PPAR activity expression agent and method for producing the same

Abstract

To provide a PPAR activity expression agent using roasted wheat, a lipid abnormality prevention / improvement agent, a fatness prevention / improvement agent, a diabetes prevention / improvement agent and production methods thereof.SOLUTION: A PPAR activity expression agent includes, as an active ingredient, roasted wheat whose L value is 60 or smaller or an extract thereof. A lipid abnormality prevention / improvement agent includes, as an active ingredient of PPAR-α activity, roasted wheat whose L value is 60 or smaller or an extract thereof. A fatness prevention / improvement agent includes, as an active ingredient of PPAR-δ activity, roasted wheat whose L value is 60 or smaller or an extract thereof. A diabetes prevention / improvement agent includes, as an active ingredient of PPAR-γ activity, roasted wheat whose L value is 60 or smaller or an extract thereof. A production method of PPAR activity expression agent has a roasting step for roasting the wheat for acquiring the roasted wheat whose L value is 60 or smaller.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent for expressing PPAR activity, an agent for preventing and improving dyslipidemia, an agent for preventing and improving obesity, an agent for preventing and improving diabetes, and methods for producing them. More specifically, the present invention relates to an agent for expressing PPAR activity, an agent for preventing and improving dyslipidemia, an agent for preventing and improving obesity, an agent for preventing and improving diabetes, and methods for producing them, each of which contains roasted barley or an extract thereof as an active ingredient. [Background technology]

[0002] The main form of consumption of staple grains such as rice, wheat, and corn is to eat their starch. In this form of consumption, they are usually heated to make them easier to eat, but they are often heated together with water for the purpose of promoting gelatinization. On the other hand, roasting staple grains and eating them is almost unknown. In Japan, there has been a long-standing culture of eating barley tea and barley tea, which are liquids extracted from roasted barley by boiling them in hot water. In this way, it can be said that it is extremely rare to use barley tea with the aim of ingesting the liquid extracted from roasted barley, rather than the starch of the staple grains. And, despite this long-standing form of intake, there are no known documents that have examined the biological effects of roasted barley from a scientific perspective. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2013-159579 A [Patent Document 2] JP 2010-150185 A [Patent Document 3] JP 2002-095443 A Summary of the Invention [Problem to be solved by the invention]

[0004] On the other hand, the above-mentioned main grains are also used for brewing, and since the purpose of this brewing is to utilize sugar, a process of saccharifying the starch contained in the main grains is required. For example, in rice brewing, this role is played by koji mold. In this regard, wheat and barley originally contain inactive saccharifying enzymes inside, and these saccharifying enzymes are activated by germination, so they are used as malt in brewing. For this reason, as with malt, there are known documents that examine its biological effects from a scientific perspective, such as the above patent document. However, as mentioned above, the biological effects of roasted barley, which is made by roasting ungerminated barley, are not known.

[0005] The present inventors conducted research on roasted barley and found that it has a unique biological effect, leading to the completion of the present invention. That is, the present invention aims to provide a PPAR activity expression agent, a lipid abnormality prevention and improvement agent, an obesity prevention and improvement agent, and a diabetes prevention and improvement agent, and a method for producing the same, using roasted barley. [Means for solving the problem]

