JP2016204342A - Lipid metabolism-improving composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly safe composition for improving lipid metabolism, improving obesity or the like.SOLUTION: According to the present invention, there is provided a composition for improving lipid metabolism and/or improving obesity or the like which contains 14-dehydroergosterol (14-DHE) or an Aspergillus oryzae fermentation product containing 14-DHE. According to the present invention, there is also provided a method for producing the composition in which a raw material derived from cereals is fermented by Aspergillus oryzae to obtain a fermentation product containing 14-DHE.SELECTED DRAWING: None

Description

  The present invention relates to a composition for improving lipid metabolism and the like, and more particularly to a composition for improving lipid metabolism and the like containing 14-dehydroergosterol or a koji mold fermentation product containing the same.

  In recent years, as the dietary habits have become westernized, the amount of fat intake per capita has also increased, and diseases called lifestyle-related diseases such as dyslipidemia (hyperlipidemia), hypertension, obesity, etc. have increased rapidly. . Under such circumstances, there is still a need for the development of drugs and foods for preventing and improving lipid metabolism abnormalities and obesity that have a great influence on the onset and worsening of lifestyle-related diseases. In particular, since lifestyle-related diseases are often prevented and treated for a long period of time, it is desired to provide highly safe drugs and foods without serious side effects.

  Under such a background, Patent Document 1 discloses that a starch raw material (brown rice) is fermented with koji molds, yeasts, and acetic acid bacteria for the purpose of increasing the content of amino acids having an anti-obesity effect. Manufacturing dietary dietary supplements is disclosed. Patent Document 2 discloses a composition useful for the treatment of lipid metabolism disorders comprising an extract of rice fermented with Aspergillus niger containing monacolin, and Patent Document 3 has a rophenol skeleton. It is described that the compound has an effect of improving insulin resistance. On the other hand, some compounds having a steroid skeleton have anti-inflammatory activity and are put into practical use as pharmaceuticals. However, these so-called steroid preparations reduce the insulin sensitivity of cells, affect lipid metabolism, etc. It is known that there is a risk of exacerbating the symptoms of these patients (Non-patent Document 1), and it is also known that obesity may be induced by appetite enhancement or the like.

  On the other hand, Patent Document 4 reports that a gonococcal fermented product of a cereal plant-derived material containing 14-dehydroergosterol has an action of suppressing an immune reaction mediated by dendritic cells. In Patent Document 5, fermented koji molds of cereal plant-derived materials containing 14-dehydroergosterol are anti-inflammatory against inflammation caused by bacterial infections such as Helicobacter bacteria, pathogenic Escherichia coli, and periodontal disease bacteria. It has been reported to have an effect.

  However, none of the documents disclose that 14-dehydroergosterol or a koji mold fermentation product containing the same has a lipid metabolism improving action or the like.

JP 2003-125732 A Special table 2014-514340 gazette WO2007 / 043306 WO2010 / 150867 JP2013-227252A

Pharmaceutical interview form "Sol Medrol (registered trademark) for intravenous injection" revised in September 2014 (11th edition)

  An object of this invention is to provide the composition for lipid metabolism improvement, obesity improvement, etc. with high safety | security.

  The present inventors have found that 14-dehydroergosterol (hereinafter sometimes simply referred to as “14-DHE”) contained in the koji mold fermentation product suppresses an increase in blood lipids. The present inventors have also found that 14-dehydroergosterol contained in the fermentation product of koji mold suppresses increase in visceral fat and accumulation of ectopic fat. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
(1) Lipid metabolism improvement, risk reduction of lipid metabolism abnormality, obesity improvement, obesity risk reduction, weight gain suppression and / or fat comprising 14-dehydroergosterol or an aspergillus fermentation product containing the same as an active ingredient Composition for suppressing accumulation.
(2) The composition according to (1) above for improving lipid metabolism.
(3) The composition according to the above (2) for use in the treatment, prevention or amelioration of a disease or symptom in which improvement of lipid metabolism is effective for the treatment, prevention or amelioration.
(4) The composition according to (3), wherein the disease and symptom are one or more selected from the group consisting of obesity, dyslipidemia, and arteriosclerosis.
(5) For improving insulin resistance, reducing insulin resistance risk, improving hyperglycemia and / or reducing hyperglycemia risk, comprising 14-dehydroergosterol or an aspergillus fermentation product containing the same as an active ingredient Composition.
(6) The composition according to the above (5) for use in the treatment, prevention or amelioration of a disease or symptom for which insulin resistance improvement and / or hyperglycemia improvement is effective in the treatment, prevention or improvement thereof.
(7) The composition according to any one of (1) to (6) above, which is used for a human having a BMI of 23.1 or more.
(8) The composition according to any one of (1) to (7), wherein the koji mold fermentation product uses a grain-derived material as a fermentation material.
(9) The composition according to any one of (1) to (8) above, wherein the koji mold is a filamentous fungus of the genus Aspergillus.
(10) The method for producing a composition according to any one of (1) to (9), comprising fermenting a grain-derived raw material with koji mold to obtain a fermentation product containing 14-dehydroergosterol. .

  Since 14-dehydroergosterol used as an active ingredient in the present invention is extracted from a koji mold fermentation product that has been used as a food for many years, the composition of the present invention can be taken over a long period of time. It is advantageous in that it has few side effects and high safety.

It is the figure which showed the HPLC chromatogram of a crude extract and a fraction (14-DHE). FIG. 3 is a graph showing changes in body weight of each group of test animals in Example 1. * In the figure means that there is a significant difference (p <0.05) from the high fat diet group. FIG. 3 is a graph showing changes in weight gain of each group of test animals in Example 1. * In the figure means that there is a significant difference (p <0.05) from the high fat diet group. ** in the figure means that there is a significant difference (p <0.01) from the high fat diet group. 2 is a graph showing the amount of fat around the kidney of each group of test animals in Example 1. FIG. * In the figure means that there is a significant difference (p <0.05) from the high fat diet group. FIG. 4 is a graph showing testicular fat mass in each group of test animals in Example 1. 2 is a graph showing the amount of neutral fat in the liver of each group of test animals in Example 1. FIG. 2 is a graph showing the amount of neutral fat in skeletal muscle of each group of test animals in Example 1. FIG. * In the figure means that there is a significant difference (p <0.05) from the high fat diet group. FIG. 3 shows gene expression of various fat synthesis proteins in skeletal muscle of each group of test animals in Example 1. ** in the figure means that there is a significant difference (p <0.01) from the high fat diet group. FIG. 2 is a graph showing gene expression of glucose transporter protein (GLUT4) in skeletal muscle of each group of test animals in Example 1. * In the figure means that there is a significant difference (p <0.05) from the high fat diet group. FIG. 3 is a view showing blood insulin concentrations in each group of test animals in Example 1. It is the figure which showed the HPLC chromatogram of 14-DHE containing birch extract. FIG. 3 is a graph showing changes in blood free fatty acid concentration in each group of test animals in Example 2. * In the figure means that there is a significant difference (p <0.05) from the control group. FIG. 4 is a graph showing changes in blood neutral fat concentration in each group of test animals in Example 2. * In the figure means that there is a significant difference (p <0.05) from the control group. FIG. 3 is a graph showing changes in blood total cholesterol concentration in each group of test animals in Example 2. * In the figure means that there is a significant difference (p <0.05) from the control group. FIG. 3 is a graph showing changes in blood insulin concentration in each group of test animals in Example 2. FIG. 4 is a diagram showing OGTT results for each group of test animals in Example 2. * In the figure means that there is a significant difference (p <0.05) from the control group. FIG. 3 is a graph showing the amount of neutral fat in skeletal muscle of each group of test animals in Example 2. * In the figure means that there is a significant difference (p <0.05) from the control group.

