JP2012171924A - Ppar-activating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peroxisomal proliferator-activated receptor (PPAR)-activating agent which is effective for preventing and improving obesity, promoting the combustion of body fats, activating the metabolism of fatty acids, preventing and improving insulin resistance, preventing and improving diabetes, preventing and improving dyslipidemia, preventing and improving fatty liver, preventing and improving arteriosclerosis, improving endurance, preventing and improving cardiomegaly, and preventing and improving ischemic heart diseases.SOLUTION: The PPARα- or PPARδ-activating agent includes as an active ingredient a hydrophobic solvent extract of Funyu (fermented tofu) which is prepared by fermenting tofu and is the generic name of Chinese traditional fermented foods.

Description

  The present invention is obesity prevention / improvement, body fat burning promotion, fatty acid metabolism activation, insulin resistance prevention / improvement, diabetes prevention / improvement, dyslipidemia prevention / improvement, fatty liver prevention / improvement, arteriosclerosis prevention / improvement, The present invention relates to an activator for peroxisome proliferator activated receptor (referred to as PPAR) that is effective in improving endurance, preventing or improving cardiac hypertrophy, and preventing or improving ischemic heart disease.

  Low-molecular-weight lipophilic ligands such as steroids, thyroid hormones, and retinoids have various physiological functions such as morphogenesis in ontogenesis, cell proliferation, differentiation, and maintenance of homeostasis through ligand-specific nuclear receptors. It is involved in regulation. PPAR is one of the nuclear receptors, and was identified in 1990 as a protein that mediates the action of increasing peroxisomes, which are intracellular organelles involved in lipolysis, meaning that it is activated by peroxisome proliferators Was named Peroxisome Proliferator Activated Receptor α (peroxisome proliferator activated receptor: PPARα). Thereafter, δ type and γ type were identified as isoform genes structurally similar to α type, and it is known that it consists of three subtypes in total.

Each subtype of PPAR is activated in a ligand-dependent manner, and forms a heterodimer with RXR (Retinoid X Receptor) having 9-cis retinoic acid as a ligand, whereby a PPAR responsive complement (PPAR responsivelement; It controls the expression of various genes having PPRE) (Non-patent Documents 1 and 2).
For example, a reporter assay using PCO of ACO (Acyl-CoA oxidase), which is known as a key enzyme for fatty acid β-oxidation, has been performed. According to this report, linoleic acid known as a PPAR ligand is each of PPARα, δ and γ. It has been reported that the ACO transcriptional activity is enhanced through the process (Non-patent Document 3).
As will be described below, in recent years it has become clear that PPAR is involved in numerous physiological and pathological phenomena.

  Specifically, the function of PPARα is considered to be widely involved in the maintenance of energy metabolism and homeostasis in the living body such as fatty acid synthesis / transport / secretion and ATP production in fat consuming organs. In particular, β-oxidation-related enzymes (ACO, HMG-CoA synthase, Acyl-CoA synthase, Medium chain acyl-CoA dehydrogenase, Fatty acid binding protein, Lipoprotein lipase, etc.) important for fatty acid metabolism are strongly dependent on PPARα activation. It has become clear that PPARα is highly expressed in the liver, heart, kidney, etc., and PPARα activators are widely recognized as being effective in activating lipid metabolism in these organs.

  Activation of fatty acid metabolism accompanying the activation of PPARα is thought to lead to degradation of liver fat, improvement of fatty liver, promotion of decomposition and burning of body fat such as visceral fat and subcutaneous fat, and suppression of obesity. Fibrate drugs known as PPARα activators have been shown to have an action of promoting fatty acid combustion, an action of increasing HDL cholesterol, and an action of increasing the expression of adiponectin receptor in recent years. Are widely used as therapeutic agents for hyperglycemia, dyslipidemia, hyperglycemia, atherosclerosis and the like (Non-patent Document 4, Patent Documents 1 and 2). In addition, it has been reported that PPARα activators are effective for cardiac hypertrophy and ischemic heart disease (Non-patent Documents 5 and 6).

  Therefore, PPARα activators prevent and improve obesity, promote burning of body fat, activate fatty acid metabolism, prevent and improve insulin resistance, prevent and improve diabetes, prevent and improve dyslipidemia, fatty liver It is considered to be widely effective in the prevention and improvement of arteriosclerosis, prevention and improvement of arteriosclerosis, prevention and improvement of cardiac hypertrophy and ischemic heart disease, etc. In recent years, the search and development of PPARα activator has been actively conducted. (Patent Documents 3 to 5).

  PPARδ (also known as PPARβ, NUC1, FAAR) has not been clarified for a long time since it was cloned in 1992, but in recent years, it has been studied through the use of genetically modified animals and the development of selective agonists for PPARδ. It has become clear that it has various physiological functions. In studies using mice overexpressing PPARδ, suppression of body weight increase due to high-fat diet load, reduction of fat weight, reduction of blood neutral fat, and suppression of fatty liver were observed (Non-patent Document 7). ). In an experiment in which GW501516, a PPARδ selective agonist, was administered to obese rhesus monkeys, HDL cholesterol was increased and neutral fat and LDL cholesterol were decreased (Non-patent Document 8).

