JP5004153B2 - Adiponectin production promoter - Google Patents

Description

  The present invention relates to an adiponectin production promoter. Specifically, this invention relates to the adiponectin production promoter which uses the compound extracted from the nutmeg fruit as an active ingredient.

  In recent years, obesity or diabetes has been rapidly increasing in Japan due to westernization and satiety of eating habits due to excessive calorie intake and lack of exercise. Furthermore, obesity is known to cause metabolic syndrome, so-called diabetes, hyperlipidemia and arteriosclerosis as complications. Obesity is a state in which adipose tissue is excessively accumulated, and it has been considered that adipose tissue is a tissue that only accumulates excess energy as a neutral lipid.

  However, in recent years, it has been found that adipose tissue is an endocrine organ that secretes a wide variety of hormones and cytokines such as adiponectin, TNF-α, leptin, and free fatty acids and plays an important role in vivo. In hypertrophic adipocytes, secretion of adiponectin, which has a TNF-α secretion inhibitory action and a hypoglycemic action and is very important for lifestyle-related diseases, decreases. Therefore, it is thought that excessive secretion of TNF-α, decrease in insulin secretion, and increase in insulin resistance lead to lifestyle-related diseases such as diabetes, hypertension, hyperlipidemia, and arteriosclerosis.

  Therefore, in order to prevent lifestyle-related diseases such as diabetes and hyperlipidemia, it is important to increase the size of adipocytes instead of hypertrophic adipocytes, and to increase the secretion of adiponectin from the sized adipocytes. It is believed that there is. Adipocyte differentiation is mainly controlled by PPARγ, a nuclear receptor transcription factor. When adiponectin secretion increases, PPARγ receptor increases and adipocyte differentiation is promoted from preadipocytes. It is thought. Thiazolidinediones (TZDs), which are thiazolidine-based drugs used as antidiabetic drugs, are potent PPARγ agonists (agonists), and also have the effect of reducing obese adipocytes by apoptosis as they differentiate into smaller adipocytes It is said. Moreover, as a cell differentiation induction promoting substance at the research level, isobutylxanthine (non-patent document 1) which is a c-AMP degradation inhibitor, indomethacin (non-patent document 2) which is a nonsteroidal anti-inflammatory agent, and the like are known. .

  Conventionally, lifestyle-related disease prevention and improvement agents have been developed, and, for example, γ-oryzanol adiponectin secretion promoter of rice bran (patent document 1) and ginger cell differentiation promoter (patent document 2) are known. . The literature describing the pharmacological action of nutmeg includes an anti-obesity agent containing nutmeg and containing a water-soluble galactomannan as a main component (Patent Document 3), an extract extracted from nutmeg using an organic solvent, or its There is a novel liver injury inhibitor (Patent Document 4) containing a fraction (main component, myristin) as an active ingredient of a liver injury inhibitor. However, there is no known compound that has an action of enhancing nutmeg adipocyte differentiation induction and promoting and enhancing adiponectin production.

  As mentioned above, the deficiency of adiponectin (proteinaceous factor), one of the cytokines released from cells and controlling biological functions, is closely related to the development of lifestyle-related diseases such as type 2 diabetes and arteriosclerosis. It is clear that they are related. And by increasing the adiponectin concentration in blood by direct injection administration of adiponectin and the introduction and expression of adenovirus vector incorporating the gene, vascular lesions leading to arteriosclerosis It has been experimentally proven that the formation of can be suppressed (Non-Patent Documents 3 to 5). In actual applications, methods using adiponectin have been developed as obesity-improving agents (Patent Document 5), anti-inflammation, monocyte cell growth inhibitors (Patent Document 6), and the like. In this way, administration of adiponectin produced outside the body is also effective, but it is very important for the prevention and treatment of lifestyle-related diseases over the long term to improve and further enhance the production of adiponectin in the body. it is conceivable that.

