JP2003095942A - Glucose endocytosis inhibitor and glucose transporter 4 translocation inhibitor in fat cell, glucose endocytosis activator in muscle cell, and fat-reduced food and drink - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new applications of an extract of tea and components thereof, based on results of studies on effects of the tea and the components on glucose endocytosis activity, functions of glucose transporter 4 (GLUT4), or the like. SOLUTION: A glucose endocytosis inhibitor in fat cells containing catechin gallate as an active ingredient and a GLUT4 translocation inhibitor in the fat cells containing any one of the catechin gallate, catechin together with a gallate ester, and the tea extract as an active ingredient are provided in the specification, respectively. The inhibitors each reduce an amount of fat cells, so that obesity and various diseases caused by the obesity are treated and prevented by the inhibitors. On the other hand, the inhibitors activate the endocytosis of the glucose in the muscle cell, so that the excess glucose is endocytosed into the muscle cell and consumed. Therefore, the inhibitors never increase sugar concentration in the blood, nor give lassitude caused by obesity control.

Description

Detailed Description of the Invention

[0001]

The present invention relates to tea (Camellia sinensi)
The present invention relates to a new use of the tea extract obtained by extracting s) and its components, particularly a new use in the uptake of glucose.

[0002]

Background of the Invention and Prior Art As the diet becomes richer, "obesity" is one of the most serious worries of modern people. Obesity is not only cosmetically undesirable,
It is known to cause various diseases such as diabetes, arteriosclerosis, hypertriacylglycerolemia, hypercholesterolemia and thrombosis.

Obesity is caused by the differentiation and hypertrophy of adipocytes or the increase of adipocyte number itself, and "glucose uptake" is deeply involved in both cases.

By the way, since glucose is a polar substance, a transport carrier (gulucose transporter: GLUT) is required in order to take glucose into each cell from the blood. At present, nine types of GLUTs (GLUT1 to 9) have been cloned, and among them, GLUT1 and GLUT4 are expressed in adipocytes that are largely involved in sugar / lipid metabolism in vivo. Among them, GLUT4 plays a major role in the glucose uptake activity on the membrane of adipocytes.

GLUT4 is an insulin-sensitive GLUT
It is usually present in intracellular vesicles in adipocytes and muscle cells, and when stimulated by insulin, it is translocated to the cell membrane and is in a state capable of taking up glucose. The translocation of GLUT4 is
Insulin binding to the receptor and autophosphorylation of the β subunit of the receptor initiates signal transduction, followed by phosphorylation of the insulin receptor substrate (IRS), activation of phosphatidylinositol 3 kinase, Akt / Protei
Translocation is completed by exocytosis from the endoplasmic reticulum to the cell membrane via the pathway of n Kinase B activation. In addition, GLUT4 acts to move more glycogen into muscle cells, and it is reported that athletes have more GLUT4 than ordinary people (“Latest findings on carbohydrate loading” Keio University Sports Medicine). Research Center Bulletin 1996).

From the above points, as a result of research on the influence of the tea extract and its components on the glucose uptake activity, GLUT4 function, etc., the present inventor was able to obtain various discoveries and the results thereof. The present invention has been accomplished based on the above.

By the way, regarding the function of tea extract, Japanese Patent Laid-Open No. 06-80580 discloses a plasma cholesterol-lowering agent containing a polysaccharide (ribose, arabinose and glucose) contained in tea leaves as an active ingredient. Kaihei 1
No. 0-158181 shows that a lipolysis promoter containing a plant extract extracted from tea promotes the reduction of adipose tissue in the whole body or locally and improves the obesity constitution, and exerts an effect in suppressing and preventing obesity. Japanese Patent Laid-Open No. 11-302168
The publication discloses that a glucose absorption inhibitor containing epicatechin gallate in a tea extract as an active ingredient suppresses glucose absorption in the intestinal tract and is effective for treatment of obesity, diabetes and the like.

[0008]

The present invention relates to a glucose uptake inhibitor and insulin stimuli-responsive glucose uptake in adipocytes, which comprises, as an active ingredient, either catechin gallate, catechin having gallate ester, or tea extract. Suggest inhibitors.

