Anti-obesity composition, composition for inhibiting differentiation into adipocytes, or composition for inhibiting triacylglycerol accumulation in adipocytes

Abstract

To provide novel means useful for obesity prevention.SOLUTION: The present invention provides an anti-obesity composition comprising Diospyros ferrea Bakhauizen var. buxifolia Bakh. extract; a composition for inhibiting differentiation into adipocytes comprising Diospyros ferrea Bakhauizen var. buxifolia Bakh. extract; or a composition for inhibiting triacylglycerol accumulation in adipocytes comprising Diospyros ferrea Bakhauizen var. buxifolia Bakh. extract.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for anti-obesity, a composition for suppressing differentiation into adipocytes, or a composition for suppressing triacylglycerol accumulation in adipocytes.

Background Art

[0002] In recent years, it has been said that the proportion of obese people has been increasing along with lifestyle changes such as Westernization of diet and lack of exercise. Obesity is a cause of many diseases including lifestyle-related diseases such as diabetes, dyslipidemia, hypertension, and cardiovascular diseases (Non-Patent Document 1). Therefore, suppression of obesity is important.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a novel means useful for anti-obesity.

Means for Solving the Problems

[0005] As a result of intensive studies, the present inventors have found that the extract of Euphorbia kurokii is useful for suppressing differentiation into adipocytes, suppressing triacylglycerol (TG) accumulation in adipocytes, and also useful for suppressing obesity, and have further conducted intensive studies. The present disclosure includes inventions represented by the following, for example. Item 1. An anti-obesity composition containing an extract of Euphorbia kurokii. Item 2. A composition for inhibiting differentiation into adipocytes, containing an extract of Euphorbia kuroshimensis Boiss. Item 3. A composition for inhibiting triacylglycerol accumulation in adipocytes, containing an extract of Euphorbia kuroshimensis Boiss. Item 4. The composition according to any one of Items 1 to 3, wherein the extract of Euphorbia kuroshimensis Boiss. is an extract with water, ethanol, or a mixture thereof. Item 5. The composition according to any one of Items 1 to 4, which is an oral composition. Item 6. The composition according to any one of Items 1 to 5, which is a pharmaceutical composition or a food composition.

Advantages of the Invention

[0006] Provide novel means useful for anti-obesity.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0008] The anti-obesity composition included in the present disclosure contains an extract of Diospyros ferrea Bakhauizen var. buxifolia Bakh. (also described as Diospyros ferea Bakh., etc.). The composition for inhibiting differentiation into adipocytes included in the present disclosure contains an extract of Diospyros ferrea Bakhauizen var. buxifolia Bakh. The composition for inhibiting triacylglycerol accumulation in adipocytes included in the present disclosure contains an extract of Diospyros ferrea Bakhauizen var. buxifolia Bakh. Hereinafter, the anti-obesity composition, the composition for inhibiting differentiation into adipocytes, and the composition for inhibiting triacylglycerol accumulation in adipocytes may be collectively referred to as "the composition of the present disclosure".

[0009] Diospyros ferrea Bakhauizen var. buxifolia Bakh. (also described as Diospyros ferea Bakh., etc.) belongs to the genus Diospyros of the Ebenaceae family, grows in groups on each island of Okinawa Prefecture, and is also called Yaeyama Kokutan. The part of Diospyros ferrea Bakhauizen var. buxifolia Bakh. used for the extract is not limited as long as the effects of the present disclosure can be obtained, and may be any of fruits, leaves, stems, trunks, seeds, etc., and preferably fruits are exemplified. The used part may be used alone or in combination of two or more kinds.

[0010] The method for producing the extract (extraction method) and extraction conditions, etc. are not limited, and may follow a conventionally known method. For example, the above-mentioned part of Diospyros ferrea Bakhauizen var. buxifolia Bakh. can be directly used, and after being cut, crushed or dried as necessary, the extract can be obtained by solvent extraction.