JP2005-68132 JP2005-75749 JP2005-126405 JP2003-091379 US Application Publication No. 2002/0132773 JP2000-256208 A.K.G.Loeffer, Horm.Metab.Res., 32,548-554 (2000) H. Ye et al., Biohcem. J., 330,803-809 (1998) Ichiro Shimomura et al., Experimental Medicine, Vol.20, No.12, 1762-1767 (2002) A. H. Berg et al., Nature Medicine, 7, 947-953 (2001) Y. Okamoto, Circulation, 106, 2767-2770 (2002)

  In this way, pharmaceuticals that increase the size of adipocytes and increase the amount of adiponectin are already known, but TZD is highly hepatotoxic and indomethacin originally has another strong pharmacological activity, safety and side effects There is a problem in terms of. Therefore, lifestyle-related diseases such as diabetes and arteriosclerosis are rapidly increasing and countermeasures are becoming a social problem. As a drug that can be easily used for the prevention and treatment of lifestyle-related diseases, miniaturized adipocytes are used. There is a demand for the development of compounds that increase and promote and enhance the production of adiponectin. Furthermore, in order to prevent and treat lifestyle-related diseases, it is considered important to take a substance that increases the size of adipocytes and increases the amount of adiponectin as a safe food by daily diet.

  An object of the present invention is to provide a substance having a promoting action on adipocyte differentiation and adiponectin production enhancing action from nutmeg, that is, an adiponectin production promoter.

  Another object of the present invention is to provide a composition for preventing, ameliorating or treating lifestyle-related diseases or obesity comprising an adiponectin production promoter.

  In such a situation, the present inventors examined plants that increase adiponectin production and enhance adiponectin production, and as a result, the compound obtained from the extract of nutmeg promoted differentiation from preadipocytes to adipocytes. And found to enhance adiponectin production. Based on such knowledge, the present invention has been completed.

(Summary of Invention)
That is, in summary, the present invention includes the following features.

The present invention includes the following formulas 1 to 9:

によって表される化合物又はその誘導体の少なくとも１つを有効成分として含むことを特徴とする、アディポネクチン産生促進剤を提供する。
その実施形態において、上記の有効成分がナツメグの実の抽出物又は処理物である。

An adiponectin production promoter characterized in that it comprises at least one of the compound represented by formula (1) or a derivative thereof as an active ingredient.
In that embodiment, the active ingredient is a nutmeg extract or processed product.

The present invention also provides a composition for preventing, ameliorating or treating a lifestyle-related disease or obesity selected from type 2 diabetes, hyperlipidemia, hypertension and arteriosclerosis, comprising the above-mentioned adiponectin production promoter. .
In that embodiment, the composition of the present invention is a pharmaceutical or food.

(Definition)
The “adiponectin production promoter” used herein has an action of promoting differentiation from preadipocytes to adipocytes and increasing, enhancing, or promoting adiponectin production / secretion from miniaturized adipocytes. Means a substance. In the present invention, the adiponectin production promoter contains the above-mentioned compound or derivative thereof contained in nutmeg as an active ingredient.

  In the present specification, the terms “adiponectin production promoter” and “adiponectin production enhancing agent” are used interchangeably.

  The adiponectin production promoter of the present invention, as defined above, has a component obtained from nutmeg as an active ingredient as a differentiation promoter from animal preadipocytes to adipocytes, and promotes cell differentiation, thereby reducing miniaturized fat. It is a substance that increases the amount of adiponectin while increasing the number of cells. Since this substance has the effect of significantly increasing the amount of adiponectin, the adiponectin production promoter of the present invention is used to prevent, ameliorate, or treat especially lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, arteriosclerosis or obesity. It can be used for the production of foods, food additives, pharmaceuticals, etc.

  While known compounds such as TZD and indomethacin, which are known as adiponectin production enhancers, have problems of safety and side effects, the compounds of the present invention are derived from nutmeg that has been eaten for many years. Therefore, it is safe and excellent for daily intake and use.

  Furthermore, as described above, adiponectin prevents or treats type 2 diabetes, a so-called lifestyle-related disease, in which insulin secretion from the pancreas is reduced due to excessive action or blood glucose level does not decrease despite the production of insulin. And has many important physiological actions such as suppression of vascular lesions leading to arteriosclerosis, improvement of obesity, anti-inflammation, suppression of monocyte cell proliferation, and suppression of liver fibrosis.

  The adiponectin production promoter contains a substance contained in nutmeg fruit as an active ingredient. As described above, such a substance is a compound represented by Formula 1 to Formula 9 (FIG. 1). In the present invention, a derivative of the above compound is also used as an active ingredient as long as it has the same effect of promoting adiponectin production. Is possible.

