KR20100055058A - The composition for the treatment of diabetes and metabolic syndrome containing synthesized obovatol and its derivatives - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing obovatol and derivative thereof is provided to enhance AMPK(AMP-activated protein kinase) activation and to treat diabetes and metabolic disorder. CONSTITUTION: A health food for preventing or treating diabetes contains obovatol of chemical formula 1 and derivative thereof as active ingredient. In chemical formula 1, R1 and R2 are independently or selectively hydrogen, straight chain or side chain alkyl of C1-C7, acetyl, alkoxy of C1-C7, or alkoxyacetyl of C1-C7. The food additionally contains phemaceutically acceptable excipeint. The obovatol is obtained by isolating and purifying from Magnolia obovata Thunberg.

Description

Composition for the treatment of diabetes and metabolic syndrome containing synthesized obovatol and its derivatives}

The present invention relates to a composition for the prevention and treatment of diabetes mellitus and metabolic diseases containing obovatol and its derivatives as an active ingredient, and more specifically, synthesized by ovobatol extracted and purified by extracting ilhubak fruit with methanol It relates to a composition for the prevention and treatment of diabetes and metabolic diseases containing one derivative as an active ingredient.

[Formula 1]

In Chemical Formula 1,

R ¹ and R ² are independently or optionally hydrogen, C ₁ -C ₇ straight or branched alkyl, acetyl, C ₁ -C ₇ alkoxy or C ₁ -C ₇ alkoxyacetyl;

R ³ is hydrogen, C ₁ -C ₇ straight or branched alkyl, C ₂ -C ₇ alkenyl or allyl.

Preferably said R ¹ and R ² are independently or optionally methyl, ethyl, acetyl, methoxy, ethoxy, methoxyacetyl or ethoxyacetyl;

R ³ is hydrogen, propyl, propenyl or allyl.

Specific compounds of the obovatol derivative represented by Formula 1 of the present invention are as follows.

1) obovatol;

2) 3- (4-propylphenoloxy) -5-propylbenzene-1,2-diol (ob-RD);

3) 3- (4-propylphenoxy) -2-methoxy-5-propylphenol (ob-Rd-2M)

4) 1- (4-propylphenoxy) -2,3-diacetyloxy-5-propylbenzene (ob-Rd-Ac)

5) 1- (4-propylphenoxy) -2,3-di-2-methoxyacetyloxy-5-propylbenzene (ob-Rd-MAc)

6) 3- (4-((E) -prop-1-enyl) phenoxy) -5-((E) -prop-1-enyl) benzene-1,2-diol (ob-RE)

7) 1- (4-((E) -prop-1-enyl) phenoxy) -2,3-diacetyloxy-5-((E) -prop-1-enyl) benzene (ob-RE -MAc)

8) 1- (4-((E) -prop-1-enyl) phenoxy) -2,3-di-2-methoxyacetyloxy-5- (E) -prop-1-enyl) benzene (ob-RE-Ac)

1 Hardie et al, FEBS Lett. 546, pp 113-120, (2003)

David Carling et al, TRENDS in Biochemical Sciences 29, pp 18-24, (2004)

Juleen R. et al, J Appl Physiol. 93, pp 773-781, (2002)

4 Holman, R. R. et al, Metabolism, 55, S2-5, 2006; Pickup, J.C. et al., Blackwell Scientific Publ. London, pp 462-476, (1991)

[5] Innerfield, R. J., New Engl. J. Med., 334, pp 1611-1613, (1996)

Schlattner U, et al, J Biol Chem. 279, pp 43940-51, (2004)

7 Somwar R et al, Clin Ther. 20, pp 125-40, (1998)

8 Halseth AE et al, Biochem Biophys Res Commun. 294, pp 798-850, (2002)

An object of the present invention relates to a composition for the prevention and treatment of diabetes and metabolic diseases containing the synthesized obovatol and single derivatives as an active ingredient.

