KR101527890B1 - Composition for Dropping Glucose Containing Dehydrozingerone - Google Patents

Abstract

The present invention relates to a composition for reducing blood sugar containing dehydrozingerone as an active ingredient, and more particularly to a composition for treating diabetes mellitus containing dihydrogingerrone, a dietary supplement health functional food, ≪ / RTI >

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for reducing blood glucose containing dihydrogingerrone (Glucose Containing Dehydrozingerone)

The present invention relates to a composition for treating diabetes mellitus containing dihydro gingerol as an active ingredient, a health functional food for improving diabetes and a composition for inhibiting obesity.

Diabetes is a chronic disease in which glucose (glucose) in the blood is higher than normal people due to the insufficient amount of insulin, thereby releasing glucose into the urine.

Diabetes is divided into two types according to insulin production. Insulin dependent diabetes (IDDM) is caused by insulin secretion deficiency of blood glucose control hormone in the blood, It is also called juvenile diabetes because it occurs in younger age groups. Type 2 diabetes (non-insulin dependent diabetes, NIDDM) occurs mainly after the age of 40, and it accounts for most of the diabetic patients in Korea. Unlike type 1, it is known as diabetes mellitus, and it is known that insulin is inactivated by insulin resistance, and it is known that genetic factors and environmental factors are involved together. Type 2 diabetes accounts for 95% of all diabetic patients, and in the 21st century, one-fourth of the world's population is expected to develop the so-called metabolic syndrome (X-syndrome), which is thought to precede the development of type 2 diabetes.

Type 2 diabetes mellitus is a chronic metabolic disease and the incidence is increasing worldwide, and statistics suggest that the number of diabetic patients will reach about 320 million by 2020. Type 2 diabetes due to insulin action and responsiveness accounts for more than 90% of all patients and effective treatment and prevention is required.

 The pathogenesis of type 2 diabetes has not been elucidated yet, but it is a complex disease characterized by hyperglycemia in the liver, insulin resistance, and reduced sugar processing capacity in muscle and adipocytes. In particular, obesity is known to be the leading cause of diabetes in the United States, with 85% of type 2 diabetes patients being obese.

Type 2 diabetes is characterized by both increased peripheral insulin resistance and abnormal insulin secretion. In addition, type 2 diabetes is a progressive disease and causes complications such as retinopathy, cataract, nephropathy, neuropathy and atherosclerosis. To reduce the incidence of such complications, patients with type 2 diabetes need to maintain blood glucose levels close to normal levels, and patients with type 2 diabetes are given a therapeutic focus to optimize and potentially normalize blood glucose control have.

As a therapeutic agent for type 2 diabetes that aims to regulate blood glucose level, metformin of biguanide series, thiazolidinedione-based pioglitazone and rosiglitazone which activate PPARγ, α-glucosidase inhibitors which delay carbohydrate absorption in the small intestine, Oral preparations such as sulfonylurea and non-sulfonylurea which control insulin secretion in pancreatic? -Cells have been used. However, these drugs have been reported to have side effects such as digestive disorders and hepatotoxicity, which may cause safety problems.

Therefore, there is a need to develop a composition or health functional food effective for the treatment or prevention of diabetes or diabetic complications from natural substances proved to be safe.

Dehydrozergerone, on the other hand, has been shown to be effective in treating inflammation (Elias G et al.Indian J Exp Biol ,26 (7): 540-2, 1988), antioxidant (Rajakumar D. V. et al.Molecular and Cellular Biochemistry, 140: 73-79, 1994) and anticancer agents (Noriko Motohashi et al.Cancer Letters ,134: 3742, 1998), but no association with diabetes has been reported.

Accordingly, the present inventors have made intensive efforts to develop a therapeutic agent for type 2 diabetes through blood glucose lowering. As a result, it has been found that dehydrozingerone structurally similar to curcumin inhibits phosphorylation of AMPK (AMP kinase) The inventors of the present invention have found that the present invention has been completed.

It is an object of the present invention to provide a composition for treating type 2 diabetes and a composition for inhibiting obesity.

Another object of the present invention is to provide a health functional food for the improvement of type 2 diabetes and a health functional food for obesity improvement.

Another object of the present invention is to provide a method of using dehydrozingerone as an insulin sensitizer and a diabetic adjuvant containing the same as an effective ingredient.

