JP2010070540A - Dgat inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DGAT inhibitor effective for prevention or amelioration of hyperlipidemia, obesity, insulin resistance and diabetes. <P>SOLUTION: The diacylglycerol acyltransferase (DGAT) inhibitor comprises ellagitannin or a derivative thereof as an effective ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

  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.

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 1 and 2), lipid metabolism-improving drugs (Non-patent) A method using documents 3 and 4) 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, it is considered that DGAT inhibition is effective for the prevention and improvement of hyperlipidemia, obesity, insulin resistance and diabetes (Non-patent Document 5), and for this purpose, the search for a substance that inhibits DGAT activity is conducted. As a food material that inhibits DGAT activity, it has been reported that flavonoids and tangeretin, which are contained in a large amount in citrus fruits, inhibit DGAT activity of human liver-derived HepG2 cells (Non-patent Document 6). 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 7). 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), both of which have structures different from the ellagitannins used in the present invention. It is.

  Elazitannin is a substance that exists widely in the plant kingdom and is known to have a strong antioxidant action. The ellagitannins include DOG type [hexahydroxydiphenoyl group (D) → oxygen (O) → galloyl group (G): ether-bonded oxygen is supplied from D side to G side], GOD type [G → O → D: Oxygen of ether bond is supplied from G side to D side] is known.

Examples of DOG-type ellagitannins include Rugosin A to G (Non-Patent Documents 8 and 9) identified from Rosa rugosa, and Isorugosin identified from Liquidambar formosana. Examples include A, B, and D (Non-patent Document 10), and Eusupinin A (Non-patent Document 11) identified from Euphorbia supina.
Among these, it has been reported that rugosin D has an anticancer effect, and rugosine AG has a hair growth action and an acne improving effect (Patent Documents 3 and 4).
Moreover, as GOD-type ellagitannins, it is identified from Sanguiin H-2 to H-11 (Non-patent Document 12) identified from the rose family plant Sanguisorba officinails, and from the rose family plant Rubus lambertianus. Lambertianin A to D (Non-patent Document 13) and Roshenin A to E (Non-patent Document 14) identified from Rosa henryi are mentioned.
Among these, it has been reported that sanguin H-4 has a hair growth action (Patent Document 5).

  However, it has not been known so far that DOG-type ellagitannins such as lugosin, isorugosine and juspinin, and GOD-type ellagitannins such as sanguiin and lambertianin have a DGAT inhibitory action.

International Publication No. 2006/082010 Pamphlet International Publication No. 2007/138304 Pamphlet JP-A-62-129220 Japanese Patent Laid-Open No. 3-188015 JP 2004-91390 A

Inoue, S. et al., Am. J. Clin. Nutr. 55, 199S-202S (1992) Ryan, D.H. et al., Obes. Res. 3, 553S-559S (1995) Monk, J.P. and Todd, P.A., Drugs. 33, 539-576 (1987) Norio Tada, Ther. Res., 21, 2320-2325 (2000) Chen, H. C. et al., J. Clin. Invest. 11, 1715-1722 (2003) Kurowska, E. M. et al., Lipids 39, 143-151 (2004) Ko, J. S. et al., Planta Med. 68, 1131-1133 (2002) Okuda, T. et al. Chem. Pharm. Bull. 30, 4230-4233 (1982) Okuda, T. et al. Chem. Pharm. Bull. 30, 4234-4237 (1982) Hatano, T. et al. Chem. Pharm. Bull. 36, 3920-3927 (1998) Agata, I. et al. Chem. Pharm. Bull. 39, 881-883 (1991) Okuda, T. et al., Phytochemistry, 32 507-521 (1993) Tanaka, T. et al., Chem. Pharm. Bull., 41 1214-1220 (1993) Yoshida, T. et al., Chem. Pharm. Bull., 40 1997 (1992)

  The present invention relates to providing a DGAT inhibitor effective in preventing or ameliorating hyperlipidemia, obesity, insulin resistance and diabetes.