AMPK (AMP-Activated Protein Kinase) is a kinase that plays a very important role in maintaining the energy balance in the body by controlling the carbohydrate metabolism and lipid synthesis metabolism in the cell by sensing the energy state in the cell. AMPK is an alpha subunit of AMPK caused by AMPK kinase when the ATP content of the cell's bioenergy is sharply lowered, that is, when the ATP: AMP ratio in the cell decreases significantly after heavy exercise or ingestion of food for a long time. The threonine residue 172 of the subunit is phosphorylated and converted into an enzyme in an activated form. As a result, beta oxidation (β-oxidation) metabolism, which is a process of glycolysis and body fat decomposition, is actively performed to generate ATP, a bioenergy (Hardie et al, FEBS Lett. 546, pp 113-120, 2003). In contrast, ATP-consuming fat synthesis and cholesterol synthesis are inhibited (David Carling et al, TRENDS in Biochemical Sciences 29, pp 18-24, 2004). In addition, when AMPK is activated, blood glucose is promoted into cells to regulate blood glucose. On the other hand, AMPK activation leads to a decrease in blood glucose because the movement of glucose uptake pathways such as GLUT4 (glucose transporter 4) increases to the cell membrane irrespective of insulin action (Juleen R. et al, J Appl Physiol. 93, pp 773). -781, 2002).

Specific metabolic changes in accordance with AMPK activation include phosphorylated activation of AMPK, a key enzyme for fatty acid synthesis, acetyl-CoA carboxylase (ACC), which produces malonyl-CoA, and HMG-CoA reductase, a key enzyme for cholesterol synthesis. Direct phosphorylation lowers the activity of these enzymes, thereby inhibiting fat and cholesterol synthesis. In addition, when the concentration of malonyl-CoA is lowered by the inhibition of ACC activity by increasing the phosphorylation of ACC, the decomposition of fat is promoted while increasing the inflow of fatty acids into the mitochondria where the beta oxidation process occurs.

Metabolic syndrome was called X syndrome because the cause was not known in the past. However, as research continues, it has been found that insulin, which plays a role in decomposing glucose to liver muscles, is not properly made or malfunctions, and various adult diseases such as diabetic hypertension and stroke heart disease occur. Insulin's inability to carry glucose properly is called insulin resistance. Metabolic syndrome refers to a complex of symptoms caused by insulin resistance. This is why metabolic syndrome is called insulin resistance syndrome. The reason is that even though insulin is present in the body, high blood sugar is not improved due to resistance, and only insulin concentration is increased. Major symptoms include diabetes due to abnormal blood glucose metabolism, increased triglycerides due to abnormal lipid metabolism, high blood pressure due to high density cholesterol, increased sodium content, and gout due to increased uric acid. However, if three or more of the five indicators, such as abdominal obesity, diabetes, high-density cholesterol, high blood pressure, and triglycerides, exceed the standard, it is considered as metabolic syndrome. Scientists learned from the 1990s that there was a close relationship between obesity, diabetes, high blood pressure, arteriosclerosis, cerebral stroke and heart disease, but they didn't know why and called it "X syndrome." Metabolic syndrome, a phenomenon in which a number of metabolic disorders occur simultaneously due to heredity or poor lifestyle, has not been known for its exact mechanism, but insulin resistance plays a major role. Hence, it is also called 'Insulin Resistance Syndrome' and in 1998, WHO defined it as metabolic syndrome. Insulin resistance is caused by congenital shaving, but is usually caused by obesity and increased body fat and lack of exercise. The most effective way to improve insulin resistance is through active weight control and regular exercise.

Diabetes, a representative metabolic syndrome, is a disease in which insulin metabolism, including hyperglycemia, persists due to insufficient insulin action, and is highly likely to cause vascular complications in the future. Diabetes is largely divided into insulin-dependent type 1 diabetes and insulin-independent type 2 diabetes.

Diabetes treatment agents currently used in clinical practice are largely known as recombinant insulin, sulfonylurea, metformin, thiazolidinedion, and alpha-glucosidase inhibitor. . However, sulfonyl urea drugs secrete insulin even when insulin is not needed in vivo, and there is a risk of hypoglycemia (Holman, RR et al, Metabolism, 55, S2-5,2006; Pickup, JC et al., Blackwell Scientific Publ). London, pp 462-476, 1991), the biguanide metformin inhibits your biosynthesis of hepatocytes and increases insulin receptors, but causes gastrointestinal disorders and toxicity to death after prolonged administration (Innerfield, RJ, New Engl. J. Med., 334, pp 1611-1613, 1996). Thiazolidinedione drugs include rosiglitazome (Avandia) and pioglitazone (Actose Actose), which help insulin function in muscle and adipose tissue and have an excellent effect on blood sugar control. However, side effects can cause hepatotoxicity, so be sure to take regular liver function tests while taking medication. Troglitazone, the first of its class, has been discontinued due to hepatotoxicity, and this class of drugs can also cause obesity. Recently released alpha-glucosidase inhibitors primarily delay the digestion and absorption of complex carbohydrates in the small intestine and reduce postprandial hyperglycemia and hyperinsulinemia, a hallmark of insulin-independent diabetes. However, abdominal bloating, nausea, and diarrhea are typical side effects. Recently, liver damage has been reported. Therefore, there is an urgent need for the development of drugs with minimized side effects.

