JP2013173719A - Agent for preventing and/or treating metabolic syndrome, using sudachitin as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for preventing and/or treating a life-style related disease (metabolic syndrome) by using an active ingredient of Citrus Sudachi.SOLUTION: It has been discovered that sudachitin of an active ingredient contained in Citrus Sudachi has effects for promoting expression of PPARγ, adiponectin and GLUT4. An expression promoter of the PPARγ, the adiponectin and the GLUT4, especially an agent for preventing and/or ameliorating a life-style related disease (metabolic syndrome) uses the sudachitin as an active ingredient based on the effects.

Description

  The present invention relates to a preventive and / or therapeutic agent for metabolic syndrome comprising sudachitin as an active ingredient. In particular, the present invention relates to a PPARγ activator containing sudachitin as an active ingredient, and to an excellent promoter of adiponectin secretion and GLUT4 production by the excellent PPARγ activator of sudachitin.

Citrus contains many polymethoxyflavonoids, and these polymethoxyflavonoids are known to have the effect of preventing and improving lifestyle-related diseases such as diabetes and hyperlipidemia. (Patent Documents 1 and 2).
Among these polymethoxyflavonoids, especially the pharmacological effect of nobiletin, which is contained in a large amount in Citrus depressa HAYATA, specially produced in Okinawa, has been actively studied and reported (Patent Documents 3 and 4).
However, little research has been done on sudachitin, which is a tridemethyl derivative at positions 4 ′, 5 and 7 of nobiletin. This is thought to be because it is rarely present in other citrus fruits and is contained only in the Tokushima special product Sudachi.
On the other hand, Patent Document 1 describes that dried dried peel / pulp koji powder of sudachi has an effect of suppressing blood sugar rise and an effect of improving blood lipids. Patent Document 5 reports that a compound extracted from sudachi has an activating action of Sirt1 / Sirt2. However, the effect of sudachitin itself is hardly mentioned, and Non-Patent Document 1 only has been known to have strong antibacterial activity.

JP 2007-77139 A WO2002 / 0875567 WO2006 / 049234 WO2010 / 134373 publication JP 2009-126799 A

J. et al. Nat. Prod. , 2006, 69 (8), 1177-1179 Biochem Biophys Res. Commun. 2007; 357 (2): 371-376

  An object of the present invention is to find a new use of sudachitin, and in particular, to provide a therapeutic agent for metabolic syndrome containing sudachitin as an active ingredient.

  The present inventors pay attention to the fact that sudachitin is a tridemethyl body at positions 4 ′, 5 and 7 of nobiletin (2) as shown in the following chemical formula (1), and there is a difference in structure (hydroxyl group and methoxy group). We examined the difference in effect.

According to the findings so far, it has been reported that nobiletin increases the gene expression of peroxisome proliferator-responsive receptor (PPARγ2), resulting in a significant increase in the secretion of adiponectin and GLUT4 from adipocytes. (Patent Documents 3 and 4). Furthermore, nobiletin activates cAMP response factor binding protein (CREB) and extracellular signal-regulated kinase (EPK), and increases the expression level of transcription factor C / EBPβ of PPARγ2. As a result, it has been reported that the expression level of PPARγ2 increases (Non-patent Document 2). PPARγ2 increased by nobiletin is thought to act as a transcription factor to promote the expression of adiponectin and GLUT4. As a result, nobiletin is known to exhibit anti-diabetic effects such as adipocyte differentiation and adiponectin secretion, resulting in improved hyperglycemia and insulin resistance (Biochem Pharmacol. 2010 jun). 1; 79 (11): 1674-83, 2010).
Therefore, the present inventors have clarified the effect and action mechanism of sudachitin in consideration of the action effect and action mechanism of the above-mentioned nobiletin, and obtained the following knowledge.
a) Effect:
Sudachitin increases the gene expression of PPARγ and increases the gene expression of adiponectin and GLUT4.
The dose response of sudachitin with respect to the gene expression level of adiponectin draws a bell-shaped curve peaking at 1 to 30 μM as shown in FIG. 3, but in the case of nobiletin, no bell-shaped curve is shown. It increases linearly in a dose-dependent manner.
b) Mechanism of action:
Although the dose correlation regarding the gene expression level of adiponectin is different between sudachitin and nobiletin, it was found that the increase of adiponectin is based on the increase of PPARγ2 like nobiletin.
The difference in the mechanism of action of sudachitin and nobiletin for the antidiabetic effect is that IL-6 (a factor that triggers insulin resistance due to chronic inflammatory reaction) and HSL (degrading enzyme of triacylglycerol and cholesterol ester) ) Was found to be different, and nobiletin suppressed gene expression, but sudachitin had no effect. Furthermore, it was revealed that the expression of Sirt1 was increased by the influence of sudachitin as shown in FIG.
Sirt1 deacetylates Foxo1 and promotes transcription of adiponectin through activation of Foxo1 transcription and promotion of complex formation with C / EBPα (J. Biol. Chem. 281: 39915-39924 (2006)). It has been known. Thus, it was revealed that sudachitin exhibits an adiponectin expression promoting effect not only in the pathway via PPARγ2 but also in the pathway via Sirt1.
Based on the above findings, the present inventors have completed the present invention.

