JPWO2006033351A1 - Composition for inducing thioredoxin expression - Google Patents

Abstract

An object of the present invention is to provide a useful and safe composition that induces the expression of thioredoxin and alleviates the effects of oxidative stress. At least one component selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract is blended to prepare a composition used in the fields of food, medicine, feed and the like.

Description

  The present invention relates to a composition that induces the expression of thioredoxin. Specifically, the present invention relates to a composition for inducing thioredoxin expression that induces thioredoxin expression and is useful for reducing oxidative stress and the like. Furthermore, the present invention relates to a method for inducing thioredoxin expression.

  Thioredoxin is a molecule that performs a reversible oxidation-reduction reaction (redox control) using two cysteine residues in its active site as a catalyst for thioredoxin reductase, which is an NADPH-dependent enzyme (for example, Non-Patent Document 1). Thioredoxin is known to have a role in protecting the cell from damage caused by oxidative stress (free radicals) and tumor necrosis factor α (TNF-α) while maintaining the intracellular reducing environment (for example, non-patented) References 2 and 3).

  Furthermore, thioredoxin transgenic mice show resistance to prolongation of life span, ischemic injury, acute lung failure, diabetes and the like (for example, Non-Patent Documents 4 to 8). Since these pathological conditions are closely related to oxidative stress, thioredoxin is considered to exhibit an important protective action against oxidative stress.

It is considered that the influence of oxidative stress can be mitigated by inducing the expression of thioredoxin using such characteristics of thioredoxin.
Holmgren A. et al., Methods Enzymol 1995; 252: 199-208. Nakamura H. et al., Immunol Lett 1994; 42: 75-80. Matsuda M. et al., J Immunol 1991; 147: 3837-3841. Mitsui A. et al., Antioxid Redox Signal 2002; 4: 693-696. Takagi Y. et al., Proc Natl Acad Sci USA 1999; 96: 4131-4136. Hoshino T. et al., Am J Respir Crit Care Med 2003; 168: 1075-1083. Hotta M. et al., J Exp Med 1998; 188: 1445-1451. Yoon BI. Et al., Arch Environ Contam Toxicol 2001; 41: 232-236.

  An object of the present invention is to provide a useful and safe composition that induces the expression of thioredoxin and alleviates the effects of oxidative stress. A further object of the present invention is to provide a method for inducing thioredoxin expression in mammals including humans.

  The present inventors have found that mugwort extract and blue diso extract have an effect of inducing the expression of thioredoxin. The present invention has been completed based on this finding.

That is, 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 disodium and blue disodium extract.
Food and drink that is.
Item 2. A composition for inducing thioredoxin expression comprising a mugwort extract and / or a blue diso extract.
Item 3. Item 3. The composition for inducing thioredoxin expression according to Item 1 or 2, which is used for alleviating oxidative stress.
Item 4. Item 3. The composition for inducing thioredoxin expression according to Item 2, wherein the total amount of mugwort extract and / or blue diso extract is 0.1 to 25% by weight based on the total amount of the composition.
Item 5. The composition for inducing thioredoxin expression according to claim 1, which is a food.
Item 6. A food for inducing thioredoxin expression, comprising at least one selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
Item 7. A food for inducing thioredoxin expression, comprising a mugwort extract and / or a blue diso extract.
Item 8. Item 8. The food for inducing thioredoxin expression according to Item 6 or 7, which is for reducing oxidative stress.
Item 9. It is displayed for improving liver damage, improving menopause, lowering blood cholesterol, lowering blood neutral lipid, lowering blood sugar, lowering blood pressure, preventing arteriosclerosis, or preventing aging. Item 8. The food for inducing thioredoxin expression according to Item 6 or 7.
Item 10. A method for producing a food for inducing thioredoxin expression, comprising adding a mugwort extract and / or a blue diso extract in an amount of 0.1 to 25% by weight in the food.
Item 11. Item 2. The composition for inducing thioredoxin expression according to Item 1, which is a pharmaceutical composition.
Item 12. For improving menopause, for lowering blood cholesterol, for lowering blood neutral lipids, for lowering blood sugar levels, for lowering blood pressure, for preventing aging, for preventing or treating arteriosclerosis, for preventing or treating diabetes, Item 2. The composition for inducing thioredoxin expression according to Item 1, which is a pharmaceutical composition for preventing or treating liver disease or for antiallergy.
Item 13. Item 2. The composition for inducing thioredoxin expression according to Item 1, which is an animal feed.
Item 14. A method for inducing 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 production of a composition for inducing thioredoxin expression, selected from the group consisting of mugwort, mugwort extract, blue disodium and blue disodium extract.
Item 16. Use of mugwort extract and / or blue diso extract for producing a composition for inducing thioredoxin expression.
Item 17. Use of mugwort extract and / or blue diso extract for production of food for inducing thioredoxin expression.
Item 18. Use of mugwort extract and / or blue diso extract for the production of a pharmaceutical composition for inducing thioredoxin expression.

