキシン発現誘導用組 成物。  Item 3. The composition for inducing thioredoxin expression according to Item 1 or 2, for reducing oxidative stress.
項 4. ョモギ抽出物及び Ζ又は青ジソ抽出物の総量力 組成物の全量に対して 0. Item 4. Total capacity of mugwort extract and persimmon or blue diso extract 0.
1〜25重量％である、項 2に記載のチォレドキシン発現誘導用組成物。 Item 3. The composition for inducing thioredoxin expression according to Item 2, which is 1 to 25% by weight.
項 5. 食品である、請求項 1に記載のチォレドキシン発現誘導用組成物。  Item 5. The composition for inducing thioredoxin expression according to claim 1, which is a food.
項 6. ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物力 なる群より選択される少な くとも 1種を含むことを特徴とする、チォレドキシン発現誘導用食品。  Item 6. A food for inducing thioredoxin expression, comprising at least one selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
項 7. ョモギ抽出物及び Ζ又は青ジソ抽出物を含む、チォレドキシン発現誘導用食 Item 7. Food for inducing thioredoxin expression, including mugwort extract and persimmon or blue diso extract
ΡΡ
ΡΡο ΡΡο
項 8. 酸化ストレス緩和用である、項 6又は 7に記載のチォレドキシン発現誘導用食 Item 8. The food for inducing thioredoxin expression according to Item 6 or 7, which is for reducing oxidative stress
ΡΡ
ΡΡο ΡΡο
項 9. 肝障害の改善、更年期障害の改善、血中コレステロールの低下、血中中性脂 質の低下、血糖値の低下、血圧の低下、動脈硬化の予防、又は老化の予防用であ る旨が表示されている、項 6又は 7に記載のチォレドキシン発現誘導用食品。 Item 9. For improvement of liver disorder, improvement of climacteric disorder, reduction of blood cholesterol, reduction of blood neutral fat, reduction of blood glucose level, reduction of blood pressure, prevention of arteriosclerosis, or prevention of aging The food for inducing thioredoxin expression according to Item 6 or 7, wherein
項 10. ョモギ抽出物及び Z又は青ジソ抽出物を、食品中に 0. 1〜25重量％となる ように添加することを特徴とする、チォレドキシン発現誘導用食品の製造方法。 項 11. 医薬組成物である、項 1に記載のチォレドキシン発現誘導用組成物。 Item 10. A method for producing a food for inducing thioredoxin expression, comprising adding a mugwort extract and a Z or blue diso extract in an amount of 0.1 to 25% by weight in the food. Item 11. The composition for inducing thioredoxin expression according to Item 1, which is a pharmaceutical composition.
項 12. 更年期障害の改善用、血中コレステロールの低下用、血中中性脂質の低下 用、血糖値低下用、血圧低下用、老化予防用、動脈硬化の予防又は治療用、糖尿 病の予防又は治療用、肝臓病の予防又は治療用、或いは抗アレルギー用の医薬組 成物である、項 1に記載のチォレドキシン発現誘導用組成物。 Item 12. For improvement of menopause, for lowering blood cholesterol, for lowering blood neutral lipid, for lowering blood glucose level, for lowering blood pressure, for preventing aging, for preventing or treating arteriosclerosis, for preventing diabetes Alternatively, the composition for inducing thioredoxin expression according to Item 1, which is a pharmaceutical composition for treatment, prevention or treatment of liver disease, or antiallergy.
項 13. 動物用飼料である、項 1に記載のチォレドキシン発現誘導用組成物。 Item 13. The composition for inducing thioredoxin expression according to Item 1, which is an animal feed.
項 14. ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物からなる群より選択される少 なくとも 1種を、ほ乳動物に投与又は摂取させることを特徴とする、チォレドキシンの 発現誘導方法。 Item 14. A method for inducing the expression of thioredoxin, comprising administering or ingesting a mammal with at least one selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
項 15. ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物からなる群より選択される少 なくとも 1種の、チォレドキシン発現誘導用組成物の製造のための使用。 Item 15. Use for the production of a composition for inducing thioredoxin expression, selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract.
項 16. ョモギ抽出物及び Z又は青ジソ抽出物の、チォレドキシン発現誘導用組成 物の製造のための使用。 Item 16. Use of mugwort extract and Z or blue diso extract for production of thioredoxin expression-inducing composition.
項 17. ョモギ抽出物及び Z又は青ジソ抽出物の、チォレドキシン発現誘導用食品 の製造のための使用。 Item 17. Use of mugwort extract and Z or blue diso extract for the production of thioredoxin expression-inducing food.
項 18. ョモギ抽出物及び Z又は青ジソ抽出物の、チォレドキシン発現誘導用医薬 糸且成物の製造のための使用。 Item 18. Use of mugwort extract and Z or blue diso extract for the production of pharmaceutical thread and compound for induction of thioredoxin expression.
発明の効果 The invention's effect
本発明のチォレドキシン発現誘導用組成物に含まれる有効成分の 1つであるョモ ギ抽出物又は青ジソ抽出物は、チォレドキシンの発現を一層効果的に誘導すること ができる。従って、本発明のチォレドキシン発現誘導用組成物は、、酸化ストレス緩和 (フリーラジカルの消去）を目的として用いることができる。チォレドキシンは、チォレド キシンペルォキシダーゼと協調して細胞内の過酸ィ匕水素を消去することで、体内の 還元状態を維持しており、この経路は細胞内の酸化ストレス消去に必須の役割を果 たしている。抗酸化作用を有する物質としては、チォレドキシンの他に、ポリフエノー ル、ビタミン C等がある。例えば、青ジソはポリフエノールの一種であるロズマリン酸を 含み、そのフエノール性水酸基力 Sヒドロキシラジカル等の捕捉にはたらくことが知られ ている。しかし、これらの抗酸化物質は、自身が酸化されると抗酸化作用を示さなくな る。 The mugwort extract or blue diso extract, which is one of the active ingredients contained in the composition for inducing thioredoxin expression of the present invention, can induce the expression of thioredoxin more effectively. Therefore, the thioredoxin expression-inducing composition of the present invention can be used for the purpose of alleviating oxidative stress (free radical elimination). Thioredoxin maintains a reduced state in the body by cooperating with choredoxin peroxidase to eliminate peroxyhydrogen in the cell, and this pathway plays an essential role in eliminating intracellular oxidative stress. It is fulfilling. Antioxidant substances include polyphenols in addition to thioredoxin. And vitamin C. For example, blue diso contains rosmarinic acid, a kind of polyphenol, and is known to act to trap phenolic hydroxyl group S-hydroxy radicals and the like. However, these antioxidants do not exhibit antioxidant effects when they are oxidized.
[0009] 一方、チォレドキシンは、二つのシスティン残基の酸化、還元によって抗酸化作用 を発揮し、ー且酸化型になっても体内の NADPH、チォレドキシンレダクターゼ等の還 元酵素により再び還元型にもどることが知られている。従って、チォレドキシンの発現 を誘導する物質を投与 Z摂取することは、細胞の内部での酸化ストレスを消去でき、 しかも生理的な酸化ストレス消去系を利用するため、効果的だと考えられる。  [0009] On the other hand, thioredoxin exerts an antioxidant effect by oxidation and reduction of two cysteine residues. Even if it becomes oxidized, it is reduced again by reducing enzymes such as NADPH and thioredoxin reductase in the body. It is known to return. Therefore, administration of a substance that induces the expression of thioredoxin Z intake is thought to be effective because it can eliminate oxidative stress inside the cell and uses a physiological oxidative stress elimination system.
[0010] 本発明によれば、チォレドキシンの発現を誘導することによって、例えば、二日酔い 等を含む肝障害の改善、更年期障害の改善、滋養強壮等に有効な食品や医薬組成 物を提供することが可能である。また、本発明のチォレドキシン発現誘導用組成物に 使用される有効成分は、食用植物由来であるため、極めて安全性の高いものである 図面の簡単な説明  [0010] According to the present invention, by inducing the expression of thioredoxin, for example, it is possible to provide a food or a pharmaceutical composition effective for improving liver disorders including hangover, improving menopause, and nutritional tonic. Is possible. In addition, since the active ingredient used in the composition for inducing thioredoxin expression of the present invention is derived from edible plants, it is extremely safe.
[0011] [図 1A]K562細胞におけるョモギ水性抽出物によるチォレドキシン遺伝子の活性ィ匕を 示す。  [0011] FIG. 1A shows the activity of thioredoxin gene by an aqueous extract of mugwort in K562 cells.
[図 1B]K562細胞における青ジソアセトン抽出物によるチォレドキシン遺伝子の活性 化を示す。  FIG. 1B shows activation of thioredoxin gene by blue disoacetone extract in K562 cells.
[図 2A]ョモギ抽出物で処理された K562細胞におけるチォレドキシン遺伝子の活性ィ匕 を示す。  FIG. 2A shows the activity of thioredoxin gene in K562 cells treated with mugwort extract.