[0006] That is, the present invention is as follows. 1. An agent for expressing PPAR activity, characterized in that the active ingredient is roasted barley or an extract thereof having an L value of 60 or less. 2. The agent for expressing PPAR activity according to 1 above, wherein the extract is an extract of water and / or an organic solvent. 3. The agent for expressing PPAR activity according to 1 or 2 above, wherein the wheat is barley. 4. An agent for preventing and improving dyslipidemia, characterized in that roasted barley or an extract thereof having an L value of 60 or less is an active ingredient with PPAR-α activity. 5. The agent for preventing and improving dyslipidemia according to 4 above, wherein the extract is an extract of water and / or an organic solvent. 6. The agent for preventing and improving dyslipidemia according to 4. or 5., wherein the wheat is barley. 7. An agent for preventing and improving obesity, characterized in that roasted barley or an extract thereof having an L value of 60 or less is an active ingredient with PPAR-δ activity. 8. The extract is water and / or organic 8. The agent for preventing and improving obesity according to 7 above, which is an extract of an organic solvent. 9. The obesity preventing and ameliorating agent according to 7 or 8 above, wherein the wheat is barley. 10. A diabetes prevention and improvement agent, characterized in that roasted barley or an extract thereof having an L value of 60 or less is an active ingredient for PPAR-γ activity. 11. The diabetes prevention and amelioration agent according to claim 10, wherein the extract is an extract of water and / or an organic solvent. 12. The diabetes prevention and amelioration agent according to claim 10 or 11, wherein the wheat is barley. 13. A method for producing the agent for expressing PPAR activity according to 1., comprising the steps of: A method for producing a PPAR activity-expressing agent, comprising a roasting step of roasting barley to obtain roasted barley having an L value of 60 or less. 14. A method for producing the agent for preventing and improving lipid abnormalities described in 4., comprising the steps of: A method for producing an agent for preventing and improving lipid abnormalities, comprising a roasting step of roasting barley to obtain roasted barley having an L value of 60 or less. 15. A method for producing the obesity prevention and amelioration agent according to the above item 7, comprising the steps of: A method for producing an obesity prevention and improvement agent, comprising a roasting step of roasting barley to obtain roasted barley having an L value of 60 or less. 16. A method for producing the diabetes prevention and amelioration agent according to claim 10, comprising the steps of: A method for producing a diabetes prevention and amelioration agent, comprising a roasting step of roasting barley to obtain roasted barley having an L value of 60 or less. Effect of the Invention

[0007] According to the agent for expressing PPAR activity of the present invention, PPAR activity can be promoted by using roasted barley or an extract thereof. Moreover, according to the method for producing the agent for expressing PPAR activity of the present invention, such an agent for expressing PPAR activity can be produced. According to the agent for preventing and improving dyslipidemia of the present invention, PPAR-α activity can be promoted by using roasted barley or an extract thereof, which can prevent and / or improve dyslipidemia. Also, according to the method for producing the agent for preventing and improving dyslipidemia of the present invention, such an agent for preventing and improving dyslipidemia can be produced. According to the obesity prevention and improvement agent of the present invention, PPAR-δ activity can be promoted by using roasted barley or its extract, and as a result, obesity can be prevented and / or improved. In addition, according to the method for producing the obesity prevention and improvement agent of the present invention, such an obesity prevention and improvement agent can be produced. According to the diabetes prevention and amelioration agent of the present invention, PPAR-γ activity can be promoted by using roasted barley or its extract, and as a result, diabetes can be prevented and / or ameliorated. Also, according to the method for producing the diabetes prevention and amelioration agent of the present invention, such a diabetes prevention and amelioration agent can be produced. [Brief description of the drawings]

[0008] [Figure 1] FIG. 2 is a graph showing PPAR-α activity in the examples of the present invention. [Diagram 2] FIG. 2 is a graph showing PPAR-β / δ activity in the examples of the present invention. [Diagram 3] FIG. 2 is a graph showing PPAR-γ activity in the examples of the present invention. [Figure 4] FIG. 2 is a graph showing the activity of each PPAR in the examples of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [1] PPAR activation agents The agent for expressing PPAR activity of the present invention is characterized in that it contains roasted barley or an extract thereof having an L value of 60 or less as an active ingredient.

[0010] The roasted barley is barley that has been roasted, and refers to barley that has been heat-treated. The type of barley is not particularly limited. For example, seeds of cereals of the Gramineae family such as barley, wheat, rye, oats, and oats can be used. These may be used alone or in combination of two or more types. Of these, barley is preferred. In addition, examples of types of barley include two-rowed barley and six-rowed barley, etc. These may be used alone or in combination of two or more types. Further, examples of two-rowed barley include varieties such as Hindmarsh, Metcalf, Scope, Commander, Houshun, and Mikamo Golden. These may be used alone or in combination of two or more. Examples of Rokujo barley include Legacy, Shunrai, Fiber Snow, Kashima Goal, etc. These may be used alone or in combination of two or more.

[0011] The part of the barley to be roasted is not limited. That is, it is sufficient if at least the aleurone layer, endosperm, germ and part of the husk are included, but it is preferable that the barley is in a state in which the husk is attached. That is, seeds (whole) in which the aleurone layer, endosperm, germ and the like are wrapped in the husk are preferable. However, the barley to be roasted may be used as it is, or may be used after being broken into smaller pieces by crushing, pulverizing or the like. Furthermore, it is also acceptable to use processed barley by soaking in water or enzyme processing.

