JP2011037720A - Dgat inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DGAT inhibitor and a TG synthesis-suppressing agent. <P>SOLUTION: The DGAT inhibitor and the TG synthesis-suppressing agent comprise raspberry or an extract thereof as an effective ingredient. Preferably, the DGAT inhibitor comprises the extract extracted with an organic solvent. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a diacylglycerol acyltransferase (hereinafter referred to as “DGAT”) inhibitor or triacylglycerol (hereinafter referred to as “TG”) effective in preventing or improving hyperlipidemia, obesity, insulin resistance and diabetes. It relates to a synthesis inhibitor.

  In recent years, the incidence of hyperlipidemia and obesity has increased due to the constant excessive intake of fat accompanying the westernization of dietary habits. Hyperlipidemia and obesity are also a major social problem in terms of maintaining and promoting the health of the people because they are associated with lifestyle-related diseases such as insulin resistance and diabetes.

  Hyperlipidemia, in particular, an increase in blood TG concentration, has been regarded as a problem as a risk of ischemic heart disease (Non-Patent Documents 1 to 3). In addition, serum lipids show an abnormal increase after meals, and the state in which this increase is prevalent is commonly called postprandial hyperlipidemia. Postprandial hyperlipidemia is associated with hypertension, ischemic heart disease, etc. It has been pointed out that there is a high risk of leading to the development of arteriosclerotic diseases (Non-Patent Documents 4 and 5).

  Obesity, particularly visceral fat-type obesity, is often associated with lifestyle-related diseases and has been shown to increase the risk of developing arteriosclerotic diseases (Non-Patent Documents 6 and 7). In adipocytes with excessive accumulation of fat, the production of adiponectin, which enhances the action of insulin, decreases, and plasminogen activator inhibitor (PAI-1), tumor necrosis factor (TNF-α), etc. that promote the onset of arteriosclerotic diseases Production of humoral factors is promoted, and this is considered to be associated with lifestyle-related diseases and onset of arteriosclerotic diseases (Non-patent Document 8).

Insulin resistance is a state in which insulin is sufficient in the blood even though there is sufficient insulin in the blood at the center of metabolic syndrome.
Insulin resistance is considered one of the causes of diabetes. Furthermore, insulin resistance and diabetes cause diabetic retinopathy, diabetic nephropathy, neuropathy, etc., as well as the risk of developing arteriosclerotic disease due to hyperlipidemia and hypertension. Has been shown by numerous epidemiological studies (Non-Patent Documents 9 and 10).

  From the above, it is generally considered that prevention and / or improvement of hyperlipidemia, obesity, insulin resistance and diabetes is important for a healthy life.

  It is also known that TG ingested as a meal is decomposed into 2-monoacylglycerol and fatty acid by the action of lipase in the small intestine and then absorbed into small intestinal epithelial cells. The absorbed lipid is re-synthesized into TG by the action of an enzyme such as monoacylglycerol acyl substrate transferase (MGAT) or DGAT in small intestinal epithelial cells. Furthermore, the synthesized TG is released into the blood as chylomicron (CM) by associating with apoprotein B48 specifically produced by small intestinal cells. In this way, DGAT plays an important role in the uptake of TG from the small intestine into the body. In addition, DGAT is necessary for the synthesis and secretion of very low density lipoprotein (VLDL) in the liver and TG accumulation in adipose tissue. Therefore, inhibition of DGAT activity is considered to be an effective means for preventing and improving hyperlipidemia and obesity.

In addition, as a result of studies using knockout mice, DGAT-deficient mice have decreased body weight, as well as decreased blood glucose levels, improved insulin sensitivity, increased adiponectin expression in adipocytes, etc. Inhibiting the activity is considered to be a good means for preventing or improving insulin resistance and diabetes (Non-patent Document 11).

  From the above, inhibition of DGAT activity makes it possible to suppress TG synthesis and to prevent or improve hyperlipidemia, obesity, insulin resistance, and diabetes.

  Insulin resistance is known to be directly affected by the free fatty acid concentration in the blood. It has been reported that an increase in blood free fatty acid causes insulin resistance in diabetic patients and non-diabetics (Non-patent Documents 12 and 13). It has been shown to inhibit and inhibit glucose uptake of skeletal muscle by insulin (Non-Patent Document 14). Therefore, the effect of suppressing the increase in free fatty acid concentration is also expected to result in improvement of insulin resistance.

  Representative examples of conventional approaches for the prevention and improvement of hyperlipidemia, obesity, insulin resistance and diabetes include diet therapy and appetite suppressants (Non-Patent Documents 15 and 16), lipid metabolism-improving drugs (Non-patent) The method using literature 17 and 18) is mentioned. However, dietary therapy is very difficult to implement in the long term, and there are concerns about side effects when taking appetite suppressants and lipid metabolism improving drugs for long periods.

On the other hand, substances that inhibit DGAT activity have been searched for the purpose of preventing or improving hyperlipidemia, obesity, insulin resistance, and diabetes, and as a food material that inhibits DGAT activity, it has been included in citrus fruits so far. Flavonoids and tangeretin have been reported to inhibit DGAT activity in human liver-derived HepG2 cells (Non-patent Document 19).
Moreover, it is reported that the quinolone alkaloid contained in the fruit of the citrus plant Goshuyu (Evodia rutaecarpa) also has a DGAT inhibitory effect (Non-patent Document 20). However, the food materials found so far are not fully satisfactory in terms of their effects.

  In addition to food materials, phenoxyacetamide derivatives and oxadiazole derivatives have been disclosed as compounds that inhibit DGAT activity (Patent Documents 1 and 2).