That is, the gist of the present invention is as follows.
(1) An expression promoter for adiponectin and GLUT4 containing sudachitin as an active ingredient.
(2) A preventive and / or ameliorating agent for metabolic syndrome, comprising the expression promoter of (1) above.
(3) The preventive and / or ameliorating agent for metabolic syndrome according to (2), wherein the metabolic syndrome is diabetes.
(4) The preventive and / or ameliorating agent for metabolic syndrome according to (2) or (3) above, wherein diabetes is type 2 diabetes.
(5) The preventive and / or ameliorating agent for tarotic syndrome according to any one of the above (2) to (4), wherein the preventive and / or improving agent for bolic syndrome is a functional food.
(6) A PPARγ2 expression promoter containing sudachitin as an active ingredient and based on increasing the promoter activity of PPARγ2.
(7) A Sirt1 expression promoter containing sudachitin as an active ingredient.
(8) A preventive and / or ameliorating agent for metabolic syndrome, comprising the expression promoter of (7) above.

  The sudachitin of the present invention promotes the expression of adiponectin and GLUT4 through promoting the expression of PPARγ2 and Sirt1. That is, by promoting secretion of adiponectin, it is possible to suppress fat cell hypertrophy and normalize fat cells that are growing. In addition, by promoting the expression of GLUT4, it is possible to normalize the uptake of glucose in blood into adipocytes. From this, sudachitin has the effect | action which can improve insulin resistance, and can improve diabetic symptoms, such as type 2 diabetes, arteriosclerosis, and obesity, by the expression promoter of this invention.