In order to achieve the above object, the present invention provides a composition for treating diabetes comprising dehydrozingerone as an active ingredient.

The present invention also provides a composition for inhibiting obesity containing dehydrozingerone as an active ingredient.

The present invention also provides a health functional food for improving diabetes containing dehydrozingerone as an active ingredient.

The present invention also provides a health functional food for improving obesity containing dehydrozingerone as an active ingredient.

The present invention also provides a method for using dehydrozingerone as an insulin sensitizer.

The present invention also provides an adjuvant therapy for diabetes, which comprises dehydrozingerone as an insulin sensitizer.

According to the present invention, the dehydrozingerone of the present invention increases the expression or activity of AMPK (AMP kinase), induces blood glucose lowering, promotes insulin sensitivity, increases insulin regulation, reduces body weight Compositions for the treatment and improvement of type 2 diabetes, obesity inhibitors and insulin sensitizers.

1 shows the inhibitory effect of dihydrojuggeron (DHZ) on the increase of body weight and adipose tissue in a high fat diet-induced obesity model. (A) Effect of dihydrogingerrone on weight control (* P <0.05 shows a statistically significant difference compared to mice, *, # P <0.05. (B) Effects of dihydrogingerrone on visceral fat (C) Effect of dihydrogingerrone on fatty liver (D) Effect of dihydrogingerrone on the peripheral fat of liver (E) Liver and epididymal fat of dihydrogingerol (LFD: blocking method, HFD: high-fat diet, DHZ: 100 mg / kg / day dihydrogingerol with high fat diet)
FIG. 2 shows the effect of inhibiting blood glucose increase by dihydro ginger (DHZ) in a high fat diet-induced obesity model. (A) Effects of dihydrogingerrone on fasting blood sugar (FBG) levels (B) Effects of dihydrogingerrone on insulin levels (C) Effect of dihydrozincerone on levels of leptin (LFD: HFD: high fat diet, DHZ: 100 mg / kg / day dihydroginger rheon provided with high fat diet * / P <0.05 Indicating a significant difference compared to this mouse)
Figure 3 shows AMPK phosphorylation by dihydrogingerrone (DHZ) in C2C12 skeletal muscle cells. (A) Phosphorylation of AMPK and ACC in cells treated with various concentrations of dehydrogingerrone for 1 hour (B) Phosphorylation of AMPK and ACC in cells treated with 30 μM dehydrogingerone
FIG. 4 shows the effect of promoting glucose uptake by dihydrogingerrone (DHZ) in L6 muscle precursor cells. (A) Effect of stimulation of glucose (2-DG) uptake by dihydrogingerrone (* p <0.05) (B) Treatment of 2 μM Compound C and 10 μM dihydrogingerone for 1 hour (P <0.05 compared to the control group, * P <0.05). (C) AMPKα2 (2-DG) (D) AMPKa expression level in cells transfected with siRNA for 48 hours (D) Promoting glucose (2-DG) uptake in cells transformed with 100 nM AMPKa2 siRNA treated with 10 μM dihydrogingerrone for 48 hours <0.05 compared with the control group, * P <0.05 compared to the DHZ-treated cell group)
Figure 5 shows activation of the p38 MAPK signaling pathway by dihydrogingerrone (DHZ) in C2C12 skeletal muscle cells. (A) Phosphorylation of p38 MAPK over time by 30 μM dihydrozinceron (B) Phosphorylation of p38 MAPK by dihydrogenzuron at various concentrations (C) Compound C and 30 μM dihydrogingerone were treated for 1 hour Phosphorylation of p38 MAPK in Cells (D) Promotion of glucose (2-DG) uptake by dihydrogingerrone in cells treated with SB 203580, a p38 MAPK inhibitor, for 20 minutes (* p <0.05 compared to the control group *, And # P < 0.05 DHZ treated cells, respectively)
Figure 6 shows the effect of dehydrogingerrone (DHZ) on GLUT4 expression in C2C12 skeletal muscle cells. (A) RT-PCR using GLUT4 primer (B) GLUT4 expression with time by dehydrogenase
Figure 7 shows the effect of dihydrogingerrone (DHZ) on promoting insulin-induced glucose uptake. (A) Promoting glucose (2-DG) uptake in L6 muscle differentiated cells treated with insulin and dehydrogenase for 1 hour (* p <0.05 compared with the control group, * P <0.05 (DHF) in the glucose tolerance test (GTT) of high fat diet-induced obesity model (LFD: inhibition method, HFD: * P <0.05 compared to mice. *, P <0.05. High-fat diet method was used in combination with high-fat diet, Compared with the control group.
Figure 8 illustrates the function of curcumin and dehydrozingerone as an insulin sensitizer.
FIG. 9 is a graph comparing the curative power of curcumin and dehydrozingerone.