In general, obesity is accompanied by hyperlipidemia and the probability of developing diabetes is very high. Increased intracellular lipid content by obesity results in insulin resistance in insulin sensitized cells, which conventionally leads to type 2 diabetes. Therefore, if an obesity treatment effect that promotes body fat breakdown is excellent, and at the same time, a substance capable of reducing blood glucose concentration independently of insulin may be developed, it may be used as a metabolic disease treatment agent such as obesity, hyperlipidemia and diabetes treatment. Therefore, the inventors of the present invention not only obovatol and its derivatives activate the AMPK enzyme, and as a result, fat synthesis and cholesterol synthesis are inhibited, and at the same time, fatty acid beta oxidation is increased to promote lipolysis, thereby reducing blood lipid and body fat. , Increasing insulin resistance according to the reduction of body fat and increasing the cell membrane movements such as GLUT4 by AMPK activation in a insulin-independent method has been found to be excellent in hypoglycemic effect The composition of the present invention is excellent in the prevention, suppression and treatment of diabetes The present invention has been completed by confirming that it can be usefully used as a component of food, medicine and feed having efficacy.

In order to achieve the above object, the present invention provides a composition for the prevention, inhibition and treatment of diabetes and metabolic syndrome containing obovatol and its derivatives represented by the following formula (1) as an active ingredient.

[Formula 1]

In Chemical Formula 1,

R ¹ and R ² are independently or optionally hydrogen, C ₁ -C ₇ straight or branched alkyl, acetyl, C ₁ -C ₇ alkoxy or C ₁ -C ₇ alkoxyacetyl;

R ³ is hydrogen, C ₁ -C ₇ straight or branched alkyl, C ₂ -C ₇ alkenyl or allyl.

Preferably said R ¹ and R ² are independently or optionally methyl, ethyl, acetyl, methoxy, ethoxy, methoxyacetyl or ethoxyacetyl;

R ³ is hydrogen, propyl, propenyl or allyl.

The present invention also provides a method for preventing, inhibiting and preventing diabetes and metabolic syndrome, comprising administering a pharmaceutically effective amount of the obovatol and its derivatives to a subject.

The present invention also provides a dietary supplement for the prevention, inhibition and improvement of diabetes and metabolic syndrome containing obovatol and its derivatives as an active ingredient.

The present invention also provides an AMP-activated protein kinase (hereinafter, referred to as "AMPK") enzyme activator comprising obovatol and its derivatives as an active ingredient.

The composition containing the synthesized ovobatol and its derivatives as an active ingredient, foods, drugs and drugs having an excellent effect on the prevention, inhibition and treatment of diabetes and metabolic syndrome by increasing the hypoglycemic effect by activating the AMPK enzyme insulin-independent method It can be usefully used as a feed additive.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetes mellitus and metabolic syndrome containing obovatol and its derivatives as a pharmaceutically acceptable active ingredient.

More specifically, it provides a pharmaceutical composition for the prevention and treatment of diabetes mellitus and metabolic syndrome through AMPK enzyme activation containing obovatol and its derivatives as a pharmaceutically acceptable active ingredient.

The present invention also provides an AMPK enzyme activator containing obovatol and its derivatives as a pharmaceutically acceptable active ingredient.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the compounds of the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients and diluents which may be used in the composition comprising the extract include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. The administration may be carried out once a day or divided into several doses. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

In addition, the present invention provides a dietary supplement for the prevention and improvement of diabetes and metabolic syndrome containing the ovobatol and a single derivative showing a pharmacological and improving effect of diabetes as a pharmaceutically acceptable active ingredient.

The compounds of the present invention can be used in various ways, such as drugs, foods and beverages for the prevention and improvement of diabetes. Examples of the food to which the compound of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The compound of the present invention may be added to food or beverage for the purpose of preventing and improving diabetes. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the compound of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 weights per 100 weights of the composition of the invention.