It is a figure showing the change of the gene expression of PPARγ2 when 3T3-L1 adipocytes were stimulated for 24 hours using 10 μM sudachitin. It is a figure showing the change of the gene expression of PPARγ2 when 3T3-L1 adipocytes were stimulated for 48 hours using 30 μM sudachitin. Compared with the result of the 24-hour stimulation in FIG. 1, the change was increased by about 2.2 times. It is a figure showing the change of adiponectin gene expression when 3T3-L1 adipocytes are stimulated for 24 hours using 1-100 μM sudachitin. It is a figure showing the change of adiponectin gene expression when 3T3-L1 adipocyte is stimulated for 48 hours using 30 μM sudachitin. It is a figure showing a change of adiponectin gene expression when stimulating 3T3-L1 adipocytes for 72 hours using 30 μM sudachitin. Compared with the result of 48 hours stimulation in FIG. 4, it was reduced to about 1/10. It is a figure showing the change of GLUT4 gene expression when 3T3-L1 adipocyte is stimulated for 48 hours using 30 μM sudachitin. It is a figure showing the change of GLUT4 gene expression when 3T3-L1 adipocyte is stimulated for 72 hours using 30 μM sudachitin. Compared with the result of 48-hour stimulation in FIG. 6, it was reduced to about 1/10. It is a figure showing the change of adiponectin gene expression when 3T3-L1 adipocytes are stimulated with GW9662 and sudachitin 30 μM (30 μM SD) is used. It is a figure showing the result of the luciferase assay test for confirming whether sudachitin is acting on the promoter of PPARγ2. When 3T3-L1 adipocytes were stimulated for 48 hours with 10 μM sudachitin, luciferase activity increased slightly, but when the same thing was done with 30 μM sudachitin, it increased significantly by a factor of 1.5. (FIG. 9A). Further, when stimulation was performed with 30 μM sudachitin for 0, 2, 6, 12, 24, and 48 hours, a significant increase in luciferase activity was observed after 12 hours (FIG. 9B). It is a figure showing the change of Sirt1 gene expression when 3T3-L1 adipocyte is stimulated for 48 hours using 30 μM sudachitin. It is a figure showing the result of producing an obese model mouse by giving a Western diet (40% high fat diet) to C57BL / 6 mice (5 weeks old) for 5 weeks and administering sudatitin to this obese model mouse. (A) represents an increase in body weight, and (B) represents a change in food intake. (C) is a diagram comparing the amount of white adipocytes of the obese model mice administered with sudachitin for 12 weeks for each organ site. For each organ of liver, subcutaneous fat, visceral fat and brown fat, it was evaluated how the amount of white adipocytes changes with and without administration of sudachitin. Liver, subcutaneous fat and visceral fat were significantly reduced by the administration of sudachitin. CC represents a group not administered with sudachitin in the control group, and CS represents a group administered with sudachitin in the control group. WC represents a group of obese model mice not administered with sudachitin, and WS represents a group of obese model mice administered with sudachitin. It is the figure which compared the white fat cell amount of the said obesity model mouse | mouth which administered sudachitin for 4 weeks for every organ site | part. For each organ of muscle, subcutaneous fat, and visceral fat, we evaluated how the amount of white adipocytes changed with or without administration of sudachitin, and also evaluated changes in body fat percentage and total fat mass. Subcutaneous and visceral fat weights were significantly reduced by the administration of sudachitin. CC represents a group not administered with sudachitin in the control group, and CS represents a group administered with sudachitin in the control group. WC represents a group of obese model mice not administered with sudachitin, and WS represents a group of obese model mice administered with sudachitin. When the cell size of the fat cells obtained in FIG. 12 was evaluated, the size of the fat cells was significantly reduced in the sudachitin administration group. It is a figure showing the result of having performed the glucose tolerance test to the said obesity model mouse which administered sudachitin for 6 weeks. In (A), the blood glucose level was improved in the group administered with sudachitin, and a significant decrease in blood glucose level was found in 30 to 90 minutes. (B) represents the change in blood glucose level after insulin administration, and shows that insulin resistance is improved in the group administered with sudachitin. It is the figure showing the result of having evaluated serum neutral fat (serum TG), serum free fatty acid (NEFA), and serum total cholesterol (T-chol) other than the blood glucose level of FIG. The free fatty acid showed a significantly lower value in the sudatin group. CC represents a group not administered with sudachitin in the control group, and CS represents a group administered with sudachitin in the control group. WC represents a group of obese model mice not administered with sudachitin, and WS represents a group of obese model mice administered with sudachitin. An obese model mouse fed a Western diet to C57BL / 6 mice (5 weeks old) for 12 weeks shows hyperinsulinemia and hyperleptinemia, but the serum insulin level and serum leptin level are significant in the group administered with sudachitin. (See (A) and (B)). Serum adiponectin concentration decreased with intake of the Western diet and aging, but was significantly improved in the statatin group (see (C)). CC represents a group not administered with sudachitin in the control group, and CS represents a group administered with sudachitin in the control group. WC represents a group of obese model mice not administered with sudachitin, and WS represents a group of obese model mice administered with sudachitin.

The “adiponectin” of the present invention is a hormone secreted from adipose tissue consisting of 244 amino acids and activates AMP kinase (AMP-activated protein kinase: AMPK) in skeletal muscle and liver. Has the effect of promoting the combustion and uptake of sugar. Adiponectin is secreted in a large amount in small adipocytes, but it is known that the amount of secretion decreases when adipocytes are enlarged.
When fat cells are enlarged due to obesity or the like, it is believed that the amount of adiponectin secreted decreases and causes lifestyle-related diseases such as type 2 diabetes and arteriosclerosis.

  “GLUT4” of the present invention is a sugar transport carrier (glucose transporter: GLUT) on the cell membrane relating to the metabolism of carbohydrates in the body, and the amount of glucose taken up is defined by the amount of glucose transporter. . The main glucose transporter in muscle and adipose tissue is glucose transporter 4 (GLUT4). This GLUT4 is usually present inside the cell, moves (translocates) onto the cell membrane by insulin stimulation, and glucose is taken into the cell through the carrier. In addition, it has been reported that transgenic mice in which GLUT4 is overexpressed even in a small amount are unlikely to become diabetic due to a high fat diet (Ikemoto, S. et al .: Proc. Natl. Acad. Sci. USA, 92: 3086-3089, 1995), and those that promote the expression of GLUT4 are considered effective for the prevention and cure of diabetes.