Since all of the compounds of the present invention have an activity of significantly increasing adiponectin production, any one of them or any mixture thereof can be used as an active ingredient of an adiponectin production promoter. Such mixtures also include nutmeg fruit extracts or processed products. Here, the extract is a substance obtained by extraction and separation of an active ingredient with a solvent, and includes a solvent extract, a concentrated solution thereof, and a dried product thereof. Processed products include substances containing one or more active ingredients that have been completely or partially purified by subjecting the extract to a further purification step, actual processed products such as nutmeg fruit juice, and the like.
Hereinafter, extraction and purification of the compound of the present invention from nutmeg will be described.

(Extraction and purification)
When producing from nutmeg, as long as there are no inconvenient impurities, it can be used as a crude extract or partially purified product for the purposes of the present invention. The agent for promoting cell differentiation and enhancing adiponectin production according to the present invention contains a component extracted from nutmeg fruit as an active ingredient. As the nutmeg fruit, a dried product can be used, and the dried product can be pulverized.

  The extraction step can be performed using a polar organic solvent such as alcohol such as methanol, ethanol, propanol and butanol, ketone such as acetone and methyl ethyl ketone, ester such as ethyl acetate, preferably ethanol or methanol, more preferably ethanol. The extraction can be performed using. The extraction can be easily performed by a known extraction method using water or supercritical carbon dioxide in addition to the organic solvent.

  The volume of the solvent may be about 2 to about 4 times the nutmeg's actual dry weight. The extraction can usually be carried out by leaving at room temperature for about 1 day, or by heating (for example, about 40 ° C. to about 80 ° C.) to shorten the time. The extraction may be performed only once, but may be performed a plurality of times, for example, 2 to 3 times under the same conditions as described above.

  The extract is then subjected to a step of removing the solvent under constant pressure or reduced pressure. Removal is usually evaporation or evaporation under heating. The oily residue obtained by removing the solvent is subjected to a purification step.

  Purification can be performed by a known purification method such as partitioning in a water / organic solvent two-phase system, activated carbon treatment, or various chromatographic operations. A preferred method is chromatography using a silica gel column as described in Example 1 below. After the oily residue is adsorbed on silica gel equilibrated with a non-polar solvent such as hexane, the compound of the present invention can be eluted by increasing the volume of the polar solvent continuously or stepwise. Elution can be performed, for example, with a gradient from 100% hexane to about 80% acetone / about 20% hexane. If further separation is required, high performance liquid chromatography can be used. The chromatographic conditions are, for example, a silica gel column (commercially available), a flow rate of 3.5 ml / min, a detection UV of 270 nm, a solvent containing 0.1% formic acid, 60%, 70%, and 80% methanol.

The compound of the present invention is developed by developing on silica gel thin layer chromatography (hexane: acetone = 3: 1 mixed solution) and showing a deep blue color by spraying a mixed solution of FeCl ₃ -K ₃ [Fe (CN) ₆ ] Is done.

(Identification of compounds)
As a result of identifying the purified compound through analysis of UV absorption, mass spectrum, ¹ H-NMR, ¹³ C-NMR, etc., the structure shown in FIG. 1 (N1 to N9; respectively, compounds of formula 1 to formula 9) It has been determined.

  As structural features of the identified compound, it has 1 or 2 phenyl groups, and 0 or 1 hydroxy group, 0, 1 or 2 methoxy group, 0 or 1 methylene diene on the phenyl group. It has a group such as an oxy group and has 0 or 1 1-propenyl group or 2-propenyl group on one phenyl group, and the phenyl group and the phenyl group are substituted with a substituent (for example, hydroxy, acetyl, methyl Etc.) which may have a cyclic ether or an aliphatic ether group.

(Derivative)
The active ingredient of the present invention includes derivatives of the compounds represented by the above formulas 1 to 9. Such a derivative has an action of promoting or enhancing adiponectin production. For example, while maintaining the above structural characteristics and skeleton, for example, the position of the double bond of the propenyl group is from the 1st position to the 2nd position. Or a derivative converted from the 2-position to the 1-position, a derivative in which a hydroxy other than a phenyl group is converted to methoxy, a derivative in which one methoxy group on the phenyl group having a propenyl group is removed, and the like Can do.

  Furthermore, the derivatives of the present invention include optically active compounds, geometric isomers, or similar compounds of the compounds represented by Formulas 1 to 9.