Detailed description of the invention

（物理化学的特性）
（１）分子量：394.63
（２）分子式：C28H42O(高分解能APCI-Orbitrap法による観測値:m/z 395.33057(M+H)⁺、理論値:395.33139)
（３）溶剤に対する溶解性：水に不溶、エタノールに難溶、クロロホルムに易溶
（４）紫外吸収スペクトル(MeCN)：391nm
（５）¹H-NMR(CD₃OD)： 6.15 (1H, m), 5.75 (1H, m), 5.65 (1H, dd, J = 2.2, 5.9Hz), 5.27 (1H, dd, J = 7.0, 15.1 Hz), 5.21 (1H, dd, J = 7.9, 15.1 Hz), 3.64 (1H, m), 2.51 (1H, ddd, J = 2.2, 5.1, 10.6 Hz), 2.30 (1H, m), 2.20 (1H, m), 2.20 (1H, dd, J = 3.2, 7.8 Hz), 2.06 (1H, m), 2.05 (1H, m), 1.94 (1H, m), 1.90 (1H, m),1.87 (2H, m), 1.87 (1H, ddd, J = 3.2, 7.0, 7.3 Hz), 1.71 (1H, m), 1.59 (1H, m),1.57 (1H, m), 1.45 (1H, m), 1.45 (1H, ddd, J = 3.2, 6.3, 6.5 Hz), 1.30 (1H, m),1.05 (3H, d, J = 6.8 Hz ), 0.93 (3H, d, J = 7.3 Hz), 0.92 (3H, s,), 0.89 (3H, s), 0.85 (3H, d, J = 6.5 Hz), 0.83 (3H, d, J = 6.3 Hz).
（６）¹³C-NMR(CD₃OD)：149.2 (s), 143.0 (s), 135.4 (s), 132.2 (s), 132.0 (s),120.5 (s), 120.4 (s), 117.4 (s), 70.4 (s), 58.1 (s), 46.3 (s), 45.4 (s), 42.8 (s), 41.0 (s), 39.0 (s), 38.9 (s), 37.8 (s), 37.0 (s), 36.0 (s), 33.1 (s), 32.0 (s), 21.1 (s), 19.9 (s), 19.7 (s), 19.6 (s), 17.6 (s), 16.8 (s), 14.5 (s). The koji mold fermentation product used in the present invention is a fermentation product produced by koji mold fermenting raw materials, and is characterized by containing 14-dehydroergosterol (14-DHE). 14-DHE is a compound represented by the following structural formula, and has the following physicochemical characteristics.
(Structural formula)

(Physicochemical properties)
(1) Molecular weight: 394.63
(2) Molecular formula: C28H42O (observed by high resolution APCI-Orbitrap method: m / z 395.33057 (M + H) ⁺ , theoretical value: 395.333139)
(3) Solubility in solvents: Insoluble in water, hardly soluble in ethanol, easily soluble in chloroform (4) Ultraviolet absorption spectrum (MeCN): 391 nm
(5) ¹ H-NMR (CD ₃ OD): 6.15 (1H, m), 5.75 (1H, m), 5.65 (1H, dd, J = 2.2, 5.9 Hz), 5.27 (1H, dd, J = 7.0 , 15.1 Hz), 5.21 (1H, dd, J = 7.9, 15.1 Hz), 3.64 (1H, m), 2.51 (1H, ddd, J = 2.2, 5.1, 10.6 Hz), 2.30 (1H, m), 2.20 (1H, m), 2.20 (1H, dd, J = 3.2, 7.8 Hz), 2.06 (1H, m), 2.05 (1H, m), 1.94 (1H, m), 1.90 (1H, m), 1.87 ( 2H, m), 1.87 (1H, ddd, J = 3.2, 7.0, 7.3 Hz), 1.71 (1H, m), 1.59 (1H, m), 1.57 (1H, m), 1.45 (1H, m), 1.45 (1H, ddd, J = 3.2, 6.3, 6.5 Hz), 1.30 (1H, m), 1.05 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 7.3 Hz), 0.92 (3H, s,), 0.89 (3H, s), 0.85 (3H, d, J = 6.5 Hz), 0.83 (3H, d, J = 6.3 Hz).
(6) ¹³ C-NMR (CD ₃ OD): 149.2 (s), 143.0 (s), 135.4 (s), 132.2 (s), 132.0 (s), 120.5 (s), 120.4 (s), 117.4 ( s), 70.4 (s), 58.1 (s), 46.3 (s), 45.4 (s), 42.8 (s), 41.0 (s), 39.0 (s), 38.9 (s), 37.8 (s), 37.0 ( s), 36.0 (s), 33.1 (s), 32.0 (s), 21.1 (s), 19.9 (s), 19.7 (s), 19.6 (s), 17.6 (s), 16.8 (s), 14.5 ( s).

  The koji mold fermentation product used in the present invention can be prepared by inoculating koji mold as a raw material and culturing, and in some cases, extracting a fermentation product containing bacterial cells using a solvent. Since 14-DHE is contained in the gonococcal fermentation product obtained by extraction using a solvent, the gonococcal fermentation extract extracted using a solvent can be preferably used in the present invention.

  The koji mold used in the present invention is not particularly limited as long as it can produce 14-DHE. Monascus anca), Aspergillus soya, soy sauce, Aspergillus kawachi, Tempe fungus (Rhizopus oligospora), and the like. In particular, Neisseria gonorrhoeae, which are filamentous fungi of the genus Aspergillus, are preferable because of high production of 14-DHE. As the koji mold used in the present invention, particularly, Aspergillus awamori and Aspergillus niger which are narrow black molds, Aspergillus kawachi which is a narrow white mold, and subspecies derived from these bacterial species are preferable. The koji mold used in the present invention is particularly preferably Aspergillus kawachi. These koji molds can be obtained from Akita Imano Co., Higuchi Moyashi Co., Nippon Brewing Industry Co., etc.

  As a raw material for fermentation using koji mold, a grain-derived material is preferable for high production of 14-DHE. As cereals, all plants classified as Gramineae in the plant classification table are listed. Examples of plants that can be used as cereals in the present invention include rice, barley, wheat, rye, millet, millet, and corn, with rice, barley, wheat, and rye being preferred. The material used as a fermentation raw material in the present invention is preferably the seeds of the cereal plants as described above, more preferably malt (especially barley malt) or germinated brown rice from which they are germinated. Alternatively, husks of those seeds, for example, whole grains of grain seed, bran of cereal plants, rice bran, etc. can be used as fermentation raw materials.

  Fermentation using koji molds is performed by inoculating koji molds as raw materials and culturing them, and can be performed using methods known to those skilled in the art. Examples of such a method include a conventional lid method, a box method, a floor method, and a mechanical method. Moreover, not only inoculating and culturing a koji mold on a solid material, fermentation may be performed by inoculating koji mold on a liquid material (liquid medium) in which a raw material component is dissolved in a solvent such as water. For details of the culture (fermentation) conditions, for example, the fermentation handbook (Bioindustry Association, edited by Fermentation and Metabolism Research Institute, 2001) can be referred to.