  The “PPARγ2” of the present invention is one type of nuclear receptor, PPAR (Peroxisome Proliferator Activated Receptor: Peroxisome Proliferator Activated Receptor), which is one γ type of a total of three subtypes. Since there are two promoters in the human and mouse γ-type genes, two types of proteins, PPARγ1 and PPARγ2 with different N-terminals, are generated due to differences in splicing. PPARγ, particularly PPARγ2, is expressed with relatively strong specificity in adipocytes, and has been shown to play a central role in adipocyte differentiation. Thus, it is suggested that lipid metabolism is improved by promoting the expression of the PPARγ2 gene, and that it also has a preventive and therapeutic action for diabetes.

The “metabolic syndrome” of the present invention means that insulin resistance (decreased insulin function) occurs due to accumulation of visceral fat, abnormal glucose metabolism (abnormal glucose tolerance, diabetes), abnormal lipid metabolism (high triglyceremia, low (HDL cholesterolemia), risk factors of arteriosclerosis such as hypertension are symptoms that accumulate in one individual, and in addition to visceral fat type obesity, combine any two or more of hyperglycemia, hypertension, lipid abnormalities A state that has a state. Specifically, it is an early symptom of diabetes or arteriosclerosis.
The “functional food” of the present invention refers to a food having an improvement effect on a disease or medical condition. The functional food of the present invention is not particularly limited as long as it contains sudachitin, and can be used even if it contains sudachitin, as long as it contains sudachitin. . Specific examples of the food according to the present invention include tablets, granules, powders, drinks and the like containing sudachitin as so-called dietary supplements (supplements). Furthermore, a seasoning containing sudachitin, a confectionery, bread, a side dish, drinking water, etc. can be mentioned. It goes without saying that the functional food of the present invention is intended for humans, but is not limited to humans and can be used for a wide range of animals. In particular, pets such as dogs and cats suffering from lifestyle-related diseases and liver dysfunction are suitable as targets.

“Sirt1” of the present invention is one of NAD + -dependent protein deacetylases present in the cell nucleus and cytoplasm, and the enzyme activity of SIRT1 is resveratrol, butein, piceanannol, fisetin, quercetin It is known that it is activated by a polyphenol having a transstilbene structure. Activation of SIRT1 has been reported to be useful for prevention of cell death, suppression of cell aging, extension of cell life, prevention of cancer development, and the like, and various SIRT1 activators have been studied.
The sudachitin of this invention can be used as a raw material of a chemical | medical agent (a pharmaceutical and a quasi-drug are included) as an expression promoter, prevention, and / or an improvement agent. For example, it can be produced by appropriately blending the sudachitin of the present invention with a raw material for a pharmaceutical preparation. In addition, the said chemical | medical agent may be used for a human and may be used for mammals other than a human. Examples of the pharmaceutical raw material that can be blended in the sudachitin of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, crystalline cellulose, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.), binders (Distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrant (sodium alginate, agar, sodium bicarbonate, calcium carbonate, Sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, gum arabic powder, gelatin, ethanol, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption accelerators (quaternary ammonium base, lauryl) Sodium sulfate), adsorbent (g Serine, starch, lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearates, polyethylene glycol, and the like) and the like.

The administration method of sudachitin according to the present invention is generally preferably administered orally in the form of tablets, pills, soft / hard capsules, fine granules, powders, granules and the like. The water-soluble preparation can be administered orally as a liquid. Furthermore, parenteral administration may be used. When administered as a parenteral agent, the sudachitin of the present invention is dispersed in an appropriate solubilizing agent such as ethanol or water, and then applied in a dosage form such as a poultice, lotion, ointment, tincture, or cream. be able to.
The dose of sudachitin can vary depending on the administration method, medical condition, patient age, etc., but in adults, about 5 to 200 mg of active ingredient per day can usually be administered.

  EXAMPLES Next, although an Example is given and this invention is further demonstrated, this invention is not limited to these.