  In addition, GW501516 was allowed to act on skeletal muscle-derived cells, and the effects on cellular gene expression were examined. As a result, fatty acid uptake and transport, expression of fatty acid metabolism-related genes such as mitochondrial β-oxidation enzymes, uncoupling proteins, etc. It has been shown to induce. Furthermore, in mice administered with GW501516, as with adipose tissue-specific PPARδ overexpressing mice, suppression of body weight increase and decrease in fat weight were observed due to high fat diet loading, and it was also revealed that insulin resistance was improved. ing. In this mouse skeletal muscle, fatty acid metabolism-related genes and induction of fatty acid β oxidation have been confirmed. Therefore, activation of PPARδ suppresses fat accumulation in peripheral tissues by increasing energy consumption of skeletal muscle. It is considered that this may improve insulin resistance (Non-patent Document 9). In addition, administration of GW501516 to genetically obese mice suppressed pancreatic islet hypertrophy, and is considered to have a protective effect on pancreatic islets (Non-patent Document 9).

  In addition, it has been reported that obesity and insulin resistance are suppressed by overexpressing PPARδ specifically in skeletal muscle. Surprisingly, the skeletal muscle of this mouse has a very high percentage of endurance muscle fibers, which are generally called red muscle and contain a lot of mitochondria. As a result, this mouse is about 2% of the control mouse. It has been shown that it is a mouse with extremely excellent endurance that can run twice as long (Non-patent Document 10). Therefore, it has been considered that the activation of PPARδ is effective in improving exercise endurance.

  Recently, it has also been reported that PPARδ regulates insulin sensitivity in the liver. As a mechanism for this, activation of PPARδ is caused by glucose from the liver via activation of the glycolytic system of the liver and the pentose phosphate cycle. It has been suggested to decrease the supply of insulin and increase insulin sensitivity (Non-patent Document 11).

Thus, the activation of PPARδ is an increase in HDL cholesterol, a decrease in LDL cholesterol, suppression of obesity, improvement of insulin resistance / insulin sensitivity, activation of fatty acid metabolism, promotion of burning of body fat, PPARδ activators are obesity prevention / amelioration agents, body fat combustion promoters, fatty acid oxidation activators, insulin resistance, etc. It is considered effective as a prophylactic / improving agent for dyslipidemia, a prophylactic / improving agent for dyslipidemia, a prophylactic / improving agent for fatty liver, a prophylactic / improving agent for arteriosclerosis, and an endurance enhancing agent.
Therefore, search and development of PPARδ activators have been actively conducted for such purposes, and flavones, phenoxyacetic acid derivatives and the like have been reported so far (Patent Documents 6 and 7).

  PPARγ is a molecule that acts on energy storage in the presence of sufficient nutrients and acts as a so-called thrifty gene. In particular, PPARγ2 is expressed with relatively strong specificity in adipocytes, and has been shown to play a central role in adipocyte differentiation. Thiazolidine derivatives, known as PPARγ ligands, are known to downsize adipocytes and increase insulin sensitivity by strongly inducing adipocyte differentiation, and are widely used as insulin resistance improvers and diabetes therapeutics. ing. However, administration of thiazolidine derivatives has been pointed out to increase the body weight and fat weight and induce obesity (Non-patent Document 12).

  Thus, PPAR activators prevent and improve obesity, promote body fat burning, activate fatty acid metabolism, prevent and improve insulin resistance, prevent and improve diabetes, prevent and improve dyslipidemia, Widely effective in preventing and improving fatty liver, preventing and improving arteriosclerosis, improving endurance, preventing and improving cardiac hypertrophy, and preventing and improving ischemic heart disease. So-called lifestyle-related diseases and visceral fat syndrome (metabolic) It is thought to be effective in preventing and improving syndrome.

Soy milk is a traditional Chinese fermented soybean food produced by fermenting tofu, and there are various types depending on the region where it is produced, but it is mainly divided into three types: red tofu milk, white tofu milk, and odor tofu. It is done. Rot milk is a food rich in nutritional value, including protein, lipid, various amino acids, carbohydrates, vitamins (such as vitamin B group), minerals (such as calcium and phosphorus), and the like.
However, it is not known that sultry is involved in PPAR activation, effective in suppressing obesity, promoting burning of body fat, activating fatty acid metabolism, and the like.

Special Table 2002-502869 Special Table 2002-533410 Publication JP 2001-354558 A JP 2002-80362 A Japanese Patent Laid-Open No. 2003-34636 JP 2007-119429 A Special Table 2007-536343

Cell: 31 (6) 218-234, 1999 J Lipid Res. 37, 907-925, 1996 J Biol Chem. 274, 23368-23377, 1999 Curr Opin Lipidol. 10, 151-159, 1999 Endcrinology 144, 4187-4194, 2003 Circulation 108, 2393-2399, 2003 Cell. 113, 159-170, 2003 Proc. Natl. Acad. Sci. USA. 98, 5306-5311, 2001 Proc. Natl. Acad. Sci. USA. 100, 15924-15929, 2003 PloS Biol. 2, e294, 2004 Proc. Natl. Acad. Sci. USA. 103, 3444-3449, 2006 Diabetes. 49, 759-767, 2000

  The present invention relates to providing a food, drug, or quasi-drug having an excellent PPAR activation action and high safety.