  The mugwort extract or blue diso extract, which is one of the active ingredients contained in the thioredoxin expression inducing composition of the present invention, can induce thioredoxin expression more effectively. Therefore, the composition for inducing thioredoxin expression of the present invention can be used for the purpose of alleviating oxidative stress (erasing free radicals). Thioredoxin maintains a reduced state in the body by eliminating intracellular hydrogen peroxide in cooperation with thioredoxin peroxidase, and this pathway plays an essential role in eliminating intracellular oxidative stress. In addition to thioredoxin, there are polyphenol, vitamin C and the like as substances having an antioxidative action. For example, blue diso is known to contain rosmarinic acid, a kind of polyphenol, and its phenolic hydroxyl group serves to trap hydroxy radicals and the like. However, these antioxidants do not exhibit antioxidant action when they are oxidized.

  On the other hand, thioredoxin exerts an antioxidant effect by oxidation and reduction of two cysteine residues, and once it becomes an oxidized form, it is known to return again to a reduced form by a reductase such as NADPH and thioredoxin reductase in the body. Yes. Therefore, administration / ingestion of a substance that induces the expression of thioredoxin is considered effective because it can eliminate oxidative stress inside the cell and uses a physiological oxidative stress extinction system.

  According to the present invention, by inducing the expression of thioredoxin, for example, it is possible to provide foods and pharmaceutical compositions effective for improving liver disorders including hangover, improving menopause, and nutritional tonic. . Moreover, since the active ingredient used for the composition for inducing thioredoxin expression of the present invention is derived from edible plants, it is extremely safe.

Figure 2 shows activation of the thioredoxin gene by mugwort aqueous extract in K562 cells. Figure 2 shows activation of the thioredoxin gene by blue disoacetone extract in K562 cells. Figure 3 shows activation of the thioredoxin gene in K562 cells treated with Artemisia extract. Figure 3 shows activation of the thioredoxin gene in K562 cells treated with blue diso extract. Figure 3 shows activation of the thioredoxin gene by mugwort extracts in a homogeneous assay. Figure 3 shows the activation of the thioredoxin gene by blue diso extract in a homogeneous assay. The result by RT-PCR of the induction | guidance | derivation of the thioredoxin mRNA expression by a mugwort extract is shown. The result of Real Time RT-PCR of the induction | guidance | derivation of thioredoxin mRNA expression by a mugwort extract is shown. The result by RT-PCR of the induction | guidance | derivation of thioredoxin mRNA expression by a blue diso extract is shown. The result of Real Time RT-PCR of the induction | guidance | derivation of the thioredoxin mRNA expression by a blue diso extract is shown. Figure 3 shows induction of thioredoxin protein expression by Artemisia extract. Figure 3 shows the induction of thioredoxin protein expression by blue diso extract. The suppression of hydrogen peroxide-induced cytotoxicity by mugwort extract is shown. The absorbance value in 2% TritonX-100 is defined as 100% cell death, and the absorbance value in medium + cells is defined as 0% cell death.

1. Composition for Inducing Thioredoxin Expression The composition for inducing thioredoxin expression of the present invention is one of mugwort, mugwort extract, blue diso extract and blue diso extract (hereinafter sometimes simply referred to as “active ingredient”). Or it contains 2 or more types, It is characterized by the above-mentioned.

  Artemisia princeps is a perennial Asteraceae Artemisia genus plant. Mugwort extract can be obtained by extracting mugwort.

  In addition, blue perilla (Perilla frutescens) is a perennial Lamiaceae plant. A blue diso extract can be obtained by extracting a blue diso.