[0012] As the roasting method for obtaining the roasted barley, a general roasting method can be adopted, and an appropriate roasting method (roasting time, roasting temperature, etc.) may be selected to obtain the desired roasted state. As the general roasting method, methods known to those skilled in the art, such as hot air roasting, direct fire roasting, sand roasting, far infrared roasting, open kettle roasting, rotary drum roasting, and medium roasting, can be adopted. These may be used alone or in combination of two or more.

[0013] The L value of roasted barley can be adjusted by adjusting the roasting conditions such as the roasting intensity of the temperature and roasting time. As an example of the roasting conditions, when a semi-hot air roasting method is adopted, it is preferable to take out 250 g of barley and roast it at a product temperature of 200 to 220° C. for 12 to 20 minutes, and the roasting temperature and roasting time can be appropriately adjusted so as to obtain the desired L value.

[0014] The roasted barley of the present invention is barley that has been subjected to a roasting treatment. In the present invention, PPAR activity is exerted by applying heat to barley. That is, roasted barley obtained by heat-treating barley (roasting treatment) has a higher PPAR activity than unroasted barley. The heating action of the roasting process causes browning reactions such as the Maillard reaction and the caramel reaction, resulting in browning of the barley. The heating state of the barley can be grasped from the browning state, and the browning state can be identified by measuring the L value of the roasted barley. By measuring the L value, the roasting state of the barley can be confirmed, and the improvement of the PPAR activity can be grasped. As the roasting progresses and the L value decreases, the PPAR activity tends to improve. That is, although PPAR activity is not substantially observed in unroasted barley (see the examples described below), roasting expresses PPAR activity, and as the L value decreases, more dominant PPAR activity is obtained. In addition, it is usually considered difficult to brown the barley to about L30 by only the Maillard reaction. Therefore, it is considered that the caramelization reaction caused by roasting contributes to the above-mentioned action. The L value can be measured by a known method using a color difference meter.

[0015] The L value of the roasted barley of the present invention is 60 or less (usually 10 or more), so that excellent PPAR activity can be obtained. In particular, when L≦60 (usually L≧10), excellent PPAR-α activity and excellent PPAR-β / δ activity can be obtained. In particular, in the PPAR-α activity, activity exceeding that of WY14643 can be obtained. In addition, when L≦55 (usually L≧10), in the PPAR-β / δ activity, activity exceeding that of WG0742 can be obtained. Furthermore, when L≦45 (usually L≧10), PPAR-α activity and PPAR-β / δ activity that are even more excellent than those described above can be obtained. Furthermore, when L≦25 (usually L≧10), in addition to excellent PPAR-α activity and excellent PPAR-β / δ activity, excellent PPAR-γ activity can be obtained. In particular, when L≦25 (usually L≧10), in the PPAR-γ activity, activity exceeding that of troglitazone can be obtained.

[0016] The roasted barley may be used as seeds or as a powdered product obtained by pulverizing the seeds. Also, an extract may be obtained from the roasted barley and used. When obtaining an extract, the roasted barley may be subjected to the extraction step as seeds or may be crushed and then subjected to the extraction step.

[0017] When obtaining an extract, water, steam, a mixed solvent of an organic solvent, water and an organic solvent (aqueous organic solvent) can be used. As the organic solvent, either a polar organic solvent or a non-polar organic solvent can be used. In addition, specific examples of the organic solvent include monohydric, dihydric or polyhydric alcohols, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, linear or cyclic ethers such as tetrahydrofuran and diethyl ether, polyethers such as polyethylene glycol, saturated or unsaturated hydrocarbons, aromatic hydrocarbons such as benzene and toluene, halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane and carbon tetrachloride, pyridines, dimethyl sulfoxide, acetonitrile, carbon dioxide, supercritical carbon dioxide, oils and fats, waxes, and other oils, among which alcohols, ketones, saturated hydrocarbons, halogenated hydrocarbons and dimethyl sulfoxide are preferred.

[0018] The alcohols are not particularly limited, but examples thereof include monohydric alcohols such as methanol, ethanol, propanol, butanol, amyl alcohol, hexanol, heptanol, and octanol; dihydric alcohols such as 1,3-butylene glycol, ethylene glycol, propylene glycol, 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol; and trihydric or higher alcohols such as glycerin. Of these, monohydric alcohols are preferred.