  The inventors of the present invention have a PPARα activation action and a PPARδ activation action in a hydrophobic solvent extract of sultry among natural product materials with abundant dietary experience, preventing and improving obesity, promoting body fat burning, fatty acid metabolism Activation, insulin resistance prevention / improvement, diabetes prevention / improvement, dyslipidemia prevention / improvement, fatty liver prevention / improvement, arteriosclerosis prevention / improvement, endurance improvement, cardiac hypertrophy prevention / improvement, ischemic heart disease prevention / improvement The present inventors have found that it is useful as a material to be blended as an active ingredient of foods, pharmaceuticals or quasi drugs that exhibit effects such as improvement.

That is, the present invention relates to the following inventions.
[1] A PPARα and / or PPARδ activator comprising a hydrophobic solvent extract of milk as an active ingredient.
[2] A prophylactic / ameliorating agent for obesity comprising a hydrophobic solvent extract of milk as an active ingredient.
[3] A body fat combustion accelerator comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[4] An activator of fatty acid metabolism comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[5] An agent for preventing / ameliorating insulin resistance comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[6] A preventive / ameliorating agent for diabetes comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[7] A prophylactic / ameliorating agent for dyslipidemia comprising a hydrophobic solvent extract of milk as an active ingredient.
[8] A prophylactic / ameliorating agent for fatty liver comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[9] An arteriosclerosis preventive / ameliorating agent comprising a hydrophobic solvent extract of humic as an active ingredient.
[10] An endurance improver comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[11] A preventive / ameliorating agent for cardiac hypertrophy comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[12] A prophylactic / ameliorating agent for ischemic heart disease comprising a hydrophobic solvent extract of sultry milk as an active ingredient.
[13] A process for producing a PPARα and / or PPARδ activator by preparing a hydrophobic organic solvent extract of milk.

  Since the PPAR activator of the present invention has an excellent PPAR activation action and is highly safe even when ingested for a long period of time, obesity prevention / improvement, body fat combustion promotion, fatty acid metabolism activation, insulin resistance Prevention and improvement of diabetes, prevention and improvement of diabetes, prevention and improvement of dyslipidemia, prevention and improvement of fatty liver, prevention and improvement of arteriosclerosis, improvement of endurance, prevention and improvement of cardiac hypertrophy, prevention and improvement of ischemic heart disease, etc. It is useful as a material to be blended as an active ingredient in foods and beverages, medicines or quasi drugs to be exhibited.

The graph which showed PPAR (alpha) activation ability. In the figure, DW is a water extract, EtOH is a 50% ethanol extract, Hex is a hexane extract, Cont is a solvent control, and Wy is Wy14643. * Indicates a significant difference P <0.05 relative to the solvent control. The graph which showed PPAR (delta) activation ability. In the figure, DW is a water extract, EtOH is a 50% ethanol extract, Hex is a hexane extract, Cont is a solvent control, and GW is GW501516. * Indicates a significant difference P <0.05 relative to the solvent control. The graph which showed PPAR (gamma) activation ability. In the figure, DW is a water extract, EtOH is a 50% ethanol extract, Hex is a hexane extract, Cont is a solvent control, and Pio is pioglitazone.

  Soy milk is a general term for traditional Chinese fermented foods produced by fermenting tofu. Rot milk is mainly classified into three types, red rot milk, white rot milk, and odor tofu, according to the production method and the type of bacteria used for fermentation, and can be further subdivided by seasoning or the like. Tofu that is eaten in the Okinawa region of Japan is the same as that produced by the same manufacturing method. Therefore, examples of the soy milk used in the present invention include red soy milk, white soy milk, and odor tofu, and may further include sara (soy) milk, dark dairy milk, tofu and the like. Soy milk may be produced, for example, by fermenting tofu according to the procedures described below, or commercially available soy milk, odor tofu, or tofu (eg, Peking Tofu Milk, Moncho Tofu Milk, Xuzhou Blue rot, blue rot, shanghai mausoleum refined magui rot (rose rot), Henan commercial hill white sugar rot, Hunan Yiyang gold flower refined rot, Sichuan 夾 jiang rot, Suining linden rot, xi'an oil rot, etc.) You may purchase and use.

Here, the fungus for producing the milk is not particularly limited as long as it is generally used for the production of the milk, for example, Monascus anka , Monascus purpereus , Monascus which are usually used for the production of the red milk. fuliginocus, Monascus rubiginosus, Monascus spp such as Monascus serorubescus; white fermented bean curd or Mucor Sufui normally used in the manufacture of stinky tofu, Mucor rouxianus, Mucor spp, such as Mucor wutengkiao; and Actinomucor elegans, and the like can be used Micrococcus roseus .

