JP2005060308A - Agent for enhancing and promoting production of adiponectin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe medicament having activities for promoting and enhancing the production of adiponectin in the body, and hardly providing side effects. <P>SOLUTION: The medicament for promoting or enhancing the production of the adiponectin in the body is obtained by using an extract extracted from a rhizome of turmeric with an organic solvent, or curcumin contained therein, or by using theophylline, caffeine or theobromine in combination therewith. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an adiponectin production enhancing / promoting agent in animal adipocytes, comprising an extract of a ginger family Curcuma plant as an active ingredient, and a method for further activating the agent.

  Recently, a protein factor called adiponectin has been identified as one of the cytokines that control the action of the living body by mediating cell-cell interactions that are released from cells, and the deficiency is related to lifestyle-related diseases such as type 2 diabetes and arteriosclerosis It became clear that it was closely related to the onset of the disease. And by increasing the adiponectin concentration in blood by direct injection and introduction / expression of adenovirus vector incorporating the gene, blood vessel lesions that reduce blood glucose levels in diabetic model mice or lead to arteriosclerosis It has been experimentally proven that the formation of can be suppressed (see Non-Patent Documents 1 to 3). In actual application, adiponectin is used as an agent for improving obesity (see Patent Document 1), an anti-inflammatory agent, a monocyte cell growth inhibitor (see Patent Document 2), a liver fibrosis inhibitor (see Patent Document 3), and the like. A method to use has been developed. In this way, administration of adiponectin produced in vitro is also useful, but it is very important for the prevention and treatment of lifestyle-related diseases over the long term to improve or further enhance its poor production in the body it is conceivable that.

  Adiponectin is mainly produced by adipocytes in adipose tissue. Adipocytes are differentiated from mesoderm-derived preadipocytes under the influence of various hormones. Induction of experimental differentiation was performed in the presence of insulin and carbohydrate glucocorticoid in the presence of cAMP-degrading enzyme inhibitor isobutylmethylxanthine (IBMX) (non-patent document 4), non-sterol anti-inflammatory agent indomethacin (non-patent document) Reference 5), thiazolidinediones (TZD) (see Patent Document 4 and Non-Patent Document 6), which are therapeutic agents for type 2 diabetes, and vasodilator prostacyclin (PGI2) (see Non-Patent Document 7) However, the production of adiponectin by actually differentiated cells was confirmed in the study of Loeffler et al. Using IBMX and the study of Maeda et al. Using TZD. In addition, Maeda et al. Confirmed that blood adiponectin levels were significantly increased in diabetic model mice in which TZD was orally ingested compared to those in which TZD was not orally consumed, and glucose tolerance decreased even in humans. Have increased blood adiponectin levels after taking TZD.

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) A. K. G. Loeffler, Horm. Metab. Res. , 32, 548-554 (2000) H. Ye et al. Biochem. J., 330, 803-809 (1998) N. Maeda et al. , Diabetes, 50, 2094-2099 (2001) R. Negrel et al. Biochem. J., 257, 399-405 (1989) US Application Publication No. 2002/0132773 JP 2000-256208 A JP 2002-363094 A US Pat. No. 6,153,432

  As described above, pharmaceuticals that increase the level of adiponectin in the body are already known, but all have problems in terms of safety and side effects. In other words, IBMX is not approved as a pharmaceutical product, and TZD is highly hepatotoxic, so it must be taken under the strict control of doctors, and indomethacin, prostacyclin, etc. also have other pharmacological activities. Therefore, it is not easily used for prevention of lifestyle-related diseases and treatment at a light stage. Therefore, as diabetes and arteriosclerosis are rapidly increasing and countermeasures have become a social problem, it is safe to promote and enhance the production of adiponectin in the body as a drug that can be easily used to prevent and treat these lifestyle-related diseases. Development of new materials is required.

Under such circumstances, as a result of intensive studies, the present inventor has discovered the target biological activity in the organic solvent extract of turmeric rhizome and has completed the present invention.
That is, the present invention relates to the following (1) to (9).
(1) An adiponectin production enhancing / promoting agent in animal adipocytes, comprising an extract obtained by extracting a rhizome of a ginger family Curcuma plant with an organic solvent as an active ingredient.
(2) Enhancement and promotion of adiponectin production in animal adipocytes, characterized by containing as an active ingredient an extract obtained by extracting a residue obtained by steam distillation of the rhizomes of Turmeric (Curcuma) plants with an organic solvent Agent.
(3) The organic solvent extract according to (1) or (2) is further adsorbed on activated carbon, and then contains an elution component obtained by elution with a hydrophobic organic solvent including a hydrophilic organic solvent as an active ingredient. An adiponectin production enhancing or promoting agent in animal adipocytes, characterized by
(4) An elution component obtained by adsorbing the organic solvent extract according to (1) or (2) on silica gel and then elution with a hydrophobic organic solvent containing a hydrophilic organic solvent is contained as an active ingredient. An adiponectin production enhancing or promoting agent in animal adipocytes.
(5) The drug according to (3) or (4), wherein the hydrophilic organic solvent is acetone and the hydrophobic organic solvent is hexane.
(6) An adiponectin production enhancing / promoting agent in animal adipocytes, comprising curcumin as an active ingredient.
(7) The drug according to any one of (1) to (5) above, further comprising any one or more of theophylline, caffeine, or theobromine.
(8) An agent for enhancing and promoting adiponectin production in animal adipocytes, comprising curcumin and at least one of theophylline, caffeine, or theobromine.
(9) A method of further activating the adiponectin production promotion and enhancing ability in the drug according to any one of (1) to (6) above, and any one of these drugs and theophylline, caffeine, or theobromine The above method characterized by using two or more in combination

Curcumin, which is a turmeric extract of the present invention or a component thereof, enhances or promotes the production of adiponectin in animal adipocytes.
On the other hand, the above-mentioned substances such as TZD, which are known as adiponectin production enhancing substances, have problems of safety and side effects, whereas turmeric has traditionally been used as a raw material for curry powder or as a health promoter. It can be said that the drug of the present invention is extremely safe.
Furthermore, as described above, adiponectin has the effect of preventing and treating type 2 diabetes, a so-called lifestyle-related disease, in which blood glucose levels do not decrease despite the production of insulin. It has many important physiological actions such as suppression of connected vascular lesions, improvement of obesity, anti-inflammation, suppression of monocyte cell proliferation, suppression of liver fibrosis, etc. The drug provided by the present invention is safe and extremely It is a useful drug.