[0019] Examples of the saturated hydrocarbons include linear, branched, or cyclic saturated hydrocarbons. For example, linear saturated hydrocarbons such as methane, ethane, propane, n-butane, n-pentane, n-hexane, n-heptane, n-octane, n-nonane, and n-decane; branched saturated hydrocarbons such as 2-methylbutane, 2,2-dimethylpropane, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, 2-methylhexane, 3-methylhexane, and 2,2,4-trimethylpentane; and cyclic saturated hydrocarbons such as cyclopentane, cyclohexane, and cycloheptane. Of these, linear saturated hydrocarbons are preferred.

[0020] The organic solvents can be used alone or in a mixture of two or more kinds. The organic solvent may be a water-containing aqueous solvent. Examples of organic solvents used in the water-containing organic solvent include hydrophilic organic solvents. Examples of hydrophilic organic solvents include, but are not limited to, the above-mentioned alcohols, acetic acid, pyridines, and other protic hydrophilic organic solvents, and ketones, acetonitrile, dimethyl sulfoxide, and other aprotic hydrophilic organic solvents, of which protic hydrophilic organic solvents are preferred. Among these, the above-mentioned alcohols, the above-mentioned monohydric and dihydric alcohols, alcohols having 1 to 4 carbon atoms, and ethanol and 1,3-butylene glycol are more preferred. These solvents can be used alone or in a mixture of two or more kinds to form a mixed liquid.

[0021] The water content in the water-containing organic solvent is not particularly limited, but for example, when the organic solvent is an alcohol, the water content can be 30% by volume or less, 10% by volume or less, or 5% by volume or less. For example, the concentration of the hydrophilic organic solvent in the water-containing organic solvent can be at least 70% by volume of the hydrophilic organic solvent.

[0022] The extraction means in the present invention is not particularly limited, and known methods can be used. For example, immersion extraction, stirring extraction, reflux extraction, shaking extraction, ultrasonic extraction, etc. can be used. Only one of these extraction methods may be used, or two or more of them may be used in combination. Furthermore, the extraction operation may be performed once, or may be performed multiple times (repeating the extraction residue obtained after the extraction operation multiple times). In this case, a batch extractor, Soxhlet extractor, etc. may be used.

[0023] The extraction conditions are not particularly limited, but the extraction temperature may be 0 to 150°C, preferably 40 to 100°C, and more preferably 40 to 80°C. The extraction time may be appropriately selected as desired, but can usually be 10 minutes to 24 hours. Furthermore, when extraction is performed with water or steam, the water is preferably warm water or boiling water, and the temperature is usually appropriately selected from 50 to 100° C. Furthermore, high-temperature, high-pressure steam can be used instead of warm water or boiling water.

[0024] The obtained roasted barley extract can be used as it is, but can also be further diluted, concentrated, freeze-dried, etc., and / or prepared into a liquid, powder, or paste form.

[0025] The roasted barley or its extract having an L value of 60 or less according to the present invention is an active ingredient of an agent for expressing PPAR activity. The agent for expressing PPAR activity according to the present invention has an effect of enhancing the transcriptional activity of genes dependent on PPAR-α, PPAR-β / δ, and PPAR-γ. PPAR-α, PPAR-β / δ, and PPAR-γ are widely involved in the maintenance of energy metabolism and homeostasis in living organisms, and in particular, gene expression of β-oxidation-related enzymes important for lipid metabolism is strongly dependent on the activation of PPAR-α and / or PPAR-β / δ, and it is known that insulin resistance and diabetes can be improved by induction of adipocyte differentiation by a ligand of PPAR-γ. Therefore, the roasted barley or extract thereof according to the present invention having an L value of 60 or less has an activating effect on PPAR-α, PPAR-β / δ, and PPAR-γ, and is therefore effective as an agent for expressing the activity of any of PPAR-α, PPAR-β / δ, and PPAR-γ, as an agent for preventing and improving dyslipidemia, as an agent for preventing and improving obesity, as an agent for preventing and improving diabetes, etc.