[図 2B]青ジソ抽出物で処理された K562細胞におけるチォレドキシン遺伝子の活性ィ匕 を示す。  FIG. 2B shows the activity of thioredoxin gene in K562 cells treated with blue diso extract.
[図 3A]ホモジ-アスアッセィにおける、ョモギ抽出物によるチォレドキシン遺伝子の活 性化を示す。  [FIG. 3A] Activation of thioredoxin gene by mugwort extract in homozygous assay.
[図 3B]ホモジ-アスアッセィにおける、青ジソ抽出物によるチォレドキシン遺伝子の 活性化を示す。  [FIG. 3B] Activation of the thioredoxin gene by blue diso extract in homozygous assay.
[図 4A]ョモギ抽出物によるチォレドキシン mRNA発現の誘導の RT-PCRによる結果を 示す。 [Figure 4A] RT-PCR results of induction of thioredoxin mRNA expression by mugwort extract Show.
[図 4B]ョモギ抽出物によるチォレドキシン mRNA発現の誘導の Real Time RT- PCRの 結果を示す。  FIG. 4B shows the results of Real Time RT-PCR for induction of thioredoxin mRNA expression by mugwort extract.
[図 4C]青ジソ抽出物によるチォレドキシン mRNA発現の誘導の RT-PCRによる結果を 示す。  FIG. 4C shows the result of RT-PCR of induction of thioredoxin mRNA expression by blue diso extract.
[図 4D]青ジソ抽出物によるチォレドキシン mRNA発現の誘導の Real Time RT- PCRの 結果を示す。  FIG. 4D shows the result of Real Time RT-PCR of induction of thioredoxin mRNA expression by blue diso extract.
[図 5A]ョモギ抽出物によるチォレドキシンタンパク質発現の誘導を示す。  FIG. 5A shows induction of thioredoxin protein expression by mugwort extracts.
[図 5B]青ジソ抽出物によるチォレドキシンタンパク質発現の誘導を示す。  FIG. 5B shows induction of thioredoxin protein expression by blue diso extract.
[図 6]ョモギ抽出物による過酸ィ匕水素誘導性細胞傷害の抑制を示す。 2% TritonX-10 FIG. 6 shows inhibition of peroxyhydrogen-induced cytotoxicity by mugwort extract. 2% TritonX-10
0での吸光度の値を 100%細胞死、培地 +細胞での吸光度の値を 0%細胞死とする。 発明を実施するための最良の形態 The absorbance value at 0 is 100% cell death, and the absorbance value at medium + cells is 0% cell death. BEST MODE FOR CARRYING OUT THE INVENTION
[0012] 1.チォレドキシン発現誘導用組成物 [0012] 1. Composition for induction of thioredoxin expression
本発明のチォレドキシン発現誘導用組成物は、ョモギ、ョモギ抽出物、青ジソ及び 青ジソ抽出物（以下、単に「有効成分」と表記することもある）の内、 1種又は 2種以上 を含むことを特徴とする。  The composition for inducing thioredoxin expression of the present invention comprises one or more of mugwort, mugwort extract, blue diso extract and blue diso extract (hereinafter sometimes simply referred to as “active ingredient”). It is characterized by that.
[0013] ョモギ（Artemisia princeps)は、多年草のキク科ョモギ属植物である。ョモギ抽出物 は、ョモギを抽出処理することにより得ることができる。 [0013] Artemisia princeps is a perennial Asteraceae plant. The mugwort extract can be obtained by extracting mugwort.
[0014] また、青ジソ（Perilla frutescens)は、 1年草のシソ科シソ属植物である。青ジソ抽出 物は、青ジソを抽出処理することにより得ることができる。 [0014] In addition, blue perilla (Perilla frutescens) is a perennial Lamiaceae plant. A blue diso extract can be obtained by extracting blue diso.
[0015] 本発明にお 、て、有効成分としてョモギ及び Z又は青ジソを使用する場合、それら を乾燥させたのも、ペースト状にしたもの、細切したもの等を用いることができる。  [0015] In the present invention, when mugwort and Z or blue disodium are used as active ingredients, those that have been dried can also be used in the form of a paste, chopped, or the like.
[0016] ョモギ抽出物又は青ジソ抽出物としては、いずれもチォレドキシン発現誘導作用を 有するものであれば、抽出される部位は特に限定されず、植物体全体 (全草)又は任 意の部分 (例えば、葉、根茎、茎等)から抽出されたものを用いることができる。  [0016] As for the mugwort extract or blue disodium extract, any part to be extracted is not particularly limited as long as it has a thioredoxin expression-inducing action, and the whole plant body (whole plant) or any part ( For example, those extracted from leaves, rhizomes, stems, etc.) can be used.
[0017] 本発明で使用するョモギ抽出物又は青ジソ抽出物は、当業者によって通常用いら れる抽出処理方法によって得ることができる。抽出処理方法としては、特に限定され るものではないが、例えば、水、有機溶媒、又はこれらの混合液を抽出溶媒として使 用して、ョモギ又は青ジソを抽出する方法が挙げられる。 [0017] The mugwort extract or green disodium extract used in the present invention can be obtained by an extraction processing method commonly used by those skilled in the art. The extraction treatment method is not particularly limited. For example, water, an organic solvent, or a mixture thereof is used as the extraction solvent. And a method of extracting mugwort or blue disodium.
[0018] 抽出に使用する溶媒としては、特に限定されないが、例えば、水、エタノール、メタ ノーノレ、 n—プロノ ノーノレ、 iso—プロパノーノレ、 n—ブタノーノレ、 iso—ブタノーノレ、 tert—ブタ ノール等の 1級アルコール、酢酸ェチル等の低級アルキルエステル、ベンゼン、へキ サン等の炭化水素、アセトン、塩化メチレン等の従来公知の溶媒、またはそれらの混 合溶液が例示される。これらの抽出溶媒は、 1種を単独で、又は 2種以上を混合して 使用してもよい。中でも好ましい溶媒としては、水、エタノール又はエタノール及び水 の混合溶液があげられる。また、超臨界 CO等の超臨界溶媒で抽出してもよい。抽出  [0018] The solvent used for the extraction is not particularly limited. For example, water, ethanol, methanol, n-prono norole, iso-propanol, n-butanol, iso-butanol, tert-butanol, etc. Examples thereof include lower alkyl esters such as alcohol and ethyl acetate, hydrocarbons such as benzene and hexane, conventionally known solvents such as acetone and methylene chloride, or a mixed solution thereof. These extraction solvents may be used alone or in combination of two or more. Among them, preferred solvents are water, ethanol or a mixed solution of ethanol and water. Alternatively, extraction may be performed with a supercritical solvent such as supercritical CO. Extraction
2  2
を行う場合、抽出回数は単回でもよぐ収率を上げるために複数回行ってもよい。  In order to increase the yield, a single extraction may be performed a plurality of times.
[0019] この様にして得られた抽出物は、ョモギ抽出物又は青ジソ抽出物としてそのまま使 用することもできるが、更に、本発明の効果を失わない範囲内で脱臭、脱色等の精製 工程に供してもよい。また、該抽出物は、必要に応じて、合成吸着剤による処理、ろ 過処理、濃縮処理等の分離，精製工程に供してもよい。合成吸着剤による処理方法 は特に限定されず、従来公知の方法でよいが、具体的には、合成吸着剤を充填した カラムに通し、水、エタノール、又はこれらの混合溶液等の溶出液等で溶出する方法 力あげられる。合成吸着剤としては、例えば、芳香族系 (架橋スチレン系)合成吸着 剤、置換芳香族系合成吸着剤、アクリル系合成吸着剤等があげられる。また、上記 抽出処理後、ョモギ抽出物又は青ジソ抽出物は液状形態で得られるが、該抽出物に ぺクチン、デキストリン等の水溶性食物繊維を賦形剤としてカ卩え、スプレードライ法等 の公知の方法により乾燥させることにより、該抽出物を粉末形態に調製してもよい。更 に、この粉末状にした抽出物を水、エタノール、プロピレングリコール、 1,3-ブチレン グリコール、グリセリン等の溶媒に再溶解して用いることもできる。 [0019] The extract obtained in this manner can be used as a mugwort extract or a blue disodium extract as it is, but further, purification such as deodorization and decolorization is carried out within a range not losing the effect of the present invention. You may use for a process. Further, the extract may be subjected to separation and purification steps such as treatment with a synthetic adsorbent, filtration treatment, concentration treatment, etc., if necessary. The treatment method using the synthetic adsorbent is not particularly limited, and a conventionally known method may be used. Specifically, the synthetic adsorbent is passed through a column filled with the synthetic adsorbent, and then with eluent such as water, ethanol, or a mixed solution thereof. Elution method Examples of the synthetic adsorbent include aromatic (cross-linked styrene) synthetic adsorbents, substituted aromatic synthetic adsorbents, and acrylic synthetic adsorbents. Further, after the above extraction treatment, mugwort extract or green disodium extract is obtained in a liquid form, and the extract is prepared using water-soluble dietary fibers such as pectin and dextrin as an excipient, spray drying method, etc. The extract may be prepared in a powder form by drying by a known method. Furthermore, the powdered extract can be used by re-dissolving in a solvent such as water, ethanol, propylene glycol, 1,3-butylene glycol, glycerin and the like.