[0011] As the solvent used for solvent extraction, without being limited as long as the effects of the present disclosure can be obtained, for example, water; lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, isopropanol, propylene glycol, 1,3-butylene glycol, and polyhydric alcohols (regardless of anhydrous or hydrous) and the like can be mentioned. Preferably, the solvent is water; alcohols such as methanol, ethanol, isopropanol, propylene glycol, 1,3-butylene glycol, more preferably water, methanol, ethanol, and even more preferably water, ethanol, and hydrous ethanol. When using hydrous ethanol as the extraction solvent, the concentration of ethanol is not limited as long as the effects of the present disclosure can be obtained and can be appropriately determined. However, in hydrous ethanol, the ethanol content is preferably exemplified by 10 to 80% by volume, more preferably 20 to 75% by volume, and 30 to 70% by volume. The solvent may be used alone or in combination of two or more.

[0012] The extract (solvent extract) obtained through solvent extraction in this way may be used as it is, or may be used after being subjected to treatments such as concentration, drying, separation of highly active fractions, etc. The treatment can be carried out according to known procedures such as concentration under reduced pressure, filtration, freeze-drying, adsorption treatment, HPLC (High performance liquid chromatography), etc. The extract may be in any of liquid, semi-solid, and solid states.

[0013] The amount of the Carica papaya extract contained in the composition of the present disclosure is not particularly limited as long as the effects of the present disclosure can be obtained, and examples include about 0.1 to 100% by mass, about 1 to 99% by mass, about 2 to 80% by mass, about 5 to 70% by mass, about 10 to 60% by mass, etc.

[0014] In addition to the extract of Okinawa black sugar, the composition of the present disclosure may optionally contain any components such as pharmaceutically acceptable components, edible components, etc. Examples of such optional components include solvents (alcohols such as water, methanol, ethanol, lower alcohols such as isopropanol, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (regardless of anhydrous or hydrous)), excipients, disintegrants, diluents, lubricants, fragrances, colorants, sweeteners, flavoring agents, suspending agents, wetting agents, emulsifiers, solubilizers, dispersants, buffers, binders, penetration enhancers, stabilizers, bulking agents, preservatives, thickeners, pH adjusters, surfactants, coating agents, absorption promoters, adsorbents, fillers, antioxidants, cooling agents, film-forming agents, gelling agents, amino acids, antioxidant components, anti-fatigue components and other various pharmacological active components and various nutritional components. These may be used alone or in combination of two or more, and their contents may also be determined appropriately.

[0015] The usage mode of the composition of the present disclosure is not limited and may be appropriately set according to the purpose. It can be used as a pharmaceutical composition, a food composition (including beverages, health functional foods (including foods for specified health uses, nutritional functional foods, foods with functional claims, etc.), supplements, foods for patients, foods for nursing care), a feed composition, etc., and also as an additive to pharmaceutical compositions, food compositions, feed compositions, etc.

[0016] The form of the composition of the present disclosure is also not limited and may be appropriately set according to the purpose, and it may be any of solid, semi-solid, and liquid forms. Therefore, the composition may be any of powders, fine granules, granules, tablets, pills, capsules (including hard capsules and soft capsules), lozenges, chewables, gels, pastes, creams, solutions, suspensions, emulsions, sprays, freeze-dried products in liquid form, etc. Further, for example, when the composition of the present disclosure is in solid form, the composition may be used after being mixed with water or the like.

[0017] The administration (intake) method of the composition of the present disclosure may be oral administration (intake) or parenteral administration (intake). Preferably exemplified are oral administration, vascular administration (especially intravenous), subcutaneous administration, etc., and oral administration is more preferably exemplified. From this perspective, the composition of the present disclosure can be preferably used as an oral preparation, an injection, a drip infusion, etc.

[0018] The composition of the present disclosure may be produced according to the usual procedures known in the art for the various forms, usage modes, etc. described above. Using the extract of Euphorbia kurokii Maxim., and if necessary, mixing the optional components within the range that does not interfere with the effects of the present disclosure, etc., may be produced.

[0019] The subject (subject animal) of the composition of the present disclosure is not limited, and examples include humans and non-human mammals, etc. Examples of non-human mammals include animals such as mice, rats, guinea pigs, rabbits, dogs, cats, monkeys, pigs, cows, etc. The composition of the present disclosure can be applied to any subject (subject animal) diagnosed as obese in a medical institution or a subject not diagnosed as obese. From this, the composition of the present disclosure can also be preferably applied to subjects concerned about obesity, subjects who want to slow down the rate of progression of obesity, subjects who want to suppress the differentiation of preadipocytes into adipocytes, subjects who want to suppress fat accumulation in cells, and subjects who want to prevent obesity.

