JP5242885B2 - Intramuscular AMPK activator - Google Patents

Description

  The present invention relates to new uses of black tea extract components, especially new uses related to obesity and diabetes.

As the diet becomes richer, "obesity" has become one of the most serious concerns of modern people. Obesity is not only cosmetically unfavorable, but is also known to cause various diseases such as diabetes, arteriosclerosis, hypertriacylglycerolemia, hypercholesterolemia, thrombosis and the like.
Obesity is caused by adipocyte differentiation / hypertrophy or an increase in the number of fat cells itself, and in either case, “glucose uptake” is deeply involved.
Since glucose is a polar substance, a glucose transporter (GLUT) is required for glucose to be taken into each cell from the blood. Currently, 9 or more types of GLUTs have been cloned, and among them, GLUT1 and GLUT4 are mainly expressed in adipocytes that are greatly involved in sugar and lipid metabolism in vivo. Among them, GLUT4 is known to play a major role in glucose uptake activity on the adipocyte membrane.
GLUT4 is called insulin-sensitive GLUT and is usually present in intracellular vesicles in adipocytes and muscle cells. When stimulated by insulin, GLUT4 moves (translocates) onto the cell membrane and makes glucose available. The translocation of GLUT4 is triggered by the binding of insulin to the receptor and the phosphorylation of the β subunit of the receptor, followed by phosphorylation of the insulin receptor substrate (IRS), phosphatidylinositol 3 kinase ( The translocation is completed by exocytosis from the intracellular endoplasmic reticulum to the cell membrane via the pathway of activation of PI3K) and activation of Akt / Protein Kinase B. In addition, GLUT4 works to move more glucose into muscle cells, and it has been reported that athletes have more GLUT4 than ordinary people ("Latest carbohydrate loading knowledge" Keio University Sports) Bulletin of Medical Research Center 1996).

Reflecting the increase in obesity, the number of diabetic patients tends to increase. Diabetes is a troublesome disease that not only is difficult to cure once it develops, but is also likely to develop complications.
As complications that specifically develop in diabetic patients, there are diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc., which are also called the three major complications of diabetes. These complications are thought to be due to microangiopathy based on fine vascular lesions. Specifically, the function of an enzyme such as protein kinase C is abnormally enhanced and cell function is decreased, or glucose is chemically bound to a protein such as enzyme due to the persistence of hyperglycemia, and the enzyme function is decreased. It is thought that sorbitol (sugar alcohol) accumulates in cells due to metabolic disorders due to blood sugar and causes cell damage, which causes abnormalities in small blood vessels and blood cells, causing complications .

  Conventionally, the following various proposals and reports have been made on the effects of tea extract and its extract components on such obesity and diabetes.

For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 06-80580) discloses a plasma cholesterol-lowering agent containing polysaccharides (ribose, arabinose and glucose) contained in tea leaves as active ingredients,
In Patent Document 2 (Japanese Patent Laid-Open No. 10-158181), a lipolysis promoter containing a plant extract extracted from tea promotes the reduction of systemic or local adipose tissue to improve obesity constitution, suppression of obesity, Disclosed that it is effective in prevention,
Patent Document 3 (Japanese Patent Application Laid-Open No. 11-302168) discloses that a glucose absorption inhibitor containing epicatechin gallate in a tea extract as an active ingredient suppresses glucose absorption in the intestine and treats obesity, diabetes, and the like. Is disclosed to be effective,
Patent Document 4 (Japanese Patent Application Laid-Open No. 2003-81825) discloses a glucose transporter 4 expression promoter composed of catechins and a packaged beverage for diabetes prevention / improvement,
Patent Document 5 (Japanese Patent Application Laid-Open No. 2003-95942) discloses a glucose uptake inhibitor and an insulin-stimulated responsive glucose uptake in adipocytes containing any one of catechin gallate, catechin with gallate ester, and tea extract as an active ingredient. Inhibitors, GLUT4 translocation inhibitors and fat-reduced foods and drinks are disclosed.

Japanese Patent Laid-Open No. 06-80580 JP-A-10-158181 JP-A-11-302168 JP 2003-81825 A Japanese Patent Laid-Open No. 2003-95942

  The present inventor has earnestly studied the influence of black tea extract ingredients on obesity and diabetes, and based on various findings obtained as a result, new uses of black tea extract ingredients, particularly new uses related to obesity and diabetes. This is a proposal.

The present invention relates to a glucose uptake activator in muscle cells, a GLUT4 translocation activator in muscle cells, a GLUT4 translocation inhibitor or a diabetic complication preventive agent in fat cells, and a The tea extract component includes theaflavin (TF), theaflavin-3-gallate (TF3-G), and theaflavin- ^3′ -gallate (TF3′-). G) and theaflavin-3,3′-digallate (TF3,3′-G) are characterized in that they contain theaflavins which are mixed components.
In the present invention, food and drink are meant to include both beverages and foods.

If the active ingredient of the present invention is ingested, glucose uptake in fat cells, particularly glucose uptake caused by insulin stimulation can be suppressed, and the amount of fat cells can be reduced. This makes it possible to treat and prevent various diseases such as an excess fat state, that is, obesity, and diseases such as diabetes associated with obesity, arteriosclerosis, hypertriacylglycerolemia, hypercholesterolemia, and thrombosis.
As a mechanism for inhibiting glucose uptake activity in adipocytes, various mechanisms such as suppression of translocation of GLUT4 present in adipocytes or binding to an insulin receptor can be considered. Based on this finding, the inventors have devised a GLUT4 translocation inhibitor in adipocytes and foods and drinks containing the same based on such findings, elucidating that the active ingredient specifically inhibits GLUT4 translocation. Is.

  The active ingredient of the present invention not only suppresses glucose uptake in fat cells, but also reversely activates glucose uptake in muscle cells, allowing excess glucose to be taken up and consumed by muscle cells. That is, the present invention is based on a combination of two or more of the group consisting of a glucose uptake activator in muscle cells, a GLUT4 translocation activator in muscle cells, a GLUT4 translocation inhibitor in adipocytes and a diabetic complication preventive agent. It can provide also as a chemical | medical agent with the use which becomes. Therefore, if the active ingredient of the present invention is administered or ingested, the blood sugar concentration will not be increased, there will be no malaise associated with obesity prevention, and the activity power can be improved. That is, if the active ingredient of the present invention is ingested, GLUT4 translocation in muscle cells can be activated, and glucose uptake activity in muscle cells can be increased. As a result, muscle cells can take in many energy sources, Muscle cell mass can be increased. Therefore, it can be effectively used for activation of muscle tissue, reduction of physical fatigue, improvement of exercise ability, enhancement / increase of muscle tissue, and subsequent remodeling, and the glucose uptake activator in muscle cells of the present invention Can also be used as a GLUT4 translocation activator or muscle activator in muscle cells.

  In addition, in this study, as a result of examining the effects of black tea extract components on diabetic rats induced by streptozotocin (STZ) administration, administration of black tea extract components to diabetic rats reduced oxidative stress and improved fat metabolism. Through these functions, it is possible to obtain knowledge that diabetic complications, ie diabetic retinopathy, diabetic nephropathy, diabetic neuropathy can be prevented, and based on such knowledge, tea extract components The present invention proposes a preventive agent for diabetic complications and a food and drink containing the same.

  Furthermore, the black tea extract component can reduce the plasma free fatty acid content by activating AMPK in muscle, maintaining a good balance of adipocytokine secretion, and promoting fatty acid combustion (β oxidation). The present invention has been newly confirmed, and the present invention also proposes an intramuscular AMPK activator, an adipocytokine secretion balance regulator, or a fatty acid combustion (β-oxidation) promoter, which contains black tea extract as an active ingredient.