  In the present invention, when mugwort and / or blue disodium is used as an active ingredient, it is possible to use those that have been dried, those that have been made into a paste, those that have been shredded, and the like.

  As 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 (whole plant) or any part (for example, leaf, Extracts from rhizomes, stems, etc.) can be used.

  The mugwort extract or green disodium extract used in the present invention can be obtained by an extraction method usually used by those skilled in the art. Although it does not specifically limit as an extraction processing method, For example, the method of extracting a mugwort or a blue disodium using water, an organic solvent, or these liquid mixture as an extraction solvent is mentioned.

The solvent used for the extraction is not particularly limited. For example, water, ethanol, methanol, n-propanol, iso-propanol, n-butanol, iso-butanol, primary alcohols such as tert-butanol, ethyl acetate, etc. Examples include lower alkyl esters, hydrocarbons such as benzene and hexane, conventionally known solvents such as acetone and methylene chloride, or mixed solutions 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 ₂ . When performing extraction, the number of extractions may be single, or multiple times in order to increase the yield.

  The extract thus obtained can be used as it is as a mugwort extract or a blue disodium extract, but it is further subjected to a purification process such as deodorization and decoloration within a range not losing the effect of the present invention. Also good. In addition, the extract may be subjected to a separation / purification process such as a treatment with a synthetic adsorbent, a filtration treatment, and a concentration treatment as 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 eluent such as water, ethanol, or a mixed solution thereof is used. The elution method is mentioned. Examples of the synthetic adsorbent include aromatic (cross-linked styrene) synthetic adsorbents, substituted aromatic synthetic adsorbents, and acrylic synthetic adsorbents. In addition, after the above extraction treatment, mugwort extract or green disodium extract is obtained in a liquid form, and water-soluble dietary fibers such as pectin and dextrin are added to the extract as an excipient, and a known method such as a spray drying method is used. The extract may be prepared in powder form by drying by the method. Furthermore, this powdery extract can be used by re-dissolving in a solvent such as water, ethanol, propylene glycol, 1,3-butylene glycol, glycerin.

  In addition, from the viewpoint of enhancing the stability of the components contained in the extract of mogi or blue diso extract, it is desirable that these extracts are in a dry state.

  The composition for inducing thioredoxin expression of the present invention only needs to contain one or more of mugwort, mugwort extract, blue diso, and blue diso 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 can be induced in mammals in the body by being applied to mammals in various forms such as internal use, ingestion, injection, infusion, and the like.

  The applied amount (intake or dose) of the composition for inducing thioredoxin expression of the present invention may be an effective amount for inducing thioredoxin expression, and the form of the composition, the applied form, the type of active ingredient, and the subject It is appropriately selected depending on the age, sex, weight, health condition, other conditions, and the degree of symptoms. As an example, when a mugwort extract and / or blue diso extract is applied to humans as a component, the amount applied is 1 to 12 in total for mugwort extract and / or blue diso 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 composition for inducing thioredoxin expression of the present invention may be applied in a single dose or in several batches.

  In the composition for inducing thioredoxin expression of the present invention, the mixing ratio of the active ingredient can be appropriately set according to the type of the composition, the type of active ingredient to be used, the amount applied per day, and the like. In the case of using mugwort extract and / or blue disodium extract as the active ingredient, as an example of the mixing ratio, mugwort extract and / or blue disodium is used with respect to the total amount of the thioredoxin expression-inducing composition. A ratio in which the extract is 0.1 to 25% by weight in total is mentioned.

  The thioredoxin expression-inducing composition of the present invention is used in the fields of food, medicine, feed and the like. That is, by preparing the thioredoxin expression-inducing composition of the present invention in the form of food, medicine, feed, etc., it becomes possible to provide a food, pharmaceutical composition, and feed capable of inducing thioredoxin expression. . Hereinafter, specific modes in the food, medicine, and feed fields will be described in detail.

By preparing thioredoxin expression inducing composition of the food present invention as a food, a food which is capable of inducing the expression of thioredoxin is provided.