  In the present invention, the tofu used as the raw material of the milk may be produced from soybeans by a usual method. Here, soybean refers to soybean seeds that are annual grasses of Fabaceae (Glycine), and any varieties or localities can be used as long as they are used as raw materials for food and processed foods. But you can.

Tofu is preliminarily adjusted to a water content of about 70% by pressing or the like. Next, it is cut into an appropriate size, preferably about 35 to 45 mm × 35 to 45 mm × 10 to 20 mm.
Next, the above-mentioned fungus is inoculated into this tofu and fermented. Although the quantity of the microbe to inoculate is not specifically limited, 0.1-3.0 mass%, Preferably 0.2-0.5 mass% inoculum is inoculated with respect to the soybean cooled after heat processing.
Although the temperature at the time of fermentation is not specifically limited, For example, it is preferable to set it as 15-30 degreeC, and it is more preferable to set it as 20-25 degreeC. The humidity during fermentation is preferably 60% to 99%, more preferably 80 to 98%.
Although fermentation time changes with temperature, the inoculation amount of a microbe, etc., it is preferable to set it as 30 to 50 hours, it is more preferable to set it as 35 to 50 hours, and it is still more preferable to set it as 40 to 48 hours.

Subsequently, the fermented tofu is salted, and a sugar solution containing alcohol as a main component is added thereto and aged. Or you may make it age | cure | ripen by adding the said sugar solution, without performing salting.
In the salting step, the tofu issued according to the above procedure is immersed in about 15 to 20% saline for about 2 to 10 days to produce tofu with a salt content of about 10 to 18% and a water content of about 50%.
The sugar solution is mainly composed of sake and may contain sugar, salt, spices, koji, sake lees, alcohol and the like. Examples of the sugar liquid include moromi (fermented Neisseria gonorrhoeae). The alcohol content of the sugar solution may be about 3 to 20%.
The aging period varies depending on the alcohol content of the sugar solution, but is about 1 to 6 months. The higher the alcohol content of the sugar solution, the longer the aging period. Preferably, the aging period when the alcohol content is about 5% is about 1-2 months, the aging time when the alcohol content is about 5-10% is about 2-4 months, and the alcohol content is 15%. In the case of exceeding ripening, the aging period is about 4 to 6 months.

  Soy milk can be obtained under the conditions described above. The said milk can be used for extraction as it is or after drying.

The hydrophobic solvent extract of rot milk used by this invention is obtained by extracting from rot milk at normal temperature (0-30 degreeC) or under heating using a hydrophobic solvent.
It is preferable to use a solvent of 1/2 to 10 parts by mass with respect to 1 part by mass of the milk, and the extraction temperature at this time is preferably 0 to 40 ° C, more preferably 10 to 30 ° C, and the extraction time is 1 / 10 to 12 hours are preferable, and 1/2 to 5 hours are more preferable.
As the extraction, means such as solid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, and the like can be used, and solid-liquid extraction with stirring is preferable.
Examples of the hydrophobic organic solvent include chloroform, dichloromethane, diethyl ether, petroleum ether, benzene, cyclohexane, n-hexane (also referred to as hexane in the present specification) and the like. Is n-hexane.

  The extract thus obtained can be used as it is after the solvent is removed, but it can be used as it is by known separation and purification means, for example, activated carbon treatment, liquid partitioning, column chromatography, liquid chromatography, gel filtration, precision distillation, etc. After that, it may be used as a diluted solution diluted with an appropriate solvent, or it may be a concentrated extract, a dry powder, or prepared in a paste form.

  As shown in Examples below, a hydrophobic solvent extract of humor prepared by extracting humor with a hydrophobic organic solvent has an action of enhancing the transcriptional activity of genes dependent on PPARα and / or PPARδ. . As described above, PPARα and / or PPARδ are widely involved in the maintenance of energy metabolism and homeostasis in the living body, and particularly β-oxidation-related enzymes (ACO, HMG-CoA synthase, Acyl-CoA synthase, Medium important for lipid metabolism). Since gene expression of chain acyl-CoA dehydrogenase, Fatty acid binding protein, Lipoprotein lipase, etc.) is strongly dependent on the activation of PPARα and / or PPARδ (Non-patent Documents 1-10, Patent Documents 1-7), Hydrophobic solvent extract of sultry is obesity prevention / improvement, body fat burning promotion, fatty acid metabolism activation, insulin resistance prevention / improvement, diabetes prevention / improvement, dyslipidemia prevention / improvement, fatty liver prevention / improvement, arteries It is widely effective in preventing and improving sclerosis, improving endurance, preventing and improving cardiac hypertrophy, and preventing and improving ischemic heart disease.

