JP2013095678A - Ppar ligand agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a PPAR ligand agent or the like, which contains a component obtained from highly safe food materials, which have long been eaten, as active ingredient.SOLUTION: Provided are a peroxisome proliferator-activated receptor ligand agent, an agent for preventing and/or improving hyperlipidemia, an agent for preventing and/or improving diabetes, an agent for preventing and/or improving obesity, and an agent for preventing and/or improving inflammation, each of which contains a dill extract as active ingredient.

Description

  The present invention relates to a peroxisome proliferator-activated receptor (PPAR) ligand agent containing dill extract as an active ingredient. The present invention further relates to a preventive and / or ameliorating agent for human or animal dyslipidemia, diabetes, obesity, inflammation, etc., containing a dill extract.

  PPAR is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily, as is the case with steroid receptors, retinoid receptors and thyroid receptors. In mammals, it is known that there are three types of receptor subtypes, α-type, γ-type, and δ-type. It is known that PPARα is expressed in the liver, heart, skeletal muscle, brown adipocyte, kidney and the like and is mainly involved in lipid metabolism. The main regulatory organ of PPARα is the liver, and activation of PPARα promotes the burning of fat in the liver, reduces the triglyceride content in blood, liver and skeletal muscle, and improves insulin resistance . It has been reported that fibrate drugs as dyslipidemic agents act on PPARα to exhibit a serum lipid lowering action and an HDL-cholesterol raising action inhibitory action that suppresses the onset of arteriosclerosis. (Non-Patent Document 1). PPARγ is specifically expressed in adipocytes, closely related to the differentiation of adipocytes, and is regarded as a target molecule for a therapeutic agent for type 2 diabetes. It has been clarified that PPARδ has a strong energy metabolism promoting action and fatty acid combustion promoting action, particularly in skeletal muscles. It has been reported that a PPPARδ ligand exhibits an anti-obesity and insulin resistance improving action (Non-patent Document 2). Thus, it has been clarified that PPAR is deeply related to lifestyle-related diseases such as diabetes, obesity, and dyslipidemia, and the biological activity in the skin in which PPAR is involved is to activate PPARα. The action (patent document 1) which suppresses an inflammatory reaction is also known.

As mentioned above, PPARs (PPARs such as PPARα, PPARγ, and PPARδ are collectively referred to as “PPARs”) are not only involved in the regulation of sugar and lipid metabolism in the body, but also obesity and lipid abnormalities. Involvement in the onset of lifestyle-related diseases such as infectious diseases, hypertension, diabetes, cancer, inflammatory diseases, arteriosclerosis, etc. (Non-patent Document 3). The PPAR ligand agent activates PPAR, promotes lipid metabolism in the living body, reduces and accumulates fat, prevents and improves obesity, and further develops insulin resistance and dyslipidemia caused by obesity It is possible to prevent and improve hypertension and diabetes (Non-patent Document 4).
Many PPAR ligand agents containing plant extracts are known.

  Patent Document 2 describes a ligand agent containing an extract of fennel belonging to the genus Felioaceae as an active ingredient. According to Patent Document 2, a PPAR ligand agent containing an extract of fennel as an active ingredient has excellent PPAR ligand activity, and is useful as an agent for preventing and / or improving dyslipidemia, diabetes, obesity, and inflammation.

Patent Document 3 describes a PPAR ligand agent comprising monoacylglycerol.
Patent Document 4 includes (A) gallic acid ester, (B) galloyl tannins, (C) quercetin or a glycoside thereof, (D) flavone or an analog thereof, (E) isoflavone or an analog thereof, ( F) PPAR ligand agents consisting of compounds selected from catechin or epicatechin are described.

Special table 2007-522259 gazette WO2004-045632 JP 2001-354558 A JP 2002-80362 A

Toshiya Tanaka, “The Future of PPAR Agonists” Metabolic Syndrome Molecular Biology of Pathology, 141-155 (2005), Nanzan-do Chaput E. et. Al., FEBS Letters, 514, 315-322 (2002) Japanese clinical volume 63 number 4 (2005-4) Journal of Clinical and Experimental Medicine (IGAKU NO AYUMI) vol.220 No.1 (2007) “PPAR and disease”

  Conventional PPAR ligand agents are not always satisfactory in terms of activity and safety. In particular, there is a need for a PPAR ligand agent containing as an active ingredient an ingredient derived from a highly safe food material rich in food experience.