  In the above culturing step, preferably, a liquid medium containing a malt extract can be inoculated with koji mold and cultured. A malt extract is an extract obtained by germinating wheat, especially barley seeds, and optionally roasting and then extracting it with a solvent such as water, and contains a large amount of reducing sugars such as maltose, glucose and fructose. It is known to contain other peptides, amino acids, purines, vitamins and the like. In addition to malt extract, the medium may contain sugars such as maltose, glucose, sucrose, and lactose, starch, or yeast extract. A liquid culture medium is used, and liquid culture is advantageous in that culture control is relatively easy, production of by-products is small, and there is a tendency to produce more 14-DHE than solid culture. It is.

  The fermentation product of Aspergillus oryzae can be extracted by a general method using a suitable solvent from a fermentation product containing bacterial cells obtained through a culture process (fermentation). Examples of the organic solvent used for solvent extraction include ethanol, methanol, n-propanol, isopropanol, methyl acetate, ethyl acetate, hexane, heptane, and the like. Of these, ethanol, methanol, ethyl acetate, and hexane are preferable. Two or more of these solvents may be used as a mixture, or two or more solvent extraction steps may be performed using two or more organic solvents. Alternatively, edible fats and oils can be used as an extraction solvent instead of or in combination with such an organic solvent. The edible fat is preferably vegetable oil, such as soybean oil, rapeseed oil, olive oil, palm oil, sesame oil, corn oil, safflower oil, cottonseed oil, peanut oil, grape seed oil, sesame oil, camellia oil, linseed oil Etc.

  More preferably, the gonococcal fermentation product used in the present invention is obtained after inoculating and culturing the koji mold on the raw material, immersing the fermentation product containing the bacterial cells in an immersion liquid containing ethanol and water, and the immersing process. It can be prepared according to a method comprising a step of adding edible fats and oils to a mixture containing the microbial cells. The gonococcal fermentation product prepared by this method is advantageous in that it contains a high concentration of 14-DHE.

  In the immersion step, the ethanol concentration in the immersion liquid containing ethanol and water for immersing the fermentation product containing bacterial cells obtained by culturing is 20 (w / w)% or more, particularly 30 (w / w)% or more, In particular, it is preferably 40 (w / w)% or more and 95 (w / w)% or less, particularly 90 (w / w)% or less, and particularly preferably 80 (w / w)% or less. The immersion liquid may be an ethanol aqueous solution composed of water and ethanol. It is considered that the immersion liquid containing ethanol and water has an effect of weakening the cell membrane or cell wall of the microbial cells contained in the fermentation product and facilitating extraction of 14-DHE stored in the microbial cells. Immersion is preferably performed over a period of 1 hour or more, particularly 6 hours or more, particularly 12 hours or more, and more preferably 24 hours or more.

  The fermentation product immersed in the immersion liquid containing ethanol and water is then subjected to an extraction process using edible fats and oils. The edible oil / fat that can be used in the extraction step may be animal oil / fat such as lard or head, but it is more preferable to use vegetable oil / fat. Specific examples of vegetable oils that can be used for extraction include soybean oil, rapeseed oil, olive oil, palm oil, sesame oil, corn oil, safflower oil, cottonseed oil, peanut oil, grape seed oil, sesame oil, camellia oil, For example, linseed oil. Edible fats and oils are preferably used in an amount of 10 mL or less, particularly 8 mL or less, particularly 6 mL or less, based on 1 g of the culture product, because the 14-DHE concentration in the resulting koji mold fermented extract increases.

  Extraction of the fermentation product immersed in the immersion liquid containing ethanol and water can be performed by directly mixing the mixture containing microbial cells obtained after the immersion step and edible fats and oils. Specifically, for example, the mixture of the immersion liquid after immersion and the fermentation product can be stirred as necessary, and then edible fats and oils can be added and further stirred to recover the oil layer. By this extraction step, the desired 14-DHE-containing koji mold fermented extract can be obtained. Therefore, the obtained koji mold fermented extract may contain, in addition to 14-DHE extracted from the fermentation product, edible oil and fat used for extraction, and ethanol and water derived from the immersion liquid. In this extraction step, ultrasonic treatment for destroying the bacterial cells is not particularly required. By soaking in an immersion liquid containing ethanol and water, the cell membrane or cell wall of the cells is sufficiently weakened, and 14-DHE stored in the cells can be extracted simply by adding edible oil and fat and stirring. . The extraction process may be repeated multiple times as necessary so that 14-DHE can be extracted more efficiently.

  The gonococcal fermentation product prepared by the above method can contain 14-DHE of 10 ng / mg or more, more preferably 50 ng / mg or more, and particularly preferably 100 ng / mg or more.

  The koji mold fermentation product used in the present invention can be directly used for foods and the like without purification.

  As an active ingredient of the composition of the present invention, a koji mold fermentation product can be used, or 14-DHE isolated from a koji mold fermentation product can also be used. Isolation of 14-DHE from the koji mold fermentation product can be performed according to a known method.

  As shown in Examples below, 14-DHE and the koji mold fermentation product have an effect of suppressing blood lipid concentration. Therefore, 14-DHE and gonococcal fermentation products can be used as a composition for improving lipid metabolism and reducing the risk of abnormal lipid metabolism, and used in a method for improving lipid metabolism and a method for reducing the risk of abnormal lipid metabolism. Can do. Moreover, 14-DHE and a gonococcal fermentation product can be used also as a lipid metabolism improving agent and a risk reduction agent of lipid metabolism abnormality. Furthermore, in the present invention, 14-DHE and gonococcal fermentation products can be used as a composition for adjusting blood lipid concentration or a regulator of blood lipid concentration, and also used in a method for adjusting blood lipid concentration. be able to. In the present specification, “adjustment of blood lipid concentration” is used in the meaning including suppression of increase in blood lipid concentration other than HDL-cholesterol and suppression of blood lipid concentration, and decrease in blood concentration of HDL-cholesterol. It is used in the sense of including suppression of blood pressure and increase in blood concentration.

  In the present specification, “improvement of lipid metabolism” means improvement of abnormal lipid metabolism. The degree of lipid metabolism improvement can be evaluated, for example, using blood lipid concentration as an index (see Examples 1 and 2). Examples of blood lipids that serve as indices include triglycerides (triglycerides), cholesterol (total cholesterol, LDL cholesterol, HDL cholesterol), free fatty acids, and the like. In the present specification, “risk reduction” means that the expression of the state is suppressed as compared to the case where no treatment is taken with respect to any physical state or physiological state.

  Moreover, as shown in the below-mentioned Example, 14-DHE and a gonococcus fermentation product have a weight gain inhibitory effect and a fat accumulation inhibitory effect. Therefore, 14-DHE and the gonococcal fermentation product can be used as a composition for inhibiting weight gain and / or fat accumulation, and can be used in a method for inhibiting weight gain and / or fat accumulation. Moreover, 14-DHE and a koji mold fermentation product can be used also as a weight gain inhibitor and a fat accumulation inhibitor.

  Moreover, weight gain is suppressed and fat accumulation is suppressed, thereby improving the obesity state and suppressing the development of obesity. Therefore, 14-DHE and a koji mold fermentation product can be used as a composition for improving obesity and reducing obesity risk, and can also be used in an obesity improving method and an obesity risk reducing method. 14-DHE and the koji mold fermentation product can also be used as an obesity improving agent and an obesity risk reducing agent.

  In the present specification, “obesity” includes not only a state that falls within the definition of medical obesity but also a state that oneself feels obese, and “improvement in obesity” means an improvement in the obesity state . The degree of obesity improvement can be evaluated using, for example, body weight or body fat amount as an index (see Examples 1 and 2). Examples of body fat include subcutaneous fat, visceral fat, and ectopic fat. Particularly, the amount of visceral fat and ectopic fat is an indicator. In the present specification, “ectopic fat” means fat that accumulates in tissues other than the subcutaneous and internal organs where normal fat is accumulated, and includes, for example, fat that accumulates in muscle, heart, liver and the like.