In the present invention, it was tried to confirm the development of dihydrojugerone (dihydrogenerone) as a therapeutic agent for type 2 diabetes through the improvement of diabetes mellitus, the control of blood glucose level and the lowering of blood glucose level.

In the present invention, fasting glucose, fasting glucose, insulin and leptin were measured in a high fat diet in order to investigate the glucose control effect of dihydrogingerrone which is structurally similar to curcumin, which is reported to be involved in glucose regulation. In the L6 muscle precursor cells, (2-DG) levels were measured, and the effects of dihydrogingerol on glucose uptake were compared and evaluated. As a result, it was confirmed that the increase in blood glucose level was suppressed by dihydrogingerolone, and that glucose uptake was increased in a concentration-dependent manner (FIGS. 2 and 4A).

That is, in one embodiment of the present invention, it was confirmed that dihydrogingerrone increases glucose uptake in a concentration-dependent manner.

Accordingly, in one aspect, the present invention relates to a composition for treating diabetes comprising dehydrozingerone as an active ingredient.

In the present invention, the dehydrozingerone is a composition for the treatment of type 1 or type 2 diabetes, or preferably a composition for the treatment of type 2 diabetes.

The composition according to the present invention has the ability to regulate blood glucose, and specifically has a blood glucose lowering ability.

The dehydrozingerone is represented by the formula C ₁₁ H ₁₂ O ₃ and is an intermediate product of curcumin biosynthesis and is known as an antiinflammatory, antioxidant and anticancer agent.

In the present invention, in order to examine the effect of AMPK (AMP kinase) protein phosphorylation in dehydrogingerrone, western blot analysis was performed using an AMPK phosphorylated antibody to examine the expression pattern of the protein. As a result, It was confirmed that the phosphorylation of AMPK, a major glycoprotein regulating protein, was increased in muscle cells depending on concentration (A) and time (B) by gingerol (Fig. 3). In addition, glucose uptake by dihydroergensin was measured after knocking down AMPK in L6 muscle cells. As a result, it was confirmed that acceleration of glucose uptake was inhibited (FIGS. 4B, 4C and 4D).

These results indicate that dihydrogingerrone-induced glucose uptake is regulated by AMPK activity and that AMPK is involved in glucose regulation.

As a result, the dihydroergensin according to the present invention increases the expression or activity of AMPK and thus has a blood glucose controlling ability.

In order to analyze the signal transduction pathways and mechanisms involved in glucose uptake by dihydroergensin, we investigated the effect of dihydrogingerrone on p38 MAPK and GLU4, which are important for glucose uptake. As a result, p38 MAPK was activated in a time- and concentration-dependent manner by dihydrogingerrone (FIGS. 5A and 5B), and GLU4 also induced expression (FIGS. 6A and 6B).

In the present invention, in order to compare the insulin sensitizers of curcumin and dihydrogingerrone, which are known to have glucose-regulating ability, curcumin and dihydrogingerrone are combined with insulin by using differentiated muscle cell lines, Were compared and analyzed. As a result, the glucose uptake ability by insulin alone was not significantly increased by the curcumin pretreatment, but was significantly higher than the effect by insulin alone in the pretreatment condition of dihydroginger lone (FIGS. 7 and 8).

These results demonstrate that dihydrogingerol has the ability to modulate insulin sensitization and can be applied to clinical prescriptions for the treatment of diabetes through combination therapy with insulin. That is, it was confirmed that dihydrogingerrone has an insulin sensitizing function.

In another aspect, the present invention relates to a method of using dihydrogingerrone as an insulin sensitizer and a diabetic adjuvant containing dihydrogenzyme as an insulin sensitizer.

In the present invention, in order to directly compare the glycemic control ability of curcumin and dihydrogingerrone, a differentiated muscle cell line is used to treat curcumin and dihydrogingerrone, and the glucose absorption capacity is measured to determine the potency Respectively. As a result, it was confirmed that the glucose absorption capacity was higher than that of curcumin, and that dihydrogingerol had strong glucose uptake effect as well as known insulin (FIG. 9).