  As a result of searching for substances having an activity of inhibiting the activity of DGAT, the present inventors have found that ellagitannin or a derivative thereof has an excellent activity of inhibiting DGAT and can be used as a DGAT inhibitor or the like.

That is, the present invention relates to the following 1) to 3).
1) A DGAT inhibitor containing ellagitannin or a derivative thereof as an active ingredient.
2) Elazitanin or a derivative thereof has the following formula (1) or (2)

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. 1) the DGAT inhibitor.
3) The DGAT inhibitor according to 1) or 2), wherein ellagitannin or a derivative thereof is one or more selected from lugosin A, lugosin B, lugosin D, euppinein A, sanguine H-6 and lambertianin C.

  Since the DGAT inhibitor of the present invention has an excellent DGAT inhibitory action, it is useful as a pharmaceutical, food, etc. for preventing or improving hyperlipidemia, obesity, insulin resistance and diabetes.

  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.

  In the present invention, the ellagitannin derivative means at least the following formula (1) or (2):

The ellagitannin related hydrolyzable tannin which has the partial structure represented by these.

Among these, from the viewpoint of DGAT inhibitory activity, as a suitable ellagitannin derivative, the partial structure represented by the above formula (1) or (2) further has a glucose residue, and the 4-position of the glucose residue is What substituted the hexahydro diphenoyl group part of the partial structure represented by the said Formula (1) or the said Formula (2) to 6th position is mentioned.
In addition, ellagitannin also has a partial structure and a glucose residue represented by the formula (1) or (2), and the glucose residue has 4- and 6-positions in the formula (1) or (2). What substituted the hexahydro diphenoyl group part of the partial structure represented is preferable.

As described above, the ellagitannin or derivative thereof of the present invention includes ellagitannin having a partial structure represented by the above formula (1) or a derivative thereof (hereinafter also referred to as “DOG type ellagitannin or a derivative thereof”) and the above formula ( And ellagitannin having a partial structure represented by 2) or a derivative thereof (hereinafter also referred to as “GOD type ellagitannin or a derivative thereof”).
Here, in general, the DOG type refers to a type (D → O → G) in which ether-linked oxygen is supplied from the hexahydroxydiphenoyl group side to the galloyl group side, and the GOD type refers to the ether type. This is a type in which the oxygen of the bond is supplied from the galloyl group side to the hexahydroxydiphenoyl group side (G → O → D).

  Of the DOG-type ellagitannins or derivatives thereof, those represented by the following formula (3) are preferred.

In Formula (3), R ¹ represents a hydrogen atom or a galloyl group. In formula (3), “˜OR ¹ ” represents the presence of α-type and / or β-type.
In formula (3), 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 (3), 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 (3), n represents 0 or 1.

  Of the GOD type ellagitannins or derivatives thereof, those represented by the following formula (4) are preferred.

In formula (4), R ⁷ has the same meaning as R ¹ . In formula (4), “˜OR ⁷ ” represents the presence of α-type and / or β-type.
In formula (4), R ⁸ and R ⁹ are synonymous with R ² and R ³ .
In formula (4), R ¹⁰ has the same meaning as R ⁴ .
In formula (4), m represents 0-2 (preferably 0 or 1).

The DOG-type ellagitannins or derivatives thereof are preferably rugosin A, rugosin B, rugosin C, rugosin D, rugosin E, rugosin F, rugosin G, and isorugosin because they exist in edible plants and have high safety. Illustrative examples include rugosins such as A, isorgocin B and isorugosine D;
Moreover, as GOD-type ellagitannin or its derivative, since it exists in an edible plant and has high safety, Sangiin H-2, Sangiin H-3, Sangiin H-6, Sangiin H-7, Sangiin H is preferable. -8, sanguin such as sanguin H-9, sanguin H-10 and sanguin H-11; lambertianine such as lambertianin A, lambertianin B, lambertianin C, lambertianin D; rochenin A, rochenin B, rochenin C, rochenin D, rochenin E Examples include rochenin and the like.
Among these examples, rugosin A, rugosin B, rugosin D, eupinin A, sanguine H-6 and lambertianin C are preferred in terms of DGAT inhibitory activity.