When the active ingredient of the present invention is ingested, glucose uptake in adipocytes, particularly glucose uptake enhanced by insulin stimulation can be suppressed, and adipocyte mass can be reduced. This makes it possible to treat and prevent various diseases such as an excess fat state, that is, obesity, and diabetes associated with obesity, arteriosclerosis, hypertriacylglycerolemia, hypercholesterolemia, thrombosis and the like. Furthermore, the active ingredient of the present invention not only suppresses glucose uptake in adipocytes, but also reversely activates glucose uptake in muscle cells and allows excess glucose to be taken up and consumed by muscle cells, Not only does it increase blood sugar levels, and there is no fatigue associated with obesity prevention,
On the contrary, it is possible to improve activity.

As a mechanism for inhibiting glucose uptake activity in adipocytes, various mechanisms such as inhibition of translocation of glucose transporter present in adipocytes or binding to insulin receptor are considered.
In this study, it was elucidated that the active ingredient of the present invention specifically inhibits the translocation of GLUT4. Therefore, the present invention proposes a GLUT4 translocation inhibitor in adipocytes, which contains, as an active ingredient, either catechin gallate, catechin with gallate ester, or tea extract.

The present invention also provides an activator of glucose uptake in muscle cells, which comprises, as an active ingredient, either catechin gallate, catechin with gallate ester, or tea extract. Ingestion of such active ingredient activates GLUT4 translocation in muscle cells,
Glucose uptake activity in muscle cells can be increased, and as a result, muscle cells can take up a large number of energy sources and increase muscle cell mass. Therefore, it can be effectively used for activation of muscle tissue, reduction of physical fatigue, improvement of exercise capacity, enhancement / increase of muscle tissue, and eventually remodeling of constitution. Therefore, GLUT4 in muscle cells containing catechin gallate, catechin with gallate ester, or tea extract as an active ingredient
It can also be provided as a translocation activator or a muscle activator.

As the active ingredient in the present invention, catechin gallate, catechin containing gallate ester,
Any of the tea extracts may be used, but catechins with gallate esters are preferable, and catechin gallate is particularly preferable because high effects can be expected in any of the inventions.

It has been confirmed by this study that the effect can be obtained by ingesting the active ingredient of the present invention either before or after insulin secretion. Therefore, the active ingredient of the present invention is The effect can be obtained by ingestion either simultaneously with food intake or after food intake.
Moreover, since the active ingredient of the present invention is a component derived from tea that has been regularly drunk for a long time and can be taken with peace of mind by anyone, it can be taken comfortably and safely for a long period of time, and chronic symptoms and It is particularly effective for the fundamental treatment and prevention of diseases and for improving physical constitution.

The catechin of the present invention means (-)-catechin, and means (-)-catechin when (-) is omitted. For example, catechin gallate means (-)-catechin gallate.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION "Insulin stimulation-responsive glucose uptake inhibitor", "glucose uptake inhibitor in adipocytes", "GLUT4 translocation inhibitor in adipocytes", "glucose uptake in muscle cells" of the present invention "Activator", "GLUT4 translocation activator in muscle cells", "muscle activator" are all "tea extract", "catechin with gallate ester", or "catechin gallate", It can be produced by blending at an appropriate concentration.

"Tea extract" means tea (Camellia sinensis
(Including tea leaves, tea buds, tea trees, etc.), raw tea leaves, fermented teas such as black tea and Puerle tea, semi-fermented teas such as oolong tea and mash tea, green tea, kettle roasted green tea, and roasted tea. It is possible to use any one of the non-fermented teas such as the above (single), the one obtained by extracting a mixture of two or more kinds of these, or the mixture obtained by extracting each of them. However, from the viewpoint of the effect of the present invention, for example, the insulin stimuli-responsive glucose uptake inhibitory action, sencha is not green tea or gyokuro among green tea, Nuara is not dimbla and uba among black tea, and daffodil is not oolong tea. It is preferable to use iron Kannon and color types.

The "tea extract" is an extract obtained by extracting tea with water, hot water or hot water, preferably hot water having a temperature of 40 ° C to 100 ° C, especially hot water having a temperature of 90 ° C to 100 ° C. More preferably, the extract is catechin by purification means such as resin adsorption, filtration such as ultrafiltration / reverse osmosis filtration, or partition extraction using ethyl acetate, etc., and in particular, catechin with gallate ester, among them (-) There may be mentioned a tea extract obtained by purifying in the direction of increasing the content of catechin gallate, or a tea extract obtained by concentrating or drying these tea extracts. As a preferable example of the green tea extract, green tea is subjected to hot water extraction treatment, and the extract is dried to have a catechin concentration of about 30% (green tea extract manufactured by ITO EN Co., Ltd .: Theafran 30A) or green tea to hot water. Extraction treatment, and this extract was treated by a column method to eliminate components other than catechin and dried to obtain a tea polyphenol concentration of about 85 to 95% green tea extract (trade name: Theafran 90S manufactured by ITO EN). Can be illustrated.