By the way, raspberry hot water extract has a lipase inhibitory action (Patent Document 3), raspberry juice has a deodorizing action (Patent Document 4), and raspberry water and / or ethanol extract has an antiallergic action. Although it is known (Patent Document 5), it is not known that raspberry or an extract thereof has a DGAT activity inhibitory action.
In addition, although plants such as raspberries are known to contain ellagitannins (Non-patent Documents 21 to 23), it has been known that ellagitannins or their derivatives have a DGAT activity inhibitory effect. It is not done.

International Publication No. 2006/082010 Pamphlet International Publication No. 2007/138304 Pamphlet JP 2005-8572 A JP 2000-279502 A JP-A-10-236965

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  The present invention relates to providing a DGAT inhibitor and the like effective for preventing or improving hyperlipidemia, obesity, insulin resistance and diabetes.

  As a result of searching for substances having a DGAT activity inhibitory action and high safety, the present inventors have found that raspberries rich in food experience or extracts thereof have excellent DGAT activity inhibitory action and TG synthesis inhibitory action. In particular, ellagitannin having a partial structure represented by the following formula (1) contained in raspberry or a derivative thereof (hereinafter also referred to as “GOD-type ellagitannin or a derivative thereof”) has a main pharmacological activity. I found out. The present inventors can use raspberries or extracts thereof as pharmaceuticals or foods for the purpose of inhibiting DGAT activity, inhibiting TG synthesis, preventing or improving hyperlipidemia, obesity, insulin resistance and diabetes. I found.

That is, the present invention relates to the following (1) to (11).
(1) A DGAT inhibitor comprising raspberry or an extract thereof as an active ingredient.
(2) The DGAT inhibitor according to (1), wherein the extract is an organic solvent extract.
(3) The DGAT inhibitor according to (2), wherein the organic solvent extract is a hydrous ethanol extract or a hydrous acetone extract.

(4) A TG synthesis inhibitor containing raspberry or an extract thereof as an active ingredient.
(5) The TG synthesis inhibitor according to (4), wherein the extract contains ellagitannin or a derivative thereof as a main component.
(6) The TG synthesis inhibitor according to (4) or (5), wherein the extract is an organic solvent extract.
(7) The TG synthesis inhibitor according to (6), wherein the organic solvent extract is a hydrous ethanol extract or a hydrous acetone extract.

(8) An agent for preventing or improving insulin resistance comprising raspberry or an extract thereof as an active ingredient.
(9) Diabetes preventive or ameliorating agent comprising raspberry or an extract thereof as an active ingredient.

(10) Formula (1) in the molecule

The ellagitannin or derivative thereof having a partial structure represented by formula (1) and a glucose residue, wherein the hexahydrodiphenoyl group of the partial structure represented by formula (1) is substituted at the 4-position and the 6-position of the glucose residue A prophylactic or ameliorating agent for symptom of hyperlipidemia, obesity, insulin resistance or diabetes, or a TG synthesis inhibitor.
(11) The agent for preventing or ameliorating each symptom of hyperlipidemia, obesity, insulin resistance or diabetes according to (12), wherein ellagitannin or a derivative thereof is one or more selected from sangiin H-6 and lambertianin C Or a TG synthesis inhibitor.

  The DGAT inhibitor, TG synthesis inhibitor, hyperlipidemia preventing / ameliorating agent, obesity preventing / ameliorating agent, insulin resistance preventing / ameliorating agent, or diabetes preventing / ameliorating agent of the present invention has an excellent DGAT inhibitory action, etc. Therefore, it is useful as a pharmaceutical or food for preventing or improving hyperlipidemia, obesity, insulin resistance and diabetes.

HPLC analysis of HP-20 fraction, solid-liquid fraction, Lambertianine C purified product, and Sangiin H-6 purified product was performed.

As shown in Examples below, raspberry or an extract thereof has excellent DGAT inhibitory action, blood TG elevation inhibitory action, and blood FFA elevation inhibitory action (hereinafter referred to as “DGAT inhibitory action, etc.”). Moreover, since raspberries have a long dietary experience, safety is extremely high even when raspberries or extracts thereof are ingested for a long period of time.
Here, inhibition of DGAT activity is an effective means for inhibiting TG synthesis, preventing or improving hyperlipidemia, preventing or improving obesity, preventing or improving insulin resistance, or preventing or improving diabetes (Non-Patent Document 11). ). Moreover, suppression of TG elevation is an effective means for suppressing hyperlipidemia, particularly postprandial hyperlipidemia, and suppression of blood FFA elevation is an effective means for preventing or improving insulin resistance and diabetes. (Non-Patent Documents 12-14).
Therefore, the raspberry of the present invention or an extract thereof is a DGAT inhibitor, a TG synthesis inhibitor, a hyperlipidemia prevention or improvement agent, an obesity prevention or improvement agent, an insulin resistance prevention or improvement agent, or a diabetes prevention or improvement. It can be used as an agent (hereinafter referred to as “DGAT inhibitor etc.”) and can be used to produce these preparations. At this time, as the DGAT inhibitor or the like, the raspberry or the extract thereof is used alone or in addition to that, which is allowed in the later-described target object to be blended, such as a carrier appropriately selected as necessary. May be. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

Here, the raspberry in the present invention means Rubus idaeus in the family Rosaceae.
The raspberry in the present invention can be used as a juice, powder, or the like by treating the seeds and / or fruits of the plant as it is, or by processing such as pressing, grinding or drying. Of these, it is preferable to use fruits.

  The extract according to the present invention includes extracting the raspberry at room temperature (for example, 4 to 50 ° C.) or warming (room temperature to solvent boiling point) or using an extraction device such as a Soxhlet extractor. It is obtained by a known extraction means. The form is not particularly limited, and any form such as various solvent extracts, diluted liquids, concentrated liquids, pastes or dry powders thereof may be used.