Examples of the ellagitannin or derivative thereof according to the present invention include those obtained by a known chemical synthesis method, plant extracts containing ellagitannin or a derivative thereof as a main component (for example, see Non-Patent Documents 8 to 14), or purified products thereof. Is included.
For example, as rugosin, it is possible to use an extract from the fruit or petal of Hermanus (Rosa rugosa) or Pink Rose (Rosa centifolia), or a concentrated or purified extract thereof. As a use pinin, Euphorbia supina ) Whole plant, pink crocodile (Rosa centifolia) fruit or petal, or a concentrated or purified extract thereof. In addition, as sanguin and lambertianin, those extracted from the fruit of raspberry (Rubus iadeus) or concentrated or purified from the extract can be used.

As an extraction solvent for extracting ellagitannin or a derivative thereof of the present invention, water; alcohols such as methanol, ethanol, propanol and butanol; and organic solvents such as acetone are preferably used. More preferably, a solvent is used. These organic solvents may contain water.
Of these extraction solvents, it is preferable to use water and / or water-soluble organic solvents, and more preferably water, ethanol, water / ethanol mixed solution, acetone or water / acetone mixed solution, water / ethanol mixed solution or water / It is more preferable to use an acetone mixed solution. The ethanol or acetone concentration in the mixed solution is preferably 20 to 80% by volume, more preferably 30 to 70% by volume, and further preferably 40 to 60% by volume.
Moreover, you may use together the method of extracting in so-called non-oxidative atmosphere, ventilating inert gas, such as boiling deaeration and nitrogen gas, and removing dissolved oxygen.
Extraction is performed using, for example, 1 to 50 parts by weight of a solvent with respect to 1 part by weight of a plant containing the desired ellagitannin or a derivative thereof. It is preferable to immerse or heat reflux for 30 minutes to 15 days, and further for 2 hours to 10 days. Examples of the separation and purification means of the extract include activated carbon treatment, liquid-liquid distribution, column chromatography, liquid chromatography, gel filtration, and precision distillation.
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.) and the like: Gel filtration chromatography filler; activated carbon and the like, among which reverse phase resins and aromatic synthetic resins are preferred.

An example of extraction means for obtaining lugosin and youthpinin from pink rose (petal) is given, but the extraction means is not limited to this.
A water / ethanol extract is obtained from a pink rose (petal) using a water / ethanol mixture (preferably an ethanol concentration of 40 to 60% by volume). The water / ethanol extract is liquid-separated with a water / hexane solvent (preferably water: hexane (volume ratio) = 1: 3 to 3: 1), and then the aqueous layer is separated. Butanol is added to the aqueous layer, and after liquid-liquid separation with a water / butanol solvent (preferably water: butanol (volume ratio) = 1: 3 to 3: 1), the butanol layer is separated. This butanol layer is subjected to chromatography using an aromatic synthetic resin (preferably, a styrene-divinylbenzene aromatic synthetic resin), and a low-concentration ethanol water mixture (preferably an ethanol concentration of 10 to 30 volumes). %) To obtain a low-concentration ethanol-eluted fraction. Furthermore, this low-concentration ethanol elution fraction is subjected to gel filtration chromatography (preferably a modified dextran filler), washed with a methanol water mixture (preferably methanol concentration 40 to 60% by volume), ethanol, Elution with ethanol / acetone mixed solution (preferably ethanol: acetone (volume ratio) = 1: 3 to 3: 1) and containing rugosine B as an active ingredient; rugosine A, rugosine D And an ethanol / acetone fraction containing Eupinin A and containing these as active main components is obtained. The ethanol fraction and the ethanol / acetone fraction are each subjected to chromatography using a reverse-phase resin (preferably octadecylated silica gel reverse-phase resin), and the concentration of acetonitrile in the trifluoroacetic acid aqueous solution / acetonitrile mixture is one step. Elution is carried out with the above gradient, and rugosin B is isolated from the ethanol fraction, and rugosin A, rugosin D, and juspinin A are isolated from the ethanol / acetone fraction.