In addition, the present invention provides an animal feed additive useful for the prevention and improvement of diabetes mellitus containing obovatol and a single derivative as a pharmaceutically acceptable active ingredient showing the prevention and improvement of diabetes and metabolic syndrome and a feed comprising the same. .

The animal feed additive compound may be 20 to 90% high concentrate or powder or granule form.

Compounds for animal feed additives of the present invention are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymeric phosphate), polyphenols, catechins (catechin) ), Alpha-tocopherol, rosemary extract (rosemary extract), vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further include any one or more than one.

Animal feed additives containing a compound of the present invention and feed containing the same as a secondary component, various supplements such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, live microbial preparations, grains, for example Milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The animal feed additive compound may be administered to an animal alone in combination with other feed additives in an edible carrier. In addition, the compound for animal feed additives can be easily administered as a top dressing or directly mixed with the animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients. Typically, single or divided daily dosages can be used as is well known in the art.

When the compound for animal feed additives is administered separately from the animal feed, as is well known in the art, the dosage forms of the compounds may be prepared in an immediate release or sustained release formulation in combination with a non-toxic pharmaceutically acceptable edible carrier. have. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the compound may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.

In addition, the animal feed in which the compound of the present invention is included as an animal feed additive may be any protein-containing organic cereal meal conventionally used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The animal feed additive may be used by adding to the animal feed by dipping, spraying or mixing. The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg, mink and fox), and Zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish, such as trout, may also be included.

In the animal feed blending method comprising the compound according to the present invention, the compound is incorporated into the animal feed in an amount of about 1 g to 100 g per kg of feed on a dry weight basis. In addition, the feed mixture is thoroughly mixed and then a viscous granulated or granular material is obtained depending on the degree of grinding of the components. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. At this time, it is preferable to add water to the animal feed and then go through the usual pelletization, expansion, or extrusion process to prevent separation during storage. Excess water may be removed to dryness.

The composition containing obovatol and its derivatives of the present invention as an active ingredient prevents and inhibits diabetes and metabolic syndrome by activating the AMPK enzyme and increasing insulin resistance by increasing insulin resistance by activating AMPK. And food, pharmaceutical and feed additives having excellent efficacy in treatment.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Reference Example 1 Reagents and Instruments

Analyzers include ¹ H-NMR (400 MHz) and ¹³ C-NMR (400 MHz) spectrometers (JNM-AL 400, JEOL Ltd., Japan), Melting pointers (Yamako, MD-S3, Japan), Mass spectrometer (MS, PE SCIX API 2000 MS / MS, Canada) was used. Various reagents were used by Aldrich Chemical Co., and other solvents were used without purification of the first or higher reagents. Silica gel (Silica gel, Merck, 230-400 mesh) was used for the purification of the synthesized materials.

Example 1 Preparation of Obovatol

1 kg of dried Japanese hawthorn fruit is added to methanol or ethanol 4L and left to stand at room temperature for 5 days. The organic solvent layer was separated, dried under reduced pressure, adsorbed onto silica gel, and hexane and ethyl acetate (9: 1 ratio) were added to purify the active fraction. 40 g of obobatol in liquid state was obtained from 1 kg of dried Japanese hawthorn berries.

Example 2 Preparation of Tetrahydro Obovatol (ob-Rd)

Obovatol (1 g) isolated in Example 1 was dissolved in acetone (200), and then palladium-charcoal (2 mg) was added at room temperature and hydrogen was passed through.

Then, an organic solvent layer containing the active material was collected and concentrated under reduced pressure. Concentrated extract (1.1 g) was dissolved in methylene chloride (100) and added to silica gel (Merck, Art No. 9385) to adsorb the active substance, and then the ratio of ethyl acetate and hexane was 10:90 to 20:80. Silica gel column chromatography with varying active fractions separated 0.9 g of colorless ob-Rd (yield: 90%).