[0020] The applicable dose of the composition of the present disclosure to the subject (subject animal) is not particularly limited, and may be appropriately set within the range where the effects of the present disclosure can be obtained according to the physique, age, symptoms, application form, etc. of the subject (subject animal). As an example, when applied orally, for an adult (body weight 60 kg), the extract of Euphorbia kurokii Maxim. (in terms of dry matter) can be exemplified as about 5 to 30,000 mg per day, and preferably exemplified as about 8 to 20,000 mg or about 10 to 15,000 mg. Here, the dry matter refers to the dry matter of the extract of Euphorbia kurokii Maxim. concentrated under reduced pressure according to the test examples described below to remove the solvent and dried. It may be administered (intaken) once a day or multiple times a day. When applied parenterally or applied to non-humans, the applicable dose, number of applications, etc. may be appropriately determined based on the description of the oral application.

[0021] Inhibition of the differentiation of preadipocytes into adipocytes suppresses the increase in the number of adipocytes. Inhibition of triacylglycerol accumulation in adipocytes suppresses the amount of fat accumulation in adipocytes. Therefore, it can be said that inhibition of differentiation into adipocytes and / or inhibition of triacylglycerol accumulation in adipocytes are useful for preventing obesity and improving obesity, that is, useful for anti-obesity.

[0022] Therefore, the present disclosure also includes a method for preventing or improving obesity, a method for inhibiting differentiation into adipocytes, or a method for inhibiting triacylglycerol accumulation in adipocytes, which comprises administering (ingesting) the composition of the present disclosure to the subject (subject animal). Each condition such as the composition of the present disclosure and the extract of Euphorbia kurokii in these methods is explained in the same manner as described above.

[0023] Hereinafter, embodiments included in the present disclosure will be described in more detail. In the present disclosure, "comprising" also includes the meanings of "consisting essentially of" and "consisting of". Further, the present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0024] In addition, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any way in identifying the subject matter included in the present disclosure. That is, the present disclosure includes all subject matters consisting of all possible combinations of the characteristics that can be combined described in this specification.

Examples

[0025] Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples.

[0026] Test Example 1 The effects of the Ryukyu kokutan extract (EDF) on cell viability and intracellular triacylglycerol (TG) accumulation were examined according to the following procedure. 1-1) Test Procedure · Preparation of Okinawan black sugar extract The fruits of Ryukyu kokutan were extracted with 50% ethanol to obtain a Ryukyu kokutan fruit extract. Specifically, the fruits of Ryukyu kokutan were pulverized and extracted with 50% ethanol (a mixed solution of water and ethanol) at room temperature (25 °C) for 2 hours. Then, it was filtered through a sieve and further filtered under reduced pressure using filter paper, concentrated under reduced pressure to remove the solvent, and dried to obtain a powder, thereby preparing a Ryukyu kokutan fruit extract. Hereinafter, the Ryukyu kokutan fruit extract thus obtained may be referred to as EDF. In the following tests, EDF was dissolved in dimethyl sulfoxide (DMSO) and used.

[0027] · Preparation of cells (pre-culture) Mouse fibroblasts (3T3-L1 cells) were purchased from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition, Japan (cell number JCRB9014) and used as preadipocytes. The 3T3-L1 cells were cultured in an incubator adjusted to 37 °C and 5% CO 2 until confluent. As the medium, Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FBS) (an appropriate amount of L(+)-glutamine and 8% aqueous sodium bicarbonate solution was added before use) was used.

[0028] · Examination of the effect of EDF on cell viability After the pre-culture, 3T3-L1 preadipocytes were inoculated into DMEM containing 10% FBS at a cell density of 1.0×10 5 ~1.0×10 6 cells / ml, EDF was added, and the cells were cultured in an incubator adjusted to 37 °C and 5% CO 2 for 24 hours. The addition amount of EDF was set to a final concentration of 25 μg / ml, 50 μg / ml, 100 μg / ml, 150 μg / ml, or 200 μg / ml in the medium. The case of culturing without adding EDF was used as a control.