[0026] [2] Agents for preventing and improving dyslipidemia, obesity, and diabetes As described above, the roasted barley or extract thereof having an L value of 60 or less according to the present invention has an activating effect on PPAR-α, PPAR-β / δ, and PPAR-γ, and can therefore be used as an agent for expressing PPAR activity for any of PPAR-α, PPAR-β / δ, and PPAR-γ, and can further be used as an agent for preventing and improving dyslipidemia, obesity, and diabetes. The above-mentioned dyslipidemia prevention and improvement agents include dyslipidemia prevention agents that exert a dyslipidemia prevention effect, dyslipidemia improvement agents that exert a dyslipidemia improvement effect, and dyslipidemia prevention and improvement agents that exert both dyslipidemia prevention and dyslipidemia improvement effects. Similarly, obesity prevention and improvement agents include obesity prevention agents that exert an obesity prevention effect, obesity improvement agents that exert an obesity improvement effect, and obesity prevention and improvement agents that exert both obesity prevention and obesity improvement effects. Furthermore, diabetes prevention and improvement agents include diabetes prevention agents that exert a diabetes prevention effect, diabetes improvement agents that exert a diabetes improvement effect, and obesity prevention and improvement agents that exert both diabetes prevention and diabetes improvement effects.

[0027] [3] Usage of the present invention The PPAR activity-expressing agent, dyslipidemia prevention / improvement agent, obesity prevention / improvement agent, and diabetes prevention / improvement agent of the present invention, as well as substances containing these (hereinafter, simply referred to as "PPAR activity-expressing agents", etc.) can be used as medicines, as well as foods and beverages such as functional foods, foods for the sick, and foods for specified health uses. The medicines include pharmaceuticals and quasi-drugs. When used as medicines, they can be in the form of tablets, pills, capsules, powders, granules, syrups, injections, suppositories, etc. Furthermore, the PPAR activity-inducing agent of the present invention can be added to various beverages and foods to impart PPAR activity to the beverages and foods. That is, the PPAR activity-inducing agent of the present invention can be used as an additive for food and beverage products to impart PPAR activity to beverages and foods.

[0028] In addition, the PPAR activity expression agent of the present invention may contain other components other than roasted barley or its extract having an L value of 60 or less, as long as the effect of the present invention is not inhibited. Examples of other components include solvents, dispersants, molding agents, binders, disintegrants, disintegration inhibitors, lubricants, carriers, bulking agents, isotonic agents, emulsifiers, suspending agents, thickeners, coating agents, absorption promoters, coagulants, preservatives (stabilizers, moisture-proofing agents, coloring inhibitors, antioxidants, etc.), flavoring agents, odorants, coloring agents, antifoaming agents, soothing agents, antistatic agents, and pH regulators. These may be used alone or in combination of two or more.

[0029] The PPAR activity-expressing agent of the present invention can be used as it is or by being added to food and drink. For example, the PPAR activity-expressing agent of the present invention may be added directly to a beverage, or may be added to the raw materials of solid foods such as biscuits, semi-liquid foods such as cream and jam, and gel foods, and can be provided as PPAR activity-expressing foods and drinks, foods and drinks for preventing and improving dyslipidemia, foods and drinks for preventing and improving obesity, and foods and drinks for preventing and improving diabetes, etc., containing the PPAR activity-expressing agent of the present invention. In addition, the PPAR activity-expressing agent of the present invention can be dissolved in, for example, water, oils and fats, alcohols such as ethanol, propylene glycol, and glycerin, and mixtures thereof, and then added to a beverage, or to a solid food, semi-liquid food, or gel food. Furthermore, gum arabic and dextrin, which act as binders, can be added as needed to form granules, etc., which can be added to a beverage, or to a solid food, semi-liquid food, or gel food. More specifically, the food and drink containing the PPAR activity-expressing agent of the present invention may be a food and drink that has a specific health effect, or a functional food and drink that utilizes the function of a bioregulatory component, etc. Specific examples of the food and drink include alcohol, carbonated drinks, fruit drinks, coffee, black tea, tea, lactic acid bacteria drinks, yogurt, ice cream, candy, gum, confectionery, bread, noodles, etc.

[0030] [4] Method for producing PPAR activation agent The method for producing an agent for expressing PPAR activity of the present invention is characterized by comprising a roasting step of roasting barley to obtain roasted barley having an L value of 60 or less. The barley used to obtain roasted barley in the production method of the present invention can be the same as the barley described above in relation to the PPAR activity expression agent.

[0031] The above-mentioned barley to be subjected to the roasting process can be used as it is after harvesting. Furthermore, for example, barley washed with water, dried in the sun or dried with hot air at an appropriate temperature can be used. These can be subjected to the roasting process as they are, but they may be pretreated to remove impurities, etc. Also, they can be subjected to the roasting process after being appropriately crushed. For the roasting in the roasting step, the explanation for barley in the above-mentioned PPAR activity expression agent can be applied.