(Activity measurement)
The active ingredient of the present invention is characterized by having adiponectin secretion / production promoting activity.
Differentiation-inducing activity from preadipocytes to adipocytes can be determined by measuring the accumulation amount of intracellular fat serving as an index of adipocytes according to the method described in Example 2. Briefly, intracellular fat accumulation is determined by staining intracellular lipid droplets using oil Red O (ORO) solution, extracting the ORO dye, and measuring the absorbance (A ₅₁₀ ). be able to.

  The production amount of adiponectin was secreted into the culture supernatant of a mouse preadipocyte cell line (for example, 3T3-L1) cultured in a medium containing the compound of the present invention according to the method described in Example 3. The amount of adiponectin can be determined by measuring with an enzyme immunoassay (ELISA) method. For example, using a “mouse / rat adiponectin ELISA kit” manufactured by Otsuka Pharmaceutical Co., Ltd., a 500-fold dilution of the supernatant can be performed according to the measurement described in the instruction manual attached to the kit. The absorbance at 450 nm is converted into the amount of adiponectin.

(Composition)
Since the adiponectin production promoter of the present invention can promote differentiation of preadipocytes into adipocytes and enhance adiponectin production, diabetes and hyperlipidemia caused by an increase in hypertrophic adipocytes can be achieved. Preventive, ameliorating, or treating effects on lifestyle-related diseases such as symptom, hypertension, arteriosclerosis or obesity.

  Specifically, adiponectin exhibits actions such as suppression of monocyte adhesion to vascular endothelial cells, macrophage lipid accumulation, suppression of foaming, smooth muscle cell proliferation, suppression of migration, etc. By increasing the production amount, it is considered to have an anti-arteriosclerotic effect. Adiponectin has the effect of increasing the insulin sensitivity of adipocytes and promoting the burning of fat. Therefore, it is considered to have an anti-diabetic action and an anti-obesity action by increasing the secretion and production of adiponectin. .

Therefore, the present invention further comprises a composition for preventing, improving or treating a lifestyle-related disease or obesity selected from type 2 diabetes, hyperlipidemia, hypertension and arteriosclerosis, comprising the adiponectin production promoter of the present invention. I will provide a.
According to an embodiment of the present invention, the composition is a pharmaceutical or a food product.

(Pharmaceutical composition)
The pharmaceutical composition of the present invention contains an adiponectin production promoter, and the dose of the active ingredient per dosage unit is an amount effective to promote differentiation into adipocytes. The amount of each of the compounds is about 1 μg to about 2 g, or the amount of the extract is about 10 mg to about 10 g, preferably about 50 mg to about 1 g, but is not limited to this range. Alternatively, the active ingredient amount is about 0.01 μg to about 100 mg per kg body weight, but is not limited to this range.

  The administration route is an oral administration or a parenteral route such as intravenous administration, intramuscular administration, suppository administration, and dermal administration.

  The preparation includes, but is not limited to, forms such as tablets, pills, pellets, powders, granules, solutions, suspensions, emulsions, capsules, ointments and the like. If necessary, it can be formulated into a form such as enteric preparation, delayed release preparation, sustained release preparation and the like. Such formulations can be made using techniques conventional in the pharmaceutical industry. For example, it can be produced using mixing, dissolution, granulation, sugar-coating, grinding, emulsification, encapsulation, confinement, lyophilization and the like.

  The pharmaceutical composition can contain a pharmaceutically acceptable carrier, excipient, additive and the like in addition to the adiponectin production promoter of the present invention.

  For liquid and semi-solid formulations, the carrier can include, for example, saline, petroleum, animal or vegetable oils, such as peanut oil, mineral oil, soybean oil, sesame oil, synthetic oil, and the like. In addition, such preparations can contain saccharides such as dextrose, glycols such as ethylene glycol, propylene glycol, and polyethylene glycol, and glycerol. In particular, in the case of an injection solution, an isotonic solution such as a sodium chloride injection solution, a Ringer injection solution, a dextrose injection solution, a dextrose / sodium chloride injection solution, or a lactic acid-added Ringer injection solution can be included. The composition further includes other additives well known in the art such as stabilizers, preservatives, buffers, antioxidants, surfactants, alcohols, pH adjusters, thickeners, dyes, and perfumes as appropriate. be able to.