As the extraction solvent used for obtaining the raspberry extract of the present invention, either a polar solvent or a nonpolar solvent can be used.
Specific examples of the extraction solvent include, for example, water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate, ethyl acetate, and the like. Aliphatic carboxylic acid esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene Organic solvents such as halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane; supercritical carbon dioxide; pyridines; and other oils such as fats and waxes, and these should be used alone or in combination of two or more. It can be.

Of these extraction solvents, an organic solvent is preferably used from the viewpoint of improving the DGAT inhibitory action and the like of the raspberry extract.
Among organic solvents, alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone from the point of improving the DGAT inhibitory action and the like of the raspberry extract One or more selected from aliphatic carboxylic acid esters such as methyl acetate and ethyl acetate are preferably used. Of these, waters such as alcohols having 1 to 4 carbon atoms, lower alcohols having 2 to 4 carbon atoms such as ethanol, propanol and butanol; ketones having 3 to 5 carbon atoms and ketones such as acetone and methyl ethyl ketone. It is preferable to use one or more selected from organic organic solvents, and it is even more preferable to use one or more selected from ethanol and acetone.
These organic solvents may contain water (hereinafter, “water-containing” is referred to as “water-containing organic solvent”).
Further, among these organic solvents, a water-containing organic solvent is preferable, a water-containing water-soluble organic solvent is more preferable, and water-containing ethanol and water-containing acetone are more preferable.

  The mixing ratio (volume ratio) of the organic solvent and water is preferably 0.001 to 100: 99.999-0, more preferably 5 to 95:95 to 5, and still more preferably 20 to 80:80 to 20. 30-70: 70-30 is still more preferable, and 40-60: 60-40 is more preferable. In the case of hydrous alcohols, the alcohol concentration is preferably 40 to 60% by volume, and in the case of hydrous acetone, the acetone concentration is preferably 40 to 80% by volume.

  As a usage-amount of a solvent, 1-100 mL is preferable with respect to 1 g of raspberries (dry mass conversion), As extraction time, 1 minute-100 days are preferable, and 1 minute-10 days are more preferable. The extraction temperature at this time is preferably 0 ° C. to the boiling point of the solvent, more preferably 5 to 80 ° C., further 5 to 50 ° C., and still more preferably 10 to 40 ° C.

Specific examples of the extraction means for obtaining the raspberry extract include solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, and stirring.
For example, as a suitable example of immersion, immersion at 4 to 50 ° C. for 5 hours to 10 days can be mentioned.
Moreover, when shortening extraction time, solid-liquid extraction with stirring is desirable. Examples of suitable conditions for this solid-liquid extraction include stirring at 1000 to 5000 rpm under 10 to 100 ° C. (preferably 10 to 50 ° C.) for 1 to 30 minutes.
In order to prevent the oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.
Furthermore, from the viewpoint of improving the pharmacological activity of the raspberry extract, a separation technique such as chromatography or liquid-liquid distribution may be used to remove inactive impurities from the extract.

Moreover, in order to remove water-soluble inactive impurities from raspberries or extracts thereof, it is preferable to wash the raspberries or extracts thereof. It is also effective to extract a residue obtained by pressing and extracting fruit juice from fresh raspberry fruits with the above-mentioned organic solvent.
As an example of washing with water, the raspberry is homogenized and pulverized, appropriately subjected to pretreatment such as drying, and then washed with water to prepare in advance a raspberry residue obtained by separation means such as centrifugation and filtration, and then And extraction with an organic solvent.
At the time of washing with water, physical means such as immersion, ultrasonic waves, microwaves, stirring, and shaking can be used. Among these, stirring is preferable and the rotation speed at this time should just be about 1000-5000 rpm.
As processing time in the case of water washing, 1 to 60 minutes are preferable, 1 to 30 minutes are more preferable, processing temperature in the case of water washing is preferably 4 to 100 ° C. 4-50 degreeC is desirable.
As usage-amount of water at this time, 1-100 mL is preferable with respect to 1 g of raspberries or its extract (dry mass conversion).
In addition, as a method of extracting with the above-mentioned organic solvent from a raspberry residue, the method of extracting the said residue and organic solvent by mixing and stirring (about 1000-5000 rpm) for 1 to 30 minutes under 4-80 degreeC is preferable. .

Further, it is preferable to subject the raspberry or an extract thereof (preferably an organic solvent extract) to chromatography from the viewpoint of improving the DGAT inhibitory action and the like of the raspberry extract.
Examples of stationary phases used in chromatography include strong and weak acidic ion exchange resins or strong and weak basic ion exchange resins; reverse phase resins such as octadecylated silica gel, octylated silica gel, butylated silica gel, and trimethylsilylated silica gel; silica gel Normal phase resins such as fluoridyl and alumina; aromatic synthetic resins such as styrene-divinylbenzene and methacrylic ester; modified dextran (eg, Sephadex (registered trademark) LH-20), hydrophilic vinyl polymer (For example, Toyopearl HW-40 etc.) Gel filtration chromatography filler; activated carbon and the like. Among these, reverse phase resins and aromatic synthetic resins, gel filtration chromatography filler, etc. are preferred.

  Method of obtaining elution fraction (hereinafter also referred to as “active fraction” or “DGAT active fraction”) having a DGAT inhibitory action by subjecting raspberry or an extract thereof (preferably a raspberry organic solvent extract) to chromatography The following method is mentioned as an example. At this time, the wavelength for detecting the active fraction is preferably 260 to 300 nm, more preferably 270 to 290 nm.