(Example 1) Effect of promoting the expression of PPARγ2 mRNA by sudachitin 3T3-L1 adipocytes 8-12 days after differentiation were cultured in a culture medium containing DMSO or sudatin, and gene expression of PPARγ2 by RT-PCR Was measured as follows.
(1) Test material:
a) Reagents: 0.25% and 0.05% solutions of FBS (Fetal bovine serum), CS (Calf serum), penicillin / streptomycin and trypsin-EDTA were purchased from Life Technologies. Troglitazone was purchased from Cayman Chemical and prepared to 1 mM with 100% DMSO.
b) Sudachitin ((3- (4-hydroxy-3-methoxyphenyl) -1- (4-hydroxyphenyl) -1-phenylpropanone)) is purified from sudachi skin by microwave irradiation (Tokushima Prefectural Industrial Technology) Provided by the center), and prepared a 10 mM sudachitin solution in which 3.06 mg was dissolved in 1 ml DMSO and a 100 mM sudachitin solution in which 30.6 mg was dissolved in 1 ml DMSO.
c) Takara Prime Script ™ RT ligand kit and RNA iso plus were purchased from Takara Bio as RT-PCR reagents. ONPG was purchased from Wako.
DMEM, insulin derived from bovine pancreas, dexamethazone, IBMX, GW9662 (2-chloro-5-nitrobenzilide) were purchased from Sigma (Sigma Chemicals), the insulin was 1 mg / ml (10 mM HCl), dexamethasone was 0 .3 mg / l (100% EtOH), IBMX was adjusted to 11.5 mg / ml (100 mM NaOH), and GW9662 was dissolved in 1.3835 mg in 1 ml DMSO to prepare a 5 mM GW9662 solution.

(2) Test method:
a) Cultured cells and differentiation:
3T3-L1 mouse fibroblasts were cultured in DMEM (including 4.5 g / l glucose and L-glutamine) supplemented with 10% CS, 100 U / ml penicillin and 100 μg / ml streptomycin using a 10 cm dish. Two days after reaching confluence, differentiation was induced by culturing the cells in a differentiation medium (DMEM 10% FBS, 1% IBMX, 0.1% dexamethasone, 1% insulin, 0.1% troglitazone) for 48 hours. Cells were DMEM (10% FBS, 8-12 days after differentiation).
100 U / ml penicillin, 100 μg / ml streptomycin) and used in the experiment.
b) cDNA synthesis by RNA extraction and reverse transcription of RNA:
Total RNA was extracted from differentiated 3T3-L1 adipocytes using RNA iso plus. RNA concentration was measured using a spectrophotometer (NANODROP 8000, Thermo Scientific, Wilmington, DE), and 0.5 μg DNA was synthesized using a reverse transcription reaction using Takara Prime Script ™ RT ligand kit.
c) Quantitative RT-PCR analysis:
Quantitative RT-PCR was performed on the Light Cycler system (Roche) using 50 ng DNA. Samples were quantified using the Light Cycler DNA Master SYBR Green I (Roche). PCR was performed under the following conditions: 95 ° C. × 15 min, 40 × (95 ° C. × 1 s, 61 ° C. × 7 s, 72 ° C. × 11 s). The base sequences of PCR primers are as shown in the following table. Quantification data was analyzed with Roter-Gene Real-time Analysis Software 6.0. 36b4 expression was used to normalize target gene expression.

(3) Evaluation results:
3T3-L1 adipocytes were stimulated with medium supplemented with 10 μM sudachitin or 0.1% DMSO (control) for 24 hours. PPARγ2 (1.47 times ± 0.11, p <0.05) was significantly increased in cells in the medium supplemented with sudachitin (see FIG. 1), compared to the control group. Furthermore, PPARγ2 (3.24 times ± 0.58, p <0.01) significantly increased when stimulated with 30 μM sudachitin for 48 hours (see FIG. 2). In the case of stimulation for 72 hours, PPARγ2 (0.83 times ± 0.26, p = 0.28) also showed a tendency to decrease although not significant.
The results were expressed as the mean ± SD standard error. The unpaired Student-t test was basically used for the significance test, and the one-way analysis ANOVA of variance according to Tukey's Multiple Comparison Test was used for the concentration-dependent reaction. A risk factor of p <0.05 was considered significant.

(Example 2) Effect of promoting expression of adiponectin, GLUT4, and Sirt1 mRNA by sudachitin In the same manner as in Example 1, gene expression of adiponectin, GLUT4, and Sirt1 was measured by RT-PCR. In addition, the base sequence of the PCR primer used for the measurement is as shown in the following table.