In addition, it has been confirmed that the active ingredient of the present invention is effective even if taken before or after insulin secretion. Therefore, the active ingredient of the present invention can be effective even if taken before, at the same time as or after food intake.
Moreover, since the active ingredient of the present invention is an ingredient derived from black tea that has been drunk on a daily basis and can be safely consumed by anyone since long ago, it can be taken comfortably and safely for a long period of time. It is particularly effective for the fundamental treatment and prevention of diseases, as well as for improving the constitution.

  Hereinafter, although the best embodiment for carrying out the present invention is described, the embodiment of the present invention is not limited to the following examples.

  “Glucose uptake activator in muscle cells”, “GLUT4 translocation activator in muscle cells”, “GLUT4 translocation inhibitor in adipocytes”, “preventive agent for diabetic complications”, “intramuscular AMPK activity” The “agent”, “adipocytokine secretion balance regulator” and “fatty acid combustion (β-oxidation) promoter” can all be produced by blending black tea extract components in appropriate concentrations.

  Black tea means so-called black tea in general, which is obtained by completely fermenting tea leaves, regardless of production area, variety, tea leaf grade, etc. For example, it may be any of Darjeeling, Assam, Nilgiri, Uba, Dimbra, Nuara, etc., and any of leaf grade, broken grade, and other tea leaf grades, Uba species are preferred.

  The black tea extract component can be obtained by extracting the extracted black tea leaf as it is or by pulverizing it with water, hot water or hot water, preferably hot water of 40 ° C to 100 ° C, particularly hot water of 90 ° C to 100 ° C. .

Examples of the components in the tea extract component include tannin (polyphenol), amino acids, caffeine, saccharides, saponin, etc. Among them, tannin (polyphenol) is the main component.
The polyphenol in the black tea extract component is characterized in that the catechins are reduced to 30 to 40% as compared with the green tea extract, and the oxidative polymer of catechins is correspondingly increased. This is because the components are oxidized or polymerized in the fermentation process of tea leaves, and theaflavins (TF (; Theaflavin, the following [Chemical Formula 1]), which is an oxidative polymer of catechins,
TF3-G (; Theaflavin-3-gallate, the following [Chemical Formula 2]),
TF3′-G (; Theaflavin- ^3′- gallate, the following [Chemical Formula 3]),
TF3,3′-G (including Theaflavin-3 ^′ , ^3′- digallate, the following [Chemical Formula 4])), thealvidin, and other catechins are characteristically characterized by polyphenols in tea extract components. Theaflavins containing TF, TF3-G, TF3′-G and TF3,3′-G are important as the active ingredient of the present invention.

  The black tea extract component used in the present invention can be the black tea extract obtained by extracting black tea leaf as described above as the active ingredient, but the black tea extract is concentrated or dried to obtain the black tea extract. In particular, a black tea extract in which the concentration of theaflavins is further increased can be used as an active ingredient.

  In addition, the black tea extract component can be used alone as an active ingredient, but already or in the future, “a glucose uptake activator in muscle cells”, “a GLUT4 translocation activator in muscle cells”, “GLUT4 in fat cells” Effective as an active ingredient such as "translocation inhibitor", "diabetic complication preventive agent", "intramuscular AMPK activator", "adipocytokine secretion balance regulator" and "fatty acid combustion (β oxidation) promoter" It is also possible to use this mixed component as an active ingredient by mixing it with a component in which water is recognized, such as green tea extract, especially catechin.

In this case, preferable examples of the green tea extract include a green tea extract (trade name: Teaflan 30A, manufactured by ITO EN), which is obtained by subjecting the green tea to hot water extraction treatment and drying the extract to a catechin concentration of about 30%. In order to remove components other than catechins, this extract was treated by a column method and dried to obtain a green tea extract having a tea polyphenol concentration of about 85 to 95% (trade name: Teafuran manufactured by ITO EN). 90S).
Further, as catechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-catechin gallate, (-)-gallocatechin gallate, or a polymer of any of these, Or the copolymer of 2 or more types of these, or the mixture of 2 or more types of these can be mentioned.

  When the black tea extract component is used alone as an active ingredient, for example, the black tea extract is dissolved in purified water or physiological saline and provided as a drug (for example, an oral administration agent, an intraperitoneal administration agent, an intracerebral administration agent, etc.). be able to.

“Glucose uptake activator in muscle cells”, “GLUT4 translocation activator in muscle cells”, “GLUT4 translocation inhibitor in adipocytes”, “preventive agent for diabetic complications”, “intramuscular AMPK activity” The “adjuvant”, “adipocytokine secretion balance regulator” and “fatty acid combustion (β-oxidation) promoter” are all oral or parenteral agents (intramuscular, intravenous, subcutaneous, rectal, Skin administration, nasal administration, etc.), and it is preferable to have a formulation and dosage form suitable for each administration.
The dosage form can be prepared in the form of liquids, tablets, powders, granules, dragees, capsules, suspensions, emulsions, pills, etc. for oral administration, and injection for parenteral administration. Preparations, ampoules, rectal administration agents, oily suppositories, water-soluble suppositories and the like.
For compounding (formulations), commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives It can be produced by conventional methods using agents, flavoring agents, soothing agents, stabilizers and the like. Also, for example, lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or salts thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene Non-toxic additives such as glycol, citric acid, sodium chloride, sodium sulfite, and sodium phosphate can be blended.

“Glucose uptake activator in muscle cells”, “GLUT4 translocation activator in muscle cells”, “GLUT4 translocation inhibitor in adipocytes”, “preventive agent for diabetic complications”, “intramuscular AMPK activity” Agents, Adipocytokine Secretion Balance Modifiers, and Fatty Acid Combustion (β-Oxidation Accelerators) are all drugs, quasi-drugs, health foods / health drinks / special health It can also be provided as a food for food, functional food, and other drugs and feeds for non-human animals.
For example, by preparing as a quasi-drug and making it into a drinking form such as a bottled drink or a form such as a tablet, capsule, granule, etc., it can be made easier to ingest.
In addition, in the case of a health food / health drink / food for specific health / functional food with a pharmacological effect, for example, the active ingredient of the present invention includes carbonic acid, an excipient (including a granulating agent), a diluent, Or further mixed with one or two or more kinds selected from sweets, flavors, flours, starches, sugars, fats and other proteins, food materials such as carbohydrate raw materials, vitamins and minerals, or Currently known foods and beverages such as sports drinks, fruit drinks, milk drinks, tea drinks, vegetable juices, dairy drinks, alcoholic drinks, jelly, jelly drinks, carbonated drinks, chewing gum, chocolate, candy, biscuits, snacks, bread, milk It can be produced by adding to products, fish paste products, livestock meat products, frozen desserts, dried foods, supplements, etc.
Among them, foods and drinks made by adding tea extract ingredients to foods and drinks rich in saccharides have both a glucose uptake activity effect in muscle cells and a GLUT4 translocation inhibitory effect in fat cells. It can be provided as reduced food or drink (in other words, diet food or drink), muscle activated food or drink, and the like.