Turmeric rhizome is widely used edible worldwide as a raw material for curry powder, and since it has long been used as a medicinal plant in China and Japan, the safety of turmeric extract is high.
In the present invention, the extract of curcuma longa rhizome or its hydrodistilled residue extracted with an organic solvent such as ethyl alcohol is used alone or in the presence of theophylline or caffeine, and is a precursor fat of humans and mice. It promotes the differentiation of cells into adipocytes, and accordingly promotes and enhances the production of adiponectin in these animal adipocytes.
Such an adiponectin production enhancing action of the turmeric extract is concentrated by fractionating the crude extract by activated carbon treatment or silica gel column chromatography treatment. In addition, components that inhibit or suppress the activity are also removed by such treatment.

The adiponectin production enhancing action of the turmeric extract in the present invention is further increased by using a xanthine derivative usually contained in tea or coffee, such as theophylline, caffeine, theobromine, etc., which is an important point of the present invention. One of them.
These xanthine derivatives exhibit various physiological actions such as smooth muscle relaxation, central nervous excitement, and diuresis, but are biochemically known to inhibit phosphodiesterase that degrades second messenger cAMP. There are many C / EBP binding motifs in the promoter part of the adiponectin gene (Das et al., Biochem. Biophys. Res. Commun., 280, 1120-1129 (2001); Schaeffler et al., Biochem. Biochim. Acta). , 1399, 187-197 (1998); Saito et al., Gene, 229, 67-73 (1999)), suppressing the degradation of cAMP, which contributes to increased expression and activation of C / EBP. Increasing the level is expected to promote transcription of the adiponectin gene. Based on this idea, among the known cAMP phosphodiesterase inhibitors, theophylline was taken up as a compound contained in normal food, and as a result, the combination of these xanthine derivatives and turmeric extract enhanced adiponectin production in cultured cells. It was found to show a marked effect.

  On the other hand, when this extract was fractionated by various column chromatography, fat accumulation promoting activity on mouse 3T3-Ll cells appeared in multiple fractions, one of which was curcumin (diferloylmethane). It was included. Curcumin is a pigment that forms the yellow color of turmeric and curry powder, but has a strong antioxidant effect (Japanese Patent Laid-Open No. 2003-064360) and also has anti-diabetic, anti-depressive, anti-tumor and anti-inflammatory effects. It is revealed that it is a physiologically active substance (JP 2003-128539, JP 2003-113117, JP 2003-055202, Chan et al., Biochem. Pharmacol., 55, 1955-1962 (1998)) Has been. Therefore, as a result of examining the adiponectin production promoting action in cultured cells using pure curcumin, we found that adiponectin production is increased by the combined use of curcumin, theophylline and caffeine, and it is one of the means to solve the problem It revealed that. However, the adiponectin production enhancing activity contained in the turmeric extract is not only due to curcumin, but is also likely to involve other unknown components.

  As the turmeric rhizome used as the raw material of the present invention, rhizomes such as spring turmeric (Curcuma aromatica) and purple turmeric (Curcuma zedoaria) are used in addition to curcuma longa called autumn turmeric. . These are cultivated as commercial crops in subtropical areas, Okinawa Prefecture, and the like, but the production area is not particularly limited when used in the present invention.

  In addition, the harvest time of these rhizomes is the time that has been conventionally harvested for the purposes of food and medicine. The harvested fresh rhizome can be extracted with ethanol etc. as it is, but from the point of extraction efficiency, it is thinly sliced and then dried in shade, etc., and further shredded and crushed Is preferred. Moreover, the residue which extract | collected essential oil from the turmeric powder by steam distillation etc. can also be used for a raw material. Examples of the solvent used for extraction include ethanol, methanol, acetone, ethyl acetate, and the like. These are added in an amount of 3 ml to 30 ml per gram of dried turmeric rhizome slices / powder, and the mixture is extracted by stirring or standing still tightly. A known extraction device such as a percolator may be used. The extraction time varies depending on the type of solvent, the form of the raw material, the raw material / solvent ratio, the presence / absence of stirring, the temperature, etc., and appropriate conditions are selected depending on the case. For example, when the dried slice is deposited in 5 times the amount of ethanol and allowed to stand, the target activity is sufficiently extracted in about 2 weeks. If the dry powder is stirred with 10 times the amount of ethanol at room temperature, it will be extracted overnight.

  The extract thus obtained is concentrated by a rotary evaporator or the like, and the solvent is removed under reduced pressure. This residual solution is used as it is as a substance for promoting and enhancing adiponectin production, but more preferably after removing unnecessary components and activity-inhibiting components by known means such as activated carbon treatment or silica gel column chromatography. For example, the concentrated extract of the crude extract is re-extracted with a mixed solvent of ethyl acetate and hexane and adsorbed on activated carbon. Non-adsorbed components are washed away with hexane, and adsorbed components are eluted and recovered with hexane containing 5% to 50% acetone, which shows stronger activity than the crude extract. Further, even if the dried rhizome powder is extracted with hexane in advance and then extracted with hexane containing 5% to 100% acetone or ethanol, an adiponectin production-enhancing substance with an increased titer can be obtained by removing the inhibitory components. Furthermore, the titer can be increased by adsorbing the crude extract onto silica gel, washing with hexane or hexane containing several percent of acetone, and then eluting the adsorbed component with hexane containing 5% to 70% of acetone. However, the purification method described above is an exemplification, and the present invention is not limited to this.

  These extracts and eluates are used to enhance the production of adiponectin after the solvent is removed under reduced pressure. The method includes dissolving in ethanol and adding it to drinking water, including surfactants such as appropriate phospholipids. In addition to suspending in water and adding to drinking water or food, there may be mentioned a method of mixing with an appropriate adjuvant and using it in the form of a tablet. When curcumin, which is one of the active ingredients, is used instead of these turmeric extracts, curcumin can be prepared from turmeric extracts by a known method such as chromatography, and chemical synthesis can also be performed by a known method. Easy to do.

On the other hand, the xanthine derivative used to further enhance the production of adiponectin in combination with these turmeric extracts is any one or a mixture of two or more of theophylline, caffeine, or theobromine. These can be extracted from tea leaves, coffee beans, cacao beans, etc., and can be easily synthesized by known methods. When used in the present invention, these may be mixed with the turmeric extract, or added alone to drinking water, etc., and then used by drinking after drinking water containing turmeric extract. Also good. The amount of turmeric extract used in the present invention is adjusted depending on the purification method and degree. When using curcumin, which is one of its active ingredients, instead of turmeric extract, the amount of addition should be 1 mg to 10 mg or less per 1 ml of drinking water or 1 g of food. The amount of xanthine derivative added is in accordance with the concentration contained in commonly used coffee and green tea. That is, caffeine is 0.1 mg to 0.5 mg (about 3 mM) per 1 ml of drinking water or 1 g of food, and theophylline is 0.01 mg to 0 per 1 ml of drinking water or 1 g of food. .05 mg (about 0.3 mM), and theobromine is 0.1 mg to 2 mg (about 10 mM) per 1 ml of drinking water or 1 g of food. Accordingly, the dosage may be increased or decreased as appropriate within a physiologically safe range.
Next, the present invention will be described in more detail based on examples.