The raspberry or extract thereof is subjected to medium / high pressure chromatography using a reverse phase resin, and then the elution solvent is gradient to obtain an active fraction.
The elution solvent at this time is preferably a gradient of formic acid: water: acetonitrile = 0-5: 100-0: 0-100, more preferably 0.01-2: 95-10: 5-90. As column temperature, 20-50 degreeC and 30-40 degreeC are more preferable. The flow rate is preferably 1 to 40 mL / min, more preferably 15 to 25 mL / min. The retention time when fractionating the active fraction is 5 to 100 minutes, and more preferably 20 to 50 minutes.

  In addition, after adsorbing the raspberry or its extract by medium / high pressure chromatography using an aromatic synthetic resin, it is washed with 100 to 1000% by volume (preferably 300 to 600% by volume) of water of the resin volume. Then, the active fraction is obtained by elution with 100 to 1000 volume% (preferably 300 to 600 volume%) of an organic solvent or a water-containing organic solvent of the resin volume. What is necessary is just to use the same organic solvent or the water-containing organic solvent as the above-mentioned.

  Furthermore, the chromatography using the above-mentioned reverse phase resin and the chromatography using an aromatic synthetic resin may be used in combination, and as the order when attaching raspberries or an extract thereof, either may be first, Raspberry or an extract thereof is subjected to chromatography using an aromatic synthetic resin to obtain an active fraction, and then the obtained active fraction is subjected to chromatography using a reverse phase resin for further pharmacological activity. It is preferred to obtain an improved active fraction.

  Furthermore, as shown in the below-mentioned Example, a DGAT active fraction is fractionated from a raspberry or its extract by chromatography, and the DGAT active fraction further contains sangiin H-6 and lambertianin C shown below. And this sanguine H-6 and lambertianine C have the DGAT inhibitory action etc. which were further superior to the raspberry extract. Therefore, sangiin H-6 and lambertianin C, that is, ellagitannin or a derivative thereof are considered to be main components of pharmacological activity such as DGAT inhibitory action in raspberry or an extract thereof.

  Accordingly, the present invention provides the following formula (1) in the molecule.

The hexahydrodiphenoyl group of the partial structure represented by the formula (1) or (2) is substituted at the 4-position and 6-position of the glucose residue. The present invention provides a DGAT inhibitor containing ellagitannin or a derivative thereof as an active ingredient.

For this reason, raspberry or an extract thereof is obtained by using the above extraction / separation technique and an appropriately known purification technique as a main component of a raspberry extract (fraction) containing GOD-type ellagitannin or a derivative thereof, and further a GOD-type ellagitannin or a derivative thereof. It is preferable to use a raspberry extract (fraction) to improve pharmacological activity.
The content of GOD-type ellagitannin or a derivative thereof in the raspberry extract (fraction) at this time is 0.01% by mass or more, more 0.5% by mass or more, and further 15% by mass or more in terms of dry mass. Furthermore, it is preferably 95% by mass or more from the viewpoint of improving pharmacological activity.

  The raspberry or the extract thereof may be used as it is, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or a dry powder, or may be prepared as a paste. Further, it can be freeze-dried and diluted with a solvent usually used for extraction at the time of use, for example, a solvent such as water, ethanol, water / ethanol mixed solution or the like. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  In the present invention, ellagitannin means a generic name for tannin that has a hexahydrodiphenoyl group and that generates ellagic acid by hydrolysis and dehydration cyclization.

  Here, ellagitannin or a derivative contained in raspberry or an extract thereof is at least the following formula (1)

The ellagitannin related hydrolyzable tannin which has the partial structure represented by these.
Among these, from the viewpoint of improving pharmacological activity, preferred ellagitannin derivatives have a glucose residue in the partial structure represented by the above formula (1), and are located at the 4th and 6th positions of the glucose residue. What substituted the hexahydro diphenoyl group part of the partial structure represented by the said Formula (1) is mentioned.

  Of the GOD type ellagitannins or derivatives thereof, those represented by the following formula (2) are preferred.

In formula (2), R ¹ represents a hydrogen atom or a galloyl group. In formula (2), “˜OR ¹ ” represents the presence of α-type and / or β-type.
In formula (2), R ² and R ³ are the same or different and each represents a hydrogen atom or a galloyl group, or together represent a hexahydroxydiphenoyl group.
In the formula (2), R ⁴ is

[Wherein, R ⁵ and R ⁶ are the same or different and each represents a hydrogen atom or a galloyl group. ] Is represented.
In formula (2), m represents 0-2 (preferably 0 or 1).

As GOD-type ellagitannin or a derivative thereof, it is present in edible plants, and from the viewpoint of high safety, sanguin H-2, sanguin H-3, sanguin H-6, sanguin H-7, sanguin H- 8, Sangiin such as Sangiin H-9, Sangiin H-10, and Sangiin H-11; Lambertianine such as Lambertianine A, Lambertianine B, Lambertianine C, Lambertianine D; Rochenin A, Rochenin B, Rochenin C, Rochenin D, Rochenin E Can be exemplified.
Of these examples, sangiin H-6 and lambertianine C are preferred from the viewpoint of DGAT inhibitory activity.