The content of the “tea extract component” which is an active ingredient of the present invention varies depending on the method of use, but in the case of a pharmaceutical product, it is 0.001 to 1% by weight, particularly 0.01 to 0.00% in terms of dry weight of theaflavin. It is preferable to mix 5% by weight, and it is preferable to mix 0.001 to 1% by weight, particularly 0.01 to 0.5% by weight in terms of dry weight of theaflavin for foods and drinks.
When preparing as food and drink, it is preferable to blend the tea extract component in the food and drink at a concentration of 0.001 to 1% by weight and prepare a concentration of 5 to 500 times that of tea normally drunk in terms of dry weight of theaflavin.
In addition, the intake is 10 to 5000 mg per day in terms of dry weight of theaflavin, preferably about 100 to 1500 mg.

<Test 1>
In this study, changes in glucose uptake in blood components and adipose tissue and muscle tissue were examined in order to examine changes in sugar metabolism and lipid metabolism when rats freely ingested black tea extract components.

  In addition, all animal experiments in Tests 1 to 4 including this test are related to “Standards for the breeding and storage of experimental animals” (Notification No. 6 of the Prime Minister's Office in March 1980) and “Animal Experiments at Kobe University” The test was conducted according to the “Guidelines”.

(Experimental animal)
For the test, 24 Wistar / ST male rats (manufactured by SLC Japan), 6 weeks old, were used. These were divided into 4 groups of 6 animals, and after acclimatization for 7 days, respectively (1) black tea extract + streptozotocin (STZ) administration (T-STZ), (2) black tea extract only administration (T-CON), (3) Water + STZ administration (W-STZ), (4) Water-only administration (W-CON) was performed.
(1) Black tea extract for group (2), distilled water as a control for groups (3) and (4), and free intake for 35 days from the start of breeding until sacrifice. I let you. During this time, both water and black tea were changed every day, and the feed was a commercially available solid feed.

(Black tea extract)
20 g of tea leaves (variety: Uba) were extracted with 1 L of ion-exchanged water at 95 ° C. for 2 minutes, and the resulting extract was filtered through a flannel filter cloth to obtain a black tea extract (hereinafter simply referred to as “tea”). .
The amount of theaflavins (TFs) in the obtained black tea extract was measured by high performance liquid chromatography and is shown in Table 1 below. Each value shown in Table 1 is an average value of three measurement results.

(Experimental operation)
On the 7th day after the start of breeding, streptozotocin (STZ; manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 0.05 M citrate buffer (pH 4.5) was added to T-STZ group and W-STZ group to 40 mg / kg / body weight. As shown, the T-CON group and W-CON were administered with citrate buffer only from the tail vein as a control to induce the onset of type I diabetes. Thereafter, the blood glucose concentration after fasting for 12 hours was measured twice a week until sacrifice, a total of 8 times, and the transition of the steady-state blood glucose level was observed.
On the 31st day of breeding, D-glucose was orally loaded to 2 g / kg · body weight, and a glucose tolerance test (Oral Glucose Tolerance Test: OGTT) was performed. 30 minutes later, the blood was sacrificed by blood sampling, and the liver, kidney, pancreas, thigh muscle, and epididymal adipose tissue were removed, and epithelial cells were collected from the small intestine.

(Measurement)
(1. Measurement of steady-state blood glucose level)
Fasting blood glucose levels before and after STZ administration (day 0) and 3, 7, 10, 14, 17, 20, 24, and 28 days after administration were measured, and blood glucose regulation during the breeding period was observed. For measurement, Dexter ZII (manufactured by Sankyo Co., Ltd.), a blood glucose measuring device, was used to measure the glucose concentration in the tail vein blood of rats that had been fasted for 12 hours, and the results are shown in FIG.

(2. Measurement of OGTT)
On the 31st day after the start of the breeding, the rats that were fasted for 12 hours were orally administered with a sonde needle so that the D-glucose aqueous solution was 2 g / kg · body weight, before sugar loading (0 minutes), and after loading 15 30, 60, 90, 120, 180 minutes later, blood was collected from the tip of the tail of the rat and collected in an Eppendorf tube. Serum was prepared from the collected blood, and the glucose concentration in the serum was measured using glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in FIG.

(Measurement of uptake activity of 3.3-O [methyl-3H] -D-glucose (3-OMG))
Immediately after sacrifice, muscle and adipose tissues were collected, and each tissue was cut to about 50 mg. Then, it is immersed in Krebs-Ringer-HEPES buffer (KRH: 50 mM HEPES (manufactured by Sigma Aldrich Japan), pH 7.4, 137 mM NaCl, 4.8 mM KCl, 1.85 mM CaCl ₂ , 1.3 mM MgSO ₄ ), 37 After incubating at 5 ° C. for 5 minutes, 3-OMG (manufactured by Moravek Biochemicals, Inc.) was added to a final concentration of 6.5 mM and 0.5 μCi. Two minutes later, each tissue was washed 6 times with KRH, solubilized in a vial using NCS II Tissue Solubilizer (Amersham Biosciences), added with Clearsol I, and then converted into 3-OMG tissue using a liquid scintillation counter. The amount of uptake was measured, the amount of uptake in muscle tissue was shown in FIG. 7, and the amount of uptake in fat tissue was shown in FIG.

(4. Measurement of lipid peroxide)
Thiobarbituric acid reactive substances (TBARS) in serum, liver, kidney and pancreas were measured and evaluated.
Each organ was homogenized with 4 times the volume of phosphate buffer (PBS (-): 10 mM Na phosphate, pH 7.4, 0.9% NaCl) under ice-cooling using a Polytron homogenizer. For organ homogenate and serum, 0.8% 2,6-tert-butyl-p-cresol (BHT) acetic acid solution, 8.1% sodium lauryl sulfate (SDS), 20% acetic acid solution (pH 3.5), 0.8 % 2-Thiobarbitur aqueous solution (TBA reagent) was added and the mixture was allowed to stand at 5 ° C. for 60 minutes. Furthermore, after incubating in a boiling bath for 60 minutes, after adding water and butanol / pyridine mixed solution (15: 1, v / v) to the place cooled to room temperature and stirring, the reaction mixture is centrifuged at 3000 rpm for 5 minutes, The fluorescence intensity (Ex535 nm-Em553 nm) of the supernatant was measured. Tetraethoxypropane was used as a standard substance for lipid peroxide.
The organ homogenate was determined by lowry method using bovine serum albumin (BSA; bovine serum albumin) as a standard substance, and the amount of TBARS was determined by dividing the amount of protein by the amount of malondialdehyde.

(5. Measurement of serum lipids)
Triglyceride amount, total cholesterol amount, free cholesterol amount, free fatty acid amount, HDL-cholesterol amount in serum collected from the heart at the time of sacrifice were triglyceride G-Test Wako, Cholesterol E-Test Wako, NEFA C-Test Wako, HDL- Cholesterol test Wako and free cholesterol E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) were used for measurement. The amount of LDL-cholesterol was fractionated using an ultracentrifugation fractionation method, and the fraction was determined by measuring with cholesterol E-Test Wako.

(6. Measurement of small intestinal disaccharide hydrolase activity)
Seven times volume of 1.15% KCl was added to the collected small intestinal cell mass and homogenized under ice-cooling. Sucrose and maltose were dissolved in a maleic acid buffer solution (50 mM, pH 6.0) to a concentration of 0.1 M to prepare a sucrose solution and a maltose solution, respectively. A sucrose solution was added to the prepared homogenate, and sucrose, which is a sucrose-degrading enzyme, was reacted in a warm bath at 37 ° C. for 0, 10, 20, and 40 minutes. Similarly, the maltose solution was reacted with maltase for 0, 5, 10, and 20 minutes. Thereafter, the enzyme reaction is stopped by incubating in a boiling bath for 5 minutes, cooled to room temperature, centrifuged at 3000 rpm for 10 minutes, and the amount of glucose in the collected supernatant was measured using Glucose CII-Test Wako. did. The amount of protein in the homogenate was determined by the Lowry method, and the amount of glucose was divided by the amount of protein to determine the sucrase activity and maltase activity per minute. The results are shown in FIGS. 10 and 11, respectively.