  For solid formulations, the carrier can include, for example, lactose, mannitol, dextrose, saccharose, cellulose, corn starch, potato starch, glucose, hydroxypropylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, magnesium aluminate metasilicate, and the like. Such formulations also include lubricants such as silica, talc, stearic acid, magnesium stearate, calcium stearate and polyethylene glycol, binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidine, starch Disintegrating agents such as alginic acid, alginate, sodium starch glycolate, calcium fibrin glycolate, sweeteners, flavoring agents, wetting agents such as lecithin, polysorbate, lauryl sulfate, solubilizing agents such as glutamic acid or aspartic acid, etc. Additives well known in the art can be included. If necessary, the tablets or pills may be coated with a sugar coating such as sucrose, gelatin or hydroxypropylmethylcellulose phthalate or a film of a gastric or enteric substance, or may be coated with two or more layers. In addition, capsules can contain absorbable materials such as gelatin.

  The formulation is specifically described in, for example, Remington: The Science and Practice of Pharmacy (1995), 9th edition, Mack Publishing Company, Pennsylvania, USA, which describes the formulation of the pharmaceutical composition of the present invention. Can be used for.

  The pharmaceutical composition of the present invention can be administered to a subject once or several times a day. For example, it can be administered every 1 to 3 days.

(Food)
The food of the present invention can contain an active ingredient with a content as described in the section of the pharmaceutical composition. The food includes, but is not limited to, solid food, beverage, gel food, semi-solid food, functional food, health food and the like. Alternatively, it may be in the form of a food additive, in which case it can be added to any food. Arbitrary food products include processed foods such as noodles, processed oils and fats such as oils and fats, processed soybeans, processed meats, processed fishery products, processed milk products, dairy products, processed fruits such as jams, confectionery, and cooked products. (Food Processing Technology Handbook (1977) Kenpakusha).

  The types, forms, and production methods of beverages are described in, for example, “Latest Soft Drinks”, the latest Soft Drinks Editorial Committee (editing), and Korin (October 2003). Can be used for manufacturing.

  EXAMPLES The present invention will be further described below with reference to examples. However, the scope of the present invention is not limited by these examples.

220 g of nutmeg (Myristica fragrans) fruit was pulverized with a mixer, immersed in 450 ml of ethanol and left at room temperature for 1 day. Ethanol was recovered, the same amount of ethanol was added and left for 1 day. The ethanol solutions were combined and concentrated with a rotary evaporator to obtain 25.6 g of an oily residue. This oily residue was adsorbed on a silica gel column (Wakogel C-300, diameter 4.5 cm, height 20 cm) packed with hexane, and 100 ml of hexane was passed, and then 10%, 20%, 25%, 33%, 50%, 77 Hexane containing% acetone was eluted sequentially with 300 ml each. The eluate was fractionated by 20 to 50 ml. The fraction solution is developed by silica gel thin-layer chromatography (hexane: acetone = 3: 1 mixture), and the fraction 2.3g which shows dark blue color by spraying FeCl ₃ -K ₃ [Fe (CN) ₆ ] mixture Got. This fraction was purified by high performance liquid chromatography (column CAPCELLPAKC18, diameter 2 cm, length 25 cm, flow rate 3.5 ml / min, detection UV 270 nm, solvent 0.1% formic acid in 60%, 70%, 80% methanol), and compound N1 (127 mg), N2 (11 mg), N3 (7 mg), N4 (52 mg), N5 (35 mg), N6 (74 mg), N7 (6 mg), N8 (13 mg), N9 (11 mg) were purified.

The properties of the purified products N1 to N9 are as follows.
N1: Myristecin colorless oil, UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₁₁ H ₁₂ O ₃ : m / z 192.2145, measured value: m / z 192.2162
N2: colorless oil, [α] D: +7.0 (c = 1.145, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₂₁ H ₂₂ O ₅ : m / z 354.4027, measured value: m / z 354.4031
N3: colorless oil, [α] D: +35.8 (c = 1.16, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₂₀ H ₁₉ O ₄ : m / z 323.2684, measured value: m / z 323.2652
N4: colorless oil, [α] D: +2 (c = 0.53, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated for C ₂₁ H ₂₆ O ₆ : m / z 374.1730, Measured value: m / z 374.1746
N5: colorless oil, [α] D: +3 (c = 0.34, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated for C ₂₃ H ₂₈ O ₇ : m / z 416.4711, Measured value: m / z 416.1798
N6: colorless oil, [α] D: +2 (c = 0.80, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₂₀ H ₂₂ O ₄ : m / z 326.3922, measured value: m / z 326.1484
N7: colorless oil, [α] D: +1 (c = 1.77, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated for C ₂₁ H ₂₆ O ₅ : m / z 358.1779, measured value: m / z 358.1779
N8: White powder, [α] D: +25 (c = 0.88, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₂₀ H ₂₄ O ₅ : m / z 344.3806, Measured value: m / z 344.1626 ([M] ⁺ )
N9: white powder, [α] D: 0 (c = 0.84, MeOH), UV (MeOH): λmax = 232
HR-EI-MS: Calculated value of C ₂₀ H ₂₄ O ₅ : m / z 344.3806, measured value: m / z 344.11631