  Accordingly, the hydrophobic solvent extract of sultry milk has PPARα activating action and / or PPARδ activating action, so PPARα and / or PPARδ activator, obesity preventing / ameliorating agent, body fat burning promoter, fatty acid metabolic activity Agent, insulin resistance prevention / amelioration agent, diabetes prevention / improvement agent, dyslipidemia prevention / improvement agent, fatty liver prevention / improvement agent, arteriosclerosis prevention / improvement agent, endurance improver, cardiac hypertrophy prevention / improvement agent It can be used as an agent for preventing / ameliorating ischemic heart disease, etc. (hereinafter referred to as “PPAR activator etc.”), and can be used for the production of PPAR activator etc. PPAR activators include PPARα or PPARδ activation, obesity prevention / improvement, body fat burning promotion, fatty acid metabolism activation, insulin resistance prevention / improvement, diabetes prevention / improvement, dyslipidemia prevention / improvement, fatty liver prevention・ Improvement, arteriosclerosis prevention / improvement, endurance improvement, cardiac hypertrophy prevention / improvement, ischemic heart disease prevention / improvement, etc. for human or animal food, pharmaceuticals, quasi drugs or cosmetics It can be used as an active ingredient. The hydrophobic solvent extract of sultry milk activates PPARα or PPARδ, obesity prevention / improvement, body fat burning promotion, fatty acid metabolism activation, insulin resistance prevention / improvement, diabetes prevention / improvement, dyslipidemia prevention / improvement , Fat liver prevention / improvement, arteriosclerosis prevention / improvement, endurance enhancement, cardiac hypertrophy prevention / improvement, ischemic heart disease prevention / improvement, etc. It can be applied to food for sick people and food for specified health use.

  Furthermore, according to this invention, the manufacturing method of the PPAR (alpha) and / or PPAR (delta) activator by preparing the hydrophobic organic-solvent extract of milk is provided. Furthermore, according to the present invention, an obesity preventive / ameliorating agent, body fat combustion accelerator, fatty acid metabolism activator, insulin resistance preventing / ameliorating agent, diabetes prevention / Manufacturing methods such as an improving agent, a dyslipidemia preventing / ameliorating agent, a fatty liver preventing / ameliorating agent, an arteriosclerosis preventing / ameliorating agent, an endurance improving agent, a cardiac hypertrophy preventing / ameliorating agent, an ischemic heart disease preventing / ameliorating agent, etc. Provided.

Examples of the dosage form when the PPAR activator of the present invention is used as an active ingredient of pharmaceuticals and quasi drugs include oral administration or injection, suppository, such as tablets, capsules, granules, powders, syrups, etc. And parenteral administration using inhalants, transdermal absorption agents, external preparations and the like.
In order to prepare formulations of such various dosage forms, the hydrophobic solvent extract of the milk of the present invention alone or other pharmaceutically acceptable excipients, binders, bulking agents, Disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be used in appropriate combinations.
Of these dosage forms, the preferred form is oral administration, and when an oral liquid preparation is prepared, it can be produced by a conventional method by adding a flavoring agent, a buffering agent, a stabilizer and the like.

  When the PPAR activator or the like of the present invention is used as an active ingredient of a cosmetic, it can be used as a skin external preparation, a cleaning agent, a bath agent, a makeup cosmetic, or the like. Beauty lotion, lotion, massage agent, lotion, emulsion, gel, cream, ointment, powder, pack, poultice, granule, foundation, lipstick, shampoo, conditioner, hair tonic, tablet, capsule, sheet-like product, etc. Can be provided in various dosage forms. In order to prepare cosmetics of such various dosage forms, an external base material, oil or oily substance, moisturizing agent, powder, which is blended alone or in the cosmetic with the hydrophobic solvent extract of the present invention. Body, pigment, emulsifier, solubilizer, cleaning agent, UV absorber, thickener, medicinal component, fragrance, resin, antibacterial and antifungal agent, alcohols, chelates, inorganic acids, organic acids, vitamins, water A suitable combination of a functional polymer, a surfactant and the like can be used.

In the case of using the PPAR activator of the present invention as an active ingredient of food, breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages, soups, etc. In addition to these various foods, the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral preparations can be mentioned.
Examples of the beverage include fruit juice beverages, carbonated beverages, tea-based beverages, near water, sports beverages, milk beverages, alcoholic beverages, and soft drinks. Moreover, a drink can be used as the container-packed drink with which the container was filled.
Edible oils include cooking oils, seasonings, mayonnaise, dressings, processed oils and fats such as margarine, and pasta sauces.
In order to prepare foods of such various forms, the PPAR activator of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, Stabilizers, colorants, antioxidants, moisturizers, thickeners, etc. are combined as appropriate to enable PPARα and / or PPARδ activation foods, obesity prevention / amelioration foods, body fat combustion promoting foods, fatty acid metabolism activation Foods for preventing and improving insulin resistance, Foods for preventing and improving diabetes, Foods for preventing and improving dyslipidemia, Foods for preventing and improving fatty liver disease, Foods for preventing and improving arteriosclerosis, Foods for improving endurance, It can be used as a food for preventing / ameliorating cardiac hypertrophy, a food for preventing / ameliorating ischemic heart disease, a pet food, and the like.