  31 g of dried turmeric rhizome slices were immersed in 160 ml of ethanol and allowed to stand in the dark for 1 month. The ethanol solution was collected by decantation, and the rhizome was washed with a small amount of ethanol. The above solution and washing solution were combined, concentrated with a rotary evaporator, and then dried under reduced pressure with an oil pump to obtain 1.42 g (Sample 1-1) of a crude extract as a liquid.

  1.4 g of the crude extract obtained in Example 1 was dissolved in 2 ml of ethyl acetate, and 100 ml of hexane was added. The precipitate was filtered off, and 1 g of activated carbon and 2 g of celite were added to the filtrate and mixed well. Using a fine glass filter, the solids containing activated carbon and celite were collected by filtration and extracted successively with 50 ml of hexane containing 2%, 20% and 50% acetone. These filtrates and extracts were each concentrated with a rotary evaporator and then dried under reduced pressure with an oil pump. The oil was 0.896 g (active carbon non-adsorbed, Sample2-1), 60 mg (2% acetone-eluted, Sample 2). -2), 119 mg (20% acetone elution, Sample 2-3), and 64 mg (50% acetone elution, Sample 2-4) were obtained, respectively.

  10 g of dried turmeric rhizome powder was mixed with 50 ml of hexane and stirred at room temperature for 12 hours with a magnetic stirrer. Turmeric powder was collected by filtration and extracted sequentially with 50 ml of hexane containing 5%, 10%, 30% and 50% acetone. These filtrates and extracts were each concentrated with a rotary evaporator and then dried under reduced pressure with an oil pump, and 0.492 g of oily matter (elution with hexane only, Sample3-1), 56 mg (elution with 5% acetone, Sample 3-2), 32 mg (10% acetone elution, Sample 3-3), 49 mg (30% acetone elution, Sample 3-4), and 48 mg (50% acetone elution, Sample 3-5) ) Respectively.

  20 g of dried powder of spring turmeric rhizome was mixed with 100 ml of ethanol and stirred at room temperature for 36 hours with a magnetic stirrer. Turmeric powder was filtered off and the ethanol solution was concentrated with a rotary evaporator and then dried under reduced pressure with an oil pump to obtain 1.09 g (Sample 4-1) of a crude extract. 1.0 g of this was dissolved in 2 ml of ethyl acetate, and 100 ml of hexane was added. The precipitate was filtered off, and 0.75 g of activated carbon and 2 g of celite were added to the filtrate and mixed well. Using a fine glass filter, the solids containing activated carbon and celite were collected by filtration and extracted sequentially with 50 ml of hexane containing 5%, 20%, and 50% acetone. These filtrates and extracts were each concentrated with a rotary evaporator, and then dried under reduced pressure with an oil pump, and 259 mg of oil (active carbon non-adsorbed, Sample 4-2), 75 mg (5% acetone elution, Sample 4-3), 96 mg (20% acetone elution, Sample 4-4) and 107 mg (50% acetone elution, Sample 4-5) were obtained, respectively.

  20 g of dry powder of purple turmeric rhizome was mixed with 100 ml of ethanol and stirred with a magnetic stirrer at room temperature for 36 hours. Turmeric powder was filtered off and the ethanol solution was concentrated with a rotary evaporator and then dried under reduced pressure with an oil pump to obtain 0.66 g of a crude extract (Sample 5-1). 0.65 g of this was treated in the same manner as in Example 4, and the oily substance 152 mg (active carbon non-adsorbed, Sample 5-2), 22 mg (5% acetone elution, Sample 5-3), 45 mg (20% Acetone elution, Sample 5-4), and 50 mg (50% acetone elution, Sample 5-5) were obtained, respectively.

  10 g of a residue obtained by steam distillation of dried turmeric rhizome powder was mixed with 50 ml of ethanol, and stirred at room temperature with a magnetic stirrer for 6 hours. The residue was filtered off and the ethanol solution was concentrated with a rotary evaporator and then dried under reduced pressure with an oil pump to obtain 0.92 g of a crude extract (Sample 6-1). 0.9 g of this was treated in the same manner as in Example 4, and 767 mg of oil (active carbon non-adsorbed, Sample 6-2), 92 mg (2% acetone elution, Sample 6-3), 80 mg (20 % Acetone elution, Sample 6-4), 28 mg (50% acetone elution, Sample 6-5), 45 mg (100% acetone elution, Sample 6-6), and 25 mg (100% ethanol elution) Sample 6-7) were obtained.

  10 g of a residue obtained by steam-distilling the dried turmeric rhizome powder was mixed with 100 ml of hexane, and the mixture was stirred at room temperature with a magnetic stirrer for 6 hours. The residue was collected by filtration and extracted sequentially with 100 ml of hexane containing 5%, 10%, 30%, and 50% acetone. These filtrates and extracts were each concentrated with a rotary evaporator and dried under reduced pressure with an oil pump. 1.0 g of oily matter (elution with hexane only, Sample 7-1), 115 mg (elution with 5% acetone, Sample 7-2), 35 mg (10% acetone elution, Sample 7-3), 70 mg (30% acetone elution, Sample 7-4), 47 mg (50% acetone elution, Sample 7-5), And 24 mg (100% acetone elution, Sample 7-6) were obtained.

  57 mg of the 30% acetone elution (Sample 7-4) obtained in Example 7 was adsorbed on a silica gel column (Wakogel C300, 2 x15 cm) and eluted with 50 ml of hexane containing 10% to 100% acetone. did. The eluate was collected in 1/3 flasks and concentrated to dryness on a rotary evaporator. Results of a thin-layer chromatography study of yellow compounds, which are the main components of 50% acetone eluate with strong fat accumulation activity, in a bioassay using mouse 3T3-Ll preadipocytes (described later) (silica gel, acetone: hexane) ＝ Developed with a mixed solvent of 2: 3, Rf 0.55), Rf value, yellow fluorescence under UV lamp irradiation of 365 nm wavelength, orange fluorescence when heated after spraying ethanol solution containing 10% concentrated sulfuric acid, etc. In this respect, it was identified as curcumin by matching with the spot of a pure product of curcumin [manufactured by BIOMOL Research Inc. (Plymouth Meeting, PA, USA)].