  Therefore, an object of the present invention is to provide a PPAR ligand agent containing as an active ingredient a component obtained from a highly safe food material rich in food experience that has been edible for a long time.

  Furthermore, an object of the present invention is to provide a preventive and / or ameliorating agent for human or animal dyslipidemia, diabetes, obesity, inflammation and the like containing the above-mentioned components.

  The present inventor has found that an extract of dill, which is a plant belonging to the family Apiaceae, has excellent activity as a PPAR ligand agent. Furthermore, the present inventors have also found that dill extract has an excellent activity as an active ingredient of a preventive and / or ameliorating agent for human or animal dyslipidemia, diabetes, obesity, inflammation and the like.

That is, the present invention provides the following (1) to (5).
(1) A peroxisome proliferator-responsive receptor ligand agent containing a dill extract as an active ingredient.
(2) A prophylactic and / or ameliorating agent for dyslipidemia in humans or animals containing a dill extract.
(3) A preventive and / or ameliorating agent for human or animal diabetes containing dill extract.
(4) A preventive and / or ameliorating agent for human or animal obesity containing a dill extract.
(5) A prophylactic and / or ameliorating agent for human or animal inflammation containing a dill extract.

  According to the present invention, a PPAR ligand agent comprising as an active ingredient an extract of dill, which is a highly safe food material that has been eaten as a spice for a long time, and human or animal dyslipidemia, diabetes, An agent for preventing and / or improving obesity or inflammation is provided.

FIG. 1 is a graph showing the PPARα ligand activity of a PPAR ligand agent containing dill extract as an active ingredient. FIG. 2 is a diagram showing a change in the weight of the KKAY mouse. FIG. 3 is a diagram showing the transition of blood glucose level in KKAY mice. 4 (A), 4 (B), and 4 (C) are diagrams showing oxygen consumption under dark conditions, oxygen consumption under light conditions, and total oxygen consumption for KKAY mice, respectively. FIG. 5 is a graph showing rectal temperature of KKAY mice. FIG. 6 shows organ weight (subcutaneous fat) of KKAY mice. FIG. 7 (A) and FIG. 7 (B) are diagrams showing the non-ester type fatty acid value in plasma and the neutral fat value in plasma, respectively.

1. Dill extract In the present invention, the term “dill” refers to the scientific name Anethum graveolens .
The dill extract is an extract extracted from at least a part of a dill plant using an extraction medium.

  The plant part of the dill for extracting the dill extract is not particularly limited, but is typically a seed. These parts can be used raw or can be appropriately dried. These parts can be used as they are or after being appropriately pulverized.

  Examples of the extraction medium include organic solvents, solvents such as water, and supercritical fluids such as supercritical carbon dioxide, and solvents are particularly preferable.

  As the solvent as the extraction medium, it is preferable to use an organic solvent such as hexane, acetone, ethanol, methanol, isopropanol.

  The conditions for preparing the dill extract are not particularly limited. When the dill extract is prepared by solvent extraction, the amount of the solvent used is not particularly limited, but for example, it can be 0.5 to 50 times the weight of dill. In the solvent extraction, the dill is immersed in a solvent, and the solvent-soluble component is eluted in the solvent by stirring or leaving as appropriate. Although extraction time is not specifically limited, It can be 30 minutes-30 days, and it is preferable that it is 30 minutes-7 days. Although extraction temperature is not specifically limited, It can be 5-80 degreeC and it is preferable that it is 15-40 degreeC. After extraction, the solvent fraction containing the solvent-soluble component and the dill residue are separated by ordinary solid-liquid separation means such as filtration and centrifugation to obtain the solvent fraction. Although the solvent fraction itself can be used as a dill extract, a solid or concentrate obtained by removing all or part of the solvent from the solvent fraction as necessary, or the solid or the concentrate can be used as a suitable solvent. Dilutions diluted with the above, and dilutions obtained by diluting the solvent fraction with an appropriate solvent can also be suitably used as the dill extract. The method for removing the solvent from the solvent fraction is not particularly limited, and examples thereof include a method for volatilizing the solvent from the solvent fraction under heating conditions and / or reduced pressure conditions.