(7. Measurement of fructosamine)
Nitroblue tetrazolium (NBT) was dissolved in a sodium carbonate buffer solution (200 mM, pH 10.3) to a concentration of 0.48 mM to obtain an NBT solution. To the serum collected at the time of sacrifice, an NBT solution in which uricase (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved to 2.5 kU / L was added and reacted at 37 ° C. From the difference in absorbance at 530 nm after 10 minutes and 15 minutes after the start of the reaction, the amount of fructosamine was determined using a fructosamine calibrator (manufactured by Sigma Diagnostics) as a standard substance for fructosamine. The results are shown in FIG.

(8. Measurement of insulin)
Rats fasted for 12 hours were orally administered with a sonde needle so that the D-glucose aqueous solution was 2 g / kg · body weight, and the amount of insulin in the serum obtained by cardiac blood sampling after 30 minutes of glucose loading was determined to be Mercodia Rat. Insulin ELISA kit (manufactured by Funakoshi) was used for measurement, and the results are shown in FIG.

(Statistical analysis)
The experimental results were compared using Student's t-test, and the case where the p-value was 0.05 or less was considered significant.

(Results and discussion)
(1. Changes in body weight, food intake, water intake)
The body weight among the four groups at the start of breeding was almost the same and almost the same. Thereafter, in the two groups administered STZ on the seventh day of breeding, characteristic symptoms of the onset of diabetes such as weight loss, increased food intake, and increased water consumption were observed immediately after (FIGS. 1 to 3). These two groups show that administration of STZ induced the onset of type I diabetes. Further, when these two groups were compared, these three symptoms were more remarkable in the water administration group than in the black tea administration group. On the other hand, between the two groups not administered with STZ, no difference was observed between black tea administration and water administration, and both showed a steady weight gain throughout the breeding period.

(2. Transition of steady-state blood glucose level)
On the day of STZ administration (corresponding to day 0 in FIG. 4), the blood glucose level between the 4 groups was almost the same, but in the 2 groups administered with STZ, the blood glucose level significantly increased due to the onset of diabetes. A rise was induced. However, between these two groups, the increase in blood glucose level was significantly suppressed by the administration of black tea, and decreased to the control level with the passage of the breeding days. On the other hand, no change was observed in the control group not administered with STZ due to black tea administration or water administration (FIG. 4).

(3. Blood glucose level change in OGTT)
In both of the two groups that developed diabetes, the blood glucose level increased rapidly after sugar loading, and the black tea group showed significantly lower values than the water group at 0, 15, and 30 minutes. However, after that, there was no significant difference between the two groups. On the other hand, in the control group, no significant difference was observed between the water administration and black tea administration groups (FIG. 5).

(4. Effect of black tea on fructosamine content)
The amount of fructosamine, a glycated protein in the blood, was significantly increased in the diabetic group compared to the control group. However, the increase was significantly suppressed by black tea administration. In the control group, there was almost no difference between water administration and black tea administration (FIG. 6).

(5. Changes in organ weight)
The proportion of liver, kidney, pancreas and epididymal adipose tissue occupying the body weight in the control group (corresponding to Non-Diabetic in Table 2) was not different between the two groups due to water or black tea. It was. On the other hand, the diabetic group (corresponding to Diabetic in Table 2) showed significantly higher values in the liver and kidney and lower values in the adipose tissue than the control group. However, the increase in the weight ratio of liver and kidney organs due to diabetes was significantly suppressed in the black tea administration group as compared to the water administration group (see Table 2).

(6.3-OMG uptake)
3-OMG uptake activity of muscle and adipose tissue, which are insulin sensitive tissues, was measured.
Compared with the control black tea administration group and the water administration group, no significant difference was observed in the adipose tissue (FIG. 8), but in the muscle tissue, the uptake amount was significantly increased by the black tea administration (FIG. 7).
On the other hand, there was no difference between the two diabetic groups. Moreover, the diabetes group showed a high value compared with the control group (FIGS. 7-8).

(7. Effect of tea on TBARS content)
When the amount of lipid peroxide in the diabetic group was compared with that in the control group, there was no difference between the liver and kidney, but it increased in serum and decreased in pancreas. However, the increase in the amount of serum lipid peroxide in this diabetic group was significantly reduced by the administration of black tea. There was no significant difference in serum and lipid peroxide levels in the serum and each organ between the control water administration group and the black tea administration group (Table 3).

(8. Effect of black tea on insulin content)
The amount of insulin, which is the only hormone in the body that has a function of lowering blood glucose levels, was significantly lower in the diabetic group than in the control group. The decrease in insulin in this diabetic group was significantly suppressed by the administration of black tea, and the amount of insulin was high. Furthermore, also in the control group, the black tea administration showed about three times the amount of insulin compared to the water administration (FIG. 9).

(9. Small intestine disaccharide hydrolase activity)
It was found that the sucrase activity was significantly increased in the diabetic group as compared with the control group, and in the case of diabetes, the activity was significantly decreased by black tea administration. On the other hand, there was no difference between the control water administration group and the black tea administration group (FIG. 10).
With regard to maltase activity, both the diabetic group and the control group showed a significant decrease in activity due to black tea administration. In addition, the activity of the diabetic black tea administration group was low as compared with the control water administration group (FIG. 11).

(10. Serum lipids)
The amount of free fatty acid (NEFA) was significantly lower after administration of black tea in both the diabetic group and the control group. Compared with the control water administration group, the diabetic water administration group showed a significantly high value, whereas the diabetic black tea administration group showed a significantly low value, which was similar to the control black tea administration group.
Neutral fat (Triacylglycerol) showed a significantly high value in the diabetic group compared to the control group, and the diabetic black tea-administered group showed a significantly low value of about one third of the water-administered group. On the other hand, there was no difference between the control water administration group and the black tea administration group.
Total cholesterol was significantly higher in the diabetic group than in the control group. There was no change in each group due to water and black tea.
Free cholesterol was significantly higher in the diabetic group than in the control group. Although there was no significant difference between the diabetic water administration group and the black tea administration group, the control group showed a significant increase by the black tea administration compared to the water administration.
HDL cholesterol was almost the same as the diabetic black tea administration group, with no difference between the control water administration group and the black tea administration group. On the other hand, the diabetic water administration group showed a significantly lower value compared to the control group.
LDL cholesterol was significantly higher in the diabetic group than in the control group, but no change was observed in both groups due to water administration and black tea administration (Table 4).

<Test 2>
In this study, in order to investigate the effect of rats fed either high-fat diet or normal diet with free intake of black tea extract component or green tea extract component, blood cholesterol, blood glucose level, glucose in fat cells and muscle cells were examined. Uptake activity and leptin adiponectin levels in blood were measured and compared.

[Test plan]
4-week-old male C57BL / 6J mice were purchased from Japan SLC. The breeding environment was 23 ± 3 ° C., and the lighting time was 9 o'clock to 21 o'clock. After 1 week of preliminary breeding, drinking group (HF-W), green tea drinking group (HF-G), tea drinking group (HF-B), and drinking group (C-W) ), Green tea drinking group (CG) and black tea drinking group (CB), and further, 6 groups of 5 each were reared in a total of 12 groups of 7 weeks and 14 weeks. In addition, it divided into groups so that each body weight might become equivalent, and the feed was given with the composition of the following Table 5.
On the last day of the breeding period, the animals were sacrificed by cardiac blood sampling, and muscle, fat, liver, and serum were collected.