  Furthermore, as shown in Examples described later, 14-DHE and the koji mold fermentation product have an insulin resistance improving action and an onset suppressing action. Therefore, 14-DHE and the gonococcal fermentation product can be used as a composition for improving insulin resistance and reducing the risk of developing insulin resistance, as well as a method for improving insulin resistance and a method for reducing the risk of developing insulin resistance. Can be used for Moreover, 14-DHE and a gonococcal fermentation product can be used also as an insulin resistance improving agent and an onset risk reduction agent of insulin resistance.

  The method for improving lipid metabolism, the method for reducing the risk of lipid metabolism abnormality, the method for adjusting blood lipid concentration, the method for suppressing weight gain, the method for suppressing fat accumulation, the method for improving obesity, the method for reducing obesity risk, the method for improving insulin resistance The method and the method for reducing the risk of developing insulin resistance can be carried out by ingesting or administering an effective amount of 14-DHE or a gonococcal fermentation product containing the same to a human or non-human animal.

  The use of 14-DHE or a gonococcal fermentation product containing the same in the present invention may be used in humans and non-human animals, and is intended for both therapeutic and non-therapeutic uses. As used herein, “non-therapeutic” means not including the act of operating, treating, or diagnosing a human (that is, a medical act on a human), and specifically, receiving a doctor or doctor's instructions It means that the person does not include a method of performing surgery, treatment or diagnosis on humans. In the present invention, when 14-DHE or a gonococcal fermentation product containing the same is used for humans, it can be preferably used for humans having a BMI of 23.1 or more.

  In the present invention, 14-DHE or Neisseria gonorrhoeae fermentation products can also be used for the treatment, prevention or amelioration of diseases and conditions for which lipid metabolism improvement is effective in the treatment, prevention or amelioration thereof.

  Diseases and symptoms for which lipid metabolism improvement is effective for treatment, prevention or improvement include obesity, dyslipidemia and arteriosclerosis (including coronary artery disease, myocardial infarction, cerebral infarction) and the like. Therefore, 14-DHE can be used as a therapeutic agent, preventive agent and ameliorating agent for these diseases and symptoms, and can also be used in a method for treating, preventing and improving these diseases and symptoms.

  In the present invention, 14-DHE or a koji mold fermentation product is further used for the treatment, prevention or amelioration of diseases and symptoms for which improvement of insulin resistance and / or improvement of hyperglycemia is effective in the treatment, prevention or amelioration thereof. Can do.

  Diseases and conditions for which improvement in insulin resistance is effective for the treatment, prevention or amelioration include diabetes. Therefore, 14-DHE can be used as a therapeutic agent, preventive agent and ameliorating agent for this disease and symptom, and can also be used in a method for treating, preventing and ameliorating this disease and symptom.

  Composition and use for improving lipid metabolism, reducing risk of abnormal lipid metabolism, inhibiting weight gain, inhibiting fat accumulation, improving obesity, reducing obesity risk, improving insulin resistance, improving hyperglycemia or reducing the risk of hyperglycemia according to the present invention The agent and the therapeutic agent and prophylactic agent of the present invention can be provided in the form of pharmaceuticals, quasi drugs, foods and drinks, feeds, etc., and can be carried out according to the following description. In addition, the lipid metabolism improvement method, lipid metabolism abnormality risk reduction method, blood lipid concentration adjustment method, weight gain suppression method, fat accumulation suppression method, obesity improvement method, obesity risk reduction method, insulin resistance improvement method of the present invention The method for reducing the risk of developing insulin resistance and the treatment and prevention methods of the present invention can be carried out according to the following description.

14-DHE which is an active ingredient of the present invention or a gonococcal fermentation product containing the same (hereinafter collectively referred to as “14-DHE etc.”) is orally or parenterally administered to humans and non-human animals. Preferably administered orally. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Examples of parenteral preparations include injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, and external preparations (for example, nasal preparations, transdermal preparations, ointments) ), Suppositories (for example, rectal suppositories, vaginal suppositories). These preparations can be formulated using a pharmaceutically acceptable carrier by a technique usually performed in this field. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. For example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter can be used as a carrier.

  The production method of the oral preparation is not particularly limited, and any method can be used. Active ingredients include excipients (eg lactose, sucrose, starch, mannitol), disintegrants (eg calcium carbonate, carboxymethylcellulose calcium), binders (eg pregelatinized starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, Hydroxypropyl cellulose), stabilizers and / or lubricants (eg, talc, magnesium stearate, polyethylene glycol 6000) and then compression molded, then, if necessary, taste masking, intestinal It can be produced by coating by a known method for the purpose of solubility or durability. As the coating agent, for example, ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, Eudragit (Rohm) and the like can be used. Capsules may be produced by using the active ingredient as it is or by blending the active ingredient with pharmaceutical additives such as stabilizers, dispersants, colorants and / or preservatives. The capsule may be a soft capsule or a hard capsule, and the material of the capsule is not particularly limited. For example, gelatin, vegetable fiber, starch, or the like may be used, and an adhesion preventing measure may be taken.

  Among parenteral preparations, injections contain active ingredients as dispersants (for example, Tween 80 (Atlas Powder)), preservatives (for example, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, phenol), isotonic agents (for example, Aqueous solvents (eg, distilled water, physiological saline, Ringer's solution) or oily solvents (eg, olive oil, sesame oil, cottonseed oil, corn oil, etc.) together with pharmaceutical additives such as sodium chloride, glycerin, sorbitol, glucose, invert sugar) In vegetable oil, propylene glycol) and the like. At this time, if desired, additives such as a solubilizing agent (for example, sodium salicylate, sodium acetate), a stabilizer (for example, human serum albumin), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride) are added. Also good.

  The active ingredient of the present invention, such as 14-DHE, can be taken orally by humans and non-human animals. When 14-DHE or the like is orally ingested, 14-DHE or the like may be in an isolated, purified or crudely purified form, or may be a food containing 14-DHE or the like or a form of a raw material of food. Good.

  When the 14-DHE or gonococcal fermentation product which is the active ingredient of the present invention is provided as a food, the 14-DHE or the gonococcal fermentation product can be directly contained in the food, and the food is effective for 14-DHE. It is a food containing a quantity. In this specification, “containing an effective amount” of 14-DHE means a content such that 14-DHE is ingested within the range described below when an amount normally consumed in each food is ingested. Say. “Food” means health food, functional food, health functional food (for example, food for specified health use, functional nutrition food, functional indication food), food for special use (for example, food for infants, food for pregnant women, disease, etc.) Food). The form of “food” is not particularly limited, and may be, for example, a drink form, a semi-liquid form or a gel form.

  Foods provided in the present invention include foods and drinks containing carbohydrates such as rice, noodles, breads and pasta; Western confectionery such as cookies and cakes; Japanese confectionery such as buns and sheepskin; candy, gums, yogurt and Various confectionery such as pudding and frozen desserts; whiskey, bourbon, spirits, liqueur, wine, fruit liquor, sake, sake, shochu, beer, non-alcoholic beer with alcohol content of 1% or less, sparkling liquor, other miscellaneous sake Alcoholic beverages such as strawberry high; fruit juice beverage, vegetable juice beverage, fruit juice and vegetable juice beverage, soft drink, milk, soy milk, milk beverage, drink type yogurt, drink type jelly, coffee, cocoa, tea Non-alcoholic beverages such as beverages, energy drinks, sports beverages and mineral water; processed products using eggs, seafood and Workpiece meat (including offal levers, etc.) and the like can be exemplified (including delicacies), but is not limited thereto.