Examples of the ellagitannin or derivative thereof according to the present invention include those obtained by known chemical synthesis methods, plant extracts containing ellagitannin or a derivative thereof as a main component (for example, see Non-Patent Documents 21 to 23), or purified products thereof. Is included. At this time, from the viewpoint of improving pharmacological activity, the plant extract containing ellagitannin or a derivative thereof as a main component contains ellagitannin or a derivative thereof in an amount of 0.5% by mass or more, more preferably 15% by mass or more in terms of dry matter. It is preferable that
For example, as sanguin and / or lambertianine, it is possible to use an extract from the fruit of raspberry (Rubus ideus) or a concentrated or purified extract thereof.
In addition, when extracting / separating / purifying the ellagitannin or its derivative of this invention from a plant, the extraction / separation / purification means from a raspberry mentioned above may be applied.
In addition, the obtained extract or fraction containing ellagitannin or a derivative thereof may be used as appropriate in the same form as the above-mentioned raspberry extract or fraction.

  Since ellagitannin or a derivative thereof of the present invention has a DGAT inhibitory action and the like, it can be used as a DGAT inhibitor and the like as described above, and can be used for producing these preparations. At this time, as the DGAT inhibitor or the like, the ellagitannin or a derivative thereof alone or in addition to this, those that are allowed in the target object to be blended, such as a carrier appropriately selected as necessary, are used. May be. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

  Therefore, the DGAT inhibitor or the like containing the raspberry of the present invention or an extract thereof, or ellagitannin or a derivative thereof as an active ingredient has DGAT inhibitory activity; TG synthesis suppression; hyperlipidemia, obesity, insulin resistance or diabetes symptoms It can be used as an active ingredient such as pharmaceuticals for humans or animals (non-humans), quasi-drugs, foods, functional foods, feeds, etc. that exhibit the effects of prevention, improvement, treatment, etc. In addition, the DGAT inhibitor and the like have a DGAT inhibitory activity; TG synthesis suppression; prevention, improvement or treatment of each symptom of hyperlipidemia, obesity, insulin resistance or diabetes, etc. It can be applied to the displayed food, functional food, food for the sick, and food for specified health use.

  When the DGAT inhibitor of the present invention is used as an active ingredient of a pharmaceutical product, the pharmaceutical product can be administered in any dosage form. Examples of the dosage form include oral, enteral, transmucosal, injection and the like. Examples of the dosage form of the preparation for oral administration include tablets, capsules, granules, powders, and syrups. Examples of parenteral administration include intravenous injection, intramuscular injection, suppository, inhalation agent, transdermal absorption agent, eye drop, nasal drop and the like.

  In such a preparation, the DGAT inhibitor of the present invention may be used alone or in combination with other pharmaceutically acceptable carriers. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet rays. Absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, corrigent, flavoring agent, extender, surfactant, dispersant, buffer, preservative, fragrance, coating agent Etc.

  Of these dosage forms, oral administration is preferred, and the content of raspberry or extract thereof in the preparation when used as an active ingredient of the preparation for oral administration is usually 0 of the total mass of the preparation in terms of dry matter. 0.04 to 100% by mass, and preferably 2.0 to 80% by mass. Moreover, content of an active ingredient ellagitannin or its derivative (s) is 0.01-80 mass% normally with respect to a formulation whole mass, and it is preferable that it is 0.5-80 mass%.

The dosage of the above formulation may vary according to the patient's condition, weight, sex, age or other factors, but the daily dosage per adult (60 kg) for oral administration is raspberry or its extraction 4 to 8000 mg, more preferably 40 to 8000 mg, and even more preferably 200 to 2000 mg, with a product (in terms of dry matter) as an active ingredient. The daily dose per adult is preferably 1 to 2000 mg, more preferably 10 to 2000 mg, more preferably 10 to 1000 mg, and even more preferably 50 to 500 mg, with ellagitannin or a derivative thereof as an active ingredient.
Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day.

When the DGAT inhibitor or the like of the present invention is used as an active ingredient of food, the form of the food may be solid, semi-solid or liquid. Examples of food include breads, noodles, confectionery, jelly, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, beverages, soups, seasonings, nutritional supplements, etc. , And their raw materials. Further, like the above-mentioned preparation for oral administration, it may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.
To prepare various forms of food, DGAT inhibitors, etc. alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers , Antioxidants, humectants, thickeners, and the like can be used in appropriate combinations.

The content of the raspberry or the extract thereof in the food varies depending on the usage form, but is usually 0.004 to 8% by mass, 0.008 to 4% by mass, and more 0.04 in the beverage form. It is preferably ˜2% by mass. Moreover, in solid food forms, such as a tablet and processed food, it is 0.04-80 mass% normally, 0.08-40 mass%, and it is preferable that it is 0.4-20 mass%.
Moreover, although content of the active ingredient ellagitannin or its derivative in a foodstuff changes with its usage forms, in the form of a drink, it is usually 0.001-2 mass%, 0.002-1 mass%, and more It is preferable that it is 0.01-0.5 mass%. Moreover, in solid food forms, such as a tablet and processed food, it is 0.01-20 mass% normally, 0.02-10 mass%, It is preferable that it is 0.1-5 mass% more.

In addition, when the DGAT inhibitor of the present invention is used as an active ingredient of feed, examples of the feed include livestock feed used for cattle, pigs, chickens, sheep, horses, rabbits, rats, mice, and the like. Examples include small animal feeds used for foods, fish foods used for tuna, eel, Thailand, yellowtail, shrimp, etc., pet foods used for dogs, cats, small birds, squirrels, and the like.
In addition, when manufacturing feed, DGAT inhibitor etc. alone or in addition, meat such as cattle, pigs, sheep, protein, grains, bran, potatoes, sugars, vegetables, vitamins, Commonly used feed raw materials such as minerals, and more commonly used gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc. This feed can be processed and manufactured.