On the other hand, "catechin with gallate ester" means any of (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-catechin gallate, and (-)-gallocatechin gallate. Or a polymer of any of these, or a copolymer of two or more kinds of these, or a mixture of two or more kinds of these, in which (-)-catechin gallate is 25 %, More preferably isolated “(−)-catechin gallate”. In addition to tea, "catechin with gallate ester" exists in many plants such as kora, rhubarb, apples, peaches, pears, cacao beans, and persimmon soup, and can be obtained by extracting these. it can. It is known that when the above catechin or a mixture thereof is sterilized by retort, a polymer or copolymer of these catechins is produced.

The above-mentioned "tea extract", "catechin with gallate ester", and "(-)-catechin gallate" can be blended as individual active ingredients. Stimulus-responsive glucose uptake inhibitory effect, adipocyte glucose uptake inhibitory effect, adipocyte GLUT4 translocation inhibitory effect, muscle cell glucose uptake activation effect, muscle cell GLUT4 translocation activation effect, or muscle activity It is also effective to mix these with a material having a chemical action and mix these as an active ingredient. When blended as a single active ingredient, for example, "tea extract", "catechin with gallate ester", "(-)-catechin gallate", respectively, dissolved in purified water or physiological saline or the like (Eg oral administration,
Intraperitoneal agents, intracerebral agents, etc.) and the like.

"Insulin stimulation-responsive glucose uptake inhibitor", "Glucose uptake inhibitor in adipocytes", "GLUT4 translocation inhibitor in adipocytes", "Glucose uptake activator in muscle cells" of the present invention “GLUT4 translocation activator in muscle cells” and “muscle activator” are all orally or parenterally administered (intramuscular injection, intravenous injection, subcutaneous administration, rectal administration, transdermal administration, nasal administration) It is preferable that the formulation and dosage form be suitable for each administration. Speaking of dosage forms, for example, for oral administration, it can be prepared in the form of solutions, tablets, powders, granules, sugar-coated tablets, capsules, suspensions, emulsions, pills, etc. Injections, ampoules,
It can be prepared in the form of rectal administration agent, oily suppository, water-soluble suppository and the like. As for formulation (formulation), commonly used excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives,
It can be produced by a conventional method using a solubilizing agent, a preservative, a flavoring agent, a soothing agent, a stabilizer and the like. Also,
For example, lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methyl cellulose, carboxymethyl cellulose or a salt thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin,
It is also possible to add non-toxic additives such as ethanol, propylene glycol, citric acid, sodium chloride, sodium sulfite, sodium phosphate and the like.

Further, the “insulin stimuli-responsive glucose uptake inhibitor”, “adipocyte glucose uptake inhibitor”, “adipocyte GLUT4 translocation inhibitor”, “muscle cell glucose uptake activity” of the present invention. "Agent""GLUT4 translocation activator in muscle cells" and "muscle activator" are all drugs, quasi-drugs, and health foods / health drinks / foods for specified health use with pharmacological effects. It can also be provided as a functional food, a drug or feed for animals other than humans. For example, it can be made easier to ingest by preparing it as a quasi-drug and making it into a drinkable form such as a bottled drink, or a tablet, capsule, granule or the like. Health foods with pharmacological effects
Examples of the health drink, the food for specified health use, and the functional food include the active ingredient of the present invention, carbonic acid, an excipient (including a granulating agent),
Diluting agent, or further sweetener, flavor, flour, starch, sugar, various proteins such as fats and oils, sugar raw materials and vitamins, mixed with one or more kinds selected from food and drink ingredients such as minerals, Alternatively, currently known food and drink, such as sports drinks, fruit drinks, milk drinks, tea drinks,
Vegetable juice, dairy drink, alcoholic drink, jelly, jelly drink, carbonated drink, chewing gum, chocolate,
It can be manufactured by adding it to candy, biscuits, snacks, bread, dairy products, fish meat products, livestock products, frozen desserts, dried foods, supplements and the like. Among them, "isolated catechin gallate" is a food and drink material that contains a lot of sugars.
Foods and drinks obtained by adding can be provided as excellent fat-reducing foods and drinks (in other words, diet foods and drinks), muscle-activating foods and drinks, and the like.