Although an example of the extraction means which obtains sanguin and lambertianin from a raspberry (fruit) is given, the extraction means is not limited to this.
A water / ethanol extract is obtained from raspberries (fruits) using a water / ethanol mixture (preferably an ethanol concentration of 40 to 60% by volume). This water / ethanol extract is subjected to chromatography using an aromatic synthetic resin (preferably, a styrene-divinylbenzene aromatic synthetic resin), washed with water, and then hydrous ethanol (preferably having an ethanol concentration of 40). To obtain a water-containing ethanol-eluted fraction containing sanguine H-6 and lambertianin C6 and containing these as active main components. Further, this water-containing ethanol elution fraction is subjected to chromatography using a reverse-phase resin (preferably an octadecylated silica gel reverse-phase resin), and the concentration of acetonitrile in the formic acid aqueous solution / acetonitrile mixture is increased by one or more steps. From elution and retention time, sanguin H-6 and lambertianin C are isolated, respectively.

  The obtained extract or fraction of ellagitannin or its derivative 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 prepared in a paste form But you can. Further, it can be freeze-dried and diluted with a solvent usually used for extraction at the time of use, for example, water, ethanol, water / ethanol mixed solution or the like. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  The ellagitannin or derivative thereof of the present invention has an excellent DGAT inhibitory activity, as shown in Examples below. Further, a substance having DGAT inhibitory activity is suspected to have side effects if its enzyme inhibition is non-specific, but ellagitannin or a derivative thereof of the present invention is HMG-CoA reductase which is a major metabolic enzyme in the liver. It was determined that it had no effect on the activity and G6Pase activity, was specific to DGAT, and had few side effects on the human body.

Here, the absorbed lipid is re-synthesized into TG by the action of an enzyme such as monoacylglycerol acyl substrate transferase (MGAT) or DGAT in the small intestinal epithelial cells. It plays an important role in the uptake of TG. In addition, since DGAT is an enzyme required for the synthesis and secretion of very low density lipoprotein (VLDL) in the liver and TG accumulation in adipose tissue, inhibition of DGAT activity suppresses TG synthesis, hyperlipidemia It is considered to be an effective means for preventing and improving 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 and improving insulin resistance and diabetes (Chen, HC et al., J. Clin. Invest. 11, 1715-1722 (2003)). .
Therefore, the ellagitannin or a derivative thereof can be used as a DGAT inhibitor, and can be further used to produce a preparation. At this time, as the DGAT inhibitor, the ellagitannin or a derivative thereof alone or in addition to this, a carrier that is appropriately selected as necessary, such as a carrier that is allowed in the later-described object to be blended, is 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.

  Such a DGAT inhibitor is a human or animal that exhibits DGAT inhibitory activity, TG synthesis inhibition, prevention or improvement of hyperlipidemia, prevention or improvement of obesity, prevention or improvement of insulin resistance or diabetes, etc. And can be used as an active ingredient of pharmaceuticals, quasi drugs, foods, or feeds. The DGAT inhibitor has a DGAT inhibitory activity, a TG synthesis inhibitory action, prevention or improvement of hyperlipidemia, prevention or improvement of obesity, prevention or improvement of insulin resistance or diabetes. If necessary, it can be applied to foods, functional foods, foods for the sick, and foods 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 when used as an active ingredient of a preparation for oral administration, the content of ellagitannin or a derivative thereof of the present invention in the preparation is usually 0.01 to It is 80 mass%, and it is preferable that it is 0.5-80 mass%.

  The dosage of the 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 usually ellagitannin or its The derivatives are more preferably 1 to 2000 mg, 10 to 1000 mg, and 50 to 500 mg. 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 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 oral administration preparation, 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 alone or other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers, Antioxidants, humectants, thickeners and the like can be used in appropriate combinations.