(Green tea extract component)
Green tea (variety: Motoyama tea from Shizuoka) was extracted with 20 g / L, ion exchange water at 95 ° C. for 2 minutes, and the resulting extract was filtered through a flannel filter cloth to obtain a green tea extract (hereinafter simply “green tea”). This was taken by the green tea drinking group (HF-G) and the green tea drinking group (CG).

(Black tea extract component)
Extract tea leaves of tea (variety: Uba) with 20g / L, ion-exchanged water at 95 ° C for 2 minutes, and filter the resulting extract with flannel filter cloth to make a black tea extract (hereinafter simply referred to as "tea"). This was ingested by the tea drinking group (HF-B) and the tea drinking group (CB).

[experimental method]
(Measure blood sugar and blood components)
Fasting blood glucose at 1, 5, 7, 10, 12, and 14 weeks after the start of breeding was measured with a commercially available kit.
In the glucose tolerance test, after glucose fasting for 18 hours, a glucose load was applied at 2 g / kg · body weight, and blood glucose levels at 0, 15, 30, 60 and 120 minutes were measured with a commercially available kit.
Blood lipid components (T-cholesterol, F-cholesterol, HDL-cholesterol, triglyceride) were similarly measured using a commercially available kit.
LDL-cholesterol was calculated by the Friedewald method.
Mice were sacrificed by blood sampling at 7 and 14 weeks after the start of breeding, and glucose uptake activity was measured using the extracted fat and muscle.

Glucose uptake activity was measured by cutting the extracted muscle and fat into sections of about 15 mg, and treating each tissue with Krebs-Ringer-HEPES buffer (KRH: 50 mM HEPES, pH 7.4, 137 mM NaCl, 4.8 mM KCl). , 1.85 mM CaCl ₂ , 1.3 mM MgSO ₄ ) for 10 minutes at 37 ° C. Next, 3-0-methyl- [ ³ H] -D-glucose (3-OMG) was added thereto so as to be 6.5 mM and 0.5 μCi, and allowed to act for 2 minutes. In order to stop the uptake, the reaction mixture was immediately removed by aspiration, and the tissue was quickly washed 6 times with ice-cooled KRH, then water was wiped off with filter paper, and the tissue was solubilized with NCSII. The radioactivity of 3-OMG incorporated by adding a scintillation cocktail to the solubilized solution was measured with a liquid scintillation counter. The non-specific uptake amount was determined by treating the tissue with 20 μM cytochalasin B, an inhibitor of glucose transporter, and measuring the uptake activity in the same manner as described above.

[result]
(Weight change)
From FIG. 12 and FIG. 13, it was confirmed that the long-term intake of the green tea extract component and the black tea extract component at the time of intake of a high fat diet suppressed the increase in body weight.
In the normal diet, there was no difference in body weight in any of the intake groups of water, green tea and black tea. On the other hand, in the high fat diet, a significant difference was observed from the 7th week in the black tea intake group, and a difference was also observed in the green tea at the 8th week.
Note that “a” and “b” in FIGS. 12 and 13 indicate that there is a significant difference of less than 5% between the green tea intake group and the black tea intake group by the Student's t-test for each water intake group.

(Tissue weight)
From Table 6 and Table 7 below, it was confirmed that the intake of the green tea extract component and the black tea extract component reduced the fat weight. That is, in the seventh week after breeding, the intake of green tea and black tea in the normal diet significantly suppressed the increase in fat weight, and that in the high fat diet is not only the increase in the total fat weight but also the increase in each fat site. Suppressed to food level. In the 14th week, the intake of green tea and black tea suppressed the increase in fat weight in both the normal diet and high fat diet groups, and the ratio was more remarkable than in the 7th week.
In the table below, “ ^* ” indicates that there is a significant difference in the water intake group by less than 5% by Student's t-test.

(Changes in fasting blood glucose over time)
From FIG. 14 and FIG. 15, it was confirmed that an increase in blood glucose level was suppressed by ingestion of the green tea extract component and the black tea extract component. That is, fasting blood glucose was measured with a commercially available kit using blood extracted from the tail vein at 1, 5, 7, 11, and 14 weeks.
Ingestion of green tea extract components and black tea extract components tended to lower blood glucose levels in both high-fat and normal diets. In the green tea drinking group (HF-G) and the black tea drinking group (CB), significant decreases were observed at the 5th and 11th weeks.
In addition, "a" and "b" in FIG.14 and FIG.15 show that there is a significant difference of less than 5% according to Student's t-test between the green tea intake group and the black tea intake group for each water intake group.

(Measurement of serum lipid components)
From Table 8, it was confirmed that the intake of the green tea extract component and the black tea extract component significantly reduced the amount of cholesterol in the blood and also decreased triglycerides by long-term intake. That is, using the serum obtained at the 7th and 14th weeks of breeding, total cholesterol (T-CHO), free cholesterol (F-CHO), HDL-cholesterol (HDL-CHO), triglyceride (TG) with a commercially available kit. ) Was measured, the intake of green tea and black tea significantly reduced the total cholesterol (T-CHO), HDL-cholesterol (HDL-CHO) and LDL-CHO levels in the high fat diet. Long-term intake (14 weeks) also reduced triglycerides (TG) in a high fat diet. From these facts, the intake of the green tea extract component and the black tea extract component has a lipid component-regulating action in serum, and thus an effect of improving lipid metabolism could be confirmed.
In addition, “ ^* ” and “ ^** ” in the table below indicate that there is a significant difference with respect to the water intake group at a risk rate of less than 5% by Student's t-test.

(Glucose tolerance test)
From FIG. 16 (A)-(D), in the glucose tolerance test, it was confirmed that the intake of the green tea extract component or the black tea extract component further promotes the effect of blood glucose level.
Referring to FIGS. 16A to 16D, (A) and (C) (open symbols) are normal diet intake groups, and (B) and (D) (filled symbols) are high fat diet intakes. The result of a group is shown, (A) and (B) show the results of the 5th week of breeding, and (C) and (D) show the results of the 12th week. Each circle is a water intake group, each square is a green tea intake group, and each triangle is a black tea intake group.
In the figure, “a” and “b” indicate that there is a significant difference of less than 5% between the green tea intake group and the black tea intake group according to the Student's t-test for each water intake group.

  In the high-fat diet intake group at the fifth week of breeding, the intake of the green tea extract component and the black tea extract component suppressed the maximum blood glucose level, and a rapid decrease in blood glucose level was observed. In the normal food intake group, significant blood glucose level effects were observed at 60 minutes and 120 minutes. Similarly, in the 12th week of breeding, significant blood glucose level effects were observed at 60 minutes and 120 minutes. From this, it is estimated that ingestion of the green tea extract component and the black tea extract component contributes to the improvement of abnormal glucose tolerance.

(Measurement of glucose uptake activity in tissues)
17A and 17B, in the measurement of glucose uptake activity, it was confirmed that the intake of the green tea extract component and the black tea extract component suppressed the activity with fat and promoted the activity with muscle. 17 (A) and 17 (B), “ ^* ” indicates that there is a significant difference with respect to each water intake group by Student's t-test with a risk rate of less than 5%.

  In adipose tissue, intake of black tea extract and green tea extract suppressed the glucose uptake activity, and in muscle, the glucose uptake activity was significantly increased, particularly in the high fat diet intake group. Therefore, ingestion of black tea extract component and green tea extract component suppresses fat cell hypertrophy by suppressing blood glucose uptake in adipose tissue, and conversely, intake of blood sugar in muscle, the largest tissue in the body, increases It was thought that blood sugar was suppressed.