Since it was shown in Example 1 that the expression level of mRNA changes depending on the addition concentration and stimulation time of sudachitin, the concentration effect of sudachitin was evaluated for adiponectin. In the stimulation for 24 hours, an increase in the gene was observed at a concentration of 10 μM, and an increase in the gene was observed up to 30 μM, but it was shown to decrease conversely at 100 μM (see FIG. 3).
Therefore, when stimulated with 30 μM sudachitin for 48 hours, adiponectin mRNA was 2.32 times ± 0.26 (p <0.05), which was significantly increased (see FIG. 4). However, when the time was extended and the stimulation was performed for 72 hours, the expression was 0.23 times ± 0.22 (p <0.01), and the gene expression was significantly decreased (see FIG. 5). .

Similarly, when GLUT4 was stimulated with 30 μM sudachitin for 48 hours, the mRNA of GLUT4 was 3.04 times ± 0.37 (p <0.05) and increased significantly (see FIG. 6). However, when the time was extended and the stimulation for 72 hours was reversed, it was 0.32 times ± 0.21 (p <0.01), indicating that the gene expression was significantly reduced (see FIG. 7). .
As for Sirt1, when stimulated with 30 μM sudachitin for 48 hours, the mRNA of Sirt1 was 1.87 times ± 0.31 (p <0.01), which was significantly increased (see FIG. 10). .

(Example 3) Confirmation test of increase in gene expression of PPARγ2 by sudachitin on increase in gene expression of adiponectin and GLUT4 Increased mRNA expression of adiponectin and Glut4 obtained by stimulation with 30 μM sudachitin was carried out via PPARγ2. In order to examine whether the expression of adiponectin or Glut4 mRNA is increased, GW9662 which is an inhibitor of PPARγ2 and sudachitin are simultaneously stimulated.
(1) Test method:
3T3-L1 adipocytes 8-12 days after differentiation were cultured for 48 hours in a medium supplemented with 0.1% DMSO (control), 0.1% DMSO + 5 μM GW9662, 30 μM sudachitin + DMSO, 30 μM sudachitin + 5 μM GW9662 and RT -Analysis was performed by PCR.
(2) Test results:
As shown in FIG. 8, the expression of adiponectin increased by stimulation with 30 μM sudachitin was significantly suppressed by GW9662. Also in Glut4, although not significant, the increase in expression by sudachitin tended to be suppressed by GW9662.
As described above, it was shown that sudachitin increases the expression of adiponectin and GLUT4 via PPARγ2.

(Example 4) Confirmation test that sudachitin increases promoter activity of PPARγ2 In order to examine whether sudachitin increases the promoter activity of PPARγ2, transformation and luciferase assay were performed using HEK293 cells.
(1) Test method:
HEK293 cells were transformed with FuGENE6 transduction reagent (Roche). That is, HEK293 cells were seeded in a 24-well dish and cultured until Opti-MEM medium 50-80% confluence. The cells were transduced using pGL3-basic, PPAR · 2 PL1 / PGL3 basic, and pCMVβ reagents. After 24 hours, the medium was replaced with DMEM supplemented with 10% FBS and sudachitin, and after culturing for 0-48 hours, the cells were measured for luciferase activity using Dual-Luciferase Reporter System (Promega). The transduction efficiency was corrected by measuring the activity of β-gal expression by ONPG transduced at the same time.
(2) Test results:
When stimulated with DMSO (control) and sudachitin (10 μM, 30 μM), respectively, as shown in FIG. 9 (A), compared with the control, 10 μM sudachitin was 1.43 times ± 0.22 (P <0. 05), 30 μM sudachitin significantly increased luciferase activity by 1.65 times ± 0.18 (p <0.01). In addition, there was no significant difference in the increase effect between 10 μM and 30 μM sudachitin concentrations (p = 0.07).
Next, when stimulated with DMSO (control) and 30 μM sudachitin (12 hr, 24 hr, 48 hr), respectively, as shown in FIG. 9 (B), 2.86 times ± 0.36 in 12 hours compared with the control. (P <0.01), 3.24 times ± 0.37 (p <0.01) in 24 hours, 2.84 times ± 0.30 (p <0.01) in 48 hours, stimulated with sudachitin It was shown that the luciferase activity of the PPARγ2 promoter was increased in the cultured cells.
From these results, it was shown that sudachitin has an action of increasing the expression of adiponectin, Glut4, PPARγ2, and Sirt1, and that the increase in the expression of adiponectin and Glut4 is associated with an increase in the promoter activity of PPARγ2.