The content of the active ingredient in the present invention varies depending on the method of use, but in the case of pharmaceuticals, it is 0.001-1% by weight in terms of dry weight of catechin, and particularly 0.01-.
It is preferable to add 0.5% by weight.
It is preferable to add 0.001 to 1% by weight, especially 0.01 to 0.5% by weight in terms of dry weight of catechin. When prepared as a fat-reduced food or drink or a muscle-activated food or drink, the isolated catechin gallate is added to the food or drink in an amount of 0.00
It is preferable to mix 1 to 1% by weight to prepare a catechin gallate concentration 5 to 500 times as much as that of tea which is usually drunk.
The intake amount is 10-5000 mg / day, preferably 100-1500 mg / day, in terms of dry weight of catechin.
A degree is preferable.

<Test 1> In this test, changes in glucose uptake in blood components and adipose tissue and muscle tissue were examined in order to examine changes in glucose metabolism and lipid metabolism when the rat was allowed to freely ingest the green tea extract. Examined.

(Rat raising of rats) Wist immediately after weaning was used for the test.
Male ar rats (3 weeks old) were used. These were divided into 2 groups of 5 animals, one group was allowed to freely ingest green tea extract ("Oi Ocha" manufactured by Itoen Co., Ltd.), and the other was ion-exchanged water for 3 weeks as a control. It was Body weight, food intake, and water intake were measured daily. Three weeks later,
Rats that had been fasted for 4 hours were anesthetized, blood was collected from the heart, abdominal laparotomy was performed, and adipose tissue and thigh muscle were excised and used for the experiment.

(Measurement of Plasma Components) The collected blood was separated into blood cells and plasma by centrifugation. Using the obtained plasma, blood glucose level, triacylglycerol amount, free fatty acid amount, total cholesterol amount and HDL cholesterol amount were measured using a measurement kit manufactured by Wako Pure Chemical Industries.

(3-O-Me in adipose tissue and muscle tissue
Incorporation of thyl-D-glucose (hereinafter referred to as "3-OMG") Adipose tissue and thigh muscle tissue were excised from the rat and then minced. Approximately 100 mg of the tissue fragment contained 5.5 mM glucose. Krebs-Ringer-Hepes buffer (KRH)
And incubated for 80 minutes. Further, it was replaced with KRH containing no glucose, incubated for 15 minutes, and labeled with ³ H, 3-OMG (6.5 mM, 0.5 mM).
μCi) was incorporated for 30 seconds. Then 0.3 mM
Uptake was stopped with a KRH solution of phloretin, washed several times with the same solution, and then completely dissolved with an NCSII solubilizing agent. After dissolution, the amount of 3-OMG incorporated into the tissue was measured with a liquid scintillation counter.

<Test 2> In order to investigate the effect of tea components on the glucose transport mechanism at the cell level, 3T3-L1 strain cells, which are mouse-derived preadipocytes, were differentiated into adipocytes. Alternatively, glucose uptake into cells when "catechin" was added, and G
The translocation of LUT4 was investigated.

(Preparation of Tea Extract) As "tea extract",
Green tea (Sencha (from Motoyama), Gyokuro (from Asahina), Bancha (from Shizuoka)), black tea (Nuara, Uba, Dimbula) and oolong tea (Tetsukannon, color variety, daffodils) 3 each
A total of 9 species were used for the experiment. Each tea extraction was carried out by adding 200 ml of boiling water at about 90 ° C. to 10 g of tea leaves, extracting for 10 minutes with appropriate stirring, concentrating the extract with an evaporator, and then freeze-drying.

5 mg / m for glucose uptake experiments
What was suspended in KRH so that it became 1
In the T4 translocation experiment, suspensions in Phosphate buffered saline (PBS) at the same concentration as in the uptake experiment were used. On the other hand, as "catechin", catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), A total of eight epigallocatechin gallates (EGCg) were used. These are 10 mM
What was dissolved in dimethylsulfoxide (DMSO) so that it was used for the uptake experiment and GLUT4 translocation experiment.