A mouse 3T3-L1 preadipocyte cell line (purchased from Dainippon Pharmaceutical Co., Ltd.) was pre-cultured in DMEM medium containing 10% fetal calf serum for 3 days, and then collected with EDTA-trypsin solution, 8.3 x 10 ⁴ cells / ml As a suspension, 0.2 ml each was seeded on a 96-well plate. After culturing at 37 ° C. for 2 days in the presence of 5% CO ₂ , the turmeric extract obtained in Examples 6 and 7 was made into a 50 mg / ml ethanol solution, added to each well at 2 μl / well, and further for 12 days. The culture was continued. The medium was removed by suction with an aspirator, and 0.1 ml of PBS containing 1% formaldehyde was added to each well to fix the cells. After fixing at room temperature for 30 minutes, followed by washing with water and then with 50% 2-propanol (0.1 ml / well), Oil Red O (ORO) solution (Ramirez-Zacarias et al., Histochemistry, 97, 493-497 (1992) )) And stained intracellular lipid droplets. After standing at room temperature for 1 hour, the dye solution was removed with an aspirator, washed with 50% 2-propanol-containing water, then with water, and air-dried for 1 hour. 100% 2-propanol was added at 0.1 ml / well, the ORO dye incorporated into the intracellular lipid droplets was extracted, and the absorbance was measured using a plate reader (wavelength: 510 nm). The results are shown in FIG. 1, and it can be seen that accumulation of intracellular fat, which is an index of adipocyte differentiation, is promoted by the turmeric extract and the crude product.

Mouse 3T3-Ll preadipocytes precultured in the same manner as in Example 9 was made into a suspension of 7 × 10 ⁴ cells / ml, and 2 ml per well was added to a 12-well multiwell plate previously coated with collagen. Implanted. After culturing for 3 days, the medium (DMEM medium containing 10% fetal calf serum) was replaced with a fresh medium (2 ml / well), and the turmeric extract and crude product obtained in Examples 1 to 7 were used. A representative sample ethanol solution (2-5 μl) selected based on the results of Example 9 was added. Addition amounts are as follows: Turmeric extract of Example 1 (Sample l-1), 200 μg; 2% acetone eluate of Example 2 (Sample 2-2), 120 μg; 10 of Example 3 % Acetone elution (Sample 3-3), 60 μg; spring turmeric extract of Example 4 (Sample 4-1), 44 μg; 5% acetone elution of Example 4 (Sample 4-3), 35 μg; Example 5 Purple turmeric 5% acetone elution (Sample 5-3), 44 μg; Activated turmeric 20% acetone elution (Sample 6-4) of turmeric rhizome steam distillation residue extract of Example 6, 150 μg; Example 7 turmeric rhizome steam distillation residue 30% acetone elution (Sample 7-4), 180 μg. As a control, 2 to 5 μ1 of pure ethanol was added. Cultivate for 7 to 10 days, discard the medium when the intracellular fat globule has been sufficiently observed by phase-contrast microscopy, and wash twice with PBS (1 ml / well). The sample was removed with a cell scraper together with ml of PBS, and collected by centrifugation (10000 rpm, 6 minutes). The supernatant was discarded, and the cells in each tube were pipetted with 100 μl of 1ysis buffer containing 1% Tween 20, and then disrupted and extracted by vortexing. This was centrifuged (12000 rpm, 20 minutes), the supernatant was recovered, and the protein concentration was measured. The supernatant was diluted with lysis buffer so that the protein concentration was 1 mg / ml, 50 μl thereof was mixed well with 50 μl of 2 × SDS buffer, and heated at 95 ° C. for 5 minutes for SDS treatment. 5 μl of each sample was subjected to electrophoresis using polyacrylamide gel, and mouse adiponectin was detected by Western blotting in a conventional manner. The primary antibodies used were mouse anti-adiponectin and mouse monoclonal antibody (Catalog No. MAB3608) manufactured by Chemicon, and the secondary antibody was rabbit anti-mouse Ig G1-HRP conjugate (Catalog No. 61- manufactured by Zymed). 0120). For detection of the band, an ECL measurement kit manufactured by Pharmacia was used. For comparison, β-actin was also detected by Western blotting. The primary antibody used was anti-β-actin (C-11, sc-1615) manufactured by Santa Cruz, and the secondary antibody was Zymed. Rabbit anti-goat Ig G (H + L) -HR conjugate (Catalog No. 81-1620). The results are shown in FIG. 2, FIG. 3, and FIG. FIG. 2 shows that various rhizome plant rhizome extracts and crudely purified products thereof have an action of enhancing adiponectin production of adipocytes differentiated from mouse 3T3-Ll preadipocytes. In addition, the production enhancement effect is concentration-dependent from FIG. 3, and in the case of the crude product obtained in Example 6 (Sample 6-4, ie, fraction eluted from activated carbon with a 20% acetone / hexane mixed solvent). It can be seen that it is appropriate to add at a concentration of around 75 μg / ml.
Further, FIG. 4 shows that the production enhancing action is time-dependent, and in the case of Sample 6-4, the production of adiponectin reaches its maximum around 11 days after the addition of the sample and the start of culture.

