JP2009046481A - Peroxisome proliferator-activated receptor ligand agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a natural peroxisome proliferator-activated receptor γ (PPARγ) ligand agent, and to provide a preventive or improving composition for insulin resistance syndrome, diabetic, adiposis and visceral fat adiposis. <P>SOLUTION: The PPARγ ligand agent is curcumin or its derivative. The composition containing the PPARγ ligand agent as an active ingredient is useful as the preventive or improving composition for insulin resistance syndrome, diabetic, adiposis and visceral fat adiposis. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention includes a peroxisome proliferator-responsive receptor ligand agent and an insulin resistance syndrome (type 2 diabetes, hyperinsulinemia, lipid) characterized by containing a peroxisome proliferator-responsive receptor ligand agent as active ingredients The present invention relates to a composition for preventing / ameliorating metabolic disorders, obesity, hypertension, arteriosclerotic diseases).

  Peroxisome proliferator-activated receptor (PPAR) is a ligand-dependent transcriptional regulator belonging to the nuclear receptor family identified as a transcriptional regulator that controls the expression of genes that maintain lipid metabolism. It is. In mammals, the presence of three subtypes of PPARα, PPARδ (PPARβ, NUC-1, FAAR), and PPARγ is known. PPARα is mainly expressed in the liver and PPARδ is universally expressed. PPARγ has two types of isoforms, PPARγ1 and PPARγ2, and PPARγ1 is expressed not only in adipose tissue but also in immune system organs, adrenal glands, and small intestine. PPARγ2 is specifically expressed in adipose tissue and is a master regulator that controls the differentiation and maturation of adipocytes (Non-patent Document 1).

  PPARγ ligands include arachidonic acid metabolites such as 15-deoxy-Δ12,14-prostaglandin J2 and Δ12-prostaglandin J2, ω-3 polyunsaturated fatty acids, α-linolenic acid, eicosapentaenoic acid (EPA). ), Unsaturated fatty acids such as docosahexaenoic acid (DHA), and eicosanoids such as 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid are known (Non-patent Document 2). Further, it is disclosed that a conjugated unsaturated fatty acid having 10 to 26 carbon atoms having a conjugated triene structure or a conjugated tetraene structure is a PPARγ ligand (Patent Document 1). Furthermore, among synthetic compounds, thiazolidine derivatives such as troglitazone, pioglitazone, and rosiglitazone are known to be PPARγ ligands.

  A thiazolidine derivative, which is a PPARγ ligand, is associated with an insulin resistance improving action because its agonistic activity and hypoglycemic action correlate with each other, and an insulin resistance improving drug for type 2 diabetes (insulin-independent diabetes: NIDDM) Developed as That is, the thiazolidine derivative, which is a PPARγ ligand, activates PPARγ, thereby increasing small adipocytes having normal functions differentiated from preadipocytes and enhancing production and secretion of TNFα and free fatty acids that induce insulin resistance. Insulin resistance is improved by reducing the number of hypertrophic fat cells by apoptosis (Non-patent Document 3). In addition, since PPARγ ligand improves insulin resistance, not only type 2 diabetes, but also insulin resistance syndromes such as hyperinsulinemia, dyslipidemia, obesity, hypertension, and arteriosclerotic diseases (Non-patent Document 4). It is also effective for prevention and improvement. As an effect on obesity, it has been reported that visceral fat is reduced by administering troglitazone to a type 2 diabetic patient (Non-patent Document 5), which is also effective in preventing and improving visceral fat obesity.

  Curcumin and its derivatives are components contained in tropical and subtropical plant groups represented by Curcuma longa. Turmeric is commonly known as turmeric, one of the spices of curry, but it is not only used for food but also as a coloring agent for food and clothing. Ayurveda in Indonesia and Jamu in Indonesia are also used for medicinal purposes because of their hemostatic, gastric, antibacterial and anti-inflammatory effects.