  Examples of tea beverages include black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted tea, oolong tea, turmeric tea, puer tea, rooibos tea, rose tea, chrysanthemum tea, herbal tea (for example, mint tea, jasmine) Tea).

  Examples of fruits used in fruit juice-containing beverages and fruit and vegetable juice-containing beverages include apples, mandarin oranges, grapes, bananas, pears, acai, blueberries, and ume. Moreover, as a vegetable used for a drink containing vegetable juice, fruit juice, and a drink containing vegetable juice, a tomato, a carrot, a celery, a cucumber, and a watermelon are mentioned, for example.

  Since 14-DHE and the like have various actions such as a lipid metabolism improving action and an insulin resistance improving action, they can be provided by being included in foods and drinks taken daily, and further in foods taken as supplements. In this case, 14-DHE or the like can be provided in a unit packaging form in which the amount taken per meal is predetermined, and examples of the unit packaging form per meal include packs, packaging, cans, bottles, etc. The form which prescribes | regulates a fixed quantity with is mentioned. In order to better exhibit various effects such as 14-DHE, it is desirable to ingest 50-100,000 μg, preferably 100-50000 μg of 14-DHE per serving.

  In the present invention, when 14-DHE or the like is provided in the form of food, for example, 14-DHE can be contained at 50 to 100000 μg, preferably 100 to 50000 μg per container. Further, when 14-DHE or the like is provided in the form of a supplement, for example, 14-DHE may be contained in an amount of 10 to 1000 µg, preferably 20 to 500 µg, more preferably 100 to 400 µg per capsule or capsule. Moreover, when setting it as a drink, 14-DHE per container can be contained 20-10000 microgram / 100-500 mL, Preferably it is 100-1000 microgram / 100-500 mL, More preferably, 200-500 microgram / 100-500 mL can be contained. When the content of 14-DHE per packaging unit does not reach the intake per meal, the intake is related so that 14-DHE is ingested 50 to 100,000 μg, preferably 100 to 50,000 μg per meal. It is preferable to provide a document or the like on which explanation items are described together.

  Since the pharmaceuticals and foods of the present invention utilize 14-DHE contained in persimmons that humans have ingested for many years as foods, they have low toxicity and mammals that require it (for example, humans, mice, rats, rabbits, Dogs, cats, cows, horses, pigs, monkeys, etc.). The intake or dose of 14-DHE can be determined depending on the recipient's sex, age and weight, symptoms, administration time, dosage form, administration route, combined drugs and the like. For example, when 14-DHE is ingested or administered to humans, it will be in the range of 1-100,000 μg / kg body weight, preferably 2-100,000 μg / kg body weight, more preferably 4-50000 μg / kg body weight per adult day. Can be taken or administered once or several times a day.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.
Example 1 Confirmation of Lipid Metabolism Improvement Effect and Obesity Improvement Effect of 14-DHE in Dietary Obese Mice Dietary obese mice produced by loading with a high fat diet have characteristics similar to obesity, ie, ectopic. This model exhibits fat accumulation and insulin resistance. In this example, dietary obese mice were used to verify the effect of 14-DHE on lipid metabolism improvement and obesity improvement.

1.14 Preparation of DHE Shirakaba ( Aspergillus kawachii NBRC4308 obtained from National Institute for Product Evaluation) was inoculated into potato dextrose agar medium (Difco, manufactured by BD) and cultured at 25 ° C. for 7 days. The spore-formed cells were suspended in a malt extract medium containing 0.01% Tween 20 (Difco, manufactured by BD; product name: Malt extract broth), and filtered to obtain a spore solution. Place 150 ml of malt extract medium after autoclave sterilization into a 500 ml Sakaguchi flask, add a spore solution to a concentration of 1.0 × 10 ⁵ spores / ml, and shake culture at 140 spm at 30 ° C. for 20 hours. What was performed was used as a preculture solution. Next, 3.0 L of malt extract medium is spread on a 5 L jar fermenter (manufactured by Maruhishi Bioengineer), sterilized by autoclave, and then 37 ml of preculture solution (1.25 × 10 ³ spores / ml) is added. The culture was performed at 30 ° C., 400 rpm, and 0.5 vvm for 144 hours. In addition, silicone oil was added and defoamed at the time of preculture and main culture, respectively.

The cells recovered from the medium are immersed in 59 (w / w)% ethanol overnight, hexane is added thereto, and the mixture is stirred at 400 rpm and centrifuged at 10,000 rpm to obtain a supernatant (hexane layer). It was. This was dried up, adjusted to 10 mg / ml with 100% ethanol, and filtered through a 0.22 μm diameter PTFE membrane to obtain an HPLC sample (crude extract). This was subjected to high performance liquid chromatography (moving bed; acetonitrile / 2-propanol = 99/1, flow rate; 1.0 mL / min, detection) using Develosil C30-UG-3 (10 × 250 mm) (manufactured by Nomura Chemical Co., Ltd.); 320 nm and 280 nm absorption). The chromatogram of the crude extract and the fraction is shown in FIG. Since absorption by 14-DHE increases at an absorption wavelength of 320 nm, and by-product absorption is often observed at an absorption wavelength of 280 nm, only the 14-DHE peak is fractionated while confirming the peak areas of both wavelengths. , 14-DHE was isolated.

2. Test conditions A 14-week-old male C57BL / 6J DIO model (C57BL / 6J mice that induced obesity by loading HFD60 for 10 weeks from 4 weeks old) was purchased from Japan Charles River Co., Ltd., and high-fat diet (Oriental) After feeding for 1 week with high fat diet HFD60 (60 kcal% fat) from Yeast Industry Co., Ltd., the group was divided into 2 groups of 10 animals per group so that the average body weight was almost the same. A high fat diet was administered to the 14-DHE diet group for 5 weeks, each of which was added with a high fat diet so that 14-DHE was at a final concentration of 0.001% (w / w). 14-DHE was dissolved in soybean oil and added to HFD 60 from which soybean oil had been removed in advance so as to have a prescribed amount.

As a control, a group of 14-week-old male C57BL / 6J purchased from Charles River Japan Co., Ltd. was bred for 6 weeks with AIN 93M as a standard feed was set (normal diet group).

Each feed was ingested in an amount of 3.0 g / animal / day (14-DHE was 30 μg / animal / day), and drinking water was ad libitum.

For the above three groups, after fasting overnight on the 5th week (day 35 of administration) from the administration start date (day 1 of administration), dissection was performed, and heart blood sampling, liver, skeletal muscle, and visceral fat were performed. Extraction of fat around testis and fat around kidney was performed.

3. Measurement Item (1) Body Weight The day before the start of administration (day 0) and the fifth day, the tenth day, the fourteenth day, the twenty-first day, the twenty-eighth day and the thirty-fifth day The body weight of each mouse was measured in the eyes (the first day of administration is the first day of administration).

(2) Measurement of visceral fat mass Wet weights of testicular fat and kidney peripheral fat were measured respectively.