[0032] [5] Method for producing an agent for preventing and improving dyslipidemia, an agent for preventing and improving obesity, and an agent for preventing and improving diabetes The methods for producing the agent for preventing and ameliorating dyslipidemia, the agent for preventing and ameliorating obesity, and the agent for preventing and ameliorating diabetes of the present invention are similar to the methods for producing the above-mentioned agent for expressing PPAR activity, and the explanation for the method for producing the agent for expressing PPAR activity can be applied thereto. EXAMPLES

[0033] The present invention will be described below based on examples, but the present invention is not limited to these examples. [Experimental Examples 1-5] [1] Preparation of roasted barley Barley (variety Kashimagol) was roasted by semi-hot air roasting at different roasting intensities (roasting temperature, roasting time) as follows to obtain roasted barley. The L value of the obtained roasted barley was measured using a color difference meter (manufactured by Nippon Denshoku Industries Co., Ltd., product name: Spectrocolorimeter, model name: SE-6000). The L values ​​of the examples and comparative examples are shown in Table 1.

[0034] <Example 1> 250g of barley (Kashima Goal variety) was placed in a rotating drum type semi-hot air roaster (manufactured by Fuji Coffee Machine Co., Ltd., product name Coffee Roaster, model name COFFEE DISCOVERY) and roasted under the following conditions: product temperature x roasting time 195℃ x 12 minutes 30 seconds to obtain roasted barley. The L value of this roasted barley was 50.0.

[0035] <Example 2> Except for roasting at 200°C for 17 minutes, roasted barley was obtained in the same manner as in Example 1. The L value of this roasted barley was 44.5.

[0036] <Example 3> Except for roasting at 210°C for 14 minutes and 30 seconds, roasted barley was obtained in the same manner as in Example 1. The L value of this roasted barley was 41.3.

[0037] <Example 4> Roasted barley was obtained in the same manner as in Example 1, except that two-stage roasting was performed: first roasting at 220-250°C for 1-2 minutes and second roasting at 260-290°C for 1-2 minutes. The L value of this roasted barley was 30.0.

[0038] <Example 5> Except for roasting at 247°C for 20 minutes and 00 seconds, roasted barley was obtained in the same manner as in Example 1. The L value of this roasted barley was 17.7.

[0039] [Table 1]

[0040] [2] Pretreatment for PPAR activity confirmation test The roasted barley of Experimental Examples 1 to 5 obtained by the above [1] Preparation of roasted barley were each individually treated as follows and subjected to the PPAR activity confirmation test described later. Roasted barley and unroasted barley were ground in a coffee mill (LaboNext, high-speed mill, HS-20) to obtain a ground product. Next, 4 g of the ground product was collected in a 50 mL plastic tube, 40 mL of solvent (acetone) was added, and ultrasonic extraction was performed for 15 minutes using an ultrasonic cleaner (Suzuki, SUC-900A). After that, the residue was removed by centrifugation (15,000 rpm, 15 minutes), and the solvent was removed using a centrifugal concentrator. The residue was then dissolved in 400 μL of dimethyl sulfoxide (hereinafter referred to as "DMSO") to obtain a test sample for PPAR activity confirmation test.

[0041] [3] Methods for confirming PPAR activity For the evaluation of PPAR-α, β / δ, and γ activities, COS-1 cell transformants were prepared using the PPAR-α (or β / δ or γ) Gal4 fusion protein expression plasmid pPPAR-α ((or β / δ or γ)-Gal4 as an expression plasmid for PPAR-α, β / δ, and γ, the plasmid pGal4-Luc in which a luciferase structural gene, a reporter gene, was linked downstream of the Gal4 protein binding sequence, and the plasmid pSEAP-control for correcting transformation efficiency, and the luminescence intensity was measured to evaluate the PPAR-α, β / δ, and γ activities. Specifically, the evaluation was performed according to the following (1) to (5).