The measurement results of ¹ H-NMR and ¹³ C-NMR of these compounds are shown in Tables 1 and 2.
The above results were analyzed, and the literature `` ACHerrera et al., Phytochemistry, 23, 9, 2025-2028 (1984), M. Hattori et al., Chem. Pharm. Bull., 35, 2, 668-674 (1987), N Nakatani et al., Phytochemistry, 27, 10, 3127-3129 (1988), HMTB Herer et al., Phytochemistry, 44, 4, 699-703 (1997), F. Filleur et al., Natural Product Letters, 16, 1, 1-7 ( 2001), H. Shimomura et al., Phytochemisrty, 6, 5, 1513-1515 (1987), XADomiguez et al., Phytochemistry, 29, 8, 2651-2653 (1990), S. Hada et al., Phytochemistry, 27, 2, 563- As a result of comparing the spectrum with the physicochemical properties described in “568 (1988)”, the compounds N1 to N9 were identified as the structures shown in FIG.

Mouse preadipocyte cell line 3T3-L1 (purchased from Dainippon Pharmaceutical Co., Ltd.) pre-cultured in Dulbecco's modified Eagle medium (DMEM medium) containing 10% fetal calf serum for 3 days in the presence of 37 ° C and 5% CO ₂ Thereafter, the above 3T3 cells were detached with an EDTA-trypsin solution, collected, and centrifuged at 1000 rpm for 4 minutes. Cells were dispensed to a 24-well multiplate at 4 × 10 ⁴ cells / ml, pre-cultured in the presence of 5% CO _{2 at} 37 ° C. for 3 days, the medium was removed, and 1 ml of medium was newly added. Next, samples N1 to N9 were added to the medium to a final concentration of 30 μM, and precultured for 14 days in the presence of 37 ° C. and 5% CO ₂ .

  In order to examine the accumulation amount of intracellular fat in mouse preadipocytes, the medium of the above 24-well multiplate was aspirated off with an aspirator, and Oil Red O (ORO) solution (Ramirez-Zacarias et al., Histochemistry, 97, 493-497 (1992)) and intracellular lipid droplets were stained. After leaving at room temperature for 1 hour, the dye solution was removed with an aspirator, washed with 50% isopropanol aqueous solution, the solution was removed with an aspirator, 100% isopropanol was added at 0.2 ml / well, and the lipid was taken up into intracellular lipid droplets The ORO dye was extracted and the absorbance was measured using a plate reader (wavelength: 510 nm).

  The result is shown in FIG. As shown in FIG. 2, even in the culture without adding insulin, it is clear that the accumulation of intracellular fat, which is an index of adipogenesis, is promoted by the N1-N9 compound from the control.

  The amount of adiponectin secreted into the culture supernatant of 3T3-L1 cells cultured as described above was measured by enzyme immunoassay (ELISA). This quantification was performed using a “mouse / rat adiponectin ELISA kit” manufactured by Otsuka Pharmaceutical Co., Ltd. according to the measurement described in the instruction manual attached to the kit for a 500-fold dilution of the supernatant. A graph obtained by converting the absorbance at 450 nm measured above into the amount of adiponectin is shown in FIG.

  As shown in FIG. 3, even in the culture without addition of insulin, compounds N1 to N9 induced differentiation of mouse preadipocytes into adipocytes, and the amount of adiponectin production was higher than that of the control.