(3) Measurement of amount of neutral fat in liver and skeletal muscle 0.1 g of frozen liver and skeletal muscle were weighed in each test tube. 4 mL chloroform / methanol (2: 1) was added thereto and homogenized for 1 minute. Further, 1 ml of 0.5% NaCl solution was added, and after stirring, the lower layer was recovered by centrifugation at 800 rpm and 20 ° C. for 20 minutes. The same operation was performed after adding 3 ml of chloroform / methanol (2: 1) again. The recovered lower layer was collected in a glass test tube, and the extract was dried at 50 ° C. while blowing nitrogen gas. After drying, it was dissolved in 1 ml of 2-propanol. The neutral fat concentration of this redissolved solution was measured using Triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The amount of neutral fat in the liver and skeletal muscle was calculated as the weight per freeze-wet weight.

(4) Ectopic fat accumulation and gene expression analysis RNA is extracted from a part of the extracted skeletal muscle using Trizol, and adipose synthesis protein (PPARγ, GDAT2, CD36 and LPL) and glucose using real-time PCR The gene expression of transporter protein (GLUT4) was analyzed. The total RNA was purified using RNAeasy Mini Kit (manufactured by QIAGEN). cDNA was synthesized from total RNA using iScript cDNA Synthesis Kit (manufactured by Bio-Rad). RT-PCR was performed with LightCycler 480 (Roche) using SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa), and expression analysis of GAPDH, PPARγ, GDAT2, CD36, LPL and GLUT4 was performed on various samples. . The primers used are shown below.

<GAPDH>
5'-AACGACCCCTTCATTGAC-3 '(forward direction) (SEQ ID NO: 1)
5'-TCCACGACATACTCAGCAC-3 '(reverse direction) (SEQ ID NO: 2)
<PPARγ>
5'-TCTGCGAGCCCTGGCAAAGCAT-3 '(forward direction) (SEQ ID NO: 3)
5'-TGGCCACCTCTTTGCTCTGCTCCT-3 '(reverse direction) (SEQ ID NO: 4)
<GDAT2>
5'-TGGCAAGAACGCAGTCACCCTGA-3 '(forward direction) (SEQ ID NO: 5)
5'-CATGGAAGATGCAGGGGGCGAAA-3 '(reverse direction) (SEQ ID NO: 6)
<CD36>
5'-TGGCACAGACGCAGCCTCCTTT-3 '(forward direction) (SEQ ID NO: 7)
5'-TGGATTCTGGAGGGGTGATGCAAA-3 '(reverse direction) (SEQ ID NO: 8)
<LPL>
5'-GTTTGGCTCCAGAGTTTGACCG-3 '(forward direction) (SEQ ID NO: 9)
5'-CATACATTCCCGTTACCGTCCAT-3 '(reverse direction) (SEQ ID NO: 10)
<GLUT4>
5'-ACCATAGGAGCTGGTGTGGTCAAT-3 '(forward direction) (SEQ ID NO: 11)
5'-GGCCAATCTCAAAGAAGGCCACAA-3 '(reverse direction) (SEQ ID NO: 12)

(5) Measurement of blood insulin concentration Blood collected in a container containing a blood coagulation inhibitor (heparin) was centrifuged at 5,000 rpm for 5 minutes to separate plasma. The insulin concentration in plasma was measured using an ultrasensitive mouse insulin measurement kit (manufactured by Morinaga Bioscience Institute).

(6) Test Regarding the results obtained, t-test was performed in the 14-DHE diet group and the high fat diet group, and the difference was significant when the difference between groups was less than 1% or less than 5%. did.

4). Results The body weight during the administration period is shown in FIG. Compared with the high fat diet group, the 14-DHE diet group showed a significant decrease in body weight on the 28th day of administration. Moreover, the weight gain from the first day of administration is shown in FIG. Compared with the high fat diet group, the 14-DHE diet group showed a significant decrease in weight gain after the 21st day of administration. From these results, it was revealed that 14-DHE has an effect of suppressing weight gain.

The amount of fat around the kidney and the amount of fat around the testis at the time of dissection are shown in FIGS. 4 and 5, respectively. Compared with the high-fat diet group, the amount of fat around the kidney was significantly decreased in the 14-DHE diet group (FIG. 4). Compared with the high-fat diet group, the 14-DHE diet group had a significant difference in the weight of fat around the testis (p = 0.096), but it tended to be suppressed (FIG. 5). Further, FIG. 6 and FIG. 7 show the amount of neutral fat in the liver and the amount of neutral fat in skeletal muscle, respectively. Compared with the high fat diet group, the amount of neutral fat in skeletal muscle was significantly reduced in the 14-DHE diet group (FIG. 7). Compared with the high-fat diet group, the 14-DHE diet group had a significant difference in the amount of triglycerides in the liver (p = 0.076), but tended to be suppressed (FIG. 6). From these facts, it is considered that peripheral fat in the kidney is equivalent to human visceral fat, and accumulation of visceral fat is suppressed by administration of 14-DHE. In addition, as obesity progresses, ectopic fat accumulation (for example, accumulation of fat in skeletal muscle and liver) occurs, and it is clear that administration of 14-DHE suppresses ectopic fat accumulation. became.

The result of examining the gene expression of fat synthesis protein in skeletal muscle is shown in FIG. In any gene, the expression was significantly lower in the 14-DHE diet group than in the high fat diet group. That is, it was revealed that the increase in gene expression of fat synthesis proteins (PPARγ, DGAT2, CD36, LPL) observed in the high fat diet group was suppressed by administration of 14-DHE.

The result of examining the gene expression of glucose transporter protein (GLUT4) in skeletal muscle is shown in FIG. The expression was significantly higher in the 14-DHE diet group than in the high fat diet group. Moreover, the blood insulin concentration is shown in FIG. Compared with the high-fat diet group, the 14-DHE diet group tended to be low although the blood insulin concentration was not significantly different (p = 0.08).

Example 2: Hyperglycemia and dyslipidemia improving action of 14-DHE and 14-DHE-containing birch extract In this example, obesity / insulin resistance model mice (KK-Ay) that spontaneously develop diabetes and dyslipidemia In the above, it was verified whether or not the onset of diabetes and dyslipidemia was suppressed by oral administration of 14-DHE and white birch extract containing the same (extract of white birch fermentation product).

1. Preparation of 14-DHE and 14-DHE-containing birch extract 14-DHE was prepared in the same manner as in Example 1.

The cultivation of white gonococcus ( Aspergillus kawachii NBRC4308) when preparing a 14-DHE-containing white cocoon extract was carried out in the same manner as in Example 1. The cells recovered from the medium were immersed in 59 (w / w)% ethanol overnight, then soybean oil was added thereto, and then the mixture was stirred at 400 rpm and centrifuged at 10,000 (oil layer). Was made into a 14-DHE-containing birch extract. This was subjected to high performance liquid chromatography (acetonitrile / 2-propanol = 99/1) using Develosil C30-UG-3 (10 × 250 mm) (manufactured by Nomura Chemical Co., Ltd.), and 14-per white birch extract was analyzed. The DHE containing concentration was calculated. FIG. 11 shows a chromatogram of the 14-DHE-containing koji extract. The degree of purification was confirmed at absorption wavelengths of 280 nm and 320 nm.

2. Test conditions Six-week-old male KK-Ay / Ta jc1 mice obtained from Clea Japan Co., Ltd. were grouped into 3 groups of 7 mice per group so that the average body weight was almost the same. AIN93G feed (produced by Oriental Yeast Co., Ltd.) which is a simple feed, AIN93G feed with 14-DHE added to a final concentration of 0.0006% (w / w) in the 14-DHE diet group, The 14-DHE-containing white rabbit extract group was administered with AIN93G feed supplemented with 14-DHE-containing white rabbit extract so that the final 14-DHE concentration was 0.0006% (w / w) for 6 weeks. Each feed was ingested in an amount of 5.0 g / animal / day (14-DHE was 30 μg / animal / day), and drinking water was ingested freely.