(Cultivation of preadipocyte cell line 3T3-L1 cells and induction of differentiation into adipocytes) 3T3-L1 cells, which are mouse-derived preadipocyte cell lines, were plated at 35 mm or 100 mm.
After seeding on a mm dish and culturing until it reached a saturated state, differentiation into adipocytes was induced. 1 μM differentiation induction
dexamethazone, 0.5 mM 3-isobuthyl-1-methylx
Cells were cultured in DMEM medium (containing 10% fetal bovine serum (FBS)) supplemented with antine, 10 μg / ml insulin and 100 μM ascorbyl phosphate for 3 days, and then containing 10 μg / ml insulin and 100 μM ascorbyl phosphate Cultured in DMEM for 2 days. afterwards,
The cells were cultured in DMEM medium containing 10% FBS for 5 to 8 days, and used in each experiment when about 90% of cells were differentiated into adipocytes by microscopic observation.

(Uptake of 3-OMG into cells) 3T3
-L1 cells were seeded on a 35 mm dish for differentiation.
Then, the cells were removed by culturing in serum-free medium for 18 hours in advance.
I made a sensitization. The effects of tea extract and catechin are
It investigated using the two methods shown. The first method is
Tea extract (final concentration 100 μg / m
l) or catechin (50 μM) was added to the cells for 15 minutes
And then 100 nM insulin for 15 minutes
rear, ³3-OMG labeled with H (6.5 mM, 0.5
μCi) was incorporated for 30 seconds. Another way is
Add 100 nM insulin to cells in advance for 15 minutes
Then, after translating GLUT4, tea extraction
Add the gifts,³Incorporation of H-3-OMG was performed.
Either method³Uptake of H-3-OMG
Is set to 30 seconds, and 0.3 mM flux, which is an inhibitor of GLUT, is used.
By rapidly washing the cells with loretin KRH solution
The reaction was stopped. In addition, add 0.3 mM phloretin to the cells.
Washed 3 times with KRH solution of 0.5% SDS
After solubilization, intracellularize with liquid scintillation counter
Captured in³The value of H was measured. Blindly,³H-
The adsorption of 3-OMG to non-specific cells was preliminarily performed.
The same measurement was performed using cells whose uptake was inhibited by retin.
It was Incorporated into cells³Blinded value was subtracted from H value
The thing was made into the true glucose uptake amount.

(Preparation of Cell Membrane and Detection of GLUT4 by Western Blotting) Detection of GLUT4 by Western blotting was carried out by desensitizing 3T3-L1 adipocytes seeded in a 100 mm dish and differentiated in the same manner as above for 15 minutes. Tea extract treatment (the above-mentioned tea extract, that is, 10 g of tea leaves, 200 ml of boiling water at about 90 ° C. was added, and the mixture was extracted for 10 minutes with appropriate stirring, and the extract was concentrated by an evaporator and then freeze-dried to obtain. 50 μg / ml of tea extract was applied) and then 1
The one that was stimulated with insulin for 5 minutes was used. These cells were homogenized and a cell membrane fraction was prepared by a density gradient ultracentrifugation method. The amount of protein in the obtained cell membrane fraction was measured, and 1 μg thereof was subjected to SDS-PAGE to separate the protein. The separated protein is PVDF
(Polyvinylidene difluoride) film transferred to 5% skim milk Tris-buffered saline-Tween (TBST: 2
0 mM Tris-HCl (pH 8.0), 0.15 M Na
Cl, 0.05% Tween 20) solution was used for blocking. After washing the membrane several times with TBST, an anti-GLUT4 antibody was reacted as a primary antibody and an anti-goat IgG antibody labeled with horseradish peroxidase was reacted as a secondary antibody. GLUT4 was detected by reacting the immune complex on the membrane with an ECL plus chemiluminescent reagent and exposing it to an X-ray film.

(Detection of Insulin Receptor Phosphorylation by Immunoprecipitation Method) The influence of tea extract on insulin receptor (IR) phosphorylation was the same as above in 3T3-L1 adipocytes seeded in 100 mm dishes and differentiated. Treatment of tea extract for 15 minutes (the above-mentioned tea extract, that is, 10 ml of tea leaves, 200 ml of boiling water at about 90 ° C. was added, and the mixture was extracted for 10 minutes with appropriate stirring, and the extract was concentrated by an evaporator, After that, the tea extract obtained by freeze-drying was made to act at 50 μg / ml.), And then 1
What was subjected to insulin stimulation for 5 minutes) was used. These cells were treated with RIPA buffer (50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 1% NP40,
The whole extract was prepared by dissolving with 0.5% deoxycholate, 0.1% SDS). The amount of protein in the obtained extract was measured, 1 mg of protein was immunoprecipitated with anti-IR-β antibody, and the IR-β subunit bound to the antibody was labeled as protein.
Adsorbed on A / G agarose. The IR-β subunit bound to the antibody was recovered by centrifugation, and SDS-PAGE was performed.
Then, the protein was separated and subjected to Western blotting using an anti-phosphotyrosine antibody as the primary antibody.
The procedure of Western blotting was performed in the same manner as above, and horseradish peroxidase-labeled anti-rabbit IgG was used as the secondary antibody.