Example 3 Preparation of 3- (4-propylphenoxy) -2-methoxy-5-propylphenol (ob-Rd-2M)

After dissolving ob-Rd (1 g) prepared in Example 2 in acetone (200), potassium carbonate (K ₂ CO ₃ ) (1.1 g) and methyl iodide (0.3 g) was added for 5 hours at room temperature Was stirred. After the reaction was completed, an organic solvent layer containing the active material was collected and concentrated under reduced pressure. Concentrated extract (1.2 g) was dissolved in methylene chloride (30) and added to silica gel (Merck, Art No. 9385) to adsorb the active substance, and then the ratio of ethyl acetate and hexane was 10:90 to 20:80. The active fractions were separated by silica gel column chromatography with changing to give a colorless 3- (4-propylphenoxy) -2-methoxy-5-propylphenol (ob-Rd-2M, 1 g) (yield: 90% ).

Example 4 Preparation of 1- (4-propylphenoxy) -2,3-diacetyloxy-5-propylbenzene (preparation of ob-Rd-Ac)

Ob-Rd (1 g) prepared in Example 2 was dissolved in acetone (200), and then potassium carbonate (K ₂ CO ₃ ) (1.2 g) and acetyl chloride (500 mg) were added and stirred at room temperature for 5 hours. A colorless 1- (4-propylphenoxy) -2,3-diacetyloxy-5-propylbenzene (1.1 g) was obtained by the same method as the method of Example 3 except for the yield. %).

Example 5 Preparation of 3- (4-((E) -prop-1-enyl) phenoxy) -5-((E) -prop-1-enyl) benzene-1,2-diol ob-RE)

Obovatol (1 g) was dissolved in methanol, then palladium chloride (PdCl ₂ ) (10 mg) was added and stirred at room temperature for 5 hours. After the reaction was completed, an organic solvent layer containing the active material was collected and concentrated under reduced pressure. Concentrated extract (1.1 g) was dissolved in methylene chloride (30) and added to silica gel (Merck, Art No. 9385) to adsorb the active substance, and then the silica gel column was changed to 20:80 ratio of ethyl acetate and hexane. Chromatography separated the active fractions. Then, the active fractions were dried to give a colorless 3- (4-((E) -prop-1-enyl) phenoxy) -5-((E) -prop-1-enyl) benzene-1, 2-diol (0.91 g) was obtained (yield: 90%).

Example 6 1- (4-((E) -prop-1-enyl) phenoxy) -2,3-di-2-methoxyacetyloxy-5-((E) -prop-1 Preparation of -enyl) benzene (ob-RE-MAc)

After dissolving the propenyl ovobatol (1 g) prepared in Example 5 in acetone (200), by adding potassium carbonate (K ₂ CO ₃ ) (1.2 g) and (methoxyacetyl chloride) (600 mg) at room temperature Colorless 1- (4-((E) -prop-1-enyl) phenoxy) -2,3-di- was carried out in the same manner as in Example 2 except that the mixture was stirred for 5 hours at. 2-methoxyacetyloxy-5-((E) -prop-1-enyl) benzene (1.15 g) was obtained (yield: 90%)

<Example 7>

1- (4-((E) -prop-1-enyl) phenoxy) -2,3-diacetyloxy-5-((E) -prop-1-enyl) benzene (ob-RE-Ac Manufacture of

After dissolving propenyl obovatol (1 g) prepared in Example 5 in acetone (200), and then added potassium carbonate (K ₂ CO ₃ ) (1.3 g) and acetyl chloride (500 mg) for 5 hours at room temperature Colorless 1- (4-((E) -prop-1-enyl) phenoxy) -2,3-diacetyloxy-5- was carried out in the same manner as in Example 2 except for stirring. ((E) -prop-1-enyl) benzene (1.0 g) was obtained (yield: 90%).

<Analysis>

The compounds prepared in Examples 1 to 7 were subjected to UV absorbance analysis, IR (infrared) absorbance analysis and high resolution mass spectrometry to determine the molecular weight and molecular formula of the pure purified compound. Specifically, UV absorbance analysis was measured by Shimadzu UV-265 spectrophotometer, and IR absorbance was measured by Bio-Rad's DigiLab Divzone FTS-80 spectrophotometer (Bio-Rad Digilab Division FTS- 80 spectrophotometer) and molecular weight and molecular formula were determined by measuring high resolution MS using VG70-SEQ mass spectrometry (MS). In addition, ¹ H, ¹³ C-NMR spectra were obtained using nuclear magnetic resonance (Varian 300, 500 NMR), and the structures were determined by comprehensive analysis of these spectra.

The analysis results are summarized in Tables 1 and 2 below.