Enhancement of adiponectin gene expression was confirmed by RT-PCR. Mouse 3T3-Ll preadipocytes precultured in the same manner as in Example 9 was made into a suspension of 4 × 10 ⁴ cells / ml, and 2 ml per well was added to a 12-Well multiwell plate previously coated with collagen. Implanted. After culturing for 3 days, the medium (DMEM medium containing 10% fetal calf serum) was replaced with a fresh medium (2 ml / well), and the turmeric rhizome steam distillation residue extract obtained in Example 6 was treated with activated carbon 20% An ethanol solution (4 μl; containing extracts 106, 159, and 212 μg) for acetone elution was added. As a control, 4 μl of pure ethanol was added. After culturing for 12 days and confirming the development of intracellular fat globules in the sample-added well by observation with a phase contrast microscope, the medium is discarded, washed once with PBS (1 ml / well), and 0.3 ml of cells in each well is removed. RNA was extracted by mixing with Isogen (manufactured by Wako Pure Chemical Industries). DNA and protein were removed from the manufacturer according to the attached instructions, total RNA was precipitated with isopropyl alcohol, washed with ethanol, air-dried, dissolved in 10 μl of water, and the RNA amount was determined from the absorbance at 260/280 nm. This RNA was converted to cDNA using the Promega reverse transcriptase system, and adiponectin gene expression was confirmed by PCR using this template. The primers used were as follows: mouse adiponectin, 5′-one AAGGACAAGGCCGTTCTCT-3 ′ and 5′-TATGGGTAGTTGCAGTCAGTTGG-3 ′; β-actin for control, 5′-GACGAGGCCCAGAGCAAGAGA-3 ′ and 5′-TAGATGGGCACAGTGTGGGTGA-3 '. PCR was performed according to the attached manual using Ampli Taq DNA Polymerase manufactured by Applied Systems. Denaturation was performed at 94 ° C. for 1 minute, annealing was performed at 64 ° C. for 1 minute, and polymerization was performed at 72 ° C. for 1 minute, which was repeated 30 times in total. The electrophoresis result of the PCR product is shown in FIG. Bands of the expected size (219 bp for adiponectin and 332 bp for actin) are observed, and the intensity of the band (adiponectin gene expression level) is clearly higher than that of the control in cells cultured with the sample added.
This result was then confirmed by quantitative PCR. Using the Corvette Rotor-Gene 2000 Real-Time Cycler, the Qiagen QuantiTect SYBR Green PCR system was used to quantify the relative expression level of the adiponectin gene relative to β-actin. The primers are the same as described above. The results are shown in FIG. It can be seen that the adiponectin gene expression is enhanced 7 to 40 times by adding the sample.

The mouse 3T3-Ll preadipocyte cell line was pre-cultured in DMEM medium containing 10% fetal bovine serum for 3 days, and then 0.2 ml of each suspension containing 1.3 × 10 ⁴ cells was added to 96-well. Implanted in a plate. After culturing for 3 days, the medium was changed to 0.2 ml each of the above fresh medium containing 0, 20, 100, 400 μM caffeine, 100, 400 μM theobromine, or 100, 400 μM theophylline, and then obtained in Example 2. Turmeric extracted crude product (50 mg / ml ethanol solution) was added to each well at 2 μ1 / well, and the culture was further continued for 12 days. Aspirate the medium with an aspirator, fix the cells with 0.1 ml of PBS containing 1% formaldehyde, stain intracellular lipid droplets with Oil Red O (ORO) solution, and stain cell nuclei with Giemsa reagent. . A micrograph of the stained cells is shown in FIG. It can be seen that the fat accumulation promoting action as an index of adipocyte differentiation is further enhanced by adding 100 μM or more of caffeine, theobromine, or theophylline in addition to the turmeric extract.

Mouse 3T3-Ll preadipocytes pre-cultured using DMEM medium containing 10% fetal calf serum in the same manner as in Example 9 was made into a suspension of 7 × 10 ⁴ cells / ml, and collagen coating was applied in advance. 2 ml per well was seeded in 12-Well multiwell plates. After culturing for 3 days, the medium was replaced with fresh medium with or without 400 μM theophylline (2 ml / well), then ethanol solution with 10, 20, or 30 nmol of curcumin or obtained in Example 4 4 μl of an ethanol solution containing the crude extract at 11 mg / ml was added. For control, 4 μl of pure ethanol was added. After culturing for 10 days, cells were collected in the same manner as in Example 10, and the protein extracted with 1ysis buffer was subjected to SDS treatment and electrophoresis through polyacrylamide gel, and adiponectin was detected by Western blotting. The results are shown in FIG. 8, and it can be seen that theophylline further enhances the enhancement of adiponectin production by turmeric extract and curcumin.

Normal human preadipocytes (manufactured by Sanko Junyaku) are pre-cultured for 4 days in PBM medium (PBM growth medium, PBM is Sanko Junyaku) supplemented with 10% FBS and 2 mM glutamine. And suspended in PBM growth medium at a rate of 4 × 10 ⁴ cells / ml, and each 2 ml was implanted in a 12-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)] (2 ml / well). To this culture, 20 mM theophylline aqueous solution was added to a final concentration of 500 μM, and then the crude turmeric extract obtained in Example 1 (Sample l-1, 200 μg) and the crude product obtained in Example 2 or 6 were used. The purified product (Sample 2-2, 100 μg or Sample 6-4, 212 μg) or curcumin (20 nmol) was added as a 4 μl ethanol solution. Only 4 μl of ethanol was added to the control. In another well, the theophylline was not added, but the same amount of water (50 μ1) was added, and then the sample was added in the same manner as described above. After incubation for 14 days, it was confirmed with a phase-contrast microscope that significant fat globules were formed in the cells in the wells to which the sample or curcumin was added in addition to theophylline. A photomicrograph is shown in FIG. A micrograph (Fig. 10) of a sample obtained by staining cells cultured and treated in a 96-well plate under the same conditions with formalin and then stained with ORO reagent and Giemsa reagent shows that when theophylline and curcumin are added simultaneously, It was confirmed that it increased remarkably. Next, the culture medium in the 12-well plate was removed by aspiration, washed twice with PBS, and the cells were collected in the same manner as in Example 10 and extracted with 1ysis buffer. After measuring the protein concentration of the extract, the protein concentration is about 1 mg / ml (in the case of samples for lanes 1 to 9 in FIG. 11) or 0.7 mg / ml (in the case of samples for lanes 10 to 12 in FIG. 11). After SDS treatment, 5 μl of each sample was subjected to electrophoresis on polyacrylamide gel, and adiponectin and β-actin were detected by Western blotting. The same antibody as in Example 10 was used except that human anti-adiponectin and mouse monoclonal antibody (Catalog No. MAB3604) manufactured by Chemicon were used as the primary antibody. The results are shown in FIG. Adiponectin production is enhanced when a turmeric extract or curcumin is added in addition to theophylline.

  Experiments were conducted in the same manner as in Example 14 except that normal human preadipocytes were used instead of theophylline and 500 μM caffeine was used, and adiponectin production enhancement by turmeric extract or curcumin in the presence of caffeine was confirmed. did. The results are shown in FIG. In addition, the sample amount added in the electrophoresis of this figure is 7.5 micrograms (lanes 3-8) or 5 micrograms (lanes 1, 2, 9-14) as a protein. The same experiment was conducted with the caffeine concentration lowered to 100 μM, and it was confirmed that adiponectin production was enhanced by the coexistence of caffeine and curcumin even at this concentration. The results are shown in FIG. The amount of sample added in the electrophoresis of this figure is 5 μg of each lane as protein. Furthermore, the experiment was conducted with the curcumin concentration of 3, 10, and 15 μM in three stages, and in the presence of caffeine, it was confirmed that the enhancement of adiponectin production depends on the curcumin concentration, but about 10 μM is the most appropriate. . The results are shown in FIG. The amount of sample added in the electrophoresis of this figure is 3.8 μg of each lane as protein. In the experiment of FIG. 14, it is confirmed that curcumin and caffeine have a synergistic effect even at different concentrations.