In addition, the content of raspberry or its extract in the feed varies depending on the form of use, but is usually 0.04 to 80% by mass, 0.08 to 40% by mass, and 0.4 in terms of dry matter. It is preferably ˜20% by mass.
Moreover, although content of the active ingredient ellagitannin or its derivative in a feed changes with the usage forms, it is 0.01-20 mass% normally, 0.02-10 mass%, more 0.1-5 mass% Is preferred.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Production Example 1: Preparation of Raspberry 50% Ethanol Extract 100 g of freeze-dried powder of raspberry (Rubus idaeus L.) fruit (FD raspberry powder, available from Kyoritsu Bussan Co., Ltd) , "%") Soaked in ethanol water (ethanol concentration 50% by volume) at room temperature (20-30 ° C) for 15 hours, filtered off insoluble matter and concentrated under reduced pressure to obtain an extract (solid content 59 g) .
In addition, content and identification of GOD type ellagitannin were calculated | required with the following method.
Method for quantification and identification of ellagitannin <HPLC analysis conditions>
Column: Inertsil ODS-3 (octadecylated silica gel, manufactured by GL Sciences, 4.6 x 250 mm, 5 mm)
Column temperature: 40 ° C
Flow rate: 1.0 mL / min
Detection: UV280nm
Eluent: A liquid 1% formic acid aqueous solution, B liquid acetonitrile A / B = (0 min) 90/10 → 90/10 (10 min) → 80/20 (70 min) → 90/10 (71 min) → 90/10 (80 minutes)

  The total content of GOD type ellagitannins in the raspberry 50% ethanol extract [solid content] (in terms of dry mass) was 1.0% by mass. As an ellagitannin, sangiin H-6 and lambertianin C were identified by HPLC analysis.

Production Example 2 Preparation of Raspberry 70% Acetone Extract 1 g of freeze-dried powder of raspberry (Rubus idaeus L.) fruit (FD raspberry powder, obtained from Kyoritsu Bussan Co., Ltd.) in 10 volumes (10 mL) of 70% acetone water ( It was immersed in acetone (70% by volume) at room temperature (20-30 ° C.) for 7 days, insoluble matter was filtered off and concentrated under reduced pressure to obtain an extract (solid content: 0.3 g).
The total content of GOD-type ellagitannins in the raspberry 70% acetone extract [solid content] (in terms of dry mass) was 0.7% by mass. Moreover, sanguine H-6 and lambertianin C were identified as ellagitannin by HPLC analysis.

Example 1: DGAT activity inhibition test 50 μg of liver microsome fraction was added to 200 μL of DGAT activity measurement buffer (250 mM Sucrose, 10 mM Tris-HCl ( pH 7.5), 10 mM MgCl ₂ , 0.8 mM EDTA, 0.1% Bovine Serum Albumin, 100 μM Oleoyl-CoA (0.05 μCi / 200 μL, 55 mCi / mmol), 1.2 mM 1,2-diolein) at 37 ° C., 20 Incubated for minutes.
Table 1 shows the DGAT activity in the presence of each test substance. The DGAT activity is expressed as a relative value with the solvent control DGAT activity as 100.

Production Example 3: Preparation of Raspberry Solid-Liquid (50% Ethanol) Fraction Product Raspberry (Rubus idaeus L.) fruit freeze-dried powder (FD raspberry powder, obtained from Kyoritsu Bussan) 10 g (400 mL) Ion exchanged water was added, and the mixture was stirred at 3000 rpm for 10 minutes at room temperature (20 to 30 ° C.) with a TK auto homomixer (manufactured by Tokushu Kika Kogyo Co.). Thereafter, the mixture was centrifuged (3000 rpm, 20 minutes), and the supernatant was removed. The water washing of this operation was repeated 3 times to remove water-soluble components, and then the residue was extracted with 50% ethanol water. Extraction is performed by using 400 mL of 50% ethanol water, stirring with TK auto homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at 3000 rpm for 10 minutes, and then obtaining a supernatant by centrifugation (3000 rpm, 20 minutes). went. This extraction operation was also repeated three times, and the supernatants obtained three times were combined and concentrated under reduced pressure to obtain a solid-liquid fraction (1.3 g).
The total content of GOD-type ellagitannins in the solid-liquid fraction (in terms of dry mass) was 23.9% by mass. As an ellagitannin, sangiin H-6 and lambertianin C were identified by HPLC analysis (see FIG. 1).

Example 2 Hyperlipidemia Inhibition Test C57BL / 6 mice (7 weeks old) purchased from Charles River Japan were used after acclimation for about 1 week. As the triacylglycerol synthesis inhibitor, the raspberry extract (solid-liquid fraction product) prepared in Production Example 3 was used. A group of 9 mice was used for the test group and the control group. Lipid loading was performed by orally administering 40 μL of lipid emulsion (10% triacylglycerol oil, 1% egg yolk lecithin, 4% bovine serum albumin) per gram of body weight using a stomach tube. To the lipid emulsion given to the test group, raspberry extract was further added to a final concentration of 1%. Blood was collected 1, 2, 4, and 6 hours after administration, and the triacylglycerol concentration and FFA concentration in plasma were measured. The TG concentration was measured by triglyceride E-Test Wako (Wako Pure Chemical Industries, Ltd.) by the glycerin elimination method. The FFA concentration was measured by an acyl-CoA synthase / acyl-CoA oxidase method using NEFA C-Test Wako (Wako Pure Chemical Industries, Ltd.). The reaction was carried out in a 96-well plate, and the absorbance at a TG concentration of OD595 nm and an FFA concentration of OD550 nm was measured using a BioRad Microplate Reader MODEL550. As for the TG concentration, the concentration of triacylglycerol contained in plasma was determined from a calibration curve prepared using a glycerin standard solution attached to the kit. The FFA concentration was determined from the calibration curve prepared using the oleic acid standard solution attached to the kit. The results of the change in TG concentration and the amount of change (Δ) are shown in Table 2 and Table 3, respectively. Tables 4 and 5 show the results of the change in FFA concentration and the amount of change (Δ), respectively.