[0020] また、モギ抽出物又は青ジソ抽出物の含有成分の安定性を高めるという観点から、 これらの抽出物は、乾燥状態であることが望ましい。  [0020] From the viewpoint of enhancing the stability of the components contained in the extract of mogi or blue diso extract, these extracts are preferably in a dry state.
[0021] 本発明のチォレドキシン発現誘導用組成物は、ョモギ、ョモギ抽出物、青ジソ、及 び青ジソ抽出物の内の 1種又は 2種以上を含んでいればよい。これらの含有成分の 中で、ョモギ抽出物及び青ジソ抽出物は、チォレドキシン発現をより効果的に誘導で きるため、本発明において好適に使用される。 [0022] 本発明のチォレドキシン発現誘導用組成物は、内服、摂取、注射、点滴等の各種 形態で、ほ乳動物に適用されることにより、該ほ乳動物に体内でチォレドキシンの発 現を誘導することができる。 [0021] The composition for inducing thioredoxin expression of the present invention may contain one or more of mugwort, mugwort extract, blue disodium and blue disodium extract. Among these components, mugwort extract and blue diso extract are preferably used in the present invention because they can induce thioredoxin expression more effectively. [0022] The composition for inducing thioredoxin expression of the present invention is applied to a mammal in various forms such as internal use, ingestion, injection, infusion, etc., thereby inducing the expression of thioredoxin in the body of the mammal. Can do.
[0023] 本発明のチォレドキシン発現誘導用組成物の適用量 (摂取量又は投与量）につい ては、チォレドキシン発現誘導にための有効量であればよぐその糸且成物の形態、適 用形態、有効成分の種類、対象者の年齢、性別、体重、健康状態、その他の条件、 症状の程度等により適宜選択される。一例として、ョモギ抽出物及び Z又は青ジソ抽 出物を含有成分としてヒトに適用する場合であれば、その適用量は、乾燥重量換算 で、ョモギ抽出物及び Z又は青ジソ抽出物が総量で 1人当り 1日当り 2〜400mg/kg程 度、好ましくは 10〜150mg/kg程度、より好ましくは 20〜80mg/kg程度となる量が挙げ られる。また、本発明のチォレドキシン発現誘導用組成物は、上記の 1日当たりの適 用量を単回で適用してもよく、数回に分けて適用してもょ 、。  [0023] The applied amount (intake or dose) of the thioredoxin expression-inducing composition of the present invention may be an effective amount for inducing thioredoxin expression, as long as it is an effective form for the formation of tyredoxin. It is selected as appropriate according to the type of active ingredient, the age, sex, weight, health status, other conditions of the subject, the degree of symptoms, etc. As an example, if mugwort extract and Z or blue dissociation extract are applied to humans as ingredients, the amount applied is the total amount of mugwort extract and Z or blue dissociation extract in terms of dry weight. The amount per person per day is about 2 to 400 mg / kg, preferably about 10 to 150 mg / kg, more preferably about 20 to 80 mg / kg. In addition, the thioredoxin expression-inducing composition of the present invention may be applied in a single dose or in several divided doses.
[0024] 本発明のチォレドキシン発現誘導用組成物において、上記有効成分の配合割合 については、該組成物の種類、使用する有効成分の種類、 1日当たりの適用量等に 応じて適宜設定することができる。上記有効成分として、ョモギ抽出物及び Z又は青 ジソ抽出物を使用する場合であれば、その配合割合の一例として、チォレドキシン発 現誘導用糸且成物の総量に対して、ョモギ抽出物及び Z又は青ジソ抽出物が総量で 0[0024] In the composition for inducing thioredoxin expression of the present invention, the blending ratio of the active ingredient may be appropriately set according to the type of the composition, the type of active ingredient used, the amount applied per day, and the like. it can. If mugwort extract and Z or blue diso extract are used as the above active ingredients, as an example of the blending ratio, mugwort extract and Z with respect to the total amount of thioredoxin expression-inducing yarn and composition. Or the total amount of blue diso extract is 0
. 1〜25重量％となる割合が挙げられる。 The ratio which becomes 1 to 25 weight% is mentioned.
[0025] 本発明のチォレドキシン発現誘導用組成物は、食品、医薬、飼料等の分野で使用 される。即ち、本発明のチォレドキシン発現誘導用組成物を、食品、医薬品、飼料等 の形態に調製することにより、チォレドキシンの発現誘導を可能とする食品、医薬組 成物、飼料を提供することが可能になる。以下、食品、医薬、及び飼料分野での具体 的態様について、詳細に説明する。  [0025] The composition for inducing thioredoxin expression of the present invention is used in the fields of food, medicine, feed and the like. That is, by preparing the composition for inducing expression of thioredoxin according to the present invention in the form of food, pharmaceuticals, feed, etc., it is possible to provide foods, pharmaceutical compositions, and feeds that enable induction of thioredoxin expression. Become. Hereinafter, specific embodiments in the food, pharmaceutical and feed fields will be described in detail.
[0026] ^  [0026] ^
本発明のチォレドキシン発現誘導用組成物を食品として調製することにより、チォ レドキシンの発現を誘導することができる食品が提供される。  By preparing the thioredoxin expression-inducing composition of the present invention as a food product, a food product capable of inducing thioredoxin expression is provided.
[0027] 当該食品としては、例えば、栄養補助食品、ノ ランス栄養食品、健康食品、栄養機 能食品、特定保健用食品、病者用食品等の飲食品が挙げられる。これらの食品の製 造方法は、網膜保護効果が得られるものであれば特に限定されない。当該食品の好 適な具体例として、粉末、顆粒、カプセル、錠剤等の形態を有するサプリメントが例示 される。この様な形態の食品には、好適には、含有成分として、ョモギ抽出物及び/ 又は青ジソ抽出物が使用される。また、上記形態以外にも、当該食品としては、ガム[0027] Examples of the food include foods and drinks such as nutritional supplements, non-nutrition nutritional foods, health foods, nutritional functional foods, foods for specified health use, and foods for the sick. Made of these foods The production method is not particularly limited as long as a retinal protective effect can be obtained. Suitable examples of the food include supplements having the form of powder, granules, capsules, tablets and the like. In such a form of food, a mugwort extract and / or a blue diso extract is preferably used as a component. In addition to the above forms, the food includes gum.
、キャンディー、グミ、錠菓、クッキー、ケーキ、チョコレート、アイスクリーム、ゼリー、ム ース、プリン、ビスケット、コーンフレーク、チユアブルタブレット、ウエハース、煎餅等 の菓子類;炭酸飲料、清涼飲料、乳飲料、コーヒー飲料、紅茶飲料、果汁飲料、栄養 飲料、アルコール飲料、ミネラルウォーター等の飲料類;粉末ジュース，粉末スープ 等の粉末飲料；ドレッシング、ソース等の調味料；パン類；麵類；力まぼこ等の練り製 品；ふりかけ等があげられる。また、経口摂取用の形態以外に、経管摂取用（流動食 等）の形態としてもよい。 Candy, candies, gummi, tablet confectionery, cookies, cakes, chocolate, ice cream, jelly, mousse, pudding, biscuits, cornflakes, chewable tablets, wafers, rice crackers, etc .; carbonated drinks, soft drinks, milk drinks, Coffee beverages, tea beverages, fruit juice beverages, beverages such as beverages, alcoholic beverages and mineral water; powdered beverages such as powdered juice and powdered soup; seasonings such as dressings and sauces; breads; Kneaded products such as sprinkles. In addition to the oral intake form, it may be in a tube intake form (liquid food, etc.).
[0028] 当該食品における有効成分の含有割合については、上記の 1日当たりの適用量、 食品の形態、食品の風味等に応じて適宜調節することができる。例えば、有効成分と して、ョモギ抽出物及び Z又は青ジソ抽出物を使用する場合、通常、該食品の総量 に対して、ョモギ抽出物及び Z又は青ジソ抽出物が総量で 0. 1〜25重量％、好まし くは 0. 5〜： LO重量％、更に好ましくは 1〜5重量％となる割合が例示される。また、チ ォレドキシンの発現誘導をより効果的に発現させるために、高含有量のョモギ抽出物 及び Z又は青ジソ抽出物を含む食品としてもよい。このように高含量のョモギ抽出物 及び Z又は青ジソ抽出物を含む食品とする場合、食品の総量に対して、ョモギ抽出 物及び Z又は青ジソ抽出物が総量で、例えば 20重量％以上、好ましくは 30〜70重 量％となる割合が挙げられる。  [0028] The content ratio of the active ingredient in the food can be appropriately adjusted according to the daily application amount, the form of the food, the flavor of the food, and the like. For example, when mugwort extract and Z or blue diso extract are used as active ingredients, the amount of mugwort extract and Z or blue diso extract is generally 0.1 to Examples include a ratio of 25% by weight, preferably 0.5 to: LO% by weight, more preferably 1 to 5% by weight. In order to more effectively express the induction of thioredoxin, the food may contain a high content of mugwort extract and Z or blue diso extract. In this way, when the food contains a high content of mugwort extract and Z or blue diso extract, the total amount of mugwort extract and Z or blue diso extract is, for example, 20% by weight or more. A proportion of 30 to 70% by weight is preferred.