[0042] (1) Transformation of COS-1 cells COS-1 cells were harvested by trypsinization, centrifuged at 4°C (1000 rpm, 3 min), and the supernatant was removed. The cells were dispersed in 2 mL of medium and plated on a 60 mm culture dish (Corning) at a density of 5 x 105 cells / well, and then cultured at 37°C for 24 hours in the presence of 5% CO2. Effectene Transfection Reagent (QIAGEN) was used for transformation. 150 μL of Buffer EC, 0.25 μg of pPPAR-α (or β / δ or γ)-Gal4, 1 μg of pGal4-Luc, and 1 μg of pSEAP-control vector were added to a 1.5 mL tube, and finally 18 μL of Enhancer was added and stirred with a vortex for 1 second. After leaving it at 25°C for 3 minutes, 25 μL of Effectene was added, stirred with a vortex for 10 seconds, and left at 25°C for 7 minutes. During this time, the medium in the 60 mm culture dish was removed and 4 mL of new medium was added for medium replacement. After 7 minutes, 1 mL of medium was added to a 1.5 mL tube, suspended by pipetting twice, and the entire amount was dropped into the 60 mm culture dish and cultured at 37°C in the presence of 5% CO2 for 16 hours.

[0043] (2) Adding the test sample to COS-1 cells The transformed cells were harvested by trypsinization and centrifuged at 4°C (1000 rpm, 3 minutes), after which the supernatant was removed and the cells were suspended in 10 mL of medium, seeded at 125 μL / well in a 96-well plate (NUNC), and cultured at 37°C for 1-2 hours in the presence of 5% CO2. Then, 1.25 μL / well of the test sample obtained in [2] above was added, and the cells were gently stirred and cultured at 37°C for 24 hours in the presence of 5% CO2.

[0044] The COS-1 cells used were those provided by the RIKEN Cell Bank. The details are as follows: The culture medium was prepared by dissolving 10.0 g of Dulbecco's Modified Eagle Medium (DMEM, Nissui) in 1.0 L of ultrapure water, adding 1.4 g of sodium bicarbonate to adjust the pH to 7.4. Then, 10% fetal bovine serum (FBS: JRH BIOSCIENCE Lot. 5H2144) was added and sterilized by filtration. The FBS was dissolved in a water bath at 37°C and inactivated by heat treatment at 56°C for 30 minutes. After inactivation, the cells were dispensed in 100 mL portions and stored at -20°C until ready for use. The cells were cultured by dissolving the suspension in a ceramic tube (stored under a liquid nitrogen layer in a medium containing 10% dimethylsulfoxide) and centrifuging at 1000 rpm, 4°C, for 3 minutes, removing the supernatant, dispersing the cells in 5mL of medium, transferring them to a 100mm culture dish (Corning) containing 15mL of medium, and culturing them overnight at 37°C in the presence of 5% CO2. After confirming that there was no contamination by bacteria, the medium was replaced and subcultured. For subculture, the medium was removed from the 100 mm culture dish, washed with PBS(-), and the cells were harvested by trypsinization. After centrifugation at 1000 rpm, 4°C, for 3 minutes, the supernatant was removed, 2 mL of medium was added to disperse the cells, and the cells were then plated on a 100 mm culture dish containing 15 mL of medium and cultured at 37°C in the presence of 5% CO2. The cells were subcultured every 3 days. Trypsin solution: Trypsin (GIBCO) (0.5 g), EDTA·3Na (0.2 g) were mixed with PBS(-) to make 1.0 L, and the mixture was stirred at 4°C for 16 hours. After confirming that the mixture was completely dissolved, the mixture was sterilized by filtration. PBS(-): NaCl (8 g), KCl (0.2 g), Na2HPO4·12H2O (2.9 g), and KH2PO4 (0.2 g) were dissolved in 1.0 L of ultrapure water. This was sterilized by autoclaving (121°C, 15 minutes).

[0045] (3) Luciferase activity measurement 24 hours after the addition of the test samples, 25 μL / well of the medium was collected from the 96-well plate and transferred to a 96-well white plate. Then, 100 μL / well of the luciferase activity measurement solution melted at 37°C was added to the remaining 100 μL / well, and the mixture was allowed to react in the dark for 35 minutes, after which the luminescence intensity of each was measured.

[0046] (4) SEAP (Secreted Alkaline Phosphatase) activity measurement 25μL / well of 1x Dilution Buffer was added to 25μL / well of medium collected from the 96-well plate, and the plate was covered with cellophane tape, then gently stirred and left at 65℃ for 30 minutes. After that, the plate was cooled to 4℃, and returned to 25℃, and 90μL / well of Assay Buffer was added and gently stirred. The plate was left at 25℃ for 5 minutes, and 10μL / well of MUP solution was added and gently stirred. After reacting for 60 minutes at 25℃ in the dark, the fluorescence intensity (Ex=360nm, Em=460nm) based on 4-methylumbelliferone was measured.