Mouse preadipocyte cell line 3T3-L1 (purchased from Dainippon Pharmaceutical Co., Ltd.) pre-cultured in Dulbecco's modified Eagle medium (DMEM medium) containing 10% fetal calf serum for 3 days in the presence of 37 ° C and 5% CO ₂ Thereafter, the above 3T3 cells were detached with an EDTA-trypsin solution, collected, and centrifuged at 1000 rpm for 4 minutes. Distribute cells to a 24-well multiplate at 4 × 10 ⁴ cells / ml, pre-incubate for 3 days in the presence of 37 ° C and 5% CO ₂ , remove the medium, and add 1 ml of the medium supplemented with insulin. (Insulin final concentration 10 μg / ml medium) was added. Next, samples N1 to N9 were added to the medium to a final concentration of 30 μM, and precultured for 14 days in the presence of 37 ° C. and 5% CO ₂ . The amount of adiponectin secreted into the culture supernatant was measured by enzyme immunoassay (ELISA). This quantification was performed using a “mouse / rat adiponectin ELISA kit” manufactured by Otsuka Pharmaceutical Co., Ltd. according to the measurement described in the instruction manual attached to the kit for a 500-fold dilution of the supernatant. FIG. 4 shows a graph obtained by converting the absorbance at 450 nm measured above into the amount of adiponectin.

  From FIG. 4, the compounds N3, N7 and N9 had adiponectin secretion promoting ability superior to indomethacin which has been conventionally known to promote adiponectin secretion.

Normal human preadipocytes (manufactured by Sanko Junyaku) are cultured in PBM medium (PBM growth medium, PBM is Sanko Junyaku) supplemented with 10% FBS and 2 mM glutamine for 4 days, and then the cells are recovered with EDTA-trypsin solution. Then, the cells were suspended in a PBM growth medium at a rate of 4 × 10 ⁴ cells / ml, and 0.2 ml was planted in a 96-well plate previously coated with collagen. After culturing at 37 ° C. for 3 days in the presence of 5% CO ₂ , the medium was replaced with PBM differentiation medium [PBM growth medium supplemented with insulin (10 μg / ml) and dexamethasone (0.1 μM)] (0.2 ml / ml). well). Samples N1 to N9 were added to this culture solution to a final concentration of 15 μM. Only 2 μl of ethanol was added to the control. After pre-culturing for 14 days, the amount of adiponectin secreted into the culture supernatant was measured by enzyme immunoassay (ELISA). This quantification was performed using a “human adiponectin ELISA kit” manufactured by Otsuka Pharmaceutical Co., Ltd. according to the measurement described in the instruction manual attached to the 6-fold diluted supernatant. FIG. 5 shows a graph in which the absorbance at 450 nm measured above is converted into the amount of adiponectin.

  As shown in FIG. 5, compounds N1 to N9 induced differentiation of human preadipocytes into adipocytes, and the adiponectin production amount was higher than that of the control.

  The adiponectin production promoter of the present invention has the activity of increasing the amount of adiponectin and increasing the amount of adiponectin by promoting differentiation induction from animal preadipocytes to adipocytes. Because of the effect of significantly increasing the amount of adiponectin, the adiponectin production promoter of the present invention is particularly effective for preventing, ameliorating or treating lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, arteriosclerosis or obesity. And has high industrial utility value.

The compound of Formula 1-Formula 9 of this invention is shown. It is the graph which evaluated the accumulation | storage amount of the intracellular fat used as a parameter | index of fat cell formation when the mouse | mouth preadipocyte was induced differentiation to the fat cell about the compound of N1-N9 in the absence of insulin. Example 2). It is the graph which evaluated the amount of adiponectin production when a mouse | mouth preadipocyte was induced to differentiate into a fat cell about the compound of compound N1-N9 in the absence of insulin (refer Example 3). It is the graph which evaluated the amount of adiponectin production when a mouse | mouth preadipocyte was differentiation-induced to a fat cell about the compound of compound N1-N9 in presence of insulin (refer Example 4). In the figure, con is EtOH (ethanol) as a negative control, and Indo is indomethacin as a positive control. It is the graph which evaluated the production amount of adiponectin when a human preadipocyte was induced to differentiate into a fat cell about the compound of compound N1-N9 (refer Example 5).

Claims (3)

The following formulas 1 to 9 as active ingredients :

The adiponectin production promoter which consists of 1 type or 2 or more types of the isolated compound represented by these. Enable adiponectin production enhancer according to claim 1 as a component, which comprises mixing with at least a pharmaceutically acceptable carrier, type 2 diabetes, hyperlipidemia, lifestyle diseases selected from hypertension and arteriosclerosis Or the manufacturing method of the pharmaceutical composition for prevention, improvement, or treatment of obesity. A food additive comprising the adiponectin production promoter according to claim 1.

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