As physiological effects of curcumin, antioxidative action, bile secretion promoting action (biliary action), visceral (liver, pancreas) function enhancing action, carcinogenesis suppressing action, lipid metabolism improving action, whitening action and the like have been clarified. P. Suresh Babu and K. Srinivasan show that blood cholesterol, triglycerides and phospholipids are reduced when diet containing 0.5% curcumin is given to diabetic rats induced with streptozotocin (Non-patent Document 6). It has been reported that kidney damage associated with diabetes is improved (Non-patent Document 7). JP-A-11-246399 (Patent Document 2) discloses an effect of enhancing the activity of acyl CoA oxidase, which is a β-oxidation-promoting enzyme in rats ingesting curcumin, and an effect of inhibiting the accumulation of triglycerides in the liver. Yes. However, it is not yet known that curcumin and / or its derivatives are PPARγ ligands, have a hypoglycemic action or a visceral fat reducing action.
JP 2000-355538 A JP-A-11-246399 Teruo Kawada, History of Medicine, 184, 519-523, 1998 J. Auwerx, Diabetologia, 42, 1033-1049, 1999 A. Okuno, et al., Journal of Clinical Investigation, 101, 1354-1361, 1998 RA Degronze, et al., Diabetes Care, 14, 173-194, 1991 IE Kelly, et al., Diabetes Care, 22, 288-293, 1999; Y. Mori, et al., Diabetes Care, 22, 908-912, 1999 Molecular and Cellular Biochemistry, 166, 169-175, 1997 Molecular and Cellular Biochemistry, 181, 87-96, 1998

  In view of the above, PPARγ ligand improves insulin resistance, insulin resistance syndrome such as type 2 diabetes, hyperinsulinemia, lipid metabolism abnormality, obesity (particularly visceral fat type obesity), hypertension, arteriosclerotic disease, etc. Has the effect of preventing and improving Therefore, an object of the present invention is to provide a prophylactic / ameliorating agent for insulin resistance syndrome, diabetes, obesity or visceral fat type obesity characterized by containing a natural product-derived PPARγ ligand and the active ingredient thereof. To do.

  The present inventors have found that the turmeric extract has a hypoglycemic action, and as a result of intensive studies, the specific component contained in turmeric has PPARγ ligand activity, and the specific component is curcumin and its derivatives. The present inventors have found that the specific component has a blood glucose lowering action and a visceral fat reducing effect, and have completed the present invention.

  That is, the present invention relates to a peroxisome proliferator-responsive receptor ligand agent comprising curcumin or a derivative thereof.

  The present invention also provides a composition for preventing / ameliorating insulin resistance syndrome, diabetes, or obesity or visceral fat type obesity, comprising at least one selected from the group consisting of curcumin and derivatives thereof as an active ingredient Related to things.

  According to the present invention, peroxisome proliferator-responsive receptor gamma (PPARγ) ligands and compositions containing the same are provided. The composition of the present invention is useful as an agent for preventing / ameliorating insulin resistance syndrome, diabetes, obesity or visceral fat type obesity.

  Hereinafter, embodiments of the present invention will be described in detail. The PPARγ ligand agent of the present invention is curcumin or a derivative thereof. In addition, a composition containing as an active ingredient at least one selected from the group consisting of curcumin and derivatives thereof of the present invention has a hypoglycemic effect and a visceral fat reducing effect, such as insulin resistance syndrome, diabetes, It is useful as a composition for preventing / ameliorating obesity or visceral fat type obesity.

  The curcumin used in the present invention is 1,7-bis (4-hydroxy-3-methoxyferni) -1,6-heptadiene-3,5-dione. Examples of the curcumin derivative (curcuminoid) include And methoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, dihydroxytetrahydrocurcumin, yaktinone A, yaktinone B and the like, and salts, oxidants, reductants, glycosides and esters of these compounds. And the like. These can also be used as plant-derived compounds or chemically synthesized compounds. Plant-derived curcumin and / or derivatives thereof include, for example, ginger family perennial grasses such as turmeric (Curcuma longa), cypress (spring turmeric: Curcuma aromatica), gadju (purple turmeric: Curcuma zedoaria), and turmeric (Curcuma xanthorrhiza). In addition, it can be extracted from plants such as mango ginger, Indian arrow root, yellow gadget, black gadget and galan girl.