[0029] また、当該食品において、有効成分としてョモギ抽出物及び Z又は青ジソ抽出物を 使用する場合には、更にョモギ及び/又は青ジソ自体を含んでいてもよい。  [0029] In addition, in the food, when mugwort extract and Z or blue biloba extract are used as active ingredients, mugwort and / or blue biloba itself may further be included.
[0030] 当該食品は、食品原料又は添加剤等を含む担体に上記有効成分を混合し、食品 形態に応じて、常法に従って調製することができる。添加剤としては、例えば、甘味剤 、着色剤、抗酸化剤、ビタミン類、香料等があげられる。  [0030] The food can be prepared according to a conventional method in accordance with the form of food by mixing the active ingredient with a carrier containing food raw materials or additives. Examples of the additive include sweeteners, colorants, antioxidants, vitamins, and fragrances.
[0031] 当該食品は、摂取したヒトの体内でチォレドキシンの発現を誘導することができる。  [0031] The food can induce the expression of thioredoxin in the human body ingested.
このチォレドキシンの発現によって、例えば、二日酔い等を含む肝障害の改善、更年 期障害の改善、血中コレステロールの低下、血中中性脂質の低下、血糖値の低下、 血圧の低下、動脈硬化の予防、老化の予防等が期待される。従って、当該食品は、 コレステロールが高めの人、血圧が高めの人（例えば、血圧が正常高値の人、軽症 高血圧の人）、肝機能が気になり始めた人、血糖値が気になり始めた人、身体の老 化が気になる人等に適用され得る。 This expression of thioredoxin can improve liver damage including hangover, etc. Expected to improve stage disorders, lower blood cholesterol, lower blood neutral lipids, lower blood glucose levels, lower blood pressure, prevent arteriosclerosis, and prevent aging. Therefore, the food contains high cholesterol, high blood pressure (for example, normal high blood pressure, mild hypertension), people who start to worry about liver function, It can be applied to people who are worried about aging.
[0032] 例えば、動脈硬化は、活性酸素種の一種である過酸ィヒ脂質の蓄積が原因とされて いる。当該食品を摂取することにより、チォレドキシンの発現が誘導され、チォレドキ シン自体ある 、はチォレドキシン依存性のペルォキシダーゼの活性酸素種消去作用 によって動脈硬化が改善されることが期待される。  [0032] For example, arteriosclerosis is caused by the accumulation of peroxylipid, which is a kind of active oxygen species. By ingesting the food, the expression of thioredoxin is induced, and it is expected that atherosclerosis is improved by the reactive oxygen species scavenging action of thioredoxin itself or thioredoxin-dependent peroxidase.
[0033] また、血糖値の過度の上昇は、脾臓ラ氏島のインシュリン産生細胞の酸化ストレス による傷害の関与がその発症の一因として考えられている。当該食品を摂取すること により、チォレドキシンの発現が誘導され、チォレドキシン自体、あるいはチォレドキ シン依存性のペルォキシダーゼの活性酸素種消去作用によってインシュリン産生細 胞の傷害が抑制されることで血糖値の低下が期待される。  [0033] In addition, it is considered that the excessive increase in blood glucose level is caused by the involvement of injury caused by oxidative stress in insulin-producing cells in the spleen La Isle. Ingestion of the food induces the expression of thioredoxin, which is expected to reduce blood glucose levels by inhibiting the injury of insulin-producing cells by thioredoxin itself or thioredoxin-dependent peroxidase scavenging action. Is done.
[0034] さらに、当該食品、体内でチォレドキシンの発現を誘導し、抗酸化作用を発揮する こと力 、（酸化)ストレスの解消や身体の酸化 ·還元（レドックス）のバランス維持に役 立ち、（酸化)ストレスが気になる人等に好適に適用することができる。  [0034] Furthermore, the ability to induce the expression of thioredoxin in the food and body and exert its antioxidant effect, helps to relieve (oxidation) stress and maintain the balance of oxidation / reduction (redox) of the body (oxidation) ) It can be suitably applied to people who are worried about stress.
[0035] また、当該食品を動脈硬化、糖尿病、肝臓病、アレルギー等の病者用食品として利 用することで、効果的にこれらの疾患を治療又は改善できる可能性がある。  [0035] Further, there is a possibility that these diseases can be effectively treated or improved by using the food as food for patients with arteriosclerosis, diabetes, liver disease, allergies and the like.
[0036] そして更に、当該食品は、滋養強壮、疲労回復等を目的として用いることもできる。 0037] .m  [0036] Furthermore, the food can also be used for the purpose of nutrition tonic, fatigue recovery, and the like. 0037] .m
本発明のチォレドキシン発現誘導用組成物は、上記有効成分と共に、薬学的に許 容される担体を配合して調製されることにより、チォレドキシン発現誘導用医薬組成 物として提供される。  The composition for inducing thioredoxin expression of the present invention is provided as a pharmaceutical composition for inducing thioredoxin expression by being prepared by blending a pharmaceutically acceptable carrier with the above active ingredient.
[0038] 当該網膜保護用医薬組成物に配合される担体としては、結合剤、崩壊剤、界面活 性剤、吸収促進剤、保湿剤、吸着剤、滑沢剤、充填剤、増量剤、付湿剤、防腐剤、 安定剤、乳化剤、可溶化剤、浸透圧を調節する塩、緩衝剤等の希釈剤又は賦形剤 を例示でき、これらは得られる製剤の投与単位形態に応じて適宜選択使用される。ま た、当該網チォレドキシン発現誘導用医薬組成物には、必要に応じて着色剤、保存 剤、香料、風味剤、甘味剤等の添加剤や他の薬理活性成分を含有させてもよい。 [0038] Carriers to be blended in the pharmaceutical composition for retinal protection include binders, disintegrants, surfactants, absorption enhancers, humectants, adsorbents, lubricants, fillers, extenders, and the like. Examples include wetting agents, preservatives, stabilizers, emulsifiers, solubilizers, salts or buffers for adjusting osmotic pressure, and diluents or excipients. used. Ma The reticuledoxin expression-inducing pharmaceutical composition may contain additives such as colorants, preservatives, fragrances, flavors, sweeteners, and other pharmacologically active ingredients as necessary.
[0039] 当該チォレドキシン発現誘導用医薬組成物は、内服剤;静脈注射、皮下注射、皮 内注射、筋肉注射及び腹腔内注射等の注射剤;点滴剤等の製剤形態で使用される  [0039] The thioredoxin expression-inducing pharmaceutical composition is used as an internal preparation; an injection such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection;
[0040] 当該網膜保護用医薬組成物の剤型としては、適用形態に応じて適宜設定されるが 、一例として、錠剤、散剤、粉末剤、顆粒剤、カプセル剤等の固形製剤;液剤、乳剤、 懸濁剤等の液状製剤が挙げられる。 [0040] The dosage form of the pharmaceutical composition for retinal protection is appropriately set according to the application form. As an example, solid preparations such as tablets, powders, powders, granules, capsules, etc .; liquids, emulsions And liquid preparations such as suspensions.
[0041] 当該チォレドキシン発現誘導用医薬組成物が、液剤、乳剤、懸濁剤等の注射剤で ある場合、これらは殺菌され且つ血液と等張であるのが好ましぐこれらの剤型に製 剤化するに際しては、希釈剤として例えば水、エチルアルコール、マクロゴール、プロ ピレンダリコール、エトキシ化イソステアリルアルコール、ポリオキシ化イソステアリルァ ルコール、ポリオキシエチレンソルビタン脂肪酸エステル類等を使用できる。なお、こ の場合、等張性の溶液を調整するに充分な量の食塩、ブドウ糖あるいはグリセリンを 本発明薬剤中に含有させてもよい。また、通常の溶解補助剤、緩衝剤、無痛化剤等 を添加してもよい。  [0041] When the pharmaceutical composition for inducing expression of thioredoxin is an injection such as a liquid, emulsion, suspension, etc., these are preferably sterilized and are preferably isotonic with blood. In the preparation, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used as a diluent. In this case, a sufficient amount of sodium chloride, glucose or glycerin for preparing an isotonic solution may be contained in the drug of the present invention. Ordinary solubilizing agents, buffering agents, soothing agents, etc. may be added.
[0042] また、当該チォレドキシン発現誘導用医薬組成物が液状製剤である場合は、凍結 保存または凍結乾燥等の状態で保存されて ヽてもよ!ヽ。凍結乾燥製状態である場合 には、使用時に注射用蒸留水等を加え、再度溶解して使用される。  [0042] When the pharmaceutical composition for induction of thioredoxin is a liquid preparation, it may be stored in a frozen or lyophilized state. If the product is freeze-dried, add distilled water for injection at the time of use and dissolve it again before use.