Table 1. Appearance, Molecular Formula, Molecular Weight and Solubility

division
color
Molecular formula
Molecular Weight Solubility Availability Insoluble Example 1 Colorless C ₁₈ H ₁₈ O ₃ 282 Alcohol, DMSO water Example 2 Colorless C ₁₈ H ₂₂ O ₃ 286 Alcohol, DMSO water Example 3 Colorless C ₁₉ H ₂₄ O ₃ 300 Alcohol, DMSO Hexane, water Example 4 Colorless C ₂₂ H ₂₆ O ₃ 370 Alcohol, DMSO water Example 5 Colorless C ₁₈ H ₁₈ O ₃ 282 Alcohol, DMSO water Example 6 Colorless C ₂₄ H ₂₆ O ₇ 426 Alcohol, DMSO water Example 7 Colorless C ₂₂ H ₂₂ O ₅ 366 Alcohol, DMSO water

Table 2. NMR data

division
NMR data

Example 1 ¹ H NMR (300 MHz, CDCl ₃ ): 7.15 (2H, d, J = 9 Hz), 6.94 (2H, d, J = 9 Hz), 6.59 (1H, d, J = 1.5 Hz), 6.30 (1H , d, J = 1.5 Hz), 5.97 (2H, m), 5.06 (4H, m), 3.38 (2H, d, J = 6.6 Hz), 3.20 (2H, d, J = 6.6 Hz) ppm
Example 2 ¹ H-NMR (CDCl ₃ ): 7.13 (2H, d, J = 8.7 Hz), 6.95 (2H, d, J = 8.7 Hz), 6.57 (1H, d, J = 1.5 Hz), 6.3 (1H, d , J = 2.1 Hz), 2.58 (2H, t, J = 8.1 Hz), 2.42 (2H, t, J = 8.1 Hz), 1.59 (4H, m), 0.92 (6H, m) ppm.
Example 3 ¹ H-NMR (CDCl ₃ ): 7.15 (2H, d, J = 9 Hz), 6.97 (2H, d, J = 9 Hz), 6.66 (1H, d, J = 1.5 Hz), 6.37 (1H, d , J = 1.5 Hz), 3.92 (3H, s), 2.61 (2H, t, J = 8.1 Hz), 2.48 (2H, t, J = 8.1 Hz), 1.65 (4H, m), 0.97 (6H, m ) ppm.
Example 4 ¹ H-NMR (CDCl ₃ ): 7.12 (2H, d, J = 8.7 Hz), 6.92 (2H, d, J = 8.7 Hz), 6.74 (1H, d, J = 2.1 Hz), 6.63 (1H, d , J = 2.1 Hz), 2.56 (2H, t, J = 8.1 Hz), 2.48 (2H, t, J = 8.1 Hz), 2.29 (3H, s), 2.19 (3H, s), 1.59 (4H, m ), 0.92 (6H, m) ppm.
Example 5 ¹ H-NMR (CDCl ₃ ): 7.28 (2H, d, J = 9 Hz), 6.95 (2H, d, J = 9 Hz), 6.73 (2H, d, J = 1.5 Hz), 6.42 (2H, d , J = 1.5 Hz), 6.37 (1H, d, J = 15.5 Hz), 6.15 (2H, m), 5.99 (1H, m), 5.42 (2H, s), 1.88 (3H, d, J = 7 Hz ), 1.80 (3H, d, J = 7 Hz).
Example 6 ¹ H-NMR (CDCl ₃ ): 7.27 (2H, d, J = 8.1 Hz), 6.95 (2H, d, J = 8.1 Hz), 6.77 (1H, d, J = 1.5 Hz), 6.17 (5H, m ), 4.27 (2H, s), 4.14 (2H, s), 3.525 (3H, s), 3.41 (3H, s), 1.88 (3H, d, J = 6.6), 1.83 (3H, d, J = 6.6 ).
Example 7 ¹ H-NMR (CDCl ₃ ): 7.27 (2H, d, J = 8.1 Hz), 6.97 (2H, d, J = 8.1 Hz), 6.88 (1H, d, J = 1.5 Hz), 6.75 (1H, d , J = 1.5 Hz), 6.37 (1H, d, J = 14.7 Hz), 6.14 (3H, m), 2.29 (3H, s), 2.18 (3H, s), 1.87 (3H, d, J = 6.6 Hz ), 1.81 (3H, d, J = 6.6 Hz).