In another embodiment of the present invention, in order to determine whether or not blood glucose can be regulated in animals, dihydroergensolone was fed to a group of three groups , Diabetes mellitus and high digestive system + dihydro ginger auron). The results showed that dihydroerginizer was more effective than diabetes mellitus in high fat diets. (Fig. 1). &Lt; / RTI &gt;

Accordingly, the present invention relates, in a further aspect, to a composition for inhibiting obesity containing the dehydrogenzyme as an active ingredient.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of the compositions.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select parenterally when injecting parenterally or intraperitoneally, intramuscularly, subcutaneously, intravenously, intramuscularly or intrasternally , But is not limited thereto.

The composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods . Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluent, liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg per day, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The dihydroergizing agent of the present invention has a blood glucose controlling ability to induce blood glucose lowering, increases activity through phosphorylation of AMPK, decreases weight gain, and increases insulin function control. Therefore, &Lt; / RTI &gt;

Accordingly, the present invention relates to a health functional food for improving diabetes containing dihydrogingerrone as an active ingredient from a different viewpoint.

In another aspect, the present invention relates to a health functional food for improving obesity containing dihydrojuggerone as an active ingredient.

The health food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of the food. Examples of the food include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, various soups, And a combination thereof, all of which include health foods in a conventional sense.

The dihydrogingerrone according to the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of dihydroxygermanium in the health food may be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g, . However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the above-mentioned dihydroxyzincoron as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

In addition, the dihydrogingerrone of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of from 0 to about 20 parts by weight per 100 parts by weight of the dihalo gingerolone of the present invention.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The term "treatment" in the present invention means all the actions of improving or alleviating the symptoms of diabetes by the administration of the composition.

As used herein, the term "improvement" is meant to include preventing, ameliorating, treating, or delaying the onset of such symptoms.

As used herein, the term "improved obesity" means any action that improves or alleviates the symptoms of obesity with the administration of the composition.

As used herein, the term "functional food" means food prepared and processed using raw materials or ingredients having functionality useful to the human body according to Law No. 6727 on health functional foods, and " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One: Dihydro Ginger Ron (DHZ) on  Of weight and fat tissue

In order to analyze the effect of dihydrogingerol on the metabolism of rats, high fat diet induced obesity and measured body weight and amount of adipose tissue. The mice were divided into the low-fat diet (LFD), high fat diet (HFD) and high fat diet + dihydroginger rheum (HFD + DHZ) groups and weighed and weighed for 13 weeks.

As a result, it was confirmed that the weight gain induced by the high fat diet was inhibited by dihydroergensin (Fig. 1A), and visceral fat was significantly reduced in the group fed with dihydroergensin as compared to the high fat diet group (Fig. 1B ). In addition, it was confirmed that the fatty liver and the peripheral fat tissue induced by the high fat diet method were significantly reduced in the group fed with dihydroergensin as compared with the high fat fat diet group (Fig. 1C, 1D). This indicates that dihydrogingerrone is effective in weight control of obese subjects.

Example  2: Dihydro Ginger Ron (DHZ) on  Inhibition of blood glucose increase by

In order to analyze the effect of dihydrozinceron on blood glucose increase, blood glucose levels in laboratory animals were measured. Fasting blood glucose (FBG), insulin, and leptin were measured by dividing the mice into the LFD, HFD, and HHD + DHZ groups.

As a result, it was confirmed that the fasting glucose level (FBG), insulin and leptin increased by the high fat diet were inhibited by dihydrogingerrone (Figs. 2A, 2B and 2C). This indicates that type 2 diabetes induced by high fat diet is inhibited by dehydrogingerrone.

Example  3: Dihydro Ginger Ron (DHZ) on  by AMPK ( AMP kinase ) Phosphorylation

In order to confirm the mechanism of dehydrogenase pathway in C2C12 skeletal muscle cells, the activity of AMPK, the core of glucose regulation, was measured. Western blotting analysis was performed using cell lysate of C2C12 skeletal muscle cells treated with dihydrojuggerone and AMPK phosphorylated antibody (Threonine 172, Millipore-Upstate).