[0043] 当該医薬組成物における有効成分の配合割合は、上記の 1日当たりの適用量や 該組成物の形態等に応じて適宜調節することができる。  [0043] The blending ratio of the active ingredient in the pharmaceutical composition can be appropriately adjusted according to the amount of application per day, the form of the composition, and the like.
[0044] 当該医薬組成物は、投与されたヒトの体内でチォレドキシンの発現を誘導し、これ によって種々の有用生理活性が発揮させることができる。具体的には、当該医薬組 成物は、更年期障害の改善、血中コレステロールの低下、血中中性脂質の低下、血 糖値低下、血圧低下、老化予防、動脈硬化の予防又は治療、糖尿病の予防又は治 療、肝臓病の予防又は治療、抗アレルギー等のために有用である。中でも特に、当 該医薬組成物は、動脈硬化の予防又は治療用、糖尿病の予防又は治療用、或いは 肝臓病の予防又は治療用の医薬組成物として好適に使用される。 [0045] 飼料 [0044] The pharmaceutical composition induces the expression of thioredoxin in the human body to which it is administered, and thereby exhibits various useful physiological activities. Specifically, the pharmaceutical composition can improve menopause, lower blood cholesterol, lower blood neutral lipid, lower blood glucose level, lower blood pressure, prevent aging, prevent or treat arteriosclerosis, diabetes It is useful for prevention or treatment of liver disease, prevention or treatment of liver disease, antiallergy and the like. In particular, the pharmaceutical composition is suitably used as a pharmaceutical composition for preventing or treating arteriosclerosis, preventing or treating diabetes, or preventing or treating liver disease. [0045] Feed
本発明のチォレドキシン発現誘導用組成物を飼料として調製することにより、チォ レドキシンの発現を誘導することができる飼料が提供される。当該飼料には、家畜や 家禽用の飼料の他、ほ乳動物用のペットフードも含まれる。  By preparing the composition for inducing thioredoxin expression of the present invention as a feed, a feed capable of inducing the expression of thioredoxin is provided. These feeds include livestock and poultry feeds as well as pet food for mammals.
[0046] 当該飼料は、飼料原料や飼料用添加物と共に、上記有効成分を配合することによ り、調製されるすることにより、調製される。当該飼料は、その形状については特に制 限されない。 [0046] The feed is prepared by being prepared by blending the active ingredient together with feed raw materials and feed additives. The feed is not particularly limited in its shape.
[0047] 当該飼料の摂取量は、上記のヒトの場合の適用量を参考にして適宜選択すること ができる。また、当該飼料中の有効成分の配合割合についても、対象動物の種類、 使用する有効成分の種類、 1日当たりの有効成分の摂取量等に応じて適宜設定する ことができる。  [0047] The intake of the feed can be appropriately selected with reference to the above-mentioned application amount for humans. Also, the mixing ratio of the active ingredient in the feed can be appropriately set according to the type of the target animal, the type of the active ingredient to be used, the intake amount of the active ingredient per day, etc.
[0048] 当該飼料は、摂取した動物の体内でチォレドキシンの発現を誘導して、酸化ストレ スによる影響を軽減することができる。従って、当該飼料は、動物の健全な生育を促 し、健康状態を維持するのに有用である。  [0048] The feed can reduce the effects of oxidative stress by inducing the expression of thioredoxin in the body of the ingested animal. Therefore, the feed is useful for promoting the healthy growth of animals and maintaining a healthy state.
[0049] 2.チォレ キシンの 謙 方法  [0049] 2. Cheole xin's humility
本発明は、更に、ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物力もなる群より選 択される少なくとも 1種の有効量を、チォレドキシンの発現の誘導が望まれているほ 乳動物に投与又は摂取させることを特徴とする、チォレドキシンの発現誘導方法を提 供する。  According to the present invention, at least one effective amount selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract is administered to a mammal in which induction of thioredoxin expression is desired Alternatively, the present invention provides a method for inducing expression of thioredoxin, which is characterized by ingestion.
[0050] 当該方法において、ほ乳動物にはヒトが含まれる。また、当該改善方法において、 使用する有効成分の種類、これらの投与又は摂取有効量、 1日当たりの投与又は適 用回数等については、前述する通りである。また、当該方法は、更年期障害の改善、 血中コレステロールの低下、血中中性脂質の低下、血糖値の低下剤、血圧の低下、 老化の予防、動脈硬化の予防又は治療、糖尿病の予防又は治療、肝臓病の予防又 は治療、アレルギー疾患の予防又は治療等に有用である。  [0050] In the method, the mammal includes a human. In the improvement method, the types of active ingredients to be used, the effective dose or intake thereof, the number of doses or application per day, etc. are as described above. In addition, the method can be used to improve menopause, lower blood cholesterol, lower blood neutral lipid, lower blood sugar, lower blood pressure, prevent aging, prevent or treat arteriosclerosis, prevent diabetes or It is useful for treatment, prevention or treatment of liver disease, prevention or treatment of allergic diseases, and the like.
実施例  Example
[0051] 以下、本発明を実施例を挙げてさらに詳しく説明するが、本発明はこれらの実施例 に限定されない。 実施例 1 [0051] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples. Example 1
[0052] (1)ョモギ抽出物 Z青ジソ抽出物の調製 I  [0052] (1) Preparation of mugwort extract Z blue diso extract I
ョモギ（Artemisia princeps)又は青ジソ（Perilla frutescens)に 2容量の滅菌水をカロえ て 3分間、 9000rpmでホモジェナイズし、濾過した後、 15000rpmで遠心分離を行った。 この上清を、解析用の乾燥粉末を得るために凍結乾燥した (水性抽出物)。  Two volumes of sterile water were added to Artemisia princeps or Perilla frutescens, homogenized at 9000 rpm for 3 minutes, filtered, and then centrifuged at 15000 rpm. The supernatant was lyophilized (aqueous extract) to obtain a dry powder for analysis.
[0053] 濾過によって得られた青ジソの濾過ケーキ（filter cake)を凍結乾燥し、 60容量のァ セトンに溶解させて薄膜フィルターで濾過し、 30°Cのエバポレーターで乾燥させた。 乾燥させた材料を、 1mlのエタノールに溶解させ (アセトン抽出物）、解析に使用した。  [0053] A filter cake of blue diso obtained by filtration was freeze-dried, dissolved in 60 volumes of acetone, filtered through a thin film filter, and dried by an evaporator at 30 ° C. The dried material was dissolved in 1 ml ethanol (acetone extract) and used for analysis.
[0054] 本実施例を通して使用したスルフオラフアンは、 LTK laboratories Inc. (製品番号： S 8046)から購入し、 10 Mの濃度で使用した。本発明者らは、スルフオラフアンが、チ ォレドキシンの発現を誘導することを確認して 、る。  [0054] The sulforan used throughout this example was purchased from LTK laboratories Inc. (product number: S 8046) and used at a concentration of 10M. The present inventors have confirmed that sulforafuan induces the expression of cheredoxin.
[0055] (2)培養細胞及び培養系  [0055] (2) Cultured cells and culture system
ヒト赤芽球様白血病細胞株 K562を、熱処理で不活性ィ匕した 10%ゥシ胎児血清 (FC S)及び抗生物質（100 IU/mlのペニシリン及び 100 (g /mlストレプトマイシン)をカ卩えた RPMI 1640培地（Life Technologies)中にて、 37°C、 5%COの湿潤条件下で培養した  Human erythroblastic leukemia cell line K562 was inoculated with 10% urinary fetal serum (FC S) and antibiotics (100 IU / ml penicillin and 100 (g / ml streptomycin) inactivated by heat treatment. Cultured in RPMI 1640 medium (Life Technologies) under humid conditions at 37 ° C and 5% CO
2  2
[0056] (3)プラスミド [0056] (3) Plasmid
pTRXCATプラスミドを、 Taniguchi Y. et al., Nucleic Acids Res 1996; 24:2746-2752 .の記載に従って作製した。 pTRXCATベクター由来の Hindlll-BamHIインサートを、 p Bluescriptll KS (+) (pTRXblue vector)にサブクローユングした。本実施例において使 用された pTRX- Lucベクターを、 Kim Y-C. J Biol Chem 2001; 276, 18399-18406.及 び Kim YC. et al., Oncogene 2003; 22:1860-1865に記載される方法に従って調製し た。 pTRX(- 1148)- Lucベクターは、 pTRXblueベクターの Kpnl/BamHIフラグメントを、 p GL3 basicベクター（Promega)の Kpnl/Bglllサイトにラーゲーシヨンさせることで作製さ れた。  The pTRXCAT plasmid was generated as described in Taniguchi Y. et al., Nucleic Acids Res 1996; 24: 2746-2752. The Hindlll-BamHI insert from the pTRXCAT vector was subcloned into p Bluescriptll KS (+) (pTRXblue vector). The pTRX-Luc vector used in this example is the method described in Kim YC. J Biol Chem 2001; 276, 18399-18406. And Kim YC. Et al., Oncogene 2003; 22: 1860-1865. Prepared according to The pTRX (-1148) -Luc vector was prepared by ligating the Kpnl / BamHI fragment of the pTRXblue vector to the Kpnl / Bglll site of the pGL3 basic vector (Promega).