2. PPAR ligand agent, prophylactic and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation, etc. The PPAR ligand agent of the present invention has the ability to bind to a PPAR ligand binding region, that is, PPAR ligand activity. Here, the PPAR ligand activity can be determined by, for example, a reporter assay (Cell, 1995, 83, p. 803-812) for evaluating the binding of a PPAR ligand-binding region and GAL4 to a fusion protein by expression of luciferase, PPAR It can be measured by a competition binding assay (Cell, 1995, 83, p.813-819) using a protein containing a ligand binding region. In these assays, samples that show higher activity than the solvent control and that are dose-dependent are evaluated as “with PPAR ligand activity”. The PPAR ligand agent of the present invention typically exhibits ligand activity against at least one PPAR of α-type, γ-type and δ-type. The PPAR ligand agent may exhibit PPAR ligand activity in an in vivo environment in a human or animal body, or may exhibit PPAR ligand activity in an in vitro environment such as a test device. . The PPAR ligand agent of the present invention that exhibits PPAR ligand activity in an in vivo environment becomes an important regulator of many physiological functions including sugar and lipid metabolism via PPAR, and is effective in treating dyslipidemia, diabetes, obesity, inflammation, etc. Can be prevented and / or ameliorated.

In the present invention, prevention of dyslipidemia refers to preventing or delaying dyslipidemia or borderline conditions. Further, the improvement of dyslipidemia refers to bringing the state of the dyslipidemia or the boundary region described above closer to the state defined as the normal region. Diabetes prevention refers to preventing or delaying diabetic or borderline conditions. Further, the improvement of diabetes refers to the approach of a state defined as a normal range from a diabetic state or a boundary state. Prevention of obesity refers to preventing or delaying the state of being considered obese or obese. Moreover, the improvement of obesity means improving the state considered to be obesity or obesity. In the invention, inflammation refers to inflammation through leukotriene B ₄ involving PPAR, inflammatory cytokines and proteins in cells such as macrophages (eg, TNF-α, IL-1β, IL-6, NO synthase, gelatinase) B, refers to inflammation caused by scavenger receptors. And prevention of inflammation refers to preventing or delaying the above symptoms of inflammation. Further, the improvement of inflammation refers to recovery or reduction of the above-mentioned inflammation symptoms.

  In the present invention, “animal” refers to a non-human animal, for example, a mammal other than a human, which means a rat, mouse, guinea pig, hamster, dog, cat, etc., and breed and age are not particularly limited.

  The PPAR ligand agent of the present invention and the prophylactic and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation and the like preferably contain a dill extract in an amount exhibiting a desired action, and the dill extract as a whole It may consist only of a thing, or may contain a dill extract and another component. In the PPAR ligand agent of the present invention, the amount of dill extract is preferably 0.01% or more, more preferably 0.01 to 100%, and still more preferably 0.02 to 80% (per total weight (wet weight) of the PPAR ligand agent). The ratio of the weight of the dill extract in terms of dry matter) can be blended. Further, in the preventive and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation, etc., the amount of dill extract is preferably 0.01% or more, preferably 0.01 to 100%, more preferably 0.02 to 80%. (The weight of the dill extract in terms of dry matter per total weight (wet weight) of the preventive and / or improving agent) can be blended.

  The PPAR ligand agent or dyslipidemia, diabetes, obesity, inflammation, etc. preventive and / or ameliorating agent of the present invention may further contain other components. Other components are not particularly limited as long as they are components allowed in the final form of foods and drinks, pharmaceuticals and the like.