The amount of PPAR activator and the like for these products varies depending on the form of use, but in the form of food, in the total composition, usually 0.0001 to 10% by mass in terms of dry matter of the hydrophobic solvent extract of sultry, Furthermore, it is preferable to set it as 0.001-5 mass%, still more 0.002-2 mass%.
For example, in the case of beverages, the PPAR activator and the like in the beverage are 0.001 to 0.5% by mass, and further 0.005 to 0.005% in terms of the dry matter of the hydrophobic solvent extract of sult in the entire composition. It is preferably 25% by mass, and more preferably 0.01 to 0.1% by mass. In the case of food tablets and / or capsules such as tablets, the PPAR activator is 0.1 to 95% by mass, further 1 to 90% in terms of the dry matter of the hydrophobic solvent extract of sult in the entire composition. Those containing 5% by mass and still 5-50% by mass are preferred.
In the case of pharmaceutical preparations other than the above, for example, oral solid preparations such as tablets, granules, capsules, and oral liquid preparations such as oral liquids, syrups, etc. In terms of conversion, it is usually preferably 0.01 to 95% by mass, more preferably 5 to 90% by mass, and still more preferably 10 to 50% by mass.
In the case of cosmetics, in the total composition, it is usually 0.00001 to 5% by mass, further 0.0001 to 3% by mass, and further 0.001 to 1% by mass in terms of dry matter of the hydrophobic solvent extract of sultry. It is preferable to do this.

The dose (effective intake) of the PPAR activator and the like is preferably 1 to 5000 mg / 60 kg body weight per day in terms of dry matter of the hydrophobic solvent extract of slaughter milk. More preferably, it is 5-3000 mg / 60 kg body weight, More preferably, it is 10-2000 mg / 60 kg body weight, and it is still more preferable to set it as 100-1000 mg / 60 kg body weight.
Administration or ingestion of the PPAR activator of the present invention activates PPARα and / or PPARδ, obesity prevention / improvement, body fat burning promotion, fatty acid metabolism activation, insulin resistance prevention / improvement, diabetes prevention / improvement It can promote prevention / improvement of dyslipidemia, prevention / improvement of fatty liver, prevention / improvement of arteriosclerosis, improvement of endurance, prevention / improvement of cardiac hypertrophy, prevention / improvement of ischemic heart disease, etc. Therefore, the PPAR activator of the present invention can be used in the method for that purpose. The subject of administration or ingestion is not particularly limited as long as it is a human or animal in need thereof, but patients with obesity, insulin resistance, diabetes, dyslipidemia, fatty liver, arteriosclerosis, cardiac hypertrophy or ischemic heart disease And their reserves, and those with reduced endurance.

(Production Example 1-1) Preparation of Hexane Hexane Extract-1
70 g of white rotted milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), finely crushed, added with 100 mL of hexane, stirred at room temperature (20 ° C.) for 2 hours, filtered the hexane phase and then rotary Concentration by an evaporator gave 2.02 g of extract. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).
(Production Example 1-2) Preparation of Hexane Hexane Extract-2
In the same procedure as in Production Example 1-1, 3.41 g of hexane extract was prepared from cocoon milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was 1% ( w / v) was dissolved in ethanol.
(Production Example 1-3) Preparation of hexane extract of rot milk-3
In the same procedure as in Production Example 1-1, 0.46 g of hexane extract was prepared from red roasted milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was 1% ( w / v) was dissolved in ethanol.
(Production Example 1-4) Preparation of hexane extract of rot milk-4
In the same procedure as in Production Example 1-1, 0.30 g of hexane extract was prepared from dark dairy milk (Beijing Wang Jiaohe Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / v). So that it was dissolved in ethanol.
(Production Example 1-5) Preparation of hexane extract of milk
In the same procedure as in Production Example 1-1, 0.20 g of hexane extract was prepared from odor tofu (Beijing Wang Jiaohe Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / v). So that it was dissolved in ethanol.

(Comparative Production Example 1-1) Preparation of 50% Ethanol Extract of Rot Milk-1
70 g of white-cured milk (Hwang Ryh Shiang CO., LTD., Purchased on the market) is finely crushed, added with 100 mL of 50% ethanol and stirred at room temperature (20 ° C.) for 2 hours, and the filtrate is rotovap To give 10.62 g of extract. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).
(Comparative Production Example 1-2) Preparation-2 of 50% ethanol extract of milk
In the same procedure as Comparative Production Example 1-1, 13.21 g of a 50% ethanol extract was prepared from cocoon milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was It melt | dissolved in ethanol so that it might become 1% (w / v).
(Comparative Production Example 1-3) Preparation of 50% ethanol extract of milk
In the same procedure as in Comparative Production Example 1-1, 10.61 g of 50% ethanol extract was prepared from red roasted milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was It melt | dissolved in ethanol so that it might become 1% (w / v).
(Comparative Production Example 1-4) Preparation of 50% ethanol extract of milk
In the same procedure as in Comparative Production Example 1-1, 11.82 g of a 50% ethanol extract was prepared from dark dairy milk (Beijing Wang Liwa Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / It was dissolved in ethanol so that v).
(Comparative Production Example 1-5) Preparation of 50% Ethanol Extract of Rot Milk-5
In the same procedure as Comparative Production Example 1-1, 5.81 g of 50% ethanol extract was prepared from odor tofu (Beijing Wang Jiaohe Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / It was dissolved in ethanol so that v).