  Examples of the food include food and drink such as nutritional supplements, balanced nutritional foods, health foods, functional nutritional foods, foods for specified health use, and foods for the sick. The method for producing these foods is not particularly limited as long as a retinal protective effect can be obtained. As a suitable specific example of the said foodstuff, the supplement which has forms, such as a powder, a granule, a capsule, a tablet, is illustrated. 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 confectionery such as gum, candy, gummi, tablet confectionery, cookies, cake, chocolate, ice cream, jelly, mousse, pudding, biscuits, cornflakes, chewable tablets, wafers, rice crackers, etc. Drinks such as carbonated drinks, soft drinks, milk drinks, coffee drinks, tea drinks, fruit juice drinks, nutrition drinks, alcoholic drinks and mineral water; powdered drinks such as powdered juice and powdered soup; seasonings such as dressings and sauces Breads; noodles; kneaded products such as kamaboko; sprinkles and the like. Moreover, it is good also as forms for tube | pipe ingestion (liquid food etc.) other than the form for oral ingestion.

  About the content rate of the active ingredient in the said foodstuff, it can adjust suitably according to said application amount per day, the form of foodstuff, the flavor of foodstuff, etc. For example, when a mugwort extract and / or a green disodium extract is used as an active ingredient, the mugwort extract and / or the green disodium extract is usually 0.1 to 25 wt. %, Preferably 0.5 to 10% by weight, and more preferably 1 to 5% by weight. Moreover, in order to express the induction | guidance | derivation induction | guidance | derivation of thioredoxin more effectively, it is good also as a foodstuff containing a high content of mugwort extract and / or a blue diso extract. Thus, when it is set as the foodstuff containing a high content of mugwort extract and / or a blue dissociation extract, a mugwort extract and / or a blue dissociation extract are the total amount with respect to the total amount of food, for example, 20 weight% or more The ratio which becomes 30 to 70 weight% preferably is mentioned.

  Moreover, in the said foodstuff, when using a mugwort extract and / or a blue disodium extract as an active ingredient, you may contain a mugwort and / or a blue disodium itself.

  The said foodstuff can be prepared according to a conventional method according to a food form by mixing the said active ingredient with the support | carrier containing a foodstuff raw material or an additive. Examples of additives include sweeteners, colorants, antioxidants, vitamins, and fragrances.

  The food can induce the expression of thioredoxin in the human body ingested. By the expression of this thioredoxin, for example, improvement of liver disorders including hangover etc., improvement of menopause, reduction of blood cholesterol, reduction of blood neutral lipid, reduction of blood glucose level, reduction of blood pressure, prevention of arteriosclerosis, It is expected to prevent aging. Therefore, the food has a high cholesterol level, a high blood pressure level (for example, a normal high blood pressure level, a mild hypertension level), a person who starts to worry about liver function, and a blood glucose level. It can be applied to a person who is afraid of aging or physical aging.

  For example, arteriosclerosis is caused by accumulation of lipid peroxide, which is a kind of reactive oxygen species. By ingesting the food, the expression of thioredoxin is induced, and it is expected that atherosclerosis is improved by thioredoxin itself or the reactive oxygen species elimination action of thioredoxin-dependent peroxidase.

  In addition, it is considered that the excessive increase in blood glucose level is caused by the involvement of injury due to oxidative stress in insulin-producing cells of the pancreatic islet. Ingestion of the food induces the expression of thioredoxin, and the blood glucose level is expected to be reduced by inhibiting the injury of insulin-producing cells by the action of thioredoxin itself or the reactive oxygen species elimination action of thioredoxin-dependent peroxidase. .

  Furthermore, it induces the expression of thioredoxin in the food and the body and exerts an antioxidant effect, which helps to eliminate (oxidation) stress and maintain the balance of body oxidation / reduction (redox). It can be suitably applied to people who become.

  Moreover, by using the food as food for patients with arteriosclerosis, diabetes, liver disease, allergies, etc., there is a possibility that these diseases can be effectively treated or improved.

  Furthermore, the food can be used for the purpose of nutrition tonic, recovery from fatigue, and the like.

Pharmaceutical Composition The composition for inducing thioredoxin expression of the present invention is prepared 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 incorporated in the pharmaceutical composition for protecting the retina include binders, disintegrants, surfactants, absorption enhancers, humectants, adsorbents, lubricants, fillers, extenders, moisturizers, antiseptics Illustrative agents, stabilizers, emulsifiers, solubilizers, diluents or excipients such as salts for adjusting osmotic pressure, buffering agents, etc., can be selected and used as appropriate according to the dosage unit form of the resulting preparation. The reticulated thioredoxin expression-inducing pharmaceutical composition may contain additives such as colorants, preservatives, fragrances, flavors, sweeteners, and other pharmacologically active ingredients as necessary.