As a result, dihydrogingerrone increased phosphorylation of AMPK in a concentration- and time-dependent manner (Figs. 3A, 3B). The maximal AMPK activity was observed after 10 min of treatment with 30 μM dihydrozincerone and the phosphorylation of AMPK and ACC was steadily increased after dihydrogingerol treatment. This means that the increase in AMPK phosphorylation in skeletal muscle cells is due to dehydrogingerrone.

Example  4: Dehydrozergerone in dehydrozingerone  Promoting glucose uptake by

L6 muscle precursor cells, which are more suitable for glucose uptake studies than C2C12 cells, were used to analyze glucose uptake by dihydrogingerrone in skeletal muscle. L6 muscle precursor cells (ATCC, Americal Type Culture Collection) were dispensed into 12-well plates and then differentiated into muscle cells in DMEM medium containing 2% FBS. The muscle cells were treated with dehydrozingerone (Sigma-Aldrich) and incubated at 37 ° C for 1 hour. To each well, 2-deoxy- [ ³ H] -D-glucose ) For 15 minutes, washed twice with PBS buffer, and dried. Lysis buffer (0.5 N NaOH + 0.5% SDS) was added to each well to dissolve the cells. 500 μl of each solution was added to the LSC vials in an amount of 450 μl each, mixed with 1 ml of the cocktail (Ultimagold), and then measured with a β-counter.

As a result, it was confirmed that the absorption of glucose (2-DG) was accelerated from the dihydrogingerol concentration of 3M and the maximum glucose uptake effect was promoted at 10 μM (FIG. 4A). Insulin 100 nM was used as a positive control.

Example  5: AMPK  ( AMP kinase ) Through inhibition Dihydro Ginger Ron (DHZ) on  Inhibition of glucose absorption

AMPK2 siRNA (NM-001013367, Dharmacon, USA) was treated with 2 μM of the AMPK inhibitor compound C (Calbiochem) (FIG. 4B) or with the L6 muscle bulb (2-DG) levels were measured by treating L6 muscle precursor cells with dihydroergensin after knocking down AMPK by transfection into cells (Fig. 4C) (Fig. 4D ).

As a result, it was confirmed that the stimulation of glucose uptake by dihydrogingerrone was blocked by the AMPK inhibitor compound C (FIG. 4B), and it was confirmed that inhibition of glucose uptake by dehydrogingerrone was inhibited in AMPK2 knockdown cells (Fig. 4D). This means that AMPK plays an important role in glucose uptake by dihydrogingerrone.

Example  6: Dihydro Ginger Ron (DHZ) on  by p38 MAPK Signal path  Activation

To investigate the signal transduction pathway involved in glucose uptake by dihydrogingerrone, the effect of dihydrogingerrone on p38 MAPK, an important glucose uptake, was investigated. Activation of p38 MAPK by AMPK, which plays an important role in glucose uptake, was investigated after treatment of C2C12 skeletal muscle cells with 30 μM dihydrozincerone and after treatment with the AMPK inhibitor compound C. Next, the MAPK inhibitor SB203580 was treated to confirm that p38 MAPK is involved in glucose uptake by dihydrogingerrone.

As a result, p38 MAPK was activated in a time- and concentration-dependent manner by dihydrogingerrone (Figs. 5A and 5B), and p38 MAPK phosphorylation by dihydrogingerrone was inhibited by compound C (Fig. 5C). In addition, the inhibited p38 MAPK inhibited glucose uptake by dihydrozincerone (Fig. 5D). This means that the activation of p38 MAPK in glucose uptake by dihydrogingerrone occurs through AMPK.

Example  7: GLUT4  For expression Dihydro Ginger Ron (DHZ)  effect

Glucose transporter GLU4 plays an important role in regulating glucose homeostasis as a mediator of glucose uptake. GLU4 expression was examined in C2C12 cells stimulated with glucose uptake by dihydrogingerrone

As a result, dihydrogingerrone increased both mRNA and protein levels of GLU4 (Fig. 6A, 6B), indicating that dihydrogingerrone could induce GLU4 expression and regulate glucose uptake.

Example  8: Promoting insulin-induced glucose uptake Dihydro Ginger Ron (DHZ)  effect

In order to analyze the effect of dihydrogingerrone on insulin-stimulated glucose uptake, it has been shown that dihydrogingerrone increases insulin-stimulated glucose uptake by simultaneously treating insulin and dihydrogingerrone in L6 muscle differentiated cells (Fig. 7A). In addition, in order to confirm that dihydrogingerrone causes blood glucose lowering through the activation of AMPK in the metabolic changes induced by high fat diet, 20% sucrose solution (2 g / kg) was used to induce intraperitoneal glucose tolerance .