  The method for preparing curcumin and its derivatives used in the present invention is not particularly limited, and conventionally known methods can be employed. For example, as a method for producing a turmeric pigment, which is a food additive (the main component is curcumin), from a dried product of turmeric rhizome, with hot ethanol, hot oil or propylene glycol, or from room temperature to hot hexane or acetone The method of extracting is mentioned. In addition, methods disclosed in Japanese Patent Application Laid-Open No. 2000-236843, Japanese Patent Application Laid-Open No. 11-235192, Japanese Patent Application Laid-Open No. 6-9479, Japanese Patent Application Laid-Open No. 11-502232, Japanese Patent Application Laid-Open No. 9-503528, etc. Can be mentioned. In the present invention, a pure compound can be used as at least one selected from curcumin and its derivatives. However, the present invention is not limited to this, and as long as it does not contain impurities that are inappropriate as pharmaceuticals or foods. Purified or crude products can also be used.

  The composition for preventing / ameliorating insulin resistance syndrome, diabetes, obesity or visceral fat obesity according to the present invention is a composition containing a PPARγ ligand, and is at least one selected from the group consisting of curcumin and its derivatives. As an active ingredient, the form is not limited, for example, as food and drinks such as health foods (specific health foods, nutritional foods) and health foods, pharmaceuticals, quasi drugs, etc. Can be used.

  When used as a food or drink, it can be directly ingested as it is, or it can be ingested in a form that is easy to take, such as capsules, tablets, and granules, using known carriers and auxiliaries. The content of the PPARγ ligand agent of the present invention in these molding agents is 0.1 to 100% by weight, preferably 10 to 90% by weight. In addition, mixed with food and drink ingredients, chewing gum, chocolate, candy, jelly, biscuits, crackers and other confectionery, ice cream, frozen desserts such as ice confectionery, tea, soft drinks, energy drinks, beauty drinks, and other udon noodles Noodles such as Chinese noodles, spaghetti, instant noodles, kneaded products such as rice cakes, bamboo rings, half pieces, seasonings such as dressing, mayonnaise, sauces, fats and oils such as margarine, butter, salad oil, bread, ham, soup, retort food, It can be used for all foods and drinks such as frozen foods. When these food and beverage compositions are ingested, the intake is 0.1 to 3000 mg / kg body weight, preferably 1 to 300 mg / kg body weight per day for each adult as the PPARγ ligand of the present invention. It can also be used as feed or pet food for livestock or pets, and its intake is preferably 0.1 to 3000 mg / kg body weight per day as the PPARγ ligand of the present invention.

  When used as a pharmaceutical, the dosage form is not particularly limited, and examples thereof include capsules, tablets, granules, injections, suppositories, and patches. In formulation, other pharmaceutically acceptable formulation materials such as excipients, disintegrants, lubricants, binders, antioxidants, colorants, anti-aggregation agents, absorption enhancers, solubilizers An agent, a stabilizer and the like can be added as appropriate. As dosages of these preparations, 0.1 to 3000 mg / kg body weight, preferably 1 to 300 mg / kg body weight per day per adult in terms of the PPARγ ligand of the present invention, is administered in 1 to several times. It can also be used as a pharmaceutical product for livestock and pets, and its dosage is preferably 0.1 to 3000 mg / kg body weight per day with the PPARγ ligand of the present invention.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.

(Example 1) Extraction of compound from turmeric, 1.0 kg of isolated turmeric powder was immersed in 8.0 L of ethanol and extracted for 2 days at room temperature and under light shielding, and then an extract was obtained by filtration. The extract was concentrated under reduced pressure to remove the solvent, and 118 g of extract was obtained. The extract was subjected to porous ion exchange resin Diaion HP-20 column chromatography (1600 ml), 30% methanol, 50% methanol, 80% methanol (1.5 L each), methanol, ethanol, ethyl acetate (each 3L) were eluted in sequence to obtain 6 fractions (fractions 1, 2, 3, 4, 5, 6). From fraction 4 (63.5 g), silica gel column chromatography A (elution solvent is hexane: acetone = 2: 1 → 3: 2 → 4: 3, v / v) and silica gel column chromatography B (elution solvent is chloroform) : Acetone = 99: 1 → 19: 1, v / v) was repeated to obtain Compound 1 (6.4 g), Compound 2 (1.2 g), and Compound 3 (1.1 g).