  The pharmaceutical composition for inducing thioredoxin expression is used in a pharmaceutical form such as an internal preparation; an injection such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection; an infusion.

  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, suspensions And liquid preparations such as agents.

  When the pharmaceutical composition for inducing thioredoxin expression is an injection such as a solution, emulsion, suspension, etc., these are preferably sterilized and isotonic with blood, and when formulated into these dosage forms As the diluent, 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. 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 and the like may be added.

  Further, when the pharmaceutical composition for inducing thioredoxin expression is a liquid preparation, it may be stored in a state such as cryopreservation or freeze-drying. In the case of a freeze-dried state, distilled water for injection is added at the time of use and dissolved 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, whereby various useful physiological activities can be exhibited. Specifically, the pharmaceutical composition can improve menopause, lower blood cholesterol, lower blood neutral lipids, lower blood sugar levels, lower blood pressure, prevent aging, prevent or treat arteriosclerosis, prevent diabetes Alternatively, it is useful for treatment, 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, for preventing or treating diabetes, or for preventing or treating liver disease.

Feed 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. The feed includes pet food for mammals in addition to feed for livestock and poultry.

  The said feed is prepared by preparing the said active ingredient with a feed raw material and a feed additive, and is prepared. 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. Moreover, the blending 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, and the like.

  The feed can reduce the influence of oxidative stress by inducing the expression of thioredoxin in the body of the ingested animal. Therefore, the feed is useful for promoting healthy growth of animals and maintaining a healthy state.

2. Method for Inducing Expression of Thioredoxin The present invention further relates to a milk in which at least one effective amount selected from the group consisting of mugwort, mugwort extract, blue diso and blue diso extract is desired to induce the expression of thioredoxin. Provided is a method for inducing thioredoxin expression, which comprises administering or ingesting an animal.

  In the method, the mammal includes a human. Moreover, in the said improvement method, the kind of active ingredient to be used, these administration or intake effective amount, the administration or frequency | count of application per day, etc. are as having mentioned above. In addition, the method includes the improvement of climacteric disorder, reduction of blood cholesterol, reduction of blood neutral lipid, blood glucose level lowering agent, reduction of blood pressure, prevention of aging, prevention or treatment of arteriosclerosis, prevention of diabetes or It is useful for treatment, prevention or treatment of liver disease, prevention or treatment of allergic diseases, and the like.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these Examples.
Example 1

(1) Preparation of Artemisia extract / Blue diso extract I
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.

  The blue diso filter cake obtained by filtration was lyophilized, dissolved in 60 volumes of acetone, filtered through a thin film filter, and dried in an evaporator at 30 ° C. The dried material was dissolved in 1 ml ethanol (acetone extract) and used for analysis.

  The sulforaphane used throughout this example was purchased from LTK laboratories Inc. (product number: S8046) and used at a concentration of 10 μM. The present inventors have confirmed that sulforaphane induces the expression of thioredoxin.

(2) Cultured cells and culture system Human erythroblast-like leukemia cell line K562 was inactivated by heat treatment with 10% fetal calf serum (FCS) and antibiotics (100 IU / ml penicillin and 100 (g / ml streptomycin). ) In RPMI1640 medium (Life Technologies) supplemented with 37) under humid conditions of 37 ° C. and 5% CO ₂ .

(3) Plasmid
The pTRXCAT plasmid was generated as described by Taniguchi Y. et al., Nucleic Acids Res 1996; 24: 2746-2752. The HindIII-BamHI insert from the pTRXCAT vector was subcloned into pBluescriptII KS (+) (pTRXblue vector). The pTRX-Luc vector used in this example was prepared according to the method described in Kim YC. J Biol Chem 2001; 276, 18399-18406. And Kim YC. Et al., Oncogene 2003; 22: 1860-1865 did. The pTRX (-1148) -Luc vector was prepared by ligating the KpnI / BamHI fragment of the pTRXblue vector to the KpnI / BglII site of the pGL3 basic vector (Promega).

  The nucleotide sequences of all the constructs used in the examples were confirmed using Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Pharimacia).