  The shape of the PPAR ligand agent or dyslipidemia, diabetes, obesity, inflammation, etc. preventive and / or ameliorating agent of the present invention is not particularly limited and may be liquid, solid or semi-solid.

  The PPAR ligand agent or dyslipidemia, diabetes, obesity, inflammation and the like preventive and / or ameliorating agent of the present invention are foods and drinks, pharmaceuticals, quasi drugs, etc., especially foods and drinks, pharmaceuticals, quasi drugs It can be used as a composition for oral consumption.

  The aspect of the said food / beverage products is not limited, It can be aspects, such as a health food, a dietary supplement, a nutritional functional food, a food for specified health. The food and drink are dill extract and other components acceptable as food and drink, such as fructose glucose liquid sugar, cyclic oligosaccharide, acidulant, thickener, inositol, fragrance, niacin, antioxidant, vitamins , Sweeteners and the like.

  The dosage form of the said pharmaceutical is not specifically limited, For example, dosage forms, such as a capsule, a tablet, a granule, an injection, a suppository, a patch, are mentioned. For pharmaceuticals or quasi-drugs, dill extract, pharmaceutically acceptable other formulation materials such as excipients, disintegrants, lubricants, binders, antioxidants, colorants, anti-aggregation It can be produced by preparing an appropriate combination with an agent, absorption promoter, solubilizer, stabilizer and the like.

  The dose or intake of the PPAR ligand agent of the present invention or a prophylactic and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation, etc. is not particularly limited, and the administration or intake route, the target human or animal It can be set as appropriate according to the age, weight, symptoms, etc. For example, when the PPAR ligand agent or the preventive and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation, etc. is in the form of food or drink, it is converted into a dill extract that is an active ingredient per day (dill extract) Of dry matter equivalent weight, hereinafter the same), preferably 0.01 to 1000 mg / kg body weight (refers to the adult body weight of humans or animals; the same applies hereinafter), more preferably 0.1 to 100 mg / kg body weight. Can do. The intake of food and drink can be performed once a day or divided into several times. When the PPAR ligand agent or the preventive and / or ameliorating agent for dyslipidemia, diabetes, obesity, inflammation, etc. is in the form of a pharmaceutical or quasi-drug, it is converted into a dill extract as an active ingredient per day, Preferably it is 0.01-1000 mg / kg body weight, More preferably, it can be 0.1-100 mg / kg body weight. Administration or ingestion of a pharmaceutical product or quasi-drug can be performed once a day or divided into several times.

[Example 1]
The PPARα ligand activity of the PPAR ligand agent containing the dill extract of the present invention as an active ingredient was confirmed.
1. Preparation of dill seed extract 10 g of dill seed was pulverized, immersed in 30 ml of acetone and extracted overnight at room temperature, and then 100 mg of an extract was obtained by filtration.

2. Measurement of PPARα Ligand Activity The PPARα activation ability of the PPAR ligand agent of the present invention was measured by Tsuyoshi Goto et al., Biochemical & Biophysical Research Communication 337 (2005) p. 440-445, was carried out by reference to the column of Material and methods Luciferase assays of "Phytol directly activates proliferator-activated receptor α (PPARα) and regulates gene expression involved in lipid metabolism in PPARα-expressing HepG2 hepatocytes". Specifically, PPARα ligand activity was measured by a reporter assay in which PPAR ligand binding region and GAL4 DNA binding region binding to the fusion protein and target gene activation were assessed by luciferase expression. The detection of luciferase activity used a reporter gene assay as described in FEBS Letters 514 (2002) 315-322. Specifically, a plasmid containing DNA encoding a fusion protein of a PPARα ligand binding region and a GAL4 DNA binding region and a reporter plasmid in which a luciferase is connected to the GAL4 binding DNA sequence are introduced into CV-1 cells, and the cells are introduced into the cells. The ligand described below was added and incubated, and then luciferase activity was detected.

  The sample was DMSO (dimethyl sulfoxide) to adjust the concentration. DMSO was used as a negative control, and PPAR ligand agent GW7647 (15 μg / ml) was used as a positive control. The results are shown in FIG.