  As a result of structural analysis, compounds 1 to 3 were known compounds, and compound 1 was identified as curcumin, compound 2 was identified as demethoxycurcumin, and compound 3 was identified as bisdemethoxycurcumin. For structural identification of these compounds, spectral data described in a report of M. Kuroyanagi et al. (Pharmaceutical Journal, 90, 1467-1470, 1970) was referred to. The structural formulas of Compounds 1 to 3 are shown in Table 1.

（実施例２） ＰＰＡＲγリガンド活性
ＣＶ−１細胞（雄性アフリカミドリザル腎臓由来の培養細胞）を９６穴培養プレートに６×１０³ｃｅｌｌｓ／ｗｅｌｌとなるように植え込み、３７℃、５％ＣＯ₂条件下で２４時間培養した。培地には、１０％ＦＢＳ（ウシ胎仔血清）、１０ｍｌ／Ｌペニシリン・ストレプトマイシン溶液（それぞれ5000IU/ml、5000μg/ml、GIBCO社）、３７ｍｇ／Ｌアスコルビン酸（和光純薬工業株式会社）を含むＤＭＥＭ（Dulbecco's Modified Eagle Medium：GIBCO社）を用いた。細胞をＯＰＴＩ−ＭＥＭ（GIBCO社）で洗浄した後、ｐＭ−ｍＰＰＡＲγと４×ＵＡＳｇ−ｌｕｃをリポフェクトアミン・プラス（GIBCO社）を用いてトランスフェクションした。なお、ｐＭ−ｍＰＰＡＲγは酵母由来転写因子ＧＡＬ４遺伝子（アミノ酸配列１〜１４７）とマウスＰＰＡＲγリガンド結合部位遺伝子（アミノ酸配列１７４〜４７５）を結合したキメラ蛋白発現用プラスミドであり、４×ＵＡＳｇ−ｌｕｃはルシフェラーゼ遺伝子の上流にＧＡＬ４の応答配列（ＵＡＳｇ）を４回組み込んだレポーター・プラスミドである。トランスフェクションの約２４時間後、サンプルを含む培地に交換し（ｎ＝４）、２４時間培養した。サンプルはジメチルスルホキシド（ＤＭＳＯ）に溶解したものを、無処置対照にはＤＭＳＯを用い、培地に１／１０００量添加した。細胞をＣａ，Ｍｇ含有リン酸緩衝生理食塩水（ＰＢＳ＋）で洗浄した後、ルックライト（Packard社）を添加し、トップカウント・マイクロプレートシンチレーション／ルミネッセンスカウンター（Packard社）にてルシフェラーゼの発光強度を測定した。

(Example 2) PPARγ ligand-active CV-1 cells (cultured cells derived from male African green monkey kidney) were implanted in a 96-well culture plate so as to be 6 × 10 ³ cells / well, and the conditions were 37 ° C. and 5% CO _2. For 24 hours. DMEM containing 10% FBS (fetal bovine serum), 10 ml / L penicillin / streptomycin solution (5000 IU / ml, 5000 μg / ml, GIBCO, respectively), 37 mg / L ascorbic acid (Wako Pure Chemical Industries, Ltd.) (Dulbecco's Modified Eagle Medium: GIBCO) was used. After washing the cells with OPTI-MEM (GIBCO), pM-mPPARγ and 4 × UASg-luc were transfected using Lipofectamine Plus (GIBCO). PM-mPPARγ is a plasmid for expression of a chimeric protein in which a yeast-derived transcription factor GAL4 gene (amino acid sequence 1-147) and a mouse PPARγ ligand binding site gene (amino acid sequence 174-475) are combined, and 4 × UASg-luc is This is a reporter plasmid in which a GAL4 response element (UASg) is incorporated four times upstream of the luciferase gene. About 24 hours after transfection, the medium was changed to a medium containing samples (n = 4) and cultured for 24 hours. Samples were dissolved in dimethyl sulfoxide (DMSO), DMSO was used as an untreated control, and 1/1000 amount was added to the medium. After washing the cells with phosphate buffered saline (PBS +) containing Ca and Mg, Looklite (Packard) was added, and the luminescence intensity of luciferase was measured using a top count microplate scintillation / luminescence counter (Packard). It was measured.