As shown in Table 2, when the lipid emulsion was orally administered, an increase in plasma TG concentration peaked at 1 to 2 hours after administration was observed, but in the test group, the plasma TG concentration after lipid loading was increased. Decline was confirmed. In the TG level 2 hours after administration, the plasma TG concentration was decreased by 23.8% in the test group as compared with the control group. Moreover, as shown in Table 3, in the test group, suppression of increase in TG concentration was also confirmed in the amount of change in plasma TG concentration after oral administration of lipid emulsion as compared with the control group. The amount of change in plasma TG concentration was 37.1% lower in the test group compared to the control group. Therefore, the raspberry extract is considered to be effective in suppressing hyperlipidemia, particularly postprandial hyperlipidemia.
Furthermore, as shown in Table 4, the FFA level after administration of the lipid emulsion was increased, peaking at 1 to 2 hours after oral administration of the lipid emulsion, similar to the TG level. A decrease in plasma FFA concentration was confirmed. As for the FFA level 2 hours after the administration, the plasma FFA concentration was decreased by 23.0% in the test group as compared with the control group. Moreover, as shown in Table 5, in the test group, suppression of increase in FFA concentration was also confirmed in the amount of change in plasma FFA concentration after oral administration of lipid emulsion compared to the control group. The amount of change in plasma FFA concentration was 81.9% lower in the test group compared to the control group. Therefore, it is considered that the raspberry extract is effective in preventing or improving insulin resistance and diabetes through decreasing the FFA concentration.

Production Example 5: Preparation of test substance (Sangiin H-6, Lambertianine C) from raspberry (1) Preparation of raspberry 50% ethanol extract The extract of Production Example 1 was used.
(2) Preparation of raspberry HP-20 fraction (hereinafter also referred to as “fraction”) 20 g of the extract of (1) above was redissolved in water, and then a styrene-divinylbenzene-based aromatic synthesis. It was made to adsorb | suck to Diaion HP-20 (1000 mL) which is an adsorption agent. HP-20 was washed with 4 L of water and then eluted with 4 L of 50% ethanol water. The obtained eluate was concentrated under reduced pressure to obtain 1.21 g of HP-20 fraction.
The total content of GOD-type ellagitannin in the HP-20 fraction product (in terms of dry mass) was 17.9% by mass.
(3) Preparation of Sangiin H-6 purified product and Lambertianin C purified product Using 400 mg of the fraction obtained in the above (2), and further fractionating by HPLC, Sangiin H-6 (43 mg), Lambertianin C (16 mg) was isolated and peak retention times were 38 to 41 minutes and 36 to 38 minutes, respectively (see FIG. 1).
<Fractionation conditions>
Column: Inertsil ODS-3 (octadecylated silica gel, 20 × 250 mm, GL Science)
Solvent: A solution 1% formic acid aqueous solution, B solution acetonitrile A / B = (0 min) 90/10 → 90/10 (10 min) → 80/20 (40 min) → 90/10 (41 min) → 90 / 10 (45 minutes)
Temperature: 40 ° C., flow rate: 18.9 mL / min, detection: UV = 280 nm
After dissolving 400 mg of sample in 20 mL of 10% ethanol, 1 mL was charged 23 times. The ¹ H and ¹³ C NMR spectra of the obtained sangiin H-6 purified product and lambertianin C purified product were obtained as described in non-patent documents 22 and 23. Consistent with the ¹ H and ¹³ C NMR spectral data of H-6 and lambertianin C.
Therefore, when the solid-liquid fraction product of Production Example 3 and the HP-20 fraction product of Production Example 3 extracted from raspberries are collected with a retention time of 31 to 46 minutes (see FIG. 1), pharmacological activity Can be further purified, and the pharmacological action of the obtained active fraction can be enhanced.

Example 3: Evaluation of inhibition of DGAT activity of sangiin H-6 and lambertianin C isolated from raspberry extract As test substances, sanguin H-6 and lambertianin C isolated from raspberry extract in the above Production Example 5 and EGCG ( Epigallocatechin gallate: source: Nagara Science).
From the mouse liver, a microsomal fraction was prepared as follows and used for DGAT activity measurement. The liver of C57BL / 6J mice (male, 6 weeks old) was homogenized in Sucrose Buffer (250 mM Sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5)), and centrifuged at 4 ° C., 12,500 × g for 15 minutes. The supernatant was collected. The supernatant was further centrifuged at 4 ° C., 100,000 × g for 60 minutes, and the precipitate was resuspended in Sucrose Buffer and used as a liver microsome fraction.
50 μg of the liver microsome fraction was added to 200 μL of DGAT activity measurement buffer (250 mM Sucrose, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 0.8 mM EDTA, 0.1) containing each test substance at the concentrations shown in the table. % Bovine Serum Albumin, 100 μM Oleoyl-CoA (0.05 μCi / 200 μL, 55 mCi / mmol), 1.2 mM 1,2-Diolein) was incubated at 37 ° C. for 20 minutes. The reaction was stopped by adding 1.5 mL of chloroform / methanol (1: 1). Next, 250 μL of PBS was added and stirred, and separated into an aqueous layer and an oil layer (lipid extraction) by centrifugation. The lower chloroform layer was collected, dried with nitrogen gas, and redissolved in chloroform.
A part of the lipid dissolved in chloroform was subjected to a liquid scintillation counter (Perkin Elmer), and the radioactivity was measured. A part of the extracted lipid of about 2000 dpm was fractionated using an HPTLC plate (20 × 10 cm Silica gel 60, Merk). As the developing solvent, hexane / diethyl ether / acetic acid (80: 20: 1) was used. Fluorography and quantitative analysis were performed using BAS2500 (Fuji Film). The ratio of the radioactivity incorporated into TG to the total radioactivity incorporated into the extracted lipid was defined as DGAT activity.
Table 6 shows the DGAT activity in the presence of each test substance. The DGAT activity is expressed as a relative value with the solvent control DGAT activity as 100.
As shown in Table 6, sanguin H-6 and lambertianin C showed significant inhibition on DGAT activity.
Thus, since ellagitannin isolated from raspberry has an excellent inhibitory action on DGAT activity, in addition to DGAT inhibition, TG synthesis inhibitor, hyperlipidemia prevention / amelioration agent, obesity prevention / amelioration agent, insulin It is considered useful as a resistance preventive / ameliorating agent and a diabetes preventing / ameliorating agent.