  In addition, the content of ellagitannin or a derivative thereof in the food varies depending on the use form, but is usually 0.001 to 2% by mass, preferably 0.002 to 1% by mass in the beverage form, 0 0.01 to 0.5 mass% is more preferable. Moreover, in solid food forms, such as a tablet and processed food, it is 0.01-20 mass% normally, 0.02-10 mass% is preferable, and 0.1-5 mass% is more preferable.

In addition, when the DGAT inhibitor of the present invention is used as an active ingredient of feed, the feed includes, for example, livestock feed used for cattle, pigs, chickens, sheep, horses, etc., for small animals used for rabbits, rats, mice, etc. Examples include feed for fish and shellfish used for feed, tuna, eel, Thailand, hamachi, shrimp, etc., and pet food used for dogs, cats, small birds, squirrels, and the like.
In addition, when producing feed, DGAT inhibitor alone or in addition, meat such as cattle, pigs, sheep, protein, grains, bran, moss, sugar, vegetables, vitamins, minerals In general, feed ingredients commonly used in foods, etc., and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc., commonly used in feeds are blended as necessary The feed can be processed and manufactured.
Moreover, although content of the ellagitannin or its derivative in a feed changes with the usage forms, it is 0.01-20 mass% normally, 0.02-10 mass% is preferable, 0.1-5 mass% Is more preferable.

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

Production Example 1 : Preparation of test substance from pink rose 600 g of petal rose (Rosa centifolia) petals in 20 volume (12 liters) 50% by volume (hereinafter referred to as “% by volume”) ethanol After dipping at room temperature for 14 days, insolubles were filtered off and concentrated under reduced pressure to obtain an extract (solid content: 260 g).
The obtained extract was liquid-partitioned in a hexane / water system, and the resulting aqueous layer was liquid-extracted with butanol. The butanol extract (51 g) obtained by concentration under reduced pressure was adsorbed on Diaion (registered trademark) HP-20 (styrene-divinylbenzene-based aromatic synthetic adsorbent) and eluted with 20% ethanol. The obtained elution fraction was concentrated under reduced pressure and freeze-dried to obtain a dried product. Furthermore, 22 g of this dried product was dissolved in 50% methanol and adsorbed on gel filtration chromatography using Sephadex (registered trademark) LH-20. After washing with 50% methanol, elution was performed with 99.5% ethanol and ethanol / acetone (1: 1). Each elution fraction was concentrated under reduced pressure and freeze-dried to obtain a 99.5% ethanol fraction (solid content 4 g) and an ethanol / acetone fraction (solid content 6.6 g).
Lugosin B (4.3 mg) was obtained by using 120 mg of the obtained 99.5% ethanol fraction and further fractionating by HPLC. Fractionation conditions are as follows.
Column: Inertsil ODS-3 (20x250mm, GL Science)
Solvent: A liquid 0.5% trifluoroacetic acid, B liquid acetonitrile A / B = 87/13 → (30 minutes) → 87/13 → (20 minutes) → 50/50
Temperature: 40 ° C., flow rate: 20 mL / min, detection: UV = 258 nm
In addition, 300 mg of the obtained ethanol / acetone fraction was used and further fractionated by HPLC to obtain Lugosin A (70 mg), Lugosin D (59 mg), and Eupinepin A (21 mg). Fractionation conditions are as follows.
Column: Inertsil ODS-3 (20x250mm, GL Science)
Solvent: A liquid 0.5% trifluoroacetic acid, B liquid acetonitrile A / B = 84/16 → (30 minutes) → 84/16 → (5 minutes) → 50/50
Temperature: 40 ° C., flow rate: 20 mL / min, detection: UV = 258 nm
In addition, when NMR analysis (JEOL-500 spectrometer; JEOL Ltd.) was performed about these, these were described in the literature, Lugosin A, Lugosin B (nonpatent literature 8), Lugosin D (nonpatent literature 9). ) And the signal of Youthpinin A (Non-Patent Document 11).