<Results and Discussion> (Effect of Green Tea on Rat Body Fat) To examine the effect of tea extract at the animal individual level, rats were allowed to freely ingest green tea for 3 weeks and changes in blood components and fat When the changes in glucose uptake activity in tissues and muscle tissues were examined, the following results were obtained.

[0035]

[Table 1]

[0036]

[Table 2]

[0037]

[Table 3]

(Change in Rat Adipose Tissue Weight Due to Ingestion of Tea) Comparison of body weight changes between a group of rats ingested with the tea extract and a control group in which only ion-exchanged water was freely ingested was observed during the breeding period and immediately before slaughter. No significant difference was observed in any of them (Fig. 1, Table 1). Also, these 2
Similarly, changes in food intake and water intake between groups were not observed (Fig. 2, Table 1). When the tissue weight was measured after slaughtering these rats, no significant difference was observed, but the adipose tissue weight was reduced by about 15%. On the other hand, there was no change in the weight of liver, spleen, thymus and kidney. In addition, macroscopic observation revealed no abnormalities in all organs including these. From these results, it was judged that the intake of the tea extract did not change the body weight of the rat, and that the effect of losing weight on animals in the normal growth period was not observed. However, the weight of adipose tissue tended to decrease. This suggests that the tea extract may suppress the induction of preadipocyte differentiation into adipocytes and may reduce the fat accumulation amount of adipocytes after differentiation.

(Changes in Plasma Components Due to Tea Ingestion) Plasma components separated from the above rats were measured. No change in blood glucose level was observed due to ingestion of tea extract. On the other hand, the amount of triacylglycerols when ingesting the tea extract was reduced by about 15% as compared with the control group (Table 2). On the other hand, the amount of free fatty acid was 35%, and the amounts of total cholesterol, HDL cholesterol and LDL cholesterol were all significantly decreased by about 20%. These results suggest that the components of the tea extract affect lipid metabolism in vivo. At least, since the main organs / tissues of triacylglycerol and cholesterol metabolism are liver, adipose tissue and small intestine, tea extract absorbs lipid components or regulates the synthesis / degradation system in these organs / tissues. It is clear that

(Effect of Tea on Glucose Uptake in Adipose Tissue and Muscle Tissue) As a result of investigating changes in ³ H-3-OMG uptake in adipose tissue and muscle tissue,
Rats in the tea group received ³ H-3-OMG in adipose tissue.
, But in muscle tissue it increased significantly (Table 3). The decrease in glucose uptake in adipose tissue of rats in the tea intake group is considered to be closely related to the tendency of decrease in lipid weight (Table 1). Further, since no increase in blood glucose level was observed in Table 2, it is considered that glucose that was not taken up by adipose tissue is actively taken up by muscle or other tissues.

(Effect of Tea Extract on Glucose Transport of 3T3-L1 Adipocytes) From the above results, it is shown that intake of tea extract reduces adipose tissue weight and affects lipid metabolism in vivo. It has been shown. Adipose tissue is a place responsible for biosynthesis of lipids from sugar. Therefore, the effect of the tea component on the sugar transport mechanism of adipose tissue at the cellular level was examined using the 3T3-L1 adipocyte cell line, and the following results were obtained.

(Effects of 9 Tea Extracts on Glucose Uptake) The 3-OMG uptake of adipocytes was increased about 3-fold by insulin stimulation, and the insulin-responsive glucose transport mechanism is present in these cells. Was confirmed. When the tea extract (100 μg / ml) was allowed to act 15 minutes before insulin, all 9 tea extracts used in this experiment had 3-OMG in the presence of insulin.
Uptake activity was reduced (Fig. 3). When the uptake of 3-OMG in the absence of insulin was also measured, oolong tea showed a tendency to promote it. Of the tea extracts that had a high inhibitory effect in the presence of insulin, the concentration dependence of the inhibitory effect was investigated focusing on sencha (Fig. 4). Sencha extract decreased the uptake in a concentration-dependent manner when added before insulin, and the effect was observed even at a low concentration of 2 μg / ml. Next, the effect of sencha extract on 3-OMG uptake was examined in cells that had been previously stimulated with insulin. Sencha extract (100 μg / ml) rapidly reduced the uptake by the action time of 1 minute (FIG. 5, left). Also, when the concentration dependence at an action time of 1 minute was examined, uptake was reduced at 5 μg / ml or more (Fig. 5, right). From the above, it became clear that the tea extract has an effect of suppressing the uptake of 3-OMG, whether it is added before or after the insulin stimulation.