  Glucose uptake in fat cells and muscle cells is known to be mostly mediated by GLUT4. Considering that the effects of black tea extract components in a high fat diet are particularly excellent as described above, black tea extraction It is suggested that the ingestion of the component suppressed the translocation of GLUT4 present in adipocytes.

(Measurement of blood adipocytokines)
In this measurement, the amounts of leptin and adiponectin secreted by adipocytes in serum were measured with a commercially available ELISA kit, and the results are shown in FIGS. 18 (A) to (D).
In these figures, “ ^* ” and “ ^** ” indicate that there is a significant difference of less than 5% by Student's t-test for the water intake group of the normal diet and the high fat diet, respectively.

When the amount of each adipocytokine in serum is considered based on the value in the water intake group of the normal diet at the 7th week, the normal diet at the 14th week, the 7th and 14th weeks in the water intake group Leptin and adiponectin increased in all high-fat diets.
On the other hand, in the ingestion group of the green tea extract component and the black tea extract component, no change was observed in the normal diet at 14 weeks and the high fat diet at 7 weeks and 14 weeks.
From these results, it was confirmed that the ingestion of green tea and black tea has an inhibitory effect on the increased secretion of leptin and adiponectin accompanying the extension of the breeding period and the increase of adipocytes due to the intake of a high fat diet.

<Test 3>
In this study, the effects of black tea extract components on glucose and lipid metabolism in insulin-sensitive tissues were examined.

[experimental method]
(Experimental animal)
The test was carried out using 6-week-old Wistar / ST male rats (Japan SLC, Inc.).
After acclimatization for 7 days, water administration only (C-W) group, tea administration only (CB) group, STZ + water administration (S-W) group, STZ + tea administration (SB) group Were divided into 5 groups (6 animals in each group).
Distilled water was supplied to the C-W group and SW group, and black tea was supplied to the CB group and SB group, respectively, and allowed to freely ingest for 35 days from the start of breeding until sacrifice. During this time, both water and black tea were changed every day, and the feed was a commercially available solid feed.

(Black tea extract component)
Extract 20 grams of tea leaves (variety: Uba) with 1 liter of ion-exchanged water at 95 ° C. for 2 minutes, and filter the tea leaves with a flannel filter cloth as a tea extract (hereinafter simply referred to as “tea”) in a water supply bottle. Administered.

(Administration of STZ)
On the 7th day from the start of black tea administration (starting on day 0), streptozotocin (STZ, Wako Pure Chemicals) dissolved in 0.05M citrate buffer (pH 4.5) in the SW group and the SB group. As a control, the CW group and the CB group were administered with citrate buffer only from the tail vein to induce the onset of type I diabetes. .
Thereafter, the blood glucose concentration after fasting for 12 hours was measured twice a week until sacrifice, a total of 8 times, and the transition of the steady-state blood glucose level was observed. On the 31st day of breeding, D-glucose was orally loaded at 2 g / kg · body weight, and a glucose tolerance test (Oral Glucose Tolerance Test: OGTT) was performed. Was orally loaded, and 30 minutes later, the blood was sacrificed by blood sampling and subjected to the following experiment.

(Measurement of blood insulin, leptin and adiponectin levels)
Serum insulin level is determined by Mercodia Rat Insulin ELISA kit (Funakoshi Co., Ltd.)
The amount of leptin was measured using a Morinagarat leptin measurement kit (Morinaga Bioscience Institute), and the amount of adiponectin was measured using a mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.).

(Preparation of cell extracts of fat, muscle and liver)
An equal amount of RIPA buffer (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 2.0% (v / v) Nonidet-P40 (registered trademark) (Wako Pure Chemical Industries, Ltd.), 1.0% (w / v) sodium deoxycholate (Wako Pure Chemical Industries, Ltd.), 0.2% (w / v) SDS, 2 mM PMSF, 20 mg / ml leupeptin (Wako Pure Chemical Industries, Ltd.) 2 mM Na ₃ VO ₄ , 100 mM NaF) and solubilized for 2 hours with occasional mixing on ice. Thereafter, the mixture was centrifuged at 19000 × g for 20 minutes at 4 ° C., and the supernatant was used as a cell extract.
After quantifying the amount of protein in this extract by the Lowry method, it was prepared so that the amount of protein was equal, converted to SDS, and subjected to SDS-PAGE using 7.5% polyacrylamide gel. Proteins separated by SDS-PAGE were transferred to a PVDF membrane. In each case, the muscle tissue was used for detection of AMPK-α (5′-AMP-activated protein kinase-α), phospho-AMPK-α, GLUT4 (glucose transporter 4), and PPAR-α (peroxisome proliferator-activated receptor α). Was used for detection of SREBP-1 (sterol regulatory binding protein-1), and liver tissue was used for detection of AMPK-α, phospho-AMPK-α, PPAR-α, and SREBP-1.

(Preparation of cell membrane fraction from muscle tissue)
Cell membrane lysis buffer (10 mM Tris-HCl, pH 7.9, 10 mM KCl, 1.5 mM MgCl ₂ .6H ₂ ) containing 0.1% (v / v) Nonidet-P40 (registered trademark) twice as much as muscle homogenate O, 1 mM PMSF, 0.5 mM DTT, 10 mg / ml leupeptin, 5 mg / ml aprotinin) and solubilized for 10 minutes with occasional mixing on ice. Then, a buffer solution containing Nonidet-P40 (registered trademark) was added to the precipitate obtained by centrifugation at 900 × g for 10 minutes at 4 ° C., mixed well, and centrifuged again at 900 × g for 10 minutes at 4 ° C. To obtain a precipitate. A buffer solution containing 1% (v / v) Nonidet-P40 (registered trademark) was added thereto and mixed well. After standing for 20 minutes on ice, the supernatant obtained by centrifugation at 19000 × g for 20 minutes was obtained as a muscle. A cell membrane extract was obtained.
The amount of protein in this extract was quantified by the Lowry method, prepared so that the amount of protein was equal, converted to SDS, and subjected to SDS-PAGE using 7.5% polyacrylamide gel. The protein separated by SDS-PAGE was transferred to a PVDF membrane (Hybond-P; Amersham Biosciences Ltd.), and the PVDF membrane was used for detection of GLUT4 and IR-β (insulin receptor β).

(Detection of protein expression by Western blotting)
The protein-transferred PVDF membrane is 30 minutes in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.06% (v / v) Tween-20) containing 1% (w / v) skim milk. Blocked. After washing the PVDF membrane with TBST buffer for a total of 4 minutes for 5 minutes, the primary antibodies diluted in TBST buffer containing 1% skim milk (anti-GLUT4 antibody 1: 1000, anti-IR-b antibody 1: 1000, anti-SREBP-1 antibody 1: 1000, anti-PPAR-α antibody 1: 1000 (manufactured by Santa Cruz Biotechnology Inc.), anti-AMPK-α antibody 1: 1000, anti-phospho-AMPK-α antibody 1: 1000 (Cell Signaling Technology, Inc.) was allowed to react for 1 hour at room temperature, and the PVDF membrane was again washed with TBST buffer for 5 minutes for a total of 4 times to wash the secondary antibody labeled with horseradish peroxidase by 0.5%. This was diluted with TBST buffer containing skim milk and allowed to react for 30 minutes, and the PVDF membrane was washed again with TBST buffer in the same manner, followed by antigen-antibody reaction on the membrane. Was developed using ECL plus (Amersham Biosciences Ltd.) and the exposed X-ray film was developed to detect protein expression.