(4) Transfection and luciferase assay
Using DMRIEC (manufactured by GIBCO), K562 cells were transfected with a luciferase reporter expression vector or a pGL3-basic vector (control) according to the instructions for use. After 4 hours of incubation, K562 cells transfected with each vector were treated with DMSO (final concentration 0.1%), 10 μM sulforaphane, blue diso acetone extract (final concentration 1%) or mugwort aqueous extract (final concentration 3%). In addition, 24 hours.

  To level the effect of transfection, Renilla luciferase gene expression was monitored using pRL-TK (Promega). Luciferase gene expression normalized by Renilla luciferase activity was analyzed 24 hours later using an assay kit (Promega). The assay was performed twice.

  According to the method described in the above (1) to (4), it was analyzed whether a vegetable extract other than the Brassicaceae plant induces 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.

Artemisia aqueous extract showed about 7.61-fold activation of thioredoxin gene expression relative to control (FIG. 1A). Moreover, the acetone extract of blue diso showed about 6.14 times the activation of thioredoxin gene expression compared to the control (FIG. 1B).
Example 2

  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.

  The pTRX-3xARE-Luc vector is a 3xARE oligonucleotide inserted into the KpnI-NheI site of the pGL3 promoter vector.

The oligonucleotides used for vector construction are as follows.
ARE: FW; 5'-AATTCGGTCACCGTTACTCAGCACTGGTCACCGTTACTCAGCACTGGTCACCGTTACTCAGCACTG-3 '(SEQ ID NO: 1)
ReV; 5'-TCGACAGTGCTGAGTAACGGTGACCAGTGCTGAGTAACGGTGACCAGTGCTGAGTAACGGTGACCG-3 '(SEQ ID NO: 2)
[Preparation of Artemisia extract / Blue diso extract II]
Mugwort or blue disodium was homogenized at 10000 rpm for 5 minutes with the addition of 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 Corporation) 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 / 100 mL). In Examples 2 to 6, a mugwort extract or blue diso extract prepared by this method was used.

  In order to test whether the degree of thioredoxin gene activation differs depending on the extract, a luciferase reporter assay was performed by the method described in Example 1 (4) using pTRX-3xARE-WT-Luc.

  Artemisia 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.

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.
Example 3

  To further test for thioredoxin activation by mugwort and blue diso extracts, the luciferase gene under the control of the thioredoxin promoter was observed by the homogeneous assay described below.

[Homogeneous assay]
K562 cells expressing the luciferase gene under the control of the thioredoxin promoter were prepared by transfection with pTRX (-1148) -Luc and pMAM2-BSD, followed by selection with blasticidin S and limiting dilution. Cells are plated in 96-well plates at a concentration of 5 × 10 ⁴ cells / well and DMSO (final concentration 0.1%), sulforaphane (10 mM), ethanol (0.2%), mugwort extract or blue diso extract ( The extract obtained in [preparation of Artemisia extract / blue diso extract II]) was added to a final concentration of 0.05, 0.1 or 0.2%, respectively, and after incubation for 24 hours, the luciferase gene Expression was analyzed using Bright-Gloluciferase assay kit (Promega). The assay was performed twice.

Mugwort extract (FIG. 3A) or blue diso extract (FIG. 3B) all activated the thioredoxin gene in a dose-dependent manner.
Example 4

  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.

(1) RT-PCR
Cells were treated with ethanol (final concentration 0.1%), mugwort extract or blue diso extract (final concentration 0.1% respectively). After 24 hours, cells were harvested and washed with phosphate buffer solution (PBS). Total RNA was isolated using 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. The RT condition was 30 minutes at 48 ° C.

  Oligonucleotide forward (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) for human thioredoxin 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. Amplification of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

(2) Real Time RT-PCR
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), sulforaphane (10 mM) were used, and DMSO (final concentration 0.1%) and ethanol (final concentration 0.1%) were used as controls. According to the instructions for use, an oligonucleotide forward (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6) and a TaqMan probe (SEQ ID NO: 7) for human thioredoxin were designed. Using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems), quantitative Real Time RT-PCR was performed in a 96-well plate using ABI PRISM7000. Each reaction mixture contained 50 ng total RNA in a final volume of 50 μl.

  RT-PCR conditions: The RT step was performed at 48 ° C. for 30 minutes, the PCR step was performed at 95 ° C. for 15 seconds and 60 ° C. for 1 minute for 40 cycles. TaqMan Ribosomal Control Reagents (Applied Biosystems) was used, and 18S rRNA was amplified as an internal control assay to standardize the amount of thioredoxin gene.