  As in the measurement group, measurement was performed using pM (a plasmid from which the PPARγ ligand binding site gene had been removed) instead of pM-mPPARγ as a control group. For each sample, the ratio (measurement group / control group) of the average value (n = 4) of the luminescence intensity of the measurement group and the control group was calculated, and the specific activity relative to the untreated control was defined as the PPARγ ligand activity of the sample. The results are shown in Table 2.

陽性対照としてトログリタゾン（三共株式会社）を用い、各化合物のＰＰＡＲγリガンド活性を比較した。表２から明らかなように、クルクミン、デメトキシクルクミン、ビスデメトキシクルクミンに、濃度依存的にＰＰＡＲγリガンド活性が認められた。

Troglitazone (Sankyo Co., Ltd.) was used as a positive control, and the PPARγ ligand activity of each compound was compared. As is clear from Table 2, curcumin, demethoxycurcumin, and bisdemethoxycurcumin showed PPARγ ligand activity in a concentration-dependent manner.

(Example 3) Effect in type 2 diabetes model mouse The effect of curcumin was evaluated using KK-Ay mice, which are genetically obese and develop type 2 diabetes. As a positive control, pioglitazone, which is a therapeutic drug for diabetes, was used.

  KK-Ay mice (female, 6 weeks old) are divided into 3 groups (5 animals each), normal diet (Oriental Yeast Co., Ltd., Table 3) is used as a basic feed, no addition group (control group), pioglitazone addition group The curcumin addition group was given by free intake for 4 weeks. Pioglitazone was prepared by pulverizing Actos Tablets 30 (containing 30 mg of pioglitazone in one tablet, Takeda Pharmaceutical Co., Ltd.) in an agate mortar and adding pioglitazone to a normal diet so that the amount of pioglitazone was 0.04%. Curcumin was added to the normal diet so that the addition amount was 0.5%.

給餌期間中１週間毎にマウス尾静脈より少量採血し、簡易式血糖測定器グルテストエース（株式会社三和化学研究所）を用いて、血糖値を測定した。

A small amount of blood was collected from the tail vein of the mouse every week during the feeding period, and the blood glucose level was measured using a simple blood glucose meter Glutest Ace (Sanwa Chemical Laboratory Co., Ltd.).

  The mouse body weight is shown in Table 4. The mouse body weights of the pioglitazone added group and the curcumin added group were almost the same as those in the non-added group (control group), and no significant difference was observed.

血糖値の測定結果を表５に示す。給餌開始時の血糖値は１３９〜１５１ｍｇ／ｄｌであり、いずれの群においても高血糖になっていなかった。無添加群（対照群）において、１週後から血糖値が上昇し、糖尿病の発症が確認された。無添加群（対照群）に比し、糖尿病治療薬であるピオグリタゾン添加群では血糖上昇が有意に抑えられており、強い血糖降下作用が認められた。クルクミン添加群においても血糖上昇が有意に抑えられており、血糖降下作用が認められた。

Table 5 shows the blood glucose level measurement results. The blood glucose level at the start of feeding was 139 to 151 mg / dl, and no hyperglycemia was observed in any group. In the non-addition group (control group), the blood glucose level increased after 1 week, and the onset of diabetes was confirmed. Compared to the non-added group (control group), the increase in blood glucose was significantly suppressed in the group added with pioglitazone, which is a therapeutic agent for diabetes, and a strong hypoglycemic effect was observed. Even in the curcumin addition group, the increase in blood glucose was significantly suppressed, and the effect of lowering blood glucose was observed.