Experimental Example 1 AMPK Activation Efficacy

In order to test the AMPK enzyme activation of the monosyntheses synthesized in Examples 1-7, the method described in the literature was applied to determine the efficacy of the compounds as follows (Schlattner U, et al, J Biol Chem. 279 , pp 43940 -51, 2004).

   Obovatol and single derivatives were treated in L6 myotube cells (American Type Culture Collection, Manassas VA.) At concentrations of 5, 10, and 20 μM as described in FIG. 2 to activate the AMPK antibody in activated form (Cell Signaling Technology, Beverly, MA) was prepared and the threonine residue 172 in the alpha (a) -subunit of AMPK was identified to confirm the increased activation of AMP-activated protein kinase (hereinafter referred to as "AMPK") enzyme according to the treatment concentration. Phosphorylation increase was shown by Western blot assay (FIG. 2).

     As a result, as shown in FIG. 2, the concentrations of ovobatol and single derivatives were increased by concentration. From the above results, it was confirmed that obovatol and single derivatives are advantageous for AMPK activation.

Experimental Example 2 Measurement of Glucose Uptake of L6 Myotube Cells

Obovatol and single derivatives synthesized in <Example 1-6> were measured by applying the method described in the literature to determine the glucose uptake in L6 myotube cells (Somwar) R et al, Clin Ther. 20 , 125-40, 1998).

Completely differentiated L6 myotube cells were incubated for 6 hours in serum-free medium (GIBCO, Auckand NZ) and then treated with 10 μM of obovatol and a single derivative for 2 hours, respectively, and 10 μM unlabeled 2-deoxyglucose, 10 The reaction was performed for 10 minutes in HEPES-saline buffer containing μM 2-deoxy- [ ³ H] -glucose (1 mCi / ml). After washing the cells three times with ice-cold PBS, the cells were lysed using 1N NaOH and cpm was measured with a scintillation counter (Packard Co. 1600PR).

In this case, non-specific glucose uptake in cells was measured by HEPES-saline buffer containing 10 μM of cytochalasin B (Sigma Chemical Co. St Louis MO) and removed from the total value.

As a result, as shown in Figure 3, it was confirmed that the glucose uptake (glucose uptake) of the obovatol and a single derivative also appeared at the same concentration.

Experimental Example 3 Measurement of Blood Glucose Level in db / db Mice

In order to measure blood glucose concentrations in db / db mice of the compounds obtained in the above examples, the efficacy of the compounds was measured as follows (Halseth AE et al, Biochem Biophys Res Commun. 294 , 798). -850, 2002).

Diabetic mice, 5 weeks old db / db mouse (The Jackson Laboratory, Bar Harbor, ME, Inc Germany), divided into 8 mice each with 5, 10 mg / kg (olive oil) obovatol-administered group and the same volume of saline-administered group (Control) was administered for 4 weeks to check the blood glucose change of the mice.

As shown in FIG. 4, the obovatol 5 mg / kg showed no significant blood glucose change in the blood glucose change as compared to the saline-administered group, but the ovobatol 10 mg / kg-administered group was 39.6% (235.75 ± 1 week). 20.9, 390.25 ± 44.1), 29.2% at 2 weeks (297.25 ± 29, 419.75 ± 28.8), 52.7% at 3 weeks (234 ± 61.5, 495 ± 86.2), 55.2% at 4 weeks (288 ± 27.6, 522 ± 78) .

Experimental Example 4 Acute Toxicity Test

In order to test the acute toxicity of the compound of the present invention, according to the regulations of the Food and Drug Administration (KFDA) was tested using male ICR mice (4 weeks old, Central Experimental Animal Co., Ltd.).

Ten mice in each group were orally administered with a compound of the present invention at a dose of 0.625-2 g / kg and observed for two weeks.

After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, blood biochemical tests were performed, and autopsy was performed to observe abnormalities of organs and thoracic organs.

As a result, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemical test and autopsy findings.

The compound of the present invention did not show a toxic change when administered orally to a dose of 2g / kg in mice, 1-1000 mg / kg orally administered to humans, 0.2-100 mg / cm ₂ when applied to the skin effect Was determined to be a substance.

Examples of the formulation of the composition are described below, but are not intended to limit the present invention but to explain in detail only.