[0057] 実施例で用いた全てのコンストラクトを、 Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Pharimacia)を用 ヽてヌクレオチド酉己歹 Uを確認した。  [0057] All the constructs used in the examples were confirmed for nucleotide nucleotides using Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Pharimacia).
[0058] (4)トランスフエクシヨン及びルシフェラーゼアツセィ DMRIEC (GIBCO製）を用い、使用説明に従って、ルシフェラーゼレポーター発現 ベクター又は pGL3-basicベクター（コントロール）を K562細胞にトランスフエクシヨンし た。 4時間のインキュベーション後、各ベクターをトランスフエクシヨンした K562細胞に DMSO (最終濃度 0.1%)、 10 Mスルフオラフアン、青ジソのアセトン抽出物（最終濃 度 1%)又はョモギ水性抽出物（最終濃度 3%)を加え、 24時間お 、た。 [0058] (4) Transformation and luciferase assembly Using DMRIEC (manufactured by GIBCO), luciferase reporter expression vector or pGL3-basic vector (control) was transfected into K562 cells according to the instructions for use. After 4 hours of incubation, K562 cells transfected with each vector were mixed with DMSO (final concentration 0.1%), 10 M sulforaphane, blue diso acetone extract (final concentration 1%) or mugwort aqueous extract (final concentration 3). %) And added 24 hours.
[0059] トランスフエクシヨンの効果を平準化するため、ゥミシィタケ（Renilla)ルシフェラーゼ 遺伝子発現を、 pRL-TK(Promega)を用いてモニターした。ゥミシィタケルシフェラー ゼ活性によって標準化されたルシフェラーゼ遺伝子発現を、アツセィキット (Promega) を用いて、 24時間後に解析を行った。アツセィは、 2度行った。  [0059] Renilla luciferase gene expression was monitored using pRL-TK (Promega) to normalize the effect of transfection. Luciferase gene expression standardized by the Renilla luciferase activity was analyzed 24 hours later using the Atsy kit (Promega). Atsey went twice.
[0060] 上記（1)〜(4)に記載の方法に従い、アブラナ科植物以外の野菜抽出物が、 K562 細胞においてチォレドキシン遺伝子の発現を誘導するかどうかについて解析を行つ た。解析には、チォレドキシン遺伝子の全制御領域を含むベクターを用いた。  [0060] According to the methods described in (1) to (4) above, an analysis was conducted as to whether vegetable extracts other than cruciferous plants induce the expression of the thioredoxin gene in K562 cells. For the analysis, a vector containing the entire regulatory region of the thioredoxin gene was used.
[0061] ョモギ水性抽出物は、コントロールに対して約 7.61倍のチォレドキシン遺伝子発現 の活性ィ匕を示した（図 1A)。また、青ジソのアセトン抽出物は、コントロールに対して 約 6.14倍のチォレドキシン遺伝子発現の活性ィ匕を示した（図 1B)。  [0061] The aqueous mugwort extract showed approximately 7.61 times the activity of thioredoxin gene expression compared to the control (Fig. 1A). In addition, the acetone extract of blue diso showed about 6.14 times the activity of thioredoxin gene expression compared to the control (Fig. 1B).
例 2  Example 2
[0062] 本発明者らは、ョモギ抽出物又は青ジソ抽出物によるチォレドキシン遺伝子発現誘 導に antioxidant responsive element (ARE)領域が関与することを見出した。従って、 以下の実施例では、野生型 3xAREを含むベクター（pTRX-3xARE-Lucベクター）を作 製し、該ベクターを用いて解析を行った。 pTRX-3xARE-Lucベクターの作製のために 使用した pcDNA3を、 Invitrogen社から購入した。  [0062] The present inventors have found that an antioxidant responsive element (ARE) region is involved in induction of thioredoxin gene expression by mugwort extract or blue diso extract. Therefore, in the following examples, a vector containing wild type 3xARE (pTRX-3xARE-Luc vector) was prepared and analyzed using the vector. pcDNA3 used for the construction of the pTRX-3xARE-Luc vector was purchased from Invitrogen.
[0063] pTRX- 3xARE- Lucベクターは、 3xAREオリゴヌクレオチドを pGL3プロモーターべク ターの Kpnl-Nhelサイトに挿入したものである。  [0063] The pTRX-3xARE-Luc vector is obtained by inserting a 3xARE oligonucleotide into the Kpnl-Nhel site of the pGL3 promoter vector.
[0064] ベクターの構築のために使用したオリゴヌクレオチドは、以下の通りである。  [0064] The oligonucleotides used for the construction of the vector are as follows.
CTGGTCACCGTTACTCAGCACTG- 3 ' (配列番号： 0 CCAGTGCTGAGTAACGGTGACCG- 3' (配列番号 2) [ョモギ抽出物 Z青ジソ抽出物の調製 π] CTGGTCACCGTTACTCAGCACTG-3 '(SEQ ID NO: 0 CCAGTGCTGAGTAACGGTGACCG-3' (SEQ ID NO: 2) [Preparation of mugwort extract Z blue diso extract π]
ョモギ又は青ジソを 2倍量の滅菌水を加えて 5分間、 lOOOOrpmでホモジェナイズした 。ホモジェネートを、 PTFE (Teflon (登録商標））メッシュフィルターに通し、 45000rpm で遠心分離した。この上清を Diaion HP20 (三菱ィ匕学株式会社製）吸着カラムに通し、 99.5%エタノールで溶出した。この溶出液を真空条件下で乾燥させ、抽出物を調製 した。さらに、この乾燥させた材料 (収率 0.1%)を最終濃度が 5% (weight/volume %;g /lOOmL)になるように、エタノールに再懸濁した。実施例 2〜6においては、この方法 で調製したョモギ抽出物又は青ジソ抽出物を使用した。  Mugwort or blue disodium was homogenized with lOOOOrpm for 5 minutes after adding 2 volumes of sterile water. The homogenate was passed through a PTFE (Teflon®) mesh filter and centrifuged at 45000 rpm. The supernatant was passed through a Diaion HP20 (Mitsubishi Chemical Co., Ltd.) adsorption column and eluted with 99.5% ethanol. The eluate was dried under vacuum to prepare an extract. Furthermore, the dried material (0.1% yield) was resuspended in ethanol to a final concentration of 5% (weight / volume%; g / lOOmL). In Examples 2-6, mugwort extract or blue diso extract prepared by this method was used.
[0065] チォレドキシン遺伝子活性ィ匕の程度が抽出物によって異なるかどうかを試験するた め、 pTRX-3xARE-WT_Lucを用いて、実施例 1 (4)に記載される方法でルシフェラー ゼレポーターアツセィを行った。  [0065] In order to test whether or not the degree of thioredoxin gene activity varies depending on the extract, the luciferase reporter assay was performed by the method described in Example 1 (4) using pTRX-3xARE-WT_Luc. went.
[0066] ョモギ抽出物（最終濃度 0.2%)は、図 2Aに示すように、ルシフェラーゼレポーター 遺伝子を約 82倍活性化した。コントロールとしてエタノール (最終濃度 0.5%)を使用し た。  [0066] The mugwort extract (final concentration 0.2%) activated the luciferase reporter gene approximately 82 times as shown in FIG. 2A. Ethanol (final concentration 0.5%) was used as a control.
[0067] 青ジソ抽出物（最終濃度 0.33%)は、図 2Bに示すように、ルシフェラーゼレポーター 遺伝子を約 32.4倍活性ィ匕した。コントロールとしてエタノール (最終濃度 0.33%)を使 用した。  [0067] The blue diso extract (final concentration 0.33%) activated the luciferase reporter gene approximately 32.4 times as shown in FIG. 2B. Ethanol (final concentration 0.33%) was used as a control.
例 3  Example 3
[0068] ョモギ抽出物及び青ジソ抽出物によるチォレドキシンの活性ィ匕についてさらに試験 するため、下記に記載のホモジ-アスアッセィにより、チォレドキシンプロモーターの 制御下でのルシフェラーゼ遺伝子を観察した。  [0068] In order to further test the activity of thioredoxin by mugwort extract and blue diso extract, the luciferase gene under the control of the thioredoxin promoter was observed by the homozygous assay described below.