[0029] After culturing, the cell viability was measured and calculated by the Neutral red method. The Neutral red method passes through an undamaged cell membrane and is taken up by the lysosomes of living cells. Utilizing this property, the amount of Neutral red taken up into the cells was measured, and the cell viability was calculated by determining the proportion of living cells. Specifically, the following procedure was followed. After the above culturing, the medium was removed, and a Neutral red solution was added to a final concentration of 50 μg / ml, and incubated at 37 °C and 5% CO 2 for 2 hours. Thereafter, the cells were washed with 2 ml of a 1% formaldehyde / 1% calcium chloride mixed solution. Further, a 1% acetic acid / 50% ethanol mixed solution was added, and the mixture was allowed to stand at room temperature for 30 minutes to extract the Neutral red taken up by the cells. The absorbance of the extract was measured at 540 nm using a spectrophotometer (V-530, JASCO Corporation).

[0030] The cell viability was calculated as a ratio relative to a control set at 100%.

[0031] · Effect of EDF on TG accumulation - Induction of differentiation into adipocytes in the presence of EDF As preadipocytes differentiate into adipocytes, lipid droplets accumulate in the cells. During the process of induction of differentiation, EDF was added to preadipocytes, and the effect of EDF on TG accumulation was examined.

[0032] Specifically, two days after the cells reached a confluent state in the pre-culture, the medium was replaced with a differentiation induction medium (this day was designated as "Day 0 of induction start"). As the differentiation induction medium, DMEM medium supplemented with 3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (0.25 mM), and bovine pancreatic-derived insulin (0.2 mM) was used. Two days later, the medium was replaced with DMEM medium supplemented only with insulin (0.2 μM), and thereafter the medium was replaced with fresh DMEM medium every two days, and the induction was completed on Day 8 of induction start. The addition of EDF to the medium was performed each time the medium was replaced on Days 0, 2, 4, and 6 of induction start. The addition amount of EDF to the medium was set to a final concentration of 50 μg / ml, 100 μg / ml, 150 μg / ml, or 200 μg / ml in the medium. The medium was replaced in the same manner except that EDF was not added to the medium, and the case of differentiation induction was used as a control.

[0033] - Measurement of intracellular TG accumulation The measurement and calculation of the intracellular TG accumulation amount were performed by staining with the conventionally known Oil red O. Oil red O is a type of azo dye and is known to dissolve into intracellular TG when it touches lipid cells. Specifically, a saturated solution of Oil red O (0.3 g) and 2-propanol (100 ml) was stirred well, and a staining solution was prepared at a ratio of saturated solution: ultrapure water = 6:4. The staining solution was filtered using No. 5C filter paper (φ185 mm) and used within 2 hours. After removing the medium from the cells after the induction was completed on the 8th day with an aspirator, the cells were washed with 2 ml of phosphate buffered saline (PBS(-)). Next, the cells were fixed with 60% ethanol, 1 ml of the staining solution was added, and the cells were left standing at room temperature for 2 hours for staining. Then, the cells were washed with 50% ethanol, washed twice with 1 ml of ultrapure water, 2 ml of ultrapure water was added, and the cells were observed under a microscope (OLYMPUS IX-70: Olympus Corporation) and photographed. Also, 1 ml of 2-propanol was added to the cell solution to which 2 ml of ultrapure water was added in this way to extract the dye of the staining solution. Next, the absorbance of the obtained extract was measured at a wavelength of 520 nm using a spectrophotometer (V-530, JASCO Corporation). Then, the TG accumulation amount was calculated and compared based on the measured values. Specifically, the TG accumulation amount was calculated as a ratio to the accumulation amount of the control being 100%.

[0034] 1-2) Results The results of cell viability are shown in Figure 1. As shown in Figure 1, no significant difference in cell viability was observed between the case where EDF was added and the control case where EDF was not added. Thus, since EDF did not affect cell viability at any of these concentrations, it was confirmed that the extracts of Euphorbia neriifolia L. did not show cytotoxicity at these concentrations.