  Experiments were performed in the same manner as in Example 14 except that normal human preadipocytes were used and cortisol (also known as hydrocortisone, 0.1 μM) was added to the PBM differentiation medium instead of dexamethasone. It was subjected to electrophoresis, and the enhancement of adiponectin production by turmeric extract or curcumin was confirmed. The results are shown in FIG.

Normal human preadipocytes were pre-cultured for 4 days in the same manner as in Example 14, and the cells were collected with EDTA-trypsin solution, suspended in PBM growth medium at a rate of ⁴ × 10 ⁴ cells / ml, and coated with collagen in advance 96- 0.2 ml was planted in each well plate. After culturing for 3 days, the medium was changed to a medium supplemented with PBM growth medium supplemented with insulin (10 μg / ml), 0.1 μM dexamethasone or hydrocortisone, and 500 μM caffeine or theobromine (0.2 ml / ml). well). To these cultures, the activated carbon-treated 2% acetone-eluted ethanol solution (5 mg / ml), curcumin (1 mM, ethanol solution) or ethanol obtained in Example 2 was added in an amount of 2 μl. After incubating for 12 days, it was confirmed with a phase contrast microscope that marked fat globules were formed in the cells in the wells to which the sample or curcumin was added. Next, the medium was removed by suction, fixed with 1% formaldehyde-PBS (++) solution, cell membrane permeabilized with 0.2% Triton X 100-PBS (++) solution, PBS containing 2% BSA-10% normal goat serum. After sequentially performing blocking treatment with (++), a primary antibody reaction was performed at 37 ° C. for 1 hour with 2% BSA-PBS (++) containing anti-human adiponectin. The cells were washed repeatedly with PBS (++), followed by a secondary antibody reaction with 2% BSA-PBS (++) containing FITC-Conjugated anti-mouse IgG (Catalog No. F0257) manufactured by Sigma for 1 hour, and after washing. And observed with a fluorescence microscope. A photomicrograph is shown in FIG. Adipose-accumulating cells appear when the sample is added with theophylline, caffeine, or theobromine, but the cells emit strong green fluorescence due to FITC bound through antibodies, and thus adiponectin is expressed. It was confirmed.

Normal human preadipocytes were cultured for 13 days in a medium in which the following compounds and samples were further added to a basic differentiation medium in which insulin (10 μg / ml) and 0.1 μM dexamethasone were added to a PBM growth medium. After removing the medium and washing, the cells in each well were extracted with 0.3 ml of isogen. RNA was purified and RT-PCR was performed in the same manner as in Example 11 to confirm the expression of the adiponectin gene. As primers for human adiponectin, 5′-TGTTGCTGGGAGCTGTTCTATG-3 ′ and 5′-ATGTCTCCCTTAGGACCAATAAG-3 ′ described in the literature (Yokota et al., J. Clin. Invest., 109, 1303-1310 (2002)) were used. -The actin primer used was the same as in Example 11 (mouse, human common part). The PCR conditions were the same as described in Example 11 except that the adiponectin was annealed at 60 ° C. and the amplification was repeated 35 times. The result of analyzing the PCR product by electrophoresis is shown in FIG. The compounds and samples added to the basic differentiation medium at the time of culture are as follows: Lanes 1 and 2, 200 μM indomethacin + 500 μM IBMX (used as a positive control); Lanes 3 and 4, 500 μM caffeine + ethanol (2 μl); Lane 5 And 6, 500 μM caffeine + 10 μM curcumin; lanes 7 and 8, ethanol (for control, 2 μl); lanes 9 and 10, 10 μM curcumin; lanes 11 and 12, ethanol (for control, 2 μl); lanes 13 and 14, 10 μM curcumin Lanes 15 and 16, 500 μM theophylline + ethanol (2 μl); Lanes 17 and 18, 500 μM theophylline + 10 μM curcumin. This result shows that the expression of the human adiponectin gene is promoted / enhanced by the synergistic action of curcumin and caffeine or theophylline. Furthermore, this result was confirmed by quantitative PCR as in Example 11. Using the Corvette Rotor-Gene 2000 Real-Time Cycler, the Qiagen QuantiTect SYBR Green PCR system was used to quantify the relative expression level of the adiponectin gene relative to β-actin. The primers are the same as described above. As a result, the gene expression of adiponectin was doubled or 4.2-fold when 10 μM curcumin was added, and 12.6 times when 500 μM caffeine and 10 μM curcumin were added simultaneously, and 500 μM theophylline and 10 μM compared to the control (corresponding to lanes 11 and 12). It was confirmed that it was enhanced 36.6 times with the simultaneous addition of curcumin.

  Normal human preadipocytes are cultured for 14 days in a medium supplemented with the following compounds and samples in a basic differentiation medium supplemented with insulin (10 μg / ml) and 0.1 μM dexamethasone or 0.1 μM hydrocortisone in a PBM growth medium. did. After removing and washing the medium, the cells in each well were extracted with 0.3 ml of isogen, and the expression of the adiponectin gene was confirmed by RT-PCR as in Example 18. The results are shown in FIG. Compounds and samples added to basal differentiation medium during culture: Lanes 1 and 2, 500 μM theophylline + ethanol (for control, 3 μl); Lanes 3 and 4, 500 μM theophylline +10 μM curcumin; Lanes 5 and 6, 200 μM indomethacin +500 μM IBMX (positive control) Lanes 7 and 8, 500 μM theophylline + ethanol (3 μl); lanes 9 and 10, 500 μM theophylline + crude product obtained in Example 6 (Sample 6-4, 75 μg / ml). However, as the glucocorticoid, 0.1 μM hydrocortisone was used for cells in lanes 1, 2, 3, and 4, and 0.1 μM dexamethasone was used for cells in other lanes. From the results of FIG. 18, it is understood that the expression of the human adiponectin gene is promoted and enhanced by the combined use of curcumin and theophylline even when the glucocorticoid is hydrocortisone. Similarly, it was confirmed that the crude extract of turmeric also promotes and enhances the expression of the human adiponectin gene.