[0069] [ホモジ-アスアッセィ]  [0069] [Homogi-Assassie]
チォレドキシンプロモーターの制御下においてルシフェラーゼ遺伝子を発現する K 562細胞を、 pTRX(-1148)-Luc及び pMAM2-BSDでトランスフエクシヨンし、その後、ブ ラストサイジン Sによる選択及び限外希釈を行うことで調製した。細胞を、 96ゥエルプレ ートに 5 X 104細胞 Zゥエルの濃度でプレーティングし、 DMSO (最終濃度 0.1%)、スル フオラフアン（10 mM)、エタノール（0.2%)、ョモギ抽出物又は青ジソ抽出物（[ョモギ抽 出物 Z青ジソ抽出物の調製 Π]で得られた抽出物）を、それぞれ最終濃度 0.05、 0.1又 は 0.2%になるように加えた）をそれぞれカ卩え、 24時間のインキュベーション後、ルシフ エラーゼ遺伝子発現を、 Bright-Glo luciferase assay kit (Promega)を用いて解析した 。アツセィは、 2度行った。 K 562 cells expressing the luciferase gene under the control of the thioredoxin promoter can be transfected with pTRX (-1148) -Luc and pMAM2-BSD, followed by selection and limiting dilution with blasticidin S. Prepared. Cells are plated in 96 uel plates at a concentration of 5 x 10 4 cell Z wels and DMSO (final concentration 0.1%), sulfaurauan (10 mM), ethanol (0.2%), mugwort extract or blue diso extract Product (the extract obtained from [Momogi extract Z blue diso extract preparation Π]) at final concentrations of 0.05, 0.1 or Was added at 0.2%), and after 24 hours of incubation, luciferase gene expression was analyzed using the Bright-Gloluciferase assay kit (Promega). Atsey went twice.
[0070] ョモギ抽出物（図 3A)又は青ジソ抽出物（図 3B)は、いずれもチォレドキシン遺伝 子を用量依存的に活性化した。 [0070] Both mugwort extract (Fig. 3A) and blue diso extract (Fig. 3B) activated the thioredoxin gene in a dose-dependent manner.
実施例 4  Example 4
[0071] 以下に記載の RT- PCR及び real time RT- PCRの方法を用いて、チォレドキシン mR NA発現に対するョモギ抽出物又は青ジソ抽出物の効果を観察した。  [0071] Using the RT-PCR and real-time RT-PCR methods described below, the effect of mugwort extract or blue diso extract on thioredoxin mRNA expression was observed.
[0072] (l) RT-PCR  [0072] (l) RT-PCR
細胞を、エタノール (最終濃度 0.1%)、ョモギ抽出物又は青ジソ抽出物（それぞれ 最終濃度 0.1%)で処理した。 24時間後、細胞を回収し、リン酸緩衝溶液 (PBS)で洗 浄した。使用説明に従って RNeasy Mini Kit (QIAGEN)を用い、トータル RNAを単離し た。 RT-PCR用の Superscript III First-strand System (QIAGEN)を用い、使用説明に 従って、 Oligo(dT)20プライマーで cDNAを合成した。 RT条件は、 48°Cで 30分であった  Cells were treated with ethanol (final concentration 0.1%), mugwort extract or blue diso extract (each final concentration 0.1%). After 24 hours, the cells were collected and washed with a phosphate buffer solution (PBS). Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the instructions for use. Using Superscript III First-strand System (QIAGEN) for RT-PCR, cDNA was synthesized with Oligo (dT) 20 primer according to the instructions for use. RT condition was 30 minutes at 48 ° C
[0073] ヒトチォレドキシンに対するオリゴヌクレオチドフォワード (配列番号 3)及びリバース プライマー (配列番号 4)を、使用説明に従って設計した。サーマルサイクラ一を使用 し、 Klen Taq DNAポリメラーゼ（Sigma)で PCRを行った。 PCRの条件は、 95°Cにて 30 秒及び 48°Cにて 30秒及び 72°Cにて 1分を 22サイクル行った。 GAPDH (Glyceraldehyd e 3 - phosphate dehydrogenase)の増幅を、内部コントロールとして使用した。 [0073] Oligonucleotide forward (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) for human cheredoxin were designed according to the instructions for use. PCR was performed with Klen Taq DNA polymerase (Sigma) using a thermal cycler. PCR conditions were 22 cycles of 95 ° C for 30 seconds, 48 ° C for 30 seconds and 72 ° C for 1 minute. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) amplification was used as an internal control.
[0074] (2) Real Time RT— PCR  [0074] (2) Real Time RT—PCR
細胞処理及びトータル RNA単離には、 RT-PCRと同じ方法を利用した。細胞処理に は、ョモギ抽出物又は青ジソ抽出物（それぞれ最終濃度 0.1%)、スルフオラフアン（10 mM)を用い、 DMSO (最終濃度 0.1 %)及びエタノール (最終濃度 0.1 %)をコントロー ルとして使用した。使用説明に従い、オリゴヌクレオチドフォワード (配列番号 5)及び リバースプライマー（配列番号 6)、ならびにヒトチォレドキシンに対する TaqManプロ一 ブ（配列番号 7)を設計した。 TaqMan One-Step RT- PCR Master Mix Reagents (Appl ied Biosystems)を使用し、 ABI PRISM7000を用いて 96ゥエルプレート中にて、定量的 Real Time RT-PCRを行った。各反応混合溶液は、最終容量 1中にの 50ngのトー タル RNAを含んだ。 The same method as RT-PCR was used for cell treatment and total RNA isolation. For cell treatment, mugwort extract or blue diso extract (final concentration 0.1% each) and sulfaurafuan (10 mM) were used, and DMSO (final concentration 0.1%) and ethanol (final concentration 0.1%) were used as controls. . The oligonucleotide forward (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6) and TaqMan probe (SEQ ID NO: 7) for human thioredoxin were designed according to the instructions for use. Using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems), quantitatively in 96 well plates using ABI PRISM7000 Real Time RT-PCR was performed. Each reaction mixture contained 50 ng of total RNA in a final volume of 1.
[0075] RT-PCR条件： RTステップは 48°Cにて 30分、 PCR^テツプは 95°Cにて 15秒及び 60°C にて 1分 40サイクノレ? Tつ 7こ。 TaqMan Ribosomal control Reagents (Applied Biosyste ms)を使用し、内部コントロールアツセィとして、 18S rRNAの増幅を行い、チォレドキ シン遺伝子の量を標準化した。  [0075] RT-PCR conditions: RT step for 30 minutes at 48 ° C, PCR step for 15 seconds at 95 ° C and 1 minute at 60 ° C for 40 cycles. Using TaqMan Ribosomal control Reagents (Applied Biosystems), 18S rRNA was amplified as an internal control assay, and the amount of thioredoxin gene was normalized.
[0076] チォレドキシンの mRNAレベルは、 K562細胞をョモギ抽出物（図 4A及び 4B)又は 青ジソ抽出物（図 4C及び 4D)で処理した 24時間後、顕著に増加した。  [0076] Thioredoxin mRNA levels markedly increased 24 hours after treatment of K562 cells with mugwort extract (FIGS. 4A and 4B) or blue diso extract (FIGS. 4C and 4D).
実施例 5  Example 5
[0077] 以下に記載するウェスタンプロット解析の方法により、チォレドキシンタンパク質発 現に対するョモギ抽出物又は青ジソ抽出物の効果を観察した。  [0077] The effect of mugwort extract or blue diso extract on cheredoxin protein expression was observed by the method of Western plot analysis described below.
[0078] [ウェスタンブロット解析]  [0078] [Western blot analysis]
細胞を、ョモギ抽出物又は青ジソ抽出物（それぞれ最終濃度 1%)、エタノール (最 終濃度 1%)で処理した。 48時間後、細胞を回収し、氷冷したリン酸緩衝溶液 (PBS) で洗浄し、その後、氷上で 30分間、細胞溶解液（solubilizing solution) (10 mM Tris- HCI (pH 7.4), 150 mM NaCl, 1% NP- 40, 1 mM EDTA, 0.1 mM PMSF, 8 (g/mlァプ 口チュン及び 2(g/mlロイぺプチン)で溶解した。この細胞溶解液を、遠心分離によつ て精製した。細胞の溶解液は、 95°Cに 5分間おき、その後、 15%SDS—ポリアクリルァ ミドゲル電気泳働で分離した (細胞溶解液 : 2.5 g)。この分離されたタンパク質を、ポ リビユリデンジフロライド膜 (PVDF膜: Millipore)に転写した。この膜を、 T- PBS (0.05% の Tween20を含む PBS)に溶解させた 10% (weight/volume)スキムミルクでー晚処理し 、抗ヒトチォレドキシンモノクローナル抗体 (レドックス ·バイオサイエンス株式会社製） で 1時間インキュベーションした。その後、ペルォキシダーゼを結合させた抗マウス Ig G (1 : 5000希釈） (Amersham Pharmacia Biotech)で 1時間インキュベーションを行った 。その後、このェピトープを、 ELCウェスタンブロット検出キット（Amersham Pharmacia Biotech)で可視化した。  Cells were treated with mugwort extract or blue diso extract (final concentration 1% each), ethanol (final concentration 1%). After 48 hours, cells were collected, washed with ice-cold phosphate buffered saline (PBS), and then on ice for 30 minutes, solubilizing solution (10 mM Tris-HCI (pH 7.4), 150 mM) The cell lysate was lysed with NaCl, 1% NP-40, 1 mM EDTA, 0.1 mM PMSF, 8 (g / ml cap tune and 2 (g / ml leupeptin). The cell lysate was allowed to stand at 95 ° C for 5 minutes, and then separated by 15% SDS-polyacrylamide gel electrophoresis (cell lysate: 2.5 g). The membrane was transferred to a ureaidene difluoride membrane (PVDF membrane: Millipore), which was treated with 10% (weight / volume) skim milk dissolved in T-PBS (PBS containing 0.05% Tween20), Incubated with human thioredoxin monoclonal antibody (Redox Biosciences) for 1 hour, then peroxida Anti-mouse Ig G conjugated with Ze.: Were incubated for 1 hour at (1 5000 dilution) (Amersham Pharmacia Biotech) after which the Epitopu were visualized ELC Western blot detection kit (Amersham Pharmacia Biotech).