  After the administration period, the mice were fasted for 6 hours, then euthanized, and blood sampling and skeletal muscle collection were performed.

3. Measurement item (1) Measurement of blood lipid concentration and blood insulin concentration Orbital blood sampling was carried out with heparinized hematocrit capillaries at 2 weeks, 4 weeks and 6 weeks after administration. The blood is collected in a container containing a blood coagulation inhibitor (heparin) and then centrifuged at 5,000 rpm for 5 minutes to separate the plasma. Free fatty acid, neutral fat (triglyceride), total cholesterol concentration, and insulin concentration in plasma Was measured. Plasma concentrations are commercially available measuring reagents, NEFA C-Test Wako (Wako Pure Chemical Industries), Triglyceride E-Test Wako (Wako Pure Chemical Industries), Cholesterol E-Test Wako (Wako Pure Chemical Industries) And it measured using ultrasensitive mouse insulin measurement kit (Morinaga Institute of Science).

(2) Oral glucose tolerance test (OGTT)
At 5 weeks after administration, an oral glucose tolerance test (OGTT) was performed to evaluate insulin resistance. That is, 0.4 g / mL glucose solution was forcibly orally administered to animals fasted overnight at 5 mL / kg (2 g / kg as glucose). Blood was collected from the tail vein into heparin-treated hematocrit capillary tubes (manufactured by Terumo) before glucose administration and 30, 90 and 180 minutes after glucose administration. Blood was centrifuged at 3,000 rpm for 15 minutes to separate plasma, and blood glucose was measured using Glucose C II-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

(3) Measurement of the amount of neutral fat in skeletal muscle The amount of neutral fat in skeletal muscle was evaluated in the same manner as in Example 1 (3).

(4) Test The results obtained are t-test between the control group for each of the 14-DHE diet group and the 14-DHE-containing birch extract group, and the difference between groups is less than 1% or less than 5% risk rate. The difference was significant.

4). Results The free fatty acid concentration, neutral fat concentration, total cholesterol concentration and insulin concentration in plasma are shown in FIG. 12, FIG. 13, FIG. 14 and FIG.

In the second week of administration, the neutral fat concentration (triglyceride) of the 14-DHE-containing birch extract group and the total cholesterol concentration of the 14-DHE-containing birch extract group and the 14-DHE diet group were compared with the control group. Was significantly lower. In the fourth week of administration, the free fatty acid concentrations in the 14-DHE-containing white rabbit extract group and the 14-DHE diet group were significantly lower than those in the control group. In the sixth week of administration, the free fatty acid concentration in the 14-DHE diet group was significantly lower than that in the control group. Moreover, the values of the 14-DHE-containing birch extract group and the 14-DHE diet group tended to be lower than those of the control group in all periods and measurement items in which no significant difference was observed. From these results, it was revealed that administration of 14-DHE or 14-DHE-containing birch extract suppresses fasting plasma triglyceride concentration, free fatty acid concentration and total cholesterol concentration after overnight fasting. Insulin concentrations were almost the same in the 3 groups at the 2nd and 4th week of administration, but at the 6th week of administration, the 14-DHE-containing white rabbit extract group and the 14-DHE diet group were not treated. In contrast, the insulin concentration tended to be low (the difference between the 14-DHE diet group and the non-administered group was not significant but p <0.1).

FIG. 16 shows changes in plasma glucose concentration during OGTT.

  In the 14-DHE-containing white birch extract group, blood glucose concentration remained unchanged even after administration of glucose, and tended to be lower than that of the control group at 0.5 hours and 1.5 hours after administration. In the 14-DHE diet group, the blood glucose level showed a slight upward trend at 0.5 hours after administration, but then decreased and remained lower than when glucose administration was disclosed. (The difference at 1.5 hours after administration was significant). The administration of 14-DHE significantly suppresses the increase in blood glucose after glucose load, and the administration of 14-DHE-containing white birch extract tends to suppress the increase in blood glucose after glucose load. It was revealed that 14-DHE and white birch extract containing it improve glucose tolerance abnormality.

The measurement result of the amount of neutral fat in skeletal muscle is shown in FIG.

  The amount of neutral fat in skeletal muscle was significantly lower in the 14-DHE-containing birch extract group compared to the control group, although there was no significant difference in the 14-DHE diet group (p = 0.090), There was a tendency to suppress (FIG. 17). From these results, it has been clarified that administration of 14-DHE or a white cocoon extract containing the same suppresses ectopic fat accumulation caused by the progression of obesity.

  From the above, 14-DHE or 14-DHE-containing birch extract reduces the risk of developing insulin resistance, improves insulin resistance, and further reduces the risk of developing hyperglycemia and dyslipidemia. At the same time, it became clear that hyperglycemia and dyslipidemia were improved or suppressed.

Example 3: Obesity improving action and lipid metabolism improving action of 14-DHE-containing birch extract in humans In this example, human tests were conducted to improve obesity improving action and lipid metabolism improvement of 14-DHE containing white rabbit extract. The effect was verified.

1. Test method This test complies with the ethical principles and the Personal Information Protection Law based on the Declaration of Helsinki, and is conducted at a third-party organization in compliance with the "Ethics Guidelines for Epidemiological Research" (Ministry of Education, Culture, Sports, Science and Technology, Ministry of Health, Labor and Welfare on June 17, 2002) did.

  Subjects were Japanese women aged 30 to under 50 who were healthy and not diagnosed with diabetes and had a self-reported BMI of 23 to 25, and were evaluated in a placebo-controlled randomized double-blind study.

  The test subjects confirmed that age and BMI were balanced among groups, and that there was no bias in the average value, and then in the group that took the test meal (hereinafter referred to as “test group”), 22 people and the group that took the control meal Twenty-two people were assigned to each (hereinafter referred to as “control group”).

  The subjects were given 1500 mg of the assigned test meal twice daily after breakfast and dinner with water or hot water every day during the intake period. In addition, when not eating, only the test meal was ingested. The test food intake period was 8 weeks.

The composition of the test meal is as follows.
(I) Test meal: Aspergillus kawachii NBRC4308 extract (prepared according to 1. in Example 1) 80% by mass and stabilizing and homogenizing agent (Rice wax S-100, manufactured by Yokoseki Yushi Kogyo Co., Ltd.) 20% by mass capsule. In 1500 mg of the test meal, 200 μg or more of 14-DHE was contained.
(Ii) Control diet: Aspergillus kawachii Same capsule as the test diet except that soybean oil is used instead of NBRC4308 extract.

  For the two groups, measurement of basic items, blood test, and questionnaire score entry were performed as shown below on a predetermined day from the intake of the test meal to the end of the intake of the test meal for 8 weeks. Hereinafter, 2 weeks before the start of intake (-2w) is taken 2 weeks before the start date of intake of the test meal, and the start date of intake (0w) is the start date of intake of the test meal before the intake of the test meal for that day. Week 4 (4w) is the 4th week from the start date of the intake of the test meal and after the intake of the test meal for that day, and the 8th week (8w) is the 8th week from the start date of the intake of the test meal. Means after taking the test meal for the day.

2. Evaluation Items (1) Basic Items As basic items, height, weight, body fat percentage, waist circumference, hips, and waist were measured. The measurement was performed at Nicoderm Research Co., Ltd. 2 weeks before the start of intake and 8 weeks after intake.