（実施例４） 食餌性肥満モデル・マウスにおける効果
Ｃ５７ＢＬ／６Ｊマウス（雌，８週齢）に高脂肪・高糖分食（オリエンタル酵母株式会社、表６）を自由摂取にて８週間与え、食餌性の肥満状態にした。その後マウスを各群７匹に分け、正常食（オリエンタル酵母株式会社、表３）を基本飼料として、無添加群（対照群）及びクルクミン添加群（添加量０．５％）を自由摂取にて４週間与えた。一晩絶食したマウスをエーテル麻酔下で開腹し、腹大動脈から採血して屠殺した。その後、子宮周辺脂肪、腎臓周辺脂肪、腸間膜周辺脂肪を摘出し、重量を測定した。子宮周辺脂肪量、腎臓周辺脂肪量、腸間膜周辺脂肪量の和を腹腔内脂肪量とした。結果を表７に示す。

(Example 4) Effect on dietary obesity model mouse C57BL / 6J mice (female, 8 weeks old) were given a high-fat and high-sugar diet (Oriental Yeast Co., Ltd., Table 6) for 8 weeks with free intake. Sexual obesity. Thereafter, the mice were divided into 7 mice in each group, and a normal diet (Oriental Yeast Co., Ltd., Table 3) was used as a basic feed, and an additive-free group (control group) and a curcumin-added group (addition amount 0.5%) were freely consumed. I gave it for 4 weeks. Mice fasted overnight were opened under ether anesthesia, and blood was collected from the abdominal aorta and sacrificed. Thereafter, peripheral uterine fat, peripheral renal fat, and peripheral mesenteric fat were extracted and weighed. The sum of the fat mass around the uterus, the fat mass around the kidney, and the fat mass around the mesentery was taken as the intraabdominal fat mass. The results are shown in Table 7.

表７から明らかなように、無添加群（対照群）に比し、クルクミン添加群での摂餌量及び体重に有意な差は認められなかったが、子宮周辺脂肪量、腎臓周辺脂肪量、腸間膜周辺脂肪量、及び腹腔内脂肪量が有意に減少した。すなわち、高脂肪・高糖分食の摂取によって蓄積した内臓脂肪が、クルクミンを含有した飼料を摂取することにより低減することが判明した。

As is clear from Table 7, no significant difference was observed in the amount of food intake and body weight in the curcumin addition group as compared to the non-addition group (control group). The mesenteric fat mass and intraperitoneal fat mass were significantly reduced. That is, it has been found that the visceral fat accumulated by ingestion of a high fat / high sugar diet is reduced by ingesting a feed containing curcumin.

(Example 5) Preparation of curcumin-containing tablets Curcumin 45 parts by weight Lactose 35 parts by weight Crystalline cellulose 15 parts by weight Sucrose fatty acid ester 5 parts by weight A food and drink tablet containing curcumin with the above composition was prepared by a conventional method. .

(Example 6) Preparation of curcumin-containing soft capsule Curcumin 40 parts by weight Sesame oil 55 parts by weight Glycerin fatty acid ester 5 parts by weight A soft capsule for eating and drinking containing curcumin was prepared by the conventional method.

(Example 7) Preparation of curcumin-containing cracker Curcumin 1 part by weight weak flour 120 parts by weight Salt 1 part by weight Baking powder 2 parts by weight Butter 30 parts by weight Water 40 parts by weight A cracker containing curcumin according to a conventional method. Was prepared.

(Example 8) Preparation of curcumin-containing udon Noodles containing curcumin 1 part by weight Strong powder 100 parts by weight Soft flour 100 parts by weight Salt 10 parts by weight Water 100 parts by weight The above composition was used to prepare udon containing curcumin by a conventional method.

(Example 9) Preparation of curcumin-containing dressing Curcumin 10 parts by weight Olive oil 80 parts by weight Vinegar 60 parts by weight Salt 3 parts by weight Pepper 1 part by weight Lemon juice 5 parts by weight The above composition contains curcumin in a conventional manner. Was prepared.

Claims (5)

Contains at least one curcumin selected from the group consisting of curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, dihydroxytetrahydrocurcumin, yaktinone A and yaktinone B as an active ingredient A composition for preventing / ameliorating visceral fat obesity (excluding pharmaceutical use). The composition according to claim 1, wherein the composition is for eating and drinking. The composition according to claim 2, which is a health functional food. The composition according to claim 3, which is a food for specified health use. The composition according to claim 1, wherein the composition is for livestock or pets.