[0035] Regarding intracellular TG accumulation, the microscopic observation results and measurement results are shown in Figure 2. In the figure, the left side shows the observation results under the microscope, and the right side shows the calculation results based on absorbance. In the figure, the part stained red on the left side is lipid. As shown in Figure 2, the intracellular TG accumulation amount significantly decreased in a concentration-dependent manner with the addition of EDF. From this, it was found that the extract of <species name> can reduce the intracellular TG accumulation amount.

[0036] Test Example 2 The effect of the addition time of EDF on the intracellular TG accumulation amount was examined according to the following procedure.

[0037] 2-1) Test Procedure · Addition of EDF, induction of differentiation into adipocytes, measurement of intracellular TG accumulation Two days after the cells reached a confluent state in the pre-culture, the medium was replaced with a differentiation induction medium (this day was designated as "Day 0 of induction start"). As the differentiation induction medium, DMEM medium supplemented with 3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (0.25 mM), and bovine pancreatic-derived insulin (0.2 mM) was used. Two days later, the medium was replaced with DMEM medium supplemented with only insulin (0.2 μM), and thereafter the medium was replaced with fresh DMEM medium every two days, and the induction was completed on Day 8 of induction start. The addition of EDF was made to be present in the medium during the periods of each arrow shown in Figure 3. For example, in case 2 in Figure 3, the cells were cultured in a differentiation induction medium containing EDF on Day 0 of induction start, and then cultured in a medium without EDF after Day 2. For example, in case 3 in Figure 3, the cells were cultured in a differentiation induction medium without EDF on Day 0 of induction start, cultured in a medium containing EDF from Day 2, and then cultured in a medium without EDF after Day 4. In case 6 in Figure 3, the cells were cultured in a medium containing EDF in all cases from Day 0 to Day 6 of induction start. The addition amount of EDF to the medium was set to a final concentration of 150 μg / ml in the medium. The medium was replaced in the same manner except that EDF was not added to the medium, and the case of differentiation induction was used as a control (1 in Figure 3). The measurement and calculation of the intracellular TG accumulation amount were performed in the same manner as in the above-mentioned "Measurement of intracellular TG accumulation amount".

[0038] 2-2) Results The results are shown in Fig. 3. As shown in Fig. 3, the intracellular TG accumulation amount significantly decreased, particularly in the cases of 2 and 6. Both 2 and 6 are the results of applying EDF at the initial stage of differentiation induction. From this, it was understood that applying EDF at the initial stage of inducing differentiation from preadipocytes to adipocytes brings about an effective reduction in the intracellular TG accumulation amount.

[0039] Test Example 3 Glycerol 3-phosphate dehydrogenase (GPDH) is the rate-limiting enzyme for TG synthesis. Since the GPDH activity increases when preadipocytes differentiate into adipocytes, it is known that GPDH can be used as an indicator of adipocyte differentiation. In this test example, the effect of EDF on GPDH activity was examined according to the following procedure.

[0040] 3-1) Test Procedure Differentiation induction was carried out in the same manner as in Test Example 1, and the GPDH activity on the 8th day (completion of induction) after the start of differentiation induction was measured. The addition amount of EDF to the medium was set to a final concentration of 75 or 150 μg / ml in the medium.

[0041] Specifically, regarding the measurement procedure of GPDH activity, the cells after the completion of induction were washed twice with 1 ml of PBS(-), 350 μl of triethanolamine / EDTA buffer (Table 1) was added, and the cells were collected with a cell scraper. Then, the cells were sonicated (middle, 6 min 15 sec, 20 sec / 10 sec for 2 times) using a sonicator BIO RUPTOR (Cosmo Bio Co., Ltd.). After centrifugation (13000 rpm, 5 min, 4°C) using a centrifuge (MX-160, Tomy Seiko Co., Ltd.), the supernatant was used as the cell lysate.

[0042] Next, triethanolamine / EDTA buffer was placed in a test tube and maintained at 28°C in a water bath. NADH (β-Nicotinamide dilithium salt), DHAP (Dihydroxyacetone phosphate dilithium salt), and 2-mercaptoethanol were each diluted with ultrapure water (Tables 2 - 4). NADH, DHAP, 2-mercaptoethanol, and cell lysate were added to the triethanolamine / EDTA buffer on the water bath to prepare a reaction mixture (Table 5), and the absorbance at 340 nm was measured using a spectrophotometer at 10-second intervals for a total of 3 minutes.