  From FIG. 1, luciferase activity was observed in a dose-dependent manner in the dill extract, and PPARα ligand activity was observed.

[Example 2]
The effect of the preventive and improving agent for dyslipidemia and the like containing the dill extract of the present invention was confirmed.
1. Preparation of Dill Seed Extract 20 kg of Dill Seed was pulverized, immersed in 35 L of hexane and extracted overnight at room temperature, and then an extract was obtained by filtration. Methanol was added to the extract and the phases were separated to obtain a methanol layer. The methanol layer was concentrated under reduced pressure to remove the solvent, and then subjected to silica gel column chromatography (0 to 80% ethyl acetate / hexane) to obtain 2.9 g of an oily crude purified fraction.

2. Effect in Diabetes Model Mouse (1) The effect of dill extract was evaluated using a KKAY mouse which is a type 2 diabetes model mouse. As the dill extract, the hexane extract of the above dill was used. As a positive control, bezafibrate, a dyslipidemic drug, was used. KKAY mice (KKAY / TaJcl, male, purchased at 4 weeks of age and individually raised) were divided into 4 groups (5 animals each), each of which was preliminarily raised for 1 week in normal breeding feed (ND), and high fat feed (HFD) ) As a basic feed, the non-added group (control group), the bezafibrate group, and the dill extract group were administered for 4 weeks and reared. Bezafibrate was added to the feed so that the amount of bezafibrate added was 0.05% (w / w). The dill extract was added to the feed so that the amount of dill extract added was 0.02% (w / w) or 0.05% (w / w).

(2) Changes in body weight and blood glucose level of KKAY mice were measured. Further, the day after the end of the experimental breeding, KKAY mice were dissected and measured for oxygen consumption, rectal temperature, organ weight (subcutaneous fat), plasma non-esteric fatty acid level, and plasma neutral fat level.

(3) Transition of the body weight of the KKAY mouse is shown in FIG. The body weights of the mice in the positive control group and the dill extract added group were almost the same as those in the non-added group (control group), and no significant difference was observed. The transition of the blood glucose level of the KKAY mouse is shown in FIG. Compared with the control group, the dill extract was 0.02%, and the increase in blood glucose was significantly suppressed, and a strong hypoglycemic effect was observed. Furthermore, a stronger blood glucose lowering effect was observed at a dill extract addition amount of 0.05%. The results of oxygen consumption under dark conditions, oxygen consumption under light conditions, and total oxygen consumption for KKAY mice are shown in FIGS. 4 (A), 4 (B), and 4 (C), respectively. Moreover, the result of the rectal temperature of the KKAY mouse is shown in FIG. Compared with the control group, an increase in oxygen consumption was observed, and an increase in rectal temperature was also observed. Furthermore, the dill extract added group showed the same effect as the positive control. This result indicates that heat production is enhanced. The results of organ weight (subcutaneous fat) in KKAY mice are shown in FIG. 6, and the results of non-ester fatty acids in plasma and neutral fats in plasma are shown in FIGS. 7 (A) and 7 (B), respectively. Organ weight (subcutaneous fat), non-ester fatty acids in plasma, and neutral fat in plasma were reduced by 0.02% for dill extract compared to the control group, and 0.05% for dill extract Confirmed the improvement in lipid metabolism equivalent to the positive control.

(4) From the above results, the same effect as that of bezafibrate as a positive control was observed in the dill extract. Furthermore, since lipid metabolism has been confirmed, it can be said that the enhancement of heat production confirmed by an increase in oxygen consumption and an increase in rectal temperature is due to lipid metabolism.

Claims (5)

  A peroxisome proliferator-responsive receptor ligand agent containing dill extract as an active ingredient.   A prophylactic and / or ameliorating agent for dyslipidemia in humans or animals containing a dill extract.   A preventive and / or ameliorating agent for diabetes in humans or animals containing a dill extract.   A preventive and / or ameliorating agent for human or animal obesity containing a dill extract.   A preventive and / or ameliorating agent for human or animal inflammation containing a dill extract.

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