Thioredoxin mRNA levels were significantly increased 24 hours after treatment of K562 cells with mugwort extract (FIGS. 4A and 4B) or blue diso extract (FIGS. 4C and 4D).
Example 5

  The effect of mugwort extract or blue diso extract on thioredoxin protein expression was observed by the method of Western blot analysis described below.

[Western blot analysis]
Cells were treated with mugwort or blue diso extract (1% final concentration, respectively) and ethanol (final concentration 1%). After 48 hours, the cells were collected and washed with ice-cold phosphate buffered saline (PBS), then on ice for 30 minutes, solubilizing solution (10 mM Tris-HCI (pH 7.4), 150 mM) Lysed with NaCl, 1% NP-40, 1 mM EDTA, 0.1 mM PMSF, 8 (g / ml aprotinin and 2 (g / ml leupeptin) This cell lysate was purified by centrifugation. Was separated at 95 ° C. for 5 minutes, and then separated by 15% SDS-polyacrylamide gel electrophoresis (cell lysate: 2.5 μg) The separated protein was separated from polyvinylidene difluoride membrane (PVDF membrane: This membrane was treated overnight with 10% (weight / volume) skim milk dissolved in T-PBS (PBS containing 0.05% Tween20) and anti-human thioredoxin monoclonal antibody (Redox Biosciences) For 1 hour, followed by Peru Anti-mouse IgG conjugated with oxidase.: It was incubated for 1 hour at (1 5000 dilution) (Amersham Pharmacia Biotech) after which the epitope were visualized ELC Western blot detection kit (Amersham Pharmacia Biotech).

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

  Since thioredoxin is an important protein for oxidative stress, we observed whether induction of thioredoxin expression by mugwort extract can suppress oxidative stress-induced injury in K562 cells. According to the method of LDH assay described below, the effect of pretreatment with mugwort extract on LDH release from injured cells was analyzed.

[LDH assay]
K562 cells were plated in 96-well plates at a concentration of 1 × 10 ⁴ cells / well. Thereafter, the cells were treated with 2% TritonX-100 (this value is converted as 100% cytotoxicity), DMSO (final concentration 0.1%), sulforaphane (10 μM), ethanol (0.2%) or mugwort extract (final concentration 0.05, 0.1 or 0.2%). After 48 hours incubation at 37 ° C., the cells were treated with 200 μM H ₂ O ₂ for 48 hours. Using LDH assay kit (Roche), LDH in a sample of 50 μl was measured according to the instructions for use.

  As shown in FIG. 6, the cells that had been pretreated for 48 hours with mugwort extract at a final concentration of 0.05, 0.1, or 0.2% were remarkably suppressed in the cytotoxicity caused by hydrogen peroxide as compared with the control.

  Therefore, it was suggested that the induction of thioredoxin by pretreatment of mugwort extract acts to protect cells against oxidative stress caused by hydrogen peroxide.

  In addition, it is expected that cytotoxicity caused by hydrogen peroxide is remarkably suppressed in cells subjected to the same test using a blue diso extract as compared with the control.

  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 an oligonucleotide forward primer used in Real Time RT-PCR.

  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.

Claims (12)

  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.   A composition for inducing thioredoxin expression comprising a mugwort extract and / or a blue diso extract.   The composition for inducing thioredoxin expression according to claim 1, which is used for alleviating oxidative stress.   The composition for inducing thioredoxin expression according to claim 2, wherein the total amount of mugwort extract and / or blue diso extract is 0.1 to 25% by weight based on the total amount of the composition.   The composition for inducing thioredoxin expression according to claim 1, which is a food.   The composition for inducing thioredoxin expression according to claim 1, which is a pharmaceutical composition.   The composition for inducing thioredoxin expression according to claim 1, which is an animal feed.   A method for producing a food for inducing thioredoxin expression, comprising adding a mugwort extract and / or a blue diso extract in an amount of 0.1 to 25% by weight in the food.   A method for inducing 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.   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.   Use of mugwort extract and / or blue diso extract for producing a composition for inducing thioredoxin expression.   Use of mugwort extract and / or blue diso extract for the production of a pharmaceutical composition for inducing thioredoxin expression.