(2) Blood test As a blood test, the concentrations of total cholesterol (TC), neutral fat (triglyceride, TG), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), and leptin were measured. Note that LDL-C is measured by a direct measurement method. The measurement was performed by collecting blood at a medical institution and then quantitatively analyzing the collected blood sample at Nippon Medical Co., Ltd. Blood collection and quantitative analysis were performed on the start date of intake and on the 8th week of intake.

(3) Questionnaire score entry Questionnaire in the form of checking any applicable score among subjects 1 to 5 (1 none, 2 almost none, 3 little, 4 moderate, 5 altitude) To fill in. The test was conducted 2 weeks before the start of intake and at 4th and 8th weeks of intake.

(4) Statistical analysis (i) The analysis was performed using IBM SPSS Statistics 19.
(Ii) Intragroup comparison (before and after comparison): Within the group, Dunett's test was performed as a before and after comparison on the start date of intake, 4 weeks after intake, and 8 weeks after intake. For items that were not measured in the 4th week of ingestion, a corresponding t-test was performed as a comparison before and after the 8th week of ingestion with 2 days before the start of ingestion or the date of ingestion.
(Iii) Comparison between groups: As for the inquiry score, Mann-Whitney test was performed based on the amount of change and the rate of change 2 weeks before the start of intake. For other items, independent samples were t-tested based on the amount of change and the rate of change 2 weeks before the start of intake. Regarding the body weight, χ ² test (chi-square test) was performed on the number of persons whose body weight increased and decreased from the 2 weeks before the start of intake to the 8th week of intake.
(Iv) The significance level was less than 5% by two-sided test.

(5) Stratified analysis The same analysis as the above-mentioned whole analysis was performed for those whose Body Math Index (Body Mass Index, hereinafter referred to as “BMI”) was 23.1 or more.

3. Results (1) Overall analysis (i) Basic items Table 1 shows the measurement results of the basic items.

  There was no significant difference between the test group and the control group on the day of ingestion.

  In the comparison analysis between groups, there was no significant difference between the test group and the control group for any item.

The results of the χ ² test for body weight are shown in Table 2.

  Those who lost body weight from 2 weeks before the start of intake to 8 weeks of intake were significantly higher in the test group than in the control group. None of the groups had any body weight change from 2 weeks before the start of intake to 8 weeks after intake.

摂取開始時において試験群と対照群に有意差の認められた項目はなかった。 (Ii) Blood test Table 3 shows the results of the blood test.

There was no significant difference between the test group and the control group at the start of ingestion.

  In the control group, the TC concentration was significantly higher at the 8th week after the start of intake. In the test group, the TG concentration was significantly low and the HDL-C concentration was significantly high at the 8th week of intake relative to the start date of intake. Moreover, the TG change amount from the intake start date to the 8th week of intake was significantly larger in the test group than in the test group.

試験群および対照群において、摂取前後で問診スコアに有意な変化は認められなかった（Ｄｕｎｎｅｔ検定）。しかしながら、摂取８週目での「太りやすい」スコアは対照群に対し試験群で有意に低く、また、摂取８週目での「やせ・体重減少」スコアは対照群に対し試験群で有意に高かった（Ｍａｎｎ―Ｗｈｉｔｎｅｙ検定）。 (Iii) Questionnaire Form Table 4 shows the analysis results of the questionnaire score.

In the test group and the control group, there was no significant change in the interview score before and after ingestion (Dunnet test). However, the “fat” score at 8 weeks after ingestion was significantly lower in the test group compared to the control group, and the “weight / weight loss” score at 8 weeks after ingestion was significantly higher in the test group than in the control group. High (Mann-Whitney test).

(2) Stratified analysis The same analysis as the overall analysis was performed for persons with a BMI of 23.1 or higher. The analysis subjects were 10 test groups and 9 control groups.

摂取開始時において試験群と対照群に有意差の認められた項目はなかった。 (I) Basic items Table 5 shows the measurement results of the basic items.

There was no significant difference between the test group and the control group at the start of ingestion.

  In the control group, the body fat percentage significantly increased at the 8th week of ingestion relative to the intake start date. On the other hand, there was no significant difference in the test group.

レプチン濃度は体脂肪率と比例関係にあることが知られるところ、対照群においては摂取開始日に対し摂取８週目でレプチン濃度が有意に高かった。一方で、試験群においては有意な差は認められなかった。さらに、両群におけるレプチン濃度の変化率を比較すると、対照群に対し試験群で抑制傾向が示された。 (Ii) Blood test Table 6 shows the results of the blood test (leptin).

It is known that the leptin concentration is proportional to the body fat percentage. In the control group, the leptin concentration was significantly higher at the 8th week of intake compared to the intake start date. On the other hand, there was no significant difference in the test group. Furthermore, when the rate of change in leptin concentration in both groups was compared, a suppression tendency was shown in the test group relative to the control group.

摂取８週目での「やせ・体重減少」スコアは、試験群では上昇し、対照群では減少しているところ、両群を比較した変化量、変化率のいずれの差も有意なものであった。 (Iii) Interview Form Table 7 shows the analysis results of the interview score.

The “lean / weight loss” score at 8 weeks after ingestion increased in the test group and decreased in the control group, but both the difference in the amount of change compared with the two groups and the rate of change were significant. It was.

4). Conclusion Compared with the intake of the control food, it was clarified that the intake of the test food for 8 weeks tends to decrease the body weight and suppress the weight increase. It was also revealed that the scores for the awareness items “easy to gain weight” and “loss / weight loss” were improved. Furthermore, the intake of the test meal significantly suppressed the TG concentration and significantly increased the HDL-C concentration, which strongly suggested that lipid metabolism was improved.

In particular, for those with a BMI of 23.1 or higher, the increase in blood leptin concentration associated with obesity is suppressed by ingestion of the test meal, and therefore the possibility that fat cell hypertrophy is suppressed by ingestion of the test meal Was suggested.

Claims (10)

  14-dehydroergosterol as an active ingredient or a koji mold fermentation product containing the same, improving lipid metabolism, reducing risk of abnormal lipid metabolism, improving obesity, reducing obesity risk, suppressing weight gain and / or suppressing fat accumulation Composition for.   The composition according to claim 1 for improving lipid metabolism.   The composition according to claim 2, for use in the treatment, prevention or amelioration of a disease or symptom for which improvement of lipid metabolism is effective in the treatment, prevention or amelioration thereof.   The composition according to claim 3, wherein the disease and symptom are one or more selected from the group consisting of obesity, dyslipidemia and arteriosclerosis.   Composition for improving insulin resistance, reducing risk of insulin resistance, improving hyperglycemia and / or reducing risk of hyperglycemia, comprising 14-dehydroergosterol as an active ingredient or a koji mold fermentation product containing the same .   The composition according to claim 5, for use in the treatment, prevention or amelioration of a disease or symptom in which improvement of insulin resistance and / or improvement of hyperglycemia is effective in the treatment, prevention or amelioration thereof.   The composition according to any one of claims 1 to 6, which is used for a human having a BMI of 23.1 or more.   The composition according to any one of claims 1 to 7, wherein the koji mold fermentation product uses a grain-derived material as a fermentation material.   The composition according to any one of claims 1 to 8, wherein the koji mold is a filamentous fungus of the genus Aspergillus. The manufacturing method of the composition as described in any one of Claims 1-9 which comprises fermenting a cereal origin raw material with a koji mold, and obtaining the fermentation product containing 14-dehydroergosterol.

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