[0079] ョモギ抽出物（図 5A)又は青ジソ抽出物（図 5B)で 48時間処理した K562細胞にお いて、チォレドキシンタンパク質の発現が増加した。 実施例 6 [0079] Expression of thioredoxin protein was increased in K562 cells treated with mugwort extract (Fig. 5A) or blue diso extract (Fig. 5B) for 48 hours. Example 6
[0080] チォレドキシンは、酸化ストレスに対して重要なはたらきをするタンパク質であること から、ョモギ抽出物によるチォレドキシン発現の誘導力 K562細胞における酸化スト レス性の傷害を抑える事ができるかどうかを観察した。以下に記載の LDHアツセィの 方法に従い、傷害された細胞からの LDH遊離に対するョモギ抽出物を用いた前処理 の効果を解析した。  [0080] Since thioredoxin is a protein that plays an important role against oxidative stress, the ability of mugwort extract to induce thioredoxin expression was observed to be able to suppress oxidative stress damage in K562 cells. . The effect of pretreatment with mugwort extract on LDH release from injured cells was analyzed according to the LDH accessory method described below.
[0081] [LDHアツセィ]  [0081] [LDH Atssey]
K562細胞を、 1 X 104細胞 Zゥエルの濃度で 96ゥエルプレートにプレーティングした。 その後、細胞を 2%TritonX-100 (この値を 100%の細胞傷害として換算する）、 DMSO ( 最終濃度 0.1%)、スルフオラフアン（10 M)、エタノール (0.2%)又はョモギ抽出物（最 終濃度 0.05、 0.1又は 0.2%)で処理した。 37°Cにて 48時間のインキュベーションの後、 この細胞を 200 μ Μの Η 0で 48時間処理した。 LDH assay kit (Roche)を用い、使用 K562 cells were plated on 96-well plates at a concentration of 1 × 10 4 cells Z-well. After that, the cells were treated with 2% TritonX-100 (this value is converted to 100% cytotoxicity), DMSO (final concentration 0.1%), sulforan (10 M), ethanol (0.2%) or mugwort extract (final concentration). 0.05, 0.1 or 0.2%). After 48 hours of incubation at 37 ° C, the cells were treated with 200 μΜ 0 for 48 hours. Use LDH assay kit (Roche)
2 2  twenty two
説明に従って、試料 1中の LDHの測定を行った。  According to the explanation, LDH in sample 1 was measured.
[0082] 図 6に示すように、最終濃度 0.05、 0.1又は 0.2%のョモギ抽出物で 48時間の前処理 を行った細胞は、コントロールに比べ、過酸ィ匕水素による細胞傷害が顕著に抑制さ れた。 [0082] As shown in FIG. 6, cells pretreated with mugwort extract at a final concentration of 0.05, 0.1, or 0.2% for 48 hours significantly inhibited cell damage due to peroxyhydrogen as compared to the control. It was done.
[0083] 従って、ョモギ抽出物の前処理によるチォレドキシンの誘導力 過酸化水素による 酸化ストレスに対して細胞保護のはたらきをすることが示唆された。  [0083] Therefore, it was suggested that thioredoxin induced by pretreatment of mugwort extract acts to protect cells against oxidative stress caused by hydrogen peroxide.
[0084] また、同様の試験を青ジソ抽出物で行った細胞においても、コントロールに比べ、 過酸ィ匕水素による細胞傷害が顕著に抑制されることが期待される。  [0084] In addition, in cells subjected to the same test using blue diso extract, it is expected that cell injury due to hydrogen peroxide is significantly suppressed as compared with the control.
配列表フリーテキスト  Sequence listing free text
[0085] 配列番号 1は、 AREフォワードプライマーである。  [0085] SEQ ID NO: 1 is an ARE forward primer.
[0086] 配列番号 2は、 AREリバースプライマーである。  [0086] SEQ ID NO: 2 is an ARE reverse primer.
[0087] 配列番号 3は、ヒトチォレドキシンに対するオリゴヌクレオチドフォワードプライマーで ある。  [0087] SEQ ID NO: 3 is an oligonucleotide forward primer for human thioredoxin.
[0088] 配列番号 4は、ヒトチォレドキシンに対するオリゴヌクレオチドリバースプライマーで ある。  [0088] SEQ ID NO: 4 is an oligonucleotide reverse primer for human thioredoxin.
[0089] 配列番号 5は、 Real Time RT-PCRで使用したオリゴヌクレオチドフォワードプライマ 一である。 [0089] SEQ ID NO: 5 is the oligonucleotide forward primer used in Real Time RT-PCR. It is one.
[0090] 配列番号 6は、 Real Time RT- PCRで使用したオリゴヌクレオチドリバースプライマー である。  [0090] SEQ ID NO: 6 is an oligonucleotide reverse primer used in Real Time RT-PCR.
[0091] 配列番号 7は、 Real Time RT-PCRで使用したヒトチォレドキシンに対する TaqManプ ローブである。  [0091] SEQ ID NO: 7 is a TaqMan probe for human thioredoxin used in Real Time RT-PCR.

Claims

請求の範囲 The scope of the claims
[I] ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物からなる群より選択される少なくとも 1種を含むことを特徴とする、チォレドキシン発現誘導用組成物。  [I] A composition for inducing thioredoxin expression, comprising at least one selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
[2] ョモギ抽出物及び Z又は青ジソ抽出物を含む、チォレドキシン発現誘導用組成物  [2] A composition for inducing thioredoxin expression comprising mugwort extract and Z or blue diso extract
[3] 酸化ストレス緩和用である、請求項 1に記載のチォレドキシン発現誘導用組成物。 [3] The composition for inducing thioredoxin expression according to claim 1, which is for reducing oxidative stress.
[4] ョモギ抽出物及び Z又は青ジソ抽出物の総量力 組成物の全量に対して 0. 1〜2[4] Total capacity of mugwort extract and Z or blue diso extract 0.1 to 2 with respect to the total amount of the composition
5重量％である、請求項 2に記載のチォレドキシン発現誘導用組成物。 The composition for inducing thioredoxin expression according to claim 2, which is 5% by weight.
[5] 食品である、請求項 1に記載のチォレドキシン発現誘導用組成物。 [5] The composition for inducing thioredoxin expression according to claim 1, which is a food.
[6] 医薬組成物である、請求項 1に記載のチォレドキシン発現誘導用組成物。 [6] The composition for inducing thioredoxin expression according to claim 1, which is a pharmaceutical composition.
[7] 動物用飼料である、請求項 1に記載のチォレドキシン発現誘導用組成物。 [7] The composition for inducing thioredoxin expression according to claim 1, which is an animal feed.
[8] ョモギ抽出物及び Z又は青ジソ抽出物を、食品中に 0. 1〜25重量％となるように 添加することを特徴とする、チォレドキシン発現誘導用食品の製造方法。 [8] A method for producing a food for inducing thioredoxin expression, comprising adding a mugwort extract and a Z or blue diso extract in an amount of 0.1 to 25% by weight in the food.
[9] ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物からなる群より選択される少なくとも[9] At least selected from the group consisting of mugwort, mugwort extract, blue biloba and blue biloba extract
1種を、ほ乳動物に投与又は摂取させることを特徴とする、チォレドキシンの発現誘 導方法。 A method for inducing the expression of thioredoxin, which comprises administering or ingesting one species to a mammal.
[10] ョモギ、ョモギ抽出物、青ジソ及び青ジソ抽出物からなる群より選択される少なくとも 1種の、チォレドキシン発現誘導用組成物の製造のための使用。  [10] Use for production of a composition for inducing thioredoxin expression, selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
[II] ョモギ抽出物及び Z又は青ジソ抽出物の、チォレドキシン発現誘導用組成物の製 造のための使用。  [II] Use of mugwort extract and Z or blue diso extract for production of thioredoxin expression-inducing composition.
[12] ョモギ抽出物及び Z又は青ジソ抽出物の、チォレドキシン発現誘導用医薬組成物 の製造のための使用。  [12] Use of mugwort extract and Z or blue diso extract for the manufacture of a pharmaceutical composition for inducing thioredoxin expression.
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