It is a graph which shows the result of having measured the amount of intracellular fat accumulation by the ORO method when the turmeric extract obtained in Example 6 and 7 or its crudely purified product was added to mouse 3T3-Ll preadipocytes and cultured. . Only solvent (ethanol) was added to Control. The explanation of the sample numbers is the same as described in Examples 6 and 7. It is a photograph which shows the result of having detected the production of adiponectin by adding the sample obtained in Example 1 to 7 to mouse 3T3-Ll preadipocytes and culturing it by Western blotting. The result of detecting β-actin for the same sample is shown in the upper part, and the detection result of adiponectin is shown in the lower part. Sample 1 used was ethanol as a solvent as a control, 2 and 3 were the crude extract obtained in Example 4 (Sample 4-1), activated carbon adsorbed and 5% acetone eluate (Sample 4-3), 4 was Samples 5-3 and 5 obtained in Example 5 are the crude extract obtained in Example 1 (Sample1-1), 6 is Samples 2-2 and 7 obtained in Example 2, and Samples obtained in Example 3 3-3 and 8 are Sample 6-4 obtained in Example 6, and 9 is Sample 7-4 obtained in Example 7. It is a photograph which shows the result of having detected the production of adiponectin by Western blotting when Sample 6-4 obtained in Example 6 was added to mouse 3T3-Ll preadipocytes at different concentrations and cultured. The concentration is shown in the figure. Experiments were performed using triplicate wells for each concentration. However, at a concentration of 0, ethanol alone (3 μ1) was added as a control. The result of detecting β-actin in the sample of each well is shown in the upper part, and the detection result of adiponectin is shown in the lower part. Sample 6-4 obtained in Example 6 was added to mouse 3T3-Ll preadipocytes and cultured, and cells were collected on days 7, 11, 14, and 21 to detect adiponectin production by Western blotting. It is a photograph which shows the result. Experiments were performed using 3 wells each day for control (added 3 μl ethanol only) and sample (added 3 μl ethanol solution). The result of detecting β-actin in the sample of each well is shown on the right side, and the detection result of the corresponding adiponectin is shown slightly down on the left side. Sample 6-4 obtained in Example 6 was added to mouse 3T3-Ll preadipocytes or ethanol alone as a control and cultured for 12 days, RNA was extracted, and mRNA of β-actin and adiponectin was amplified by RT-PCR 2 is a photograph showing the results of detection of PCR products (predicted to be 332 bp and 219 bp, respectively) by agar gel electrophoresis. + Indicates that the reaction was performed with the addition of reverse transcriptase (RT), and − indicates that the reaction was performed without the addition of the enzyme. Sample 6-4 obtained in Example 6 was added to mouse 3T3-Ll preadipocytes at different concentrations (concentration 0 was added only ethanol as a control), and cultured for 12 days. It is a graph which shows the result of having calculated | required the relative expression level of the adiponectin gene. Experiments were performed using triplicate wells for the control and each concentration sample. The bar graph is the average value, and the error bar is the standard deviation. In addition to Sample 2-2 (50 μg / ml) obtained in Example 2, caffeine, theobromine, or theophylline was added to mouse 3T3-Ll preadipocytes at different concentrations and cultured for 12 days. It is a photomicrograph of a specimen stained with ORO (red) and then cell nuclei with Giemsa (red purple) (magnification 57 times). The concentrations of caffeine, theobromine and theophylline are shown in the figure. In addition to Sample4-1 (44 μg / ml) or curcumin (0-15 μM) obtained in Example 4, theophylline (0, 100, or 400 μM) was added to mouse 3T3-Ll preadipocytes and cultured for 10 days. Is a photograph showing the results of detecting adiponectin production by Western blotting. The result of detecting standard β-actin for the same sample is shown on the right side, and the detection result of the corresponding adiponectin is shown slightly on the left side. Normal human preadipocytes in the presence of insulin and dexamethasone, theophylline and sample (A to D) or sample without theophylline (E to H) are added, and the resulting intracellular fat globule is phase-shifted after 13 days of culture. It is the microscope picture observed and recorded with the microscope (magnification 60 times). A and E are controls (only ethanol is added), B and F are crude turmeric extracts obtained in Example 1 (Sample 1-1, 100 μg / ml), and C and G are obtained in Example 2. To the crude product (Sample 2-2, 50 μg / ml), D and H were added 10 μM curcumin. A normal human preadipocyte is a micrograph of cells in which theophylline and / or curcumin was added and cultured in a 96-well plate in the presence of insulin and dexamethasone, and after 13 days, intracellular fat globules were stained with ORO. About 45 times). After ORO staining, the whole was stained with Giemsa. A was added with 10 μM curcumin, B was added with the same amount (2 μl) of ethanol as the control, C was added with 10 μM curcumin and 500 μM theophylline, and D was added with 500 μM theophylline and ethanol (2 μl). Samples and / or theophylline (500 μM) are added to normal human preadipocytes in the presence of insulin (10 μg / ml) and 0.1 μM dexamethasone, cultured for 13 days, and the cells are collected to produce adiponectin by Western blotting It is a photograph which shows the result detected by. Additives: 1, ethanol (4 μl, control); 2, crude product obtained in Example 6 (Sample 6-4, 4 μl, final concentration 106 μg / ml); 3, 10 μM curcumin; 4-6, 500 μM Theophylline + ethanol (4 μl); 7-9, 500 μM theophylline + Sample 6-4; 10-12, 500 μM theophylline + 10 μM curcumin. The results of detecting β-actin for the samples in each well are shown in the upper part, and the results of detecting adiponectin are shown in the lower part. Samples and / or caffeine (500 μM) are added to normal human preadipocytes in the presence of insulin (10 μg / ml) and 0.1 μM dexamethasone, cultured for 13 days, and the cells are collected to produce adiponectin by Western blotting It is a photograph which shows the result detected by. Additives: 1, ethanol (3 μl, control); 2,500 μM caffeine + ethanol (3 μl); 3 and 4, crude product obtained in Example 2 (Sample 2-2, 3 μl, final concentration 50 μg / ml) 5-8, 500 μM caffeine + Sample 2-2; 9 and 10, 10 μM curcumin; 11-14, 500 μM caffeine + 10 μM curcumin. The result of detecting β-actin in the sample of each well is shown in the upper part, and the detection result of adiponectin is shown in the lower part. Normal human preadipocytes are supplemented with 3-15 μM curcumin and / or caffeine (500 μM) in the presence of insulin (10 μg / ml) and 0.1 μM dexamethasone, cultured for 13 days, and the cells are recovered to produce adiponectin It is a photograph which shows the result of having detected by Western blotting. Additives: 1, ethanol (3 μl, control); 2 and 3, 100 μM caffeine + ethanol (2 μl); 4 and 5, 500 μM caffeine + ethanol (3 μl); 6 and 7, 10 μM curcumin; 8-10, 100 μM Caffeine + 10 μM curcumin; 11-13, 500 μM caffeine + 10 μM curcumin. The result of detecting β-actin in the sample of each well is shown in the upper part, and the detection result of adiponectin is shown in the lower part. Normal human preadipocytes are supplemented with 3-15 μM curcumin and / or caffeine (500 μM) in the presence of insulin (10 μg / ml) and 0.1 μM dexamethasone, cultured for 13 days, and the cells are recovered to produce adiponectin It is a photograph which shows the result of having detected by Western blotting. Additives: 1, ethanol (2 μl, control); 2, 3 μM curcumin; 3 and 4, 10 μM curcumin; 5 and 6, 15 μM curcumin; 7, 500 μM caffeine + ethanol (2 μl); 8 and 9, 3 μM curcumin + 500 μM cafe In; 10 and 11, 10 μM curcumin + 500 μM caffeine; 12 and 13, 15 μM curcumin + 500 μM caffeine. The result of detecting β-actin in the sample of each well is shown in the upper part, and the detection result of adiponectin is shown in the lower part. Samples were added to normal human preadipocytes in the presence of insulin (10 μg / ml), 0.1 μM cortisol (hydrocortisone) and 500 μM theophylline, cultured for 14 days, and the cells were recovered and the production of adiponectin was detected by Western blotting It is a photograph which shows the result. Additives: 1, ethanol (3 μl, control); 2 and 3, crude product obtained in Example 6 (Sample 6-4, 75 μg / ml); 4, 10 μM curcumin. The result of detecting β-actin in the sample of each well is shown in the upper part, and the detection result of adiponectin is shown in the lower part. The following compounds and 10 μM curcumin were added to normal human preadipocytes in the presence of insulin and cultured for 12 days. The cells were fixed and stained with an adiponectin fluorescently labeled antibody. While observing and recording this with a fluorescence microscope (A, C, E, G), microscopic photographs (B, D, F, H) of intracellular fat globules in the same visual field observed and recorded with a phase contrast microscope. (Approximately 60 times magnification). A and B, C and D, E and F, and G and H are photographs of the same field of view. In addition to 10 μM curcumin, 500 μM theophylline + 0.1 μM dexamethasone, 500 μM theobromine + 0.1 μM hydrocortisone, 500 μM theobromine + 0. 1 μM dexamethasone, 500 μM caffeine + 0.1 μM hydrocortisone were added and cultured. Normal human preadipocytes are supplemented with 500 μM caffeine or theophylline and / or 10 μM curcumin (10 mM ethanol solution, 2 μl / well) in the presence of insulin and dexamethasone, cultured for 13 days, RNA extracted, and β-actin And adiponectin mRNA are amplified by RT-PCR, and PCR products (predicted to be 332 bp and 235 bp in length, respectively) are detected by agar gel electrophoresis. Samples and compounds added in the culture are as follows: 1 and 2, 200 μM indomethacin + 500 μM IBMX (positive control); 3 and 4, 500 μM caffeine + ethanol (2 μl); 5 and 6, 500 μM caffeine + 10 μM curcumin; 7 and 8, ethanol (control, 2 μl); 9 and 10, 10 μM curcumin; 11 and 12, ethanol (control, 2 μl); 13 and 14, 10 μM curcumin; 15 and 16, 500 μM theophylline + ethanol (2 μl); 18, 500 μM theophylline + 10 μM curcumin. Here, + indicates that the reaction was performed by adding reverse transcriptase (RT), and − indicates that the reaction was performed without adding the enzyme. To normal human preadipocytes, 10 μM curcumin or turmeric extract purified product obtained in Example 6 (Sample 6-4) in the presence of insulin and hydrocortisone or dexamethasone is added together with the following compound, and after 14 days of culture, RNA is extracted. 2 is a photograph showing the results of amplification of β-actin and adiponectin mRNA by RT-PCR and detection of PCR products by agar gel electrophoresis. Samples and compounds added in culture are as follows: 1 and 2, ethanol (3 μl) +0.1 μM hydrocortisone + 500 μM theophylline; 3 and 4, 10 μM curcumin + 0.1 μM hydrocortisone + 500 μM theophylline; 5 and 6, 200 μM indomethacin +500 μM IBMX (positive control); 7 and 8, ethanol (3 μl) +0.1 μM dexamethasone + 500 μM theophylline; 9 and 10, crude product obtained in Example 6 (Sample 6-4, 75 μg / ml) +0.1 μM dexamethasone + 500 μM theophylline . Here, + indicates that the reaction was performed by adding reverse transcriptase (RT), and − indicates that the reaction was performed without adding the enzyme.