[0043] For protein quantification of the cell lysate used in the measurement of GPDH activity, Pierce (registered trademark) BCA Protein Assay Kit (Thermo Fisher Scientific K.K.) was used according to the procedure in the instruction manual. As the standard solution, bovine serum albumin (BSA) dissolved in ultrapure water to a concentration of 1 mg / ml and stored frozen at -20°C was used.

[0044] The activity of GPDH was calculated using the extinction coefficient of NADH (6.22 mM -1 cm -1 ), and was calculated based on the change in the amount of NADH per 3 minutes. The enzyme activity was expressed as a ratio to the value in the control, with the control value set as 100%.

[0045]

Table 1

[0046]

Table 2

[0047]

Table 3

[0048]

Table 4

[0049]

Table 5

[0050] 3-2) Results The results are shown in Fig. 4. As shown in Fig. 4, it was confirmed that the GPDH activity of the cells after differentiation induction decreased depending on the amount of EDF added. From this, it was understood that the differentiation of preadipocytes into adipocytes was suppressed in the presence of EDF. From this, it was shown that the extract of Ipomoea batatas has an effect of suppressing the differentiation of preadipocytes into adipocytes.

[0051] Test Example 4 For the differentiation of preadipocytes into adipocytes, transcriptional regulatory factors such as PPARγ (Peroxisome proliferator-activated receptor γ) and the C / EBP family play important roles. In the real-time PCR method, the gene expression level in a sample can be quantified by detecting this amplification product in real time. In this test example, the effect of EDF on the gene expression levels of transcriptional regulatory factors (PPARγ, C / EBPα) was examined using the real-time PCR method. In addition, the effect of EDF on the protein expression levels of the transcriptional regulatory factors was examined using the Western blotting method.

[0052] 4-1) Test Procedure Differentiation induction was carried out in the same manner as in Test Example 1, and the gene expression level and protein expression level on the second day after the start of differentiation induction were measured. The addition amount of EDF to the medium was set to a final concentration of 150 μg / ml in the medium. Specifically, the measurement was performed according to the following procedure.

[0053] · Measurement of gene expression level (real-time PCR method) Total RNA was extracted from the medium after the induction was completed using the High Pure RNA Isolation Kit (Roche). The centrifuge used was the MX-100 (Tommy Seiko Co., Ltd.). The quality of the total RNA was evaluated using the Agilent 2100 Bioanalyzer electrophoresis system (Agilent Technologies, Inc.). cDNA synthesis was performed using ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.) and the MJ mini thermal cycler (Bio-Rad Laboratories, Inc.). Quantification of the synthesized cDNA by real-time PCR was carried out using GeneAce SYBR (registered trademark) qPCR Mix α Low ROX (Nippon Gene Co., Ltd.) and the Stratagene Mx3005P (Agilent Technologies, Inc.). More details about these are as shown in Table 6 below. All were used according to the instruction manuals. Also, the primer sequences used in this test example are shown in Table 7 (the primers were purchased from the Fasmac Bio Research Support Division, Co., Ltd.). In this test example, Actinβ was used as the control gene. With the gene expression level of PPARγ or C / EBPα relative to Actinβ in the control as 1, the gene expression levels of PPARγ or C / EBPα relative to Actinβ at the time of EDF addition were calculated and compared.

[0054]

Table 6

[0055]

Table 7

[0056] · Measurement of protein amount (Western blotting method) Using the Western blotting method, the protein amount of the transcription regulator in the cells was measured and calculated. The Western blotting method is a conventionally known method in which proteins are separated by electrophoresis, transferred to a hydrophobic membrane, and the target protein is detected using an antibody.