(Effect of Tea Extract on GLUT4 Translocation) In adipocytes, GLUT4 is translocated by insulin stimulation.
However, since the uptake of glucose was increased, it was expected that the tea extract would act on this translocation signaling pathway. Therefore, the effect of the tea extract on the translocation of GLUT4 and the effect of the tea extract on the phosphorylation of the IR-β subunit, which is the most upstream part, were examined. When GLUT4 translocation was examined, insulin was found to be a GLUT on the cell membrane.
4 The amount of protein was significantly increased. When three kinds of green tea extract are allowed to act before insulin stimulation, insulin-induced G
It suppressed the translocation of LUT4 to the cell membrane (Fig. 6). The control effect of sencha and gyokuro was stronger than that of bancha. In black tea, Nuara showed the same inhibitory effect in oolong tea, and in oolong tea, color seeds and daffodils showed similar effects, and in tea, Dimbula and Uba, and oolong tea had no effect on translocation. Next, when the effect of sencha extract on the phosphorylation of IR-β subunit was examined by immunoprecipitation method, cells treated with sencha showed no β-subunit phosphorylation by insulin as in the control cells. (Fig. 7). Furthermore, the amount of IR / β subunit itself in the cells treated with sencha was the same as that in the control cells, and no change was observed. Furthermore, when the phosphorylation of tyrosine residues was examined using the whole cell protein, a band around 95 kDa corresponding to the molecular weight of IR-β subunit was phosphorylated by insulin. However, no difference in the degree of phosphorylation was found between the green tea extracts. From the above results,
It was found that the sencha extract did not change the phosphorylation of IR. Therefore, the tea extract does not affect the binding of insulin to the receptor and specifically suppresses the translocation of GLUT4, that is,
It was suggested that tea extract regulates glucose transport without showing an early signal to insulin.

(Effect of catechin on glucose uptake) Glucose uptake and GLU shown by tea extract
Comparing the effects of T4 on translocation, the behavior differs depending on the type of tea. Therefore, the difference in the composition and content of the components such as catechin contained in each extract contributes to the behavior change of the glucose transport mechanism. Was thought. So 8 catechins are ³ H-3-OMG
When the effect on uptake was investigated, the following results were obtained. Of the catechins, four catechins with gallate esters (Cg, ECg, GCg, EG
Cg) reduced glucose uptake in the presence of insulin, but not the other four (C, EC, GC, EGC) (FIG. 8). The effect of Cg was the strongest among the four types of suppression. From these, catechin in the tea extract, especially catechin having a gallate ester, especially catechin gallate (Cg) is 3T3-.
It was suggested to act on the glucose transport mechanism of L1 adipocytes. Also, black tea contains abundant theaflavin, which is a polymer.
When the -OMG uptake was examined, a fairly strong inhibitory effect was observed.

<Summary> When the tea extract was ingested in rats, the body weight did not change but the adipose tissue weight showed a tendency to decrease. Adipose tissue is a place for biosynthesis of lipids from sugars, and if the sugar transport activity is reduced or the lipolytic activity such as lipoprotein lipase or hormone sensitive lipase is increased, the weight of adipose tissue is reduced. Since the accumulation of fat is involved in the development of lifestyle-related diseases such as diabetes, the intake of tea extract can be expected to have a preventive effect on lifestyle-related diseases. In addition, since normal rats (3 years old) immediately after weaning were allowed to ingest tea,
The tendency of the decrease in adipose tissue weight is likely due to the suppression of adipocyte differentiation through the suppression of sugar transport activity. Since tea extract suppresses glucose transport activity in the presence of insulin in cultured cell lines, tea extract is converted to adipose tissue in the state where blood glucose level increases with food intake and insulin from pancreatic β cells increases. It was thought to suppress the sugar transport of the plant and prevent the supply of excess energy. On the other hand, along with this inhibitory effect, the adverse effect of hyperglycemia was expected, but no change was observed in the blood glucose level in the tea-fed rats, and the uptake of glucose in muscle tissue was enhanced. From this, it can be considered that the tea sends excess energy to the motor organs and acts to improve the activity. This suggests that muscle and adipose tissue are differentiated from the same endoderm and both express GLUT4, but that tea extract shows the opposite behavior of the same GLUT isoform in different organs / tissues. It is a thing. It was also recognized that catechins among tea extracts, especially catechins with gallate esters, and among them, catechin gallates, were particularly effective as the active ingredients having the above effects.