(Comparative Production Example 2-1) Preparation of water extract of rot milk-1
70 g of white rot milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), finely crushed, add 100 mL of water and stir at room temperature (20 ° C.) for 2 hours, and concentrate the filtrate by lyophilization 14.34 g of extract was obtained. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).
(Comparative Production Example 2-2) Preparation of water extract of rot milk -2
In accordance with the same procedure as Comparative Production Example 2-1, 14.40 g of water extract was prepared from cocoon milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was 1%. (W / v) was dissolved in ethanol.
(Comparative Production Example 2-3) Preparation of a water extract of sultry milk-3
According to the same procedure as Comparative Production Example 2-1, 4.22 g of water extract was prepared from red roasted milk (Hwang Ryh Shiang CO., LTD., Purchased commercially), and the concentration was 1%. (W / v) was dissolved in ethanol.
(Comparative Production Example 2-4) Preparation of water extract of rot milk-4
In accordance with the same procedure as Comparative Production Example 2-1, 11.27 g of water extract was prepared from dark dairy milk (Beijing Wang Liwa Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / v) So that it was dissolved in ethanol.
(Comparative Production Example 2-5) Preparation of water extract of milk
In the same procedure as Comparative Production Example 2-1, 7.38 g of water extract was prepared from stinky tofu (Beijing Wang Liwa Food Group Co., Ltd., purchased commercially), and the concentration was 1% (w / v) So that it was dissolved in ethanol.

(Comparative Production Example 3-1) Preparation of hexane extract of soybeans 50 mL of hexane was added to 20 g of cooked soybeans, stirred at room temperature (20 ° C) for 2 hours, the hexane phase was filtered, and then concentrated by a rotary evaporator to give 0.10 g. An extract of was obtained. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).
(Comparative Production Example 3-2) Preparation of 50% ethanol extract of soybeans 50 mL of 50% ethanol was added to 20 g of cooked soybeans and stirred at room temperature (20 ° C.) for 2 hours, and the filtrate was concentrated by a rotary evaporator. .64 g of extract was obtained. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).
(Comparative Production Example 3-3) Preparation of water extract of soybeans 50 mL of water was added to 20 g of steamed soybeans, stirred at room temperature (20 ° C.) for 2 hours, and the filtrate was concentrated by freeze-drying to extract 0.68 g. I got a thing. Next, the obtained extract was dissolved in ethanol so as to have a concentration of 1% (w / v).

  The extracts of Production Examples 1 to 5 and Comparative Production Examples 1-1 to 3-3 were used as test substances of Examples 1 to 3 below.

Example 1: PPARα activation test Using human colon total RNA (Clontech), PCR was performed to amplify a PPARα ligand binding site (NCBI RefSeq NM_001001928, nt183-1586, SEQ ID NO: 1). The PCR amplification product was cloned into pCR Blunt (Invitrogen), and a DNA fragment was prepared by restriction enzyme (MluI, KpnI; Takara) treatment. The prepared DNA fragment was inserted into the multicloning site (MluI / KpnI) of pBIND vector (Promega) to obtain pBIND-PPARα LBD. When this vector is introduced into a cell, it expresses a fusion protein of a PPARα ligand binding site and a GAL4 DNA binding site, and the fusion protein binds to a PPARα ligand to bind to a GAL4 binding sequence and activates transcription of a downstream gene. It is to become. In addition, a Renilla luciferase gene is separately incorporated into this vector, and the efficiency of vector introduction can be determined.
African green monkey kidney cell line CV-1 was plated on a 24-well plate and cultured in DMEM (5% charcoal-treated fetal bovine serum) for 1 day. Using a transfection reagent (Superfect transfection reagent; QIAGEN) so that the reporter plasmid (pG5-Luc; Promega) containing the GAL4 binding sequence upstream of the firefly luciferase gene and pBIND-PPARα LBD are each simultaneously 0.2 μg / well. Introduced. Thereafter, the culture medium was replaced with a DMEM (-fetal bovine serum) medium containing 0.005% (w / v) of the test substance, and further cultured for 1 day. As test substances, the extracts obtained in Production Examples 1-1 to 1-5 and Comparative Production Examples 1-1 to 2-5 and 3-1 to 3-3 were used, and the same amount was used as a control. The solvent (ethanol) was used. As a positive control, 10 μM Wy14643 (obtained from BIOMOL (Plymouth Meeting DA)) was used.
After washing with PBS, cells were lysed using a dual luciferase assay system (Promega), a substrate solution containing luciferin was added to the lysate, and firefly and Renilla luciferase activities were measured using a luminometer.
In this experimental system, PPARα-activating substances were searched for by measuring the transcriptional activity (luciferase activity) of PPARα-dependent genes. The transcriptional activity (luciferase activity) of the PPARα-dependent gene was defined as follows.

  PPARα-dependent gene transcription activity (luciferase activity) = (firefly luciferase activity by pG5-Luc) / (renilla luciferase activity by pBIND-PPARα LBD)

  The PPARα activation ability of each test substance is shown in FIG. The PPARα-dependent transcriptional activity in the control is taken as 1, and the relative value is shown.