As a result, the concentration of blood glucose significantly decreased in the group fed with dihydrojuggerone than the high-fat diet group after 30 to 60 minutes (Fig. 7B). This indicates that AMPK is activated by dihydrogingerrone to promote blood glucose intensification, and it means that dihydrogingerrone plays an important role in promoting glucose uptake induced by insulin.

Example  9: Curcumin and Dihydro Ginger Ron (DHZ)  insulin Sensitivity ( insulin sensitizer ) compare

We compared the pattern of increased glucose uptake by insulin by treating curcumin and dihydrogingerrone with insulin, respectively, using differentiated muscle cell lines, and compared the possibility of insulin sensitizers to overcome insulin resistance Respectively. L6 muscle precursor cells were plated in 12 well plates and differentiated into muscle cells in DMEM medium supplemented with 2% FBS. The muscle cells were treated with 1 μl each of dehydrogingerrone and curcumin for 24 hours, 15 minutes after treatment with insulin (10 ng / ml), the glucose derivative 2-DG (2-deoxy- [ ³ H] -D-glucose) was treated for 15 minutes in each well and washed twice with PBS buffer . Lysis buffer (0.5 N NaOH + 0.5% SDS) was added to each well to dissolve the cells in 500 μl of each well. Then, the cells were transferred into LSC vials 450 times, mixed with 1 ml of cocktail (Ultimagold), and measured with a β-counter.

As a result, as shown in FIG. 8, the glucose uptake by insulin alone was not significantly increased by the curcumin pretreatment, whereas when pretreated with dihydroergensin, the glucose uptake was significantly higher than that by insulin alone Effect. This result shows that dihydrogingerrone has a function as an insulin sensitizer, which means that the combined treatment of insulin can improve the effectiveness of diabetes treatment.

Example  10: Curcumin and Dihydro Ginger Ron (DHZ)  Comparison of blood glucose control ability

In order to compare the curative effect of curcumin and dihydrogingerone, insulin was used as a positive control to treat curcumin and dihydrogingerone in differentiated muscle cell lines, and glucose uptake ability was measured The potency was compared. L6 muscle precursor cells were plated in 12-well plates and differentiated into muscle cells in DMEM medium containing 2% FBS. The cells were treated with 3 μl each of dihydrogingerone and curcumin for 3 hours and insulin was treated with 100 ng / ml for 15 min. The glucose derivative 2-DG (2-deoxy- [ ³ H] -D-glucose ) For 15 minutes, washed twice with PBS buffer, and dried. Lysis buffer (0.5 N NaOH + 0.5% SDS) was added to each well to dissolve the cells. 500 μl of each solution was added to 450 μl of LSC vial, mixed with 1 ml of cocktail (Ultimagold), and measured with a β-counter.

As a result, as shown in FIG. 9, it was confirmed that the glucose uptake capacity was slightly higher than that of curcumin, and that dihydrogingerol had a glucose uptake effect as strong as that of well-known insulin.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A method of screening for a diabetes therapeutic agent comprising the steps of:
(a) treating C2C12 skeletal muscle cells with a diabetic therapeutic agent candidate;
(b) measuring the AMPK (AMP kinase) phosphorylation level of the test group treated with the candidate substance and the control group treated with dehydrozingerone; And
(c) selecting a candidate substance for treating diabetes mellitus when the AMPK phosphorylation level is increased compared to the control group treated with the dehydrogenzyme.
2. The method of claim 1, wherein the diabetes is type 2 diabetes.
2. The method of claim 1, wherein the AMPK (AMP kinase) phosphorylation level is measured by Western blot analysis using an AMPK phosphorylated antibody.
delete delete delete delete delete A method for screening a substance for preventing or improving obesity comprising the steps of:
(a) treating C2C12 skeletal muscle cells with a candidate substance for prevention or amelioration;
(b) measuring and comparing the AMPK phosphorylation level of the test group treated with the candidate substance and the control group treated with dehydrozingerone; And
(c) selecting a candidate substance as an agent for preventing or ameliorating obesity when the level of AMPK phosphorylation is increased as compared with a control group treated with the dehydroergizing agent.
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