Preparation Example 1 Preparation of a Pharmaceutical Formulation

1-1. Manufacture of powder

Example 1 2 g of compound

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

100 mg of the compound of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of Capsules

100 mg of the compound of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Manufacture of rings

1 g of compound of Example 1

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

1-5. Preparation of Granules

150 mg of the compound of Example 1

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

Production Example 2 Preparation of Food

Foods comprising the composite of the present invention were prepared as follows.

2-1. Preparation of Cooking Seasonings

20 to 95 parts by weight of the compound of Example 2 of the present invention was prepared for cooking spices for health promotion.

2-2. Preparation of Tomato Ketchup and Sauce

Health care tomato ketchup or sauce was prepared by adding 0.2 to 1.0 parts by weight of the compound of Example 3 to tomato ketchup or sauce.

2-3. Manufacture of Flour Food

0.5 to 5.0 parts by weight of the compound of Example 4 of the present invention was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

2-4. Preparation of soups and gravy

0.1 to 5.0 parts by weight of the compound of Example 5 of the present invention was added to soups and broth to prepare meat products for health promotion, soups and noodles of noodles.

2-5. Preparation of Ground Beef

10 parts by weight of the compound of Example 6 of the present invention was added to the ground beef to prepare a ground beef for health promotion.

2-6. Manufacture of dairy products

5 to 10 parts by weight of the compound of Example 2 of the present invention was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

2-7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The compound of Example 3 of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the dry powder of the compound of Example 3 prepared above were blended in the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Dry powder (3 parts by weight) of the compound of Example 1,

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

2-8. Manufacturing of dietary supplements

1000 mg of one compound of Examples 1-6

Vitamin mixture proper amount

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture quantity

Ferrous Sulfate 1.75 mg

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Preparation Example 3 Preparation of Beverage

3-1. Manufacture of health drinks

1000 mg of one compound of Examples 1-6

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

3-2. Preparation of Vegetable Juice

5 g of one of the compounds of Examples 1-6 of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3. Preparation of Fruit Juice

1 g of one of the compounds of Examples 1-6 of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

1 is a diagram showing an NMR spectrum of tetrahydro obovatol (ob-Rd),

FIG. 2 shows residues of threonine 172 present in the alpha (a) -subunit of AMPK to confirm increased activation of AMPK enzyme according to the treatment concentration of the sample in mouse-derived L6 muscle cells. Phosphorylation is confirmed by the antibody staining method,

Figure 3 is a diagram showing the increase in glucose uptake (glucose uptake) in the cells when the sample was treated for 2 hours to L6 muscle cells,

Figure 4 is a diagram showing the change in blood glucose when oral administration of the sample to the db / db mice model of diabetic mice.

Claims (6)

A health functional food for preventing or improving diabetes containing obovatol and its derivatives represented by the following Chemical Formula 1 as an active ingredient. [Formula 1]

In Chemical Formula 1, R ¹ and R ² are independently or optionally hydrogen, C ₁ -C ₇ straight or branched alkyl, acetyl, C ₁ -C ₇ alkoxy or C ₁ -C ₇ alkoxyacetyl; R ³ is hydrogen, C ₁ -C ₇ straight or branched alkyl, C ₂ -C ₇ alkenyl or allyl. The method of claim 1, The food is a health functional food for preventing or improving diabetes, characterized in that it further comprises a pharmaceutically acceptable excipient in the form of food or edible beverages. A pharmaceutical composition for the prevention or treatment of diabetes, comprising obovatol and its derivatives represented by the following formula (1) as an active ingredient. [Formula 1]

In Chemical Formula 1, R ¹ and R ² are independently or optionally hydrogen, C ₁ -C ₇ straight or branched alkyl, acetyl, C ₁ -C ₇ alkoxy or C ₁ -C ₇ alkoxyacetyl; R ³ is hydrogen, C ₁ -C ₇ straight or branched alkyl, C ₂ -C ₇ alkenyl or allyl. The method of claim 3, wherein Pharmaceutical composition characterized in that it contains a pharmaceutically acceptable active ingredient thereof obovatol and its derivatives as an active ingredient. The method of claim 3, wherein Pharmaceutical composition, characterized in that to increase the activity of the AMP-activated protein kinase (AMPK) enzyme having an obovatol and its derivatives as an active ingredient. The manufacturing method according to claim 1 or 3, wherein the obovatol is obtained by separating and purifying from Japanese fruit trees.

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