[0057] Specifically, after completion of the induction, the cells were washed twice with 1 ml of PBS(-), and then the cells were collected using a cell scraper with RIPA buffer (Table 8) according to a conventionally known procedure. After collection, the cells were allowed to stand on ice for 30 minutes, stirred with a vortex mixer, sonicated (middle, 10 min, 30 sec / 30 sec for 2 times), and centrifuged (15000 rpm, 10 min, 4°C). The supernatant was collected, ×3 Sample buffer (Table 9) was added, heated at 95°C for 5 minutes, and then rapidly cooled to obtain a measurement sample. The same sample was stored at -80°C until use. For quantification of the protein amount, Pierce (registered trademark) BCA Protein Assay Kit (Thermo Fisher Scientific K.K.) was used. As a standard solution, albumin (derived from bovine serum, pH 5.2) dissolved in ultrapure water to 1 mg / ml and cryopreserved at -20°C was used.

[0058]

Table 8

[0059]

Table 9

[0060] For the obtained samples, electrophoresis by SDS-PAGE method and transfer to a PVDF membrane were performed according to the conventional method. After transfer, the membrane was blocked with 5% skim milk / TBS-T, washed, and then a primary antibody was added and reacted at room temperature for 1 hour while shaking. A parafilm was placed on the membrane and further reacted overnight at 4°C. Then, after washing with TBS-T, a secondary antibody was added and reacted at room temperature for 1 hour. Then, it was washed with TBS-T, an enzyme-labeled antibody was added, reacted at room temperature for 1 hour, and washed with TBS. In this test, an anti-GAPDH antibody was used as a loading control. The antibodies used here are shown in Table 10 below.

[0061]

Table 10

[0062] The membrane was immersed in a chemiluminescent reagent for HRP (Ez West Lumi plus (Atto Corporation)) and sandwiched in a clear pocket. Bubbles were removed with a roller, the membrane was sealed, and images were taken using a chemiluminescent imaging device (AE-9300 Ez-Capture MG (Atto Corporation)) and imaging software (Imaging software: Image Saver5 for Ez-Capture MG (Atto Corporation)). Then, the bands were quantified using analysis software (CS Analyzer ver3.0 (Atto Corporation)). As shown in Figure 6, for PPARγ, with the protein expression level of PPARγ relative to β-Actin in the control set as a relative value of 1, the protein expression level of PPARγ relative to β-Actin upon addition of EDF was calculated and compared. Also, for C / EBPα, with the protein expression level of C / EBPα relative to GAPDH in the control set as a relative value of 1, the protein expression level of C / EBPα relative to GAPDH upon addition of EDF was calculated and compared. In Figure 6, the results for PPARγ were calculated based on the total value of PPARγ with molecular weights of 57 and 54, and the results for C / EBPα were calculated based on the total value of C / EBPα with molecular weights of 42 and 30.

[0063] 4-2) Results The results of the gene expression levels of PPARγ and C / EBPα are shown in Figure 5, and the results of the protein expression levels are shown in Figure 6. As shown in Figures 5 and 6, when EDF was added, the expression level of PPARγ, which is one of the regulatory factors responsible for suppressing the differentiation of preadipocytes into adipocytes, was maintained at the same level as the expression level in the control, both at the gene and protein levels. In contrast, as shown in Figures 5 and 6, when EDF was added, the expression level of C / EBPα, which is one of the regulatory factors responsible for promoting the differentiation of preadipocytes into adipocytes, was significantly reduced compared to the expression level in the control, both at the gene and protein levels. From this, it was shown that the Okinawan kokutan extract has the effect of suppressing the differentiation of preadipocytes into adipocytes at the protein level and further at the gene level.

[0064] From the above, it was found that the Okinawan kokutan extract has the effect of suppressing triacylglycerol accumulation in adipocytes and the effect of suppressing the differentiation of preadipocytes into adipocytes. From this, it was found that the Okinawan kokutan extract is useful for preventing and improving obesity.

Claims

1. An anti-obesity composition containing Ryukyu Kokutang extract.

2. A composition for inhibiting differentiation into adipocytes, comprising an extract of Ryukyu Kokutai.

3. A composition for inhibiting triacylglycerol accumulation in adipocytes, comprising an extract of Ryukyu Kokutai.

4. The composition according to any one of claims 1 to 3, wherein the Ryukyu Kokutai extract is an extract using water, ethanol or a mixture thereof.

5. The composition according to any one of claims 1 to 3, which is an oral composition.

6. The composition according to any one of claims 1 to 3, which is a pharmaceutical composition or a food composition.

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