[Experimental result]
(Effects on serum adiponectin and leptin levels)
Blood concentrations of adiponectin (FIG. 19) and leptin (FIG. 20) secreted from adipocytes and deeply involved in the pathology of diabetes were measured. In these figures, “ND” indicates not detectable, “white bar” indicates the water intake group, and “black bar” indicates the black tea intake group.
The amount of adiponectin was significantly decreased in the two groups administered with STZ compared to the control. However, the amount of adiponectin was significantly increased by black tea administration in any of the two groups of the control group and the STZ administration group. In addition, the amount of leptin was below the detection limit in both groups administered with STZ. Between the two control groups, the CB group to which black tea was administered showed significantly higher leptin levels than the C-W group.

(Expression level of AMPK-α and its active phospho-AMPK-α in muscle and liver tissues)
In both liver and muscle tissue, there was no difference in the expression level of AMPK-α that was resident in the 4 groups (FIGS. 21A and 21B). AMPK-α is phosphorylated to become an active form. When the expression level of the phosphorylated AMPK-α (phospho-AMPK-α) was detected (FIGS. 21A and 21B), phospho-AMPK- The expression level of α increased in the CB group in both muscle and liver tissues, and was a significant increase compared to the CW group. When compared between the two groups treated with STZ, the expression level of phospho-AMPK-α in the muscle was significantly decreased by the administration of black tea, and also slightly decreased in the liver.

As a result, it was found that AMPK-α of the muscle is activated by ingesting the black tea extract component. In muscle, it is known that AMPK-α is phosphorylated (activated) by exercise to promote translocation of GLUT4. In addition, the above-mentioned adiponectin and leptin have also been shown to be factors that activate AMPK-α. Therefore, it is considered that in the normal animal water intake group (CW group), increased secretion of adipocytokines may promote AMPK-α activation and lead to translocation of GLUT4. In rats administered with STZ, the increase in the water intake group (SW group) compared to the normal animal water intake group (CW group) is due to the fact that AMPK-α functions as a fuel sensor. The activation is an activation corresponding to a crisis situation, suggesting that the energy supply in the diabetes-tea intake group (SB group) may not be as critical as the water intake group (SW group). . In fact, in the SW group, the plasma free fatty acid showed a high value, which indicates that lipid oxidation is promoted as an alternative energy to replace sugar.
Overall, in normal (healthy animals), the intake of black tea extract maintains a good balance of adipocytokine secretion and promotes fatty acid burning (β oxidation) to reduce plasma free fatty acid content On the other hand, in diabetes-induced rats induced by STZ, all physiological parameters of sugar metabolism and lipid metabolism are inclined toward energy consumption, and it was confirmed that the tea extract component leads to normalization of modulation by STZ. It was.

(PPAR-α expression level in muscle and liver tissue)
When the amount of PPAR-α protein expression in the liver and muscle, which are insulin-sensitive tissues, was detected, the amount of PPAR-α expression in muscle was clearly increased in the CB group compared to the C-W group ( 22A and 22B). The SW group had the highest expression level among the 4 groups, but the SB group was comparable to the CB group. The expression level of PPAR-α in the liver was almost the same in the CW group and the CB group, but increased in the two groups administered with STZ (FIGS. 22A and 22B). In the SW group, the expression level was remarkably increased.

The above-mentioned adiponectin is known to induce fatty acid oxidation not only through activation of AMPK-α but also through activation of PPAR-α. PPAR-α forms a heterodimer with RXR and acts as a transcription factor. Here, α type expressed in muscle and adipose tissue involved in fatty acid β oxidation was examined (γ type in fat).
In the case of normal animals, the expression level of PPAR-α is clearly increased in muscle in the black tea intake group (C-B group) compared to the water intake group (C-W group), and this expression is induced by adiponectin, resulting in β-oxidation. It was thought that it led to an increase.
In the case of diabetic animals, as described above, STZ administration causes β-oxidation in an attempt to supply alternative energy via fatty acid oxidation because of insufficient energy supply by glucose to insulin-sensitive tissues as in AMPK-α. The protein expression of PPAR-α, which governs the expression of various enzymes involved in the increase, increased, and this action was alleviated in the black tea intake group (SB group).

(Influence on expression level of GLUT4 transferred to cells and their cell membranes)
There was no difference in the expression level of GLUT4 resident in muscle cells among the 4 groups (FIG. 23 (A)). Therefore, when the expression level of GLUT4 transferred to the cell membrane having the ability to take glucose into the cells was verified among the four groups, the GLUT4 transfer to the cell membrane was almost less in the two groups administered with STZ than in the control group. It was not recognized (FIG. 23 (B)). When compared between the two STZ-administered groups, a slight shift to the cell membrane was observed in the SB group compared to the SW group, which was hardly detectable. Further, when the two control groups were compared, it was found that the transfer of GLUT4 to the cell membrane was promoted more in the CB group than in the C-W group. On the other hand, the expression level of insulin receptor (IR-β) present on the cell membrane was not different among the four groups (FIG. 23B). From these facts, in both the control group and the STZ group, the expression level of GLUT4 transferred from the intracellular endoplasmic reticulum to the cell membrane was increased by black tea administration.

(Expression level of SREBP-1 in fat and liver tissue)
The protein expression level of SREBP-1 in adipose tissue, which is an organ having lipid synthesis ability, and liver was detected (FIGS. 24A and 24B).
The expression level of SREBP-1 in the adipose tissue was decreased in the CB group and significantly increased in the SW group as compared to the C-W group (FIG. 24A). The SB group also showed an increasing tendency compared to the C-W group. When compared between the two groups administered with STZ, the SB group showed a clear decrease in the expression level compared to the SW group.
The expression level of SREBP-1 in the liver was confirmed to be slightly decreased in the CB group compared to the C-W group, and the expression levels in the SW group and SB group were increased ( FIG. 24 (B)). In particular, the increase in the SW group was remarkable, but this decrease in expression was observed in the SB group compared to the SW group.

In tissues with fatty acid synthesis systems, such as liver and adipose tissue, the activity of fatty acid synthesis enzymes increases with the excess supply of sugar-derived energy, and the fatty acids are synthesized to convert energy into triglycerides and accumulate them. It is known. SREBPs control the synthesis of fatty acids (SREBP-1) and cholesterol (SREBP-2).
As described above, in the case of normal animals, there was no significant change between the black tea intake group (C-B group) and the water intake group (C-W group), but the expression level tended to decrease particularly in adipose tissue. This may be due to a decrease in sugar influx into the adipose tissue.
In the case of diabetic animals, the water intake group (SW group) increased significantly compared to normal animals (CW group). In the liver of an animal whose insulin secretory ability has already been extremely reduced by STZ, the expression of SREBP-1 is induced by the rapid influx of carbohydrates due to hyperglycemia, and that glucose is involved in the expression induction. It is speculated that hyperglycemia due to rapid glucose influx after fasting for 12 hours was involved in SREBP-1 expression.

[Summary]
It was suggested that administration of the black tea extract component alleviates the decrease in insulin secretion due to STZ in diabetic model rats. This could be verified from the fact that GLUT4 transferred to the cell membrane by stimulation with insulin could be detected in the black tea intake group and that the secretion of leptin and adiponectin from adipocytes was maintained by reducing the decrease in adipose tissue. Furthermore, since the decrease in the expression of phosphorylated AMPK-a, PPAR-α, and SREBP-1 was observed with black tea administration, it was considered that the energy deficiency in the tissue resulting from insulin deficiency was suppressed.
On the other hand, in control rats that did not induce diabetes, expression of phosphorylated AMPK-α was increased by black tea administration, and as a result, an increase in GLUT4 transferred to the cell membrane and a decrease in SREBP-1 were induced. This suggests that β-oxidation of fatty acids is promoted and synthesis is reduced.