Production Example 2 : Preparation of test substance from raspberry 100 g of freeze-dried powder of raspberry (Rubus idaeus L.) fruit (FD raspberry powder, available from Kyoritsu Bussan) in 10 volumes (1 L) of 50% ethanol water at room temperature For 15 hours, the insoluble material was filtered off and concentrated under reduced pressure to obtain an extract (solid content 59 g).
20 g of the obtained extract was redissolved in water and adsorbed on Diaion (registered trademark) HP-20 (styrene-divinylbenzene-based aromatic synthetic adsorbent). HP-20 was washed with water and then eluted with 50% ethanol. The obtained eluate was concentrated under reduced pressure to obtain 1.21 g of HP-20 fraction.
Further, 400 mg of the obtained fraction was further used, and further fractionated from the retention time (Sangiin H-6: 38 to 41 minutes / Lambertianine C: 36 to 38 minutes) by HPLC. (43 mg) and lambertianin C (16 mg) were obtained. Fractionation conditions are as follows.
Column: Inertsil ODS-3 (20x250mm, GL Science)
Solvent: A liquid 1% formic acid aqueous solution, B liquid acetonitrile A / B = 90/10 → (10 minutes) → 90/10 → (30 minutes) → 80/20
Temperature: 40 ° C., flow rate: 18.9 mL / min, detection: UV = 280 nm
In addition, when NMR analysis (Bruker Avance III 600; Bruker Biospin Co., Ltd.) was performed on these, these were Sangiin H-6 (Non-patent Document 12) and Lambertianine C (Non-Patent Document) described in the literature. It was consistent with the signal of 13).

Example 1 : Evaluation of inhibition of DGAT activity As test substances, Lugosin A, Lugosin B, Lugosin D, Euspinin A, Sangiin H-6, and Lambertianine C prepared in Production Examples 1 and 2 above, and EGCG (epigallocatechin gallate) : Obtained from Nagara Science Co., Ltd.).
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 5 μM or 10 μM test substance. Incubation was carried out at 37 ° C. for 20 minutes in% Bovine Serum Albumin, 100 μM Oleoyl-CoA (0.05 μCi / 200 μl, 55 mCi / mmol), 1.2 mM 1,2-Diolein). 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 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, Merck). 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.

Tables 1 and 2 show 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 Tables 1 and 2, Lugosin A, Lugosin B, Lugosin D, Euspinin A, Sangiin H-6, and Lambertianine C showed significant inhibition of DGAT activity. On the other hand, epigallocatechin gallate (EGCG) as a comparative control did not show an inhibitory effect on DGAT activity.
Thus, since ellagitannin exhibits an excellent inhibitory action on DGAT activity, TGAT inhibitors further include TG synthesis inhibitors, hyperlipidemia prevention / improving agents, obesity prevention / improving agents, insulin resistance prevention / improving agents, and It is thought to be useful as an agent for improving and preventing diabetes.

Example 2 : Evaluation of inhibition of other enzyme activities Using the mouse liver microsome fraction prepared as shown in Example 1, measurement of glucose 6-phosphate phosphatase (G6Pase) activity, 3-hydroxy-3-methyl Glutaryl coenzyme A (HMG-CoA) reductase activity was measured.
In the measurement of G6Pase activity, a mouse liver microsome fraction (50 μg) was added in the presence of 5 μM or 10 μM of a test substance in 100 μl of a reaction solution (50 mM HEPES buffer (pH 7.2), 100 mM KCl, 2.5 Incubation was carried out at 37 ° C. for 30 minutes in mM EDTA, 2.5 mM MgCl ₂ , 1 mM DTT, 10 mM glucose-6-phosphate, 2 mM EDTA). Next, 400 μl of a stop solution (0.42% ammonia molybdenum tetrahydrate in 1N H ₂ SO ₄ , 10% SDS, 10% ascorbic acid 6: 2: 1 mixture) was added and incubated at 50 ° C. for 30 minutes. did. 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 mixture (0.128 mM HMG-CoA ( ¹⁴⁾ containing ¹⁴ C-labeled HMG-CoA in the presence of 5 μM or 10 μM test substance 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 (0.08 nCi} ), 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, and HMG-CoA reductase activity was calculated from the radioactivity contained in mevalonolactone.
Tables 3 to 6 show the HMG-CoA reductase activity and G6Pase activity in the presence of each test substance. These enzyme activities are expressed as relative values with the enzyme activity of the solvent control as 100.