Example 1 Glucose uptake inhibitor in adipocytes, insulin stimuli-responsive glucose uptake inhibitor, GLUT4 translocation inhibitor in adipocytes, glucose uptake activator in muscle cells in the following formulation: Tablets were prepared as either GLUT4 translocation activator or muscle activator in muscle cells.

[0047] Tea extract (Theafuran 90S or Theafuran 30A)… 120mg Vitamin C ... 50 mg Emulsified oligosaccharides ... 90 mg Granulating agent: 60 mg Crystalline cellulose ... 80mg Reduced maltose syrup ... 90mg Sucrose ... 100mg Fragrance ... Appropriate amount

Example 2 A beverage as a fat-reduced food or drink or a muscle-active food or drink was prepared according to the following formulation.

[0049] Catechin gallate ... 50mg Vitamin C 50 mg Fructose grape sugar… 10g Water-soluble dietary fiber: 500 mg Fragrance ... Appropriate amount Ion-exchanged water ... 100 mL

[Brief description of drawings]

FIG. 1 is a graph showing changes in body weight of rats after the start of ingestion of tea extract in Test 1 over time.

FIG. 2 is a graph showing changes in food intake and water intake of rats in Test 1 over time.

FIG. 3 is a graph showing 3-OMG uptake activity in adipocytes when various types of tea extracts were ingested in Test 2.

FIG. 4 is a graph showing 3-OMG uptake activity in adipocytes when a green tea extract was ingested before insulin stimulation in Test 2.

FIG. 5 is a graph showing the 3-OMG uptake activity in adipocytes when the green tea extract was ingested after insulin stimulation in Test 2.

FIG. 6 is an X-ray film photograph showing the effect of green tea extract on GLUT4 translocation in Test 2.

FIG. 7 is a photograph of an X-ray film showing the effect of green tea extract on phosphorylation of insulin receptor (IR) in Test 2.

FIG. 8 is a graph showing the 3-OMG uptake activity in adipocytes when various catechins were ingested in Test 2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/06 A61P 3/06 3/10 3/10 7/02 7/02 9/10 101 9/10 101 // C07D 311/62 C07D 311/62 (72) Inventor Hitoshi Ashida 3-chome, Kamikotakamaru, Tarumi-ku, Kobe-shi, Hyogo Prefecture 11-2-54 (72) Kazuki Kanazawa 5 in Naka, Koyo-cho, Higashinada-ku, Kobe-shi, Hyogo 5-chome 533-409 (72) Genichi Danno 3-45-34 Akibadai, Nishi-ku, Kobe-shi, Hyogo (72) Inventor Takashi Furuyashiki 1-23-9 Kitaura-cho, Seto-shi, Aichi (72) Sayaka Terashima 3-1-12 Hoshigaoka, Tarumi-ku, Kobe-shi, Hyogo (72) Inventor Hironori Nagayasu 293-1 Uemura, Tokuyama-shi, Yamaguchi F-reference (reference) 4B018 LB08 MD59 ME04 MF01 4C062 FF56 4C086 AA01 AA02 BA08 MA01 MA04 NA14 ZA45 ZA54 ZA70 ZC33 ZC35 4C088 AB45 AC05 BA09 BA3 2 NA14 ZA45 ZA54 ZA70 ZC33 ZC35

Claims (5)

[Claims] 1. A glucose uptake inhibitor in adipocytes, which comprises catechin gallate as an active ingredient. 2. An insulin stimulation-responsive glucose uptake inhibitor containing catechin gallate as an active ingredient. 3. A GLUT4 translocation inhibitor in adipocytes, which comprises, as an active ingredient, either catechin gallate, catechin having a gallate ester, or tea extract. 4. An activator of glucose uptake in muscle cells, which comprises, as an active ingredient, either catechin gallate, catechin with gallate ester, or tea extract. 5. A fat-reducing food or drink obtained by adding the isolated catechin gallate.