[0047] (5) Evaluation method and comparative examples For the evaluation of PPAR activity, luciferase activity was corrected by SEAP activity. The results of each PPAR activity confirmation test are shown in Figure 1 for PPAR-α activity, Figure 2 for PPAR-β / δ activity, and Figure 3 for PPAR-γ activity. In addition, in evaluating the activity, WY14643 (Reference Example 3) at 50 μM, GW0742 (Reference Example 4) at 10 μM, and Troglitazone (Reference Example 5) at 10 μM were used as control substances for PPAR-α, PPAR-β / δ, and PPAR-γ, respectively. As comparative examples for all of PPAR-α, PPAR-β / δ, and PPAR-γ, a sample containing only the extraction solvent (DMSO) (Reference Example 2) and the unroasted barley used in the experimental examples (unroasted, L value 65-70) (Reference Example 1) were used. In addition, roasted whole barley obtained by roasting whole barley (barley with the husk removed) under the same conditions as in Experimental Example 3 was also used. These were also treated in the same manner as in the above experimental examples to prepare test samples, and activity evaluation was performed as described above. The vertical axis in Figures 1 to 4 indicates luciferase activity corrected with SEAP activity.

[0048] [4] Effects of the embodiment From the results of Table 1 and Figure 1, it can be seen that the smaller the L value (higher roasting intensity) of roasted barley, the higher the PPAR-α activity. In other words, it can be seen that PPAR-α activity can be improved by applying heat to barley. It can also be seen that Experimental Example 1 (L = 50.0) exhibits PPAR-α activity exceeding that of Reference Example 3 (control substance = WY14643), and Experimental Example 1 (L = 50.0) exhibits extremely high PPAR-α activity, 2.00 times that of Reference Example 1 (unroasted barley).

[0049] Furthermore, from the results of Table 1 and Figure 2, it can be seen that the smaller the L value (higher roasting intensity) of roasted barley, the higher the PPAR-β / δ activity. In other words, it can be seen that the activity of PPAR-β / δ can be improved by applying heat to barley. It can also be seen that Experimental Example 1 (L = 50.0) exhibits PPAR-β / δ activity exceeding that of Reference Example 4 (control substance = GW0742), and Experimental Example 1 (L = 50.0) exhibits extremely high PPAR-α activity, 1.86 times that of Reference Example 1 (unroasted barley).

[0050] Furthermore, from the results of Table 1 and Figure 3, it can be seen that the smaller the L value (higher roasting intensity) of roasted barley, the higher the PPAR-γ activity. In other words, it can be seen that the activity of PPAR-γ can be improved by applying heat to barley. And it can be seen that Experimental Example 1 (L = 50.0) shows extremely high PPAR-α activity, 1.79 times that of Reference Example 1 (unroasted barley).

[0051] Furthermore, Figure 4 (the luminescence intensities of whole barley are as follows: PPAR-α activity -526,134, PPAR-β / δ activity -1,071,257, PPAR-γ activity -590,358) shows that roasted barley with husks has significantly higher PPAR activity compared to roasted whole barley with the husks removed.

[0052] Incidentally, the present invention is not limited to the above-mentioned specific examples, and various modifications can be made within the scope of the present invention according to the purpose and application. [Industrial Applicability]

[0053] The agents for expressing PPAR activity, agents for preventing and improving dyslipidemia, agents for preventing and improving obesity, and agents for preventing and improving diabetes of the present invention, as well as substances containing them, can be used as medicines, as well as foods and beverages such as functional foods, foods for the sick, and foods for specified health uses.

Claims

1. A PPAR activation agent comprising roasted barley (excluding black hulled barley) or an extract thereof as an active ingredient, the L value of which is 10 or more and 17.7 or less, The wheat is barley, the extract is an aqueous and / or organic solvent extract, The agent for expressing PPAR activity, wherein the PPAR activity is PPAR-γ activity.

2. An agent for preventing and improving dyslipidemia, comprising the agent for expressing PPAR activity described in claim 1.

3. An agent for preventing and improving obesity, comprising an agent for expressing PPAR activity as described in claim 1.

4. A diabetes prevention and improvement agent comprising an agent for expressing PPAR activity as described in claim 1.

5. A method for producing the agent for expressing PPAR activity according to claim 1, comprising the steps of: A method for producing an agent for expressing PPAR activity, comprising a roasting step of roasting barley to obtain roasted barley (excluding black hulled barley) having an L value of 10 or more and 17.7 or less.

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