(Example 5) Effect of administration of sudachitin in obese model mice It was evaluated that sudachitin improves lipid metabolism and sugar metabolism by administering sudachitin to obese model mice.
(1) Evaluation test of lipid metabolism improvement:
When a Western diet (40% high fat diet) was given to C57BL / 6 mice from the age of 5 weeks, weight gain was significantly increased from the 5th week of administration compared to the normal chow group. In the group administered with 5 mg / kg of sudachitin once a day while giving a high fat diet, weight gain was suppressed, and the value was significantly lower than that of the control group from the 5th week of administration of sudachitin (FIG. 11 (A)). See). On the other hand, in the normal chow group, there was no change in body weight even when sudachitin was administered. Regarding the amount of food intake, there was no difference in food intake between the normal chow group and the western group, so the energy intake increased in the western group. There was no change in food intake by the administration of sudachitin in both the normal chow group and the western group (see FIG. 11B).
White fat weight at the time of dissection after 12 weeks of administration of sudachitin was not affected by the administration of sudachitin in the normal chow group, but in the western group, subcutaneous fat and visceral fat, which are liver, white adipose tissue, were significantly decreased by administration of sudachitin. However, there was no difference in brown fat (see FIG. 11C).
Furthermore, we investigated that administration of sudachitin under a high fat diet suppressed the increase in weight by suppressing the increase in white fat. When X-ray CT scans were performed on mice at 4 weeks after administration of sudachitin, there was a significant difference in the weight of subcutaneous fat and visceral fat (see FIG. 12). Furthermore, when comparing the size of adipocytes, there was a tendency for the infiltration to be significantly smaller in the sudatin group (see FIGS. 13A and 13B).
(2) Evaluation test for improved glucose metabolism:
Sudachitin was administered to the above obese model mice for 6 weeks, and a glucose tolerance test was performed. Glucose tolerance abnormalities were shown by ingestion of the Western diet, and there was a tendency of improvement in the sudatin group, and a significant decrease in blood glucose level was observed at 30 minutes and 90 minutes (see FIG. 14A). Furthermore, in the insulin tolerance test, the blood glucose level at 15, 30, 60, and 90 minutes after insulin administration was significantly lower in the sudatin group, and insulin resistance induced by ingestion of the Western diet was improved by administration of sudatin. This was suggested (see FIG. 14B). In addition, when serum neutral fat and neutral fat in liver and muscle tissue were evaluated, it showed a low value in the sudatin group, while no significant decrease was observed in serum total cholesterol, but free fatty acid (NEFA) ) Showed a significantly low value in the group treated with sudachitin (FIG. 15).
Furthermore, serum insulin levels and leptin levels were measured in order to clarify that the obesity status was improved. The mice showed hyperinsulinemia and hyperleptinemia after 12 weeks of ingestion of the Western diet, both of which significantly decreased with administration of sudachitin (see FIGS. 16A and 16B). Conversely, the adiponectin concentration, which is considered to be inversely correlated with visceral fat mass, decreased with the intake of the Western diet and aging, but was significantly improved by the administration of sudachitin (see FIG. 16C).

The sudachitin used in the present invention is contained only in Tokushima special product sudachi, and since it has been eaten for many years, there is no problem in safety and there is not much bitterness. Therefore, the adiponectin and GLUT4 expression promoter containing sudachitin of the present invention as an active ingredient can be taken for a long time as a therapeutic agent for preventing and / or improving metabolic syndrome, which is a lifestyle-related disease. Furthermore, since there is little bitterness, it can be ingested as a functional food by patients with metabolic syndrome for a long time.

Claims (8)

  An adiponectin and GLUT4 expression promoter containing sudachitin as an active ingredient.   A preventive and / or ameliorating agent for metabolic syndrome, comprising the expression promoter of claim 1.   The preventive and / or ameliorating agent for metabolic syndrome according to claim 2, wherein the metabolic syndrome is diabetes.   The preventive and / or ameliorating agent for metabolic syndrome according to claim 2 or 3, wherein the diabetes is type 2 diabetes.   The preventive and / or ameliorating agent for tarotic syndrome according to any one of claims 2 to 4, wherein the prophylactic and / or ameliorating agent for tarolic syndrome is a functional food.   A PPARγ expression promoter based on increasing PPARγ promoter activity, comprising sudachitin as an active ingredient.   A Sirt1 expression promoter containing sudachitin as an active ingredient. A preventive and / or ameliorating agent for metabolic syndrome, comprising the expression promoter of claim 7.