<Test 4>
In this study, the effect of black tea extract components on sugar absorption from the small intestine was examined.

[experimental method]
(Experimental animal)
6-week-old Wistar / SD male rats (Japan SLC Co., Ltd.) were used and reared under the same conditions as in Test 2 above, and both water and food were freely consumed during the rearing period. The drinking water was changed every day, and the feed was a commercial solid feed.

(Black tea extract component)
The same black tea extract as in Test 3 was used in the following experiment.

(Glucose tolerance test)
Rats were fasted for 12 hours and 1 ml of black tea extract was orally administered with a sonde needle 5 minutes before loading with various sugars. As a control, distilled water was orally administered in the same manner.
Five minutes after the administration of black tea or water, glucose, sucrose, or an aqueous maltose solution was orally administered using a sonde needle so as to be 2 g / kg · body weight / 1 ml. The blood glucose level was measured using glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.) before sugar loading (0 minutes) and after 15, 30, 60, 90, 120, and 180 minutes after loading.

[Experimental result]
25-26, ○ in each figure (A) indicates a water intake group, ● indicates a black tea intake group, “white bar” in each figure (B) indicates a water intake group, “black” "Bar" indicates the black tea intake group.

(Glucose tolerance test by oral glucose load)
When water or black tea extract was administered to rats 5 minutes before oral glucose loading, no change was observed between the two groups in the blood glucose level for 120 minutes, and no effect on glucose metabolism was observed (Fig. 25 (A)). In addition, no difference was observed between the two groups in the area under the line of the blood sugar level graph (FIG. 25B).

(Glucose tolerance test by oral sucrose loading)
When water or black tea extract was administered 5 minutes prior to oral loading of sucrose to rats, a tendency to suppress an increase in blood glucose level was observed by administering black tea in advance (FIG. 26 (A)). The area under the line of the blood glucose level graph was represented as a bar graph (FIG. 26B). No effect of water or black tea on the change in blood glucose level over 120 minutes was observed.

(Glucose tolerance test by oral maltose load)
Even when water or black tea extract was administered 5 minutes before maltose was orally loaded, an increase in blood glucose level was observed by administering black tea in advance (FIG. 27 (A)). In particular, the tendency was recognized after the 30 minute value. When the area under the line of the blood glucose level graph was represented as a bar graph, the blood glucose level for 120 minutes was significantly suppressed by the black tea administration compared to the water administration (FIG. 27 (B)).

[Summary]
The black tea extract component had no effect on the absorption and glucose metabolism of glucose, a simple sugar, from the intestinal tract. Disaccharides such as sucrose and fructose are hydrolyzed by enzymes in the small intestine and absorbed as monosaccharides. By administering the black tea extract component before the disaccharide was orally administered, there was a tendency to suppress an increase in blood glucose concentration. In particular, when maltose composed of two glucose molecules was orally administered, the blood glucose level for 120 minutes after administration was significantly suppressed. This suggests that the black tea extract component has a moderate effect but inhibits the activity of the disaccharide hydrolase in the small intestine.

It is the graph which showed the change of the body weight of the rat after the tea intake start in the test 1 with time. It is the graph which showed the change of the food intake of the rat after the tea intake start in the test 1 with time. It is the graph which showed the change of the water intake of the rat after the tea intake start in the test 1 with time. 2 is a graph showing changes in steady-state blood glucose level of rats in Test 1 over time. In Test 1, it is the graph which showed change of a blood glucose level in OGTT with time. 2 is a graph showing the amount of blood fructosamine at the end of Test 1. 3 is a graph showing the amount of 3-OMG uptake in muscle tissue in Test 1. FIG. 3 is a graph showing the amount of 3-OMG uptake in adipose tissue in Test 1. FIG. 3 is a graph showing the amount of serum insulin in Test 1. In Test 1, it is a graph which shows the influence which the black tea extraction component has on the hydrolytic activity of sucrase in a small intestine. In Test 1, it is a graph which shows the influence which the black tea extraction component has on the hydrolase activity of maltase in a small intestine. In Test 2, it is the graph which showed the body weight change of the high fat diet-fed rat over time. In the test 2, it is the graph which showed the weight change of the normal food breeding rat with time. In Test 2, it is the graph which showed with time the fasting blood glucose of the high fat diet-fed rat. In Test 2, it is the graph which showed with time the fasting blood glucose of a normal food breeding rat. It is the graph which showed the result of the glucose tolerance test in Test 2, (A) is the glucose tolerance test result of the normal food breeding of the fifth week of breeding, (B) is the high fat diet breeding of the fifth week of breeding The results of the glucose tolerance test, (C) shows the glucose tolerance test result of the normal food breeding at 12 weeks of breeding, and (D) shows the glucose tolerance test result of the high fat diet breeding of the 12th week of breeding. In Test 2, it is a graph which shows the result of having measured the glucose uptake activity in a structure | tissue, (A) shows the glucose uptake activity in an adipocyte tissue, (B) shows the glucose uptake activity in a muscle cell tissue. ing. In Test 2, it is a graph which shows the result of having measured the amount of serum adipocytokines, (A) is the graph which showed the amount of serum leptin in the 7th week of breeding divided into a normal diet group and a high fat diet group. Yes, (B) is a graph showing the serum leptin level in the 14th week of breeding divided into a normal diet group and a high fat diet group, and (C) is the serum adiponectin level in the 7th week of breeding. The graph shows the normal diet group and the high fat diet group separately, and (D) is the graph showing the serum adiponectin level in the 14th week of the breeding divided into the normal diet group and the high fat diet group. . In Test 3, it is a graph which shows the result of having measured the amount of adiponectin in serum. In Test 3, it is a graph which shows the result of having measured the amount of leptin in serum. In Test 3, it is a spectrum figure which shows the result of having measured the expression level of AMPK-a in a liver or muscle tissue, (A) is a muscle tissue, (B) is the result of a liver. In Test 3, it is a spectrum figure which shows the result of having measured the expression level of PPAR- (alpha) in a liver or muscle tissue, (A) is a muscle tissue, (B) is the result of a liver. In the test 3, it is a spectrum figure which shows the result of having measured the expression level of GLUT4 in a muscle tissue, (A) shows the expression level of GLUT4 which exists steadily in a muscle cell, (B) is on a cell membrane. The expression level of the transferred GLUT4 is shown. In Test 3, it is a spectrum figure which shows the result of having measured the protein expression level of SREBP-1 in a liver or a muscle tissue, (A) shows the protein expression level of SREBP-1 in a fat tissue, (B) is in a liver. The protein expression level of SREBP-1 is shown. In Test 4, it is the graph which showed the result of the glucose tolerance test by glucose oral load, (B) shows the area under the line of the graph of a blood glucose level with a bar graph. In Test 4, it is the graph which showed the result of the glucose tolerance test by sucrose oral load, (B) shows the area under the line of the graph of a blood glucose level with a bar graph. In Test 4, it is the graph which showed the result of the glucose tolerance test by the maltose oral load, (B) shows the area under the line of the graph of a blood glucose level with a bar graph.

Claims (1)

  An intramuscular AMPK activator comprising, as an active ingredient, a tea extract component containing theaflavins, which is a mixed component of theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate and theaflavin-3,3′-digallate.