Claims (9)

  An adiponectin production enhancing / promoting agent in animal adipocytes, comprising as an active ingredient an extract obtained by extracting a rhizome of a ginger family Curcuma plant with an organic solvent.   An agent for enhancing and promoting adiponectin production in animal adipocytes, comprising, as an active ingredient, an extract obtained by extracting a residue obtained by steam distillation of a rhizome of a turmeric genus (Curcuma genus) plant with an organic solvent.   The organic solvent extract according to claim 1 or 2 is further adsorbed on activated carbon, and then contains an elution component obtained by elution with a hydrophobic organic solvent containing a hydrophilic organic solvent as an active ingredient, Adiponectin production enhancer and promoter in animal adipocytes.   An elution component obtained by adsorbing the organic solvent extract according to claim 1 or 2 on silica gel and then elution with a hydrophobic organic solvent containing a hydrophilic organic solvent is contained as an active ingredient. Adiponectin production enhancer and promoter in adipocytes.   The drug according to claim 3 or 4, wherein the hydrophilic organic solvent is acetone and the hydrophobic organic solvent is hexane.   A potentiator and enhancer of adiponectin production in animal adipocytes, comprising curcumin as an active ingredient.   Furthermore, any 1 or more types of theophylline, caffeine, or theobromine are contained, The chemical | medical agent in any one of Claims 1-5 characterized by the above-mentioned.   A potentiator and enhancer of adiponectin production in animal adipocytes, comprising curcumin and at least one of theophylline, caffeine, or theobromine.   A method of further activating the adiponectin production promotion and enhancement ability of animal drugs in animal adipocytes according to any one of claims 1 to 6, wherein any one or more of these drugs and theophylline, caffeine, or theobromine In combination with the above-mentioned method

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