As shown in Table 3, Table 4, Table 5, and Table 6, none of the ellagitannins showed an inhibitory effect on HMG-CoA reductase activity and G6Pase activity.
Thus, ellagitannin shows an excellent inhibitory action on DGAT activity, but does not show an inhibitory action on other enzyme activities, indicating that it is a compound having high specificity for DGAT activity. This can be considered to mean that ellagitannin is a compound with few side effects.

Example 3
The following ingredients were mixed and then filled into a gelatin capsule to obtain a soft capsule of 250 mg per tablet.
(Composition) (%)
Lugosin B 2.5
Soy lecithin 10
Rape oil 87.5

Example 4
The following ingredients were mixed and then filled into a gelatin capsule to obtain a soft capsule of 250 mg per tablet.
(Composition) (%)
Lugosin B 0.5
Youth Pinin A 2.5
Soy lecithin 10
Safflower oil 87

Example 5
One tablet of 200 mg was produced according to a conventional method using the following components.
(Composition) (%)
Lugosin D or Youthpinin A 2.5
Hydroxypropylcellulose 45.5
Light anhydrous silicic acid 5
Lactose 13
Crystalline cellulose 13
Talc 13
Diacylglycerol 8

Example 6
One tablet of 200 mg was produced according to a conventional method using the following components.
(Composition) (%)
Lugosin B or Youthpinin A 5
Starch 90
Magnesium stearate 5

Example 7
30 g of green tea leaves were extracted with 1000 mL of water at 65 ° C. for 5 minutes and filtered to obtain a green tea liquid. The following composition was mixed using this green tea liquid, and then adjusted to pH 6.0 using sodium bicarbonate to produce a green tea drink for obesity / diabetes prevention / improvement.
(composition)
Lugosin B 50mg
Vitamin C 1000mg
Green tea liquid 1000mL

Example 8
The following composition was mixed to produce a juice drink for obesity / diabetes prevention / improvement.
(composition)
Lugosin B 50mg
Vitamin C 500mg
Lemon juice 5mL
475 mL of carbonated water
Fragrance Some amount Aspartame 5g

Example 9
The following composition was tableted to produce a chewable tablet food of 1000 mg per tablet.
(Composition) (%)
Youth Pinin A 2.5
Maltose 11
Lactose 30
Glucose 15
Vitamin C 20
Vitamin E 1
Cellulose 10
Xylitol 10
Fragrance 0.5

Example 10
Using the following ingredients, a pet food of 1,000 mg per tablet was produced according to a conventional method.
(Composition) (%)
Sangiin H-6 or Lambertianine C 1
Barley 38
Chicken meal 18
Corn Grunten Meal 18
Sorghum (Red Mylo) 10
Rapeseed, soybean mixed oil 5
Peat pulp 5
Vitamin mix 3
Mineral mix 2
Chicken oil 1

Claims (3)

  A DGAT inhibitor comprising ellagitannin or a derivative thereof as an active ingredient. Ellagitannin or a derivative thereof has the following formula (1) or (2)

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 DGAT inhibitor according to claim 1, wherein   The DGAT inhibitor according to claim 1 or 2, wherein the ellagitannin or a derivative thereof is at least one selected from rugosin A, rugosin B, rugosin D, euppinein A, sanguine H-6 and lambertianin C.