  From FIG. 1, the hexane extract of humus has PPARα activation ability, while the water extract of humor and 50% ethanol extract have no activation ability. In addition, none of the soybean extracts had activation ability.

Example 2: PPARδ activation test A PPARδ activation test was conducted in the same manner as in Example 1.
PCR was performed using Rat IEC-6 total RNA to amplify the PPARδ ligand binding site (NCBI RefSeq NM — 011145, nt690-1595, SEQ ID NO: 2). The PCR amplification product was cloned into pCR Blunt (Invitrogen), and a DNA fragment was prepared by restriction enzyme (MluI, KpnI; Takara) treatment. The prepared DNA fragment was inserted into the multicloning site (MluI / KpnI) of pBIND vector (Promega) to obtain pBIND-PPARδ LBD.
Cells introduced with the vector in the same procedure as in Example 1 were cultured with a test substance, luciferase activity was measured, and a PPARδ activator was searched. As a positive control, GW501516 (ALEXIS BIOCHEMICALS) 1 nM was used. PPARδ-dependent gene transcriptional activity (luciferase activity) was defined as follows.

  PPARδ-dependent gene transcription activity (luciferase activity) = (firefly luciferase activity by pG5-Luc) / (renilla luciferase activity by pBIND-PPARδ LBD)

  FIG. 2 shows the PPARδ activation ability of each test substance. The PPARδ-dependent transcriptional activity in the control is set to 1, and the relative value is shown.

  As shown in FIG. 2, the hexane extract of the milk has PPARδ activation ability, whereas the water extract of the milk and many 50% ethanol extracts of the milk have no activation ability. The 50% ethanol extract of odor rot milk had an activation ability, but was weaker than the hexane extract. In addition, none of the soybean extracts had activation ability.

Example 3 PPARγ Activation Test A PPARγ activation test was conducted in the same manner as in Example 1.
PCR was performed using Human colon total RNA (Clontech) to amplify the PPARγ2 ligand binding site (NCBI RefSeq NM — 015869, nt703-1606, SEQ ID NO: 3). The PCR amplification product was cloned into pCR Blunt (Invitrogen), and a DNA fragment was prepared by restriction enzyme (MluI, KpnI; Takara) treatment. The prepared DNA fragment was inserted into the multicloning site (MluI / KpnI) of pBIND vector (Promega) to obtain pBIND-PPARγ LBD.
Cells introduced with the vector in the same procedure as in Example 1 were cultured with a test substance, luciferase activity was measured, and a PPARγ activator was searched. Pioglitazone (CAYMAN) 10 μM was used as a positive control. PPARγ-dependent gene transcription activity (luciferase activity) was defined as follows.

  PPARγ-dependent gene transcriptional activity (luciferase activity) = (firefly luciferase activity by pG5-Luc) / (renilla luciferase activity by pBIND-PPARγ LBD)

  FIG. 3 shows the PPARγ activation ability of each test substance. The PPARγ-dependent transcriptional activity in the control is taken as 1, and the relative value is shown.

  FIG. 3 shows that the milk extract does not activate PPARγ.

As described above, it can be seen that the hydrophobic solvent extract of milk has PPARα activating action and PPARδ activating action but not PPARγ activating action, and has high specificity for PPARα and PPARδ. That is, it can be used as a PPARα and PPARδ selective activator.
Furthermore, since the hydrophobic solvent extract of humic milk has PPARα or PPARδ activation action, as described above, prevention and improvement of obesity, promotion of burning of body fat, activation of fatty acid metabolism, prevention and improvement of insulin resistance , Prevention and improvement of diabetes, prevention and improvement of dyslipidemia, prevention and improvement of fatty liver, prevention and improvement of arteriosclerosis, improvement of endurance, prevention and improvement of cardiac hypertrophy and prevention and improvement of ischemic heart disease, etc. It is considered effective.

Claims (13)

  A PPARα and / or PPARδ activator comprising a hydrophobic solvent extract of sorghum as an active ingredient.   An obesity-preventing / ameliorating agent comprising a hydrophobic solvent extract of milk as an active ingredient.   A body fat burning accelerator comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   A fatty acid metabolism activator comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   An agent for preventing and / or improving insulin resistance comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   Diabetes preventive / ameliorating agent comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   A prophylactic / ameliorating agent for dyslipidemia comprising a hydrophobic solvent extract of milk as an active ingredient.   Fatty liver preventive / improving agent comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   An agent for preventing or improving arteriosclerosis comprising a hydrophobic solvent extract of humor as an active ingredient.   An endurance improver comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   A preventive / ameliorating agent for cardiac hypertrophy containing a hydrophobic solvent extract of sultry milk as an active ingredient.   A prophylactic / ameliorating agent for ischemic heart disease comprising a hydrophobic solvent extract of sultry milk as an active ingredient.   A method for producing a PPARα and / or PPARδ activator by preparing a hydrophobic organic solvent extract of humic milk.

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