Example 4 Evaluation of Inhibition of Other Enzyme Activities of Sangioin H-6 and Lambertianine C Isolated from Raspberry Extract The test substances used in Example 3 were used.
Using the mouse liver microsome fraction prepared as shown in Example 3 above, measurement of glucose 6-phosphate dephosphorylating enzyme (G6Pase) activity, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) ) Reductase activity was measured.
In the measurement of G6Pase activity, a mouse liver microsome fraction (50 μg) was prepared by adding 100 μL of a reaction solution (50 mM HEPES buffer (pH 7.2), 100 mM KCl, 2.5 mM) in the presence of the test substance at the concentration shown in the table. EDTA, 2.5 mM MgCl ₂ , 1 mM DTT, 10 mM glucose-6-phosphate, 2 mM EDTA) and incubated at 37 ° C. for 30 minutes. Next, 400 μL of a reaction stop solution (0.42% ammononium molybdate tetrahydrate in 1N H ₂ SO ₄ , 10% SDS, 10% ascorbic acid 6: 2: 1 mixed solution) was added and incubated at 50 ° C. for 30 minutes. The resulting blue dye was subjected to absorbance measurement (OD ₈₂₀ ), and G6Pase activity was calculated by calculating the concentration of the resulting phosphoric acid from a standard curve prepared using an inorganic phosphoric acid solution.
HMG-CoA reductase activity was measured using a reaction solution (0.128 mM HMG-CoA ( ¹⁴⁾ containing ¹⁴ C-labeled HMG-CoA in the presence of a test substance at the concentration shown in the table in the mouse liver microsome fraction (50 μg). C-HMG-CoA, 72 MBq / mmol), 1 mM NADPH, 10 mM DTT, 10 mM EDTA in 0.12 mM phosphate buffer (pH 7.2)), and incubated at 37 ° C. for 30 minutes. Next, one-tenth volume (20 μL) of 5N HCl was added and incubated for an additional 30 minutes (acid treatment). After addition of the internal standard ^{4- 14 C-testosterone (0.08nCi)} , conversion from the reaction products mevalonate HMG-CoA by an acidic treatment, the resulting mevalonolactone was extracted with an equal volume of ethyl acetate. A part of the extract was subjected to TLC fractionation using benzene-acetone (1: 1) as a developing solvent. Fluorography and quantitative analysis were performed using BAS2500 (Fuji Film), and HMG-CoA reductase activity was calculated from the radioactivity contained in mevalonolactone.
Tables 7 and 8 show HMG-CoA reductase activity and G6Pase activity in the presence of each test substance, respectively. These enzyme activities are expressed as relative values with the enzyme activity of the solvent control as 100.

As shown in Tables 7 and 8, none of the raspberry-derived ellagitannins showed an inhibitory effect on HMG-CoA reductase activity and G6Pase activity.
Thus, raspberry-derived ellagitannin showed excellent inhibitory action on DGAT activity, but did not show inhibitory action on other enzyme activities, indicating that it is a compound with high specificity for DGAT activity. . This can be considered to mean that raspberry-derived ellagitannin is a compound with few side effects.

Claims (11)

  A DGAT inhibitor comprising raspberry or an extract thereof as an active ingredient.   The DGAT inhibitor according to claim 1, wherein the extract is an organic solvent extract.   The DGAT inhibitor according to claim 2, wherein the organic solvent extract is a hydrous ethanol extract or a hydrous acetone extract.   A TG synthesis inhibitor comprising raspberry or an extract thereof as an active ingredient.   The TG synthesis inhibitor according to claim 4, wherein the extract contains ellagitannin or a derivative thereof as a main component.   The TG synthesis inhibitor according to claim 4 or 5, wherein the extract is an organic solvent extract.   The TG synthesis inhibitor according to claim 6, wherein the organic solvent extract is a hydrous ethanol extract or a hydrous acetone extract.   An agent for preventing or improving insulin resistance comprising raspberry or an extract thereof as an active ingredient.   A diabetes preventive or ameliorating agent comprising raspberry or an extract thereof as an active ingredient. Formula (1) in the molecule

The ellagitannin or derivative thereof having a partial structure represented by formula (1) and a glucose residue, wherein the hexahydrodiphenoyl group of the partial structure represented by formula (1) is substituted at the 4-position and the 6-position of the glucose residue A prophylactic or ameliorating agent for symptom of hyperlipidemia, obesity, insulin resistance or diabetes, or a TG synthesis inhibitor.   The agent for preventing or improving hyperlipidemia, obesity, insulin resistance or diabetes, or TG synthesis according to claim 12, wherein the ellagitannin or a derivative thereof is one or more selected from sanguine H-6 and lambertianin C. Inhibitor.

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