KR20110131821A - Composition comprising sauchinone as an active ingredient for preventing or treating insulin resistance - Google Patents

Abstract

PURPOSE: A composition containing Saururus chinensis BAILL. or sauchinone is provided to suppress glycosylation and to reduce blood glucose and body weight. CONSTITUTION: A pharmaceutical composition for preventing or treating insulin resistance, insulin resistance-associated disease or insulin resistance syndrome contains 0.01-99.9 % of sauchinone or Saururus chinensis BAILL. extract fraction as an active ingredient. A method for preparing the Saururus chinensis BAILL. extract fraction comprises: a step of extracting Saururus chinensis BAILL. with non-polar solvent and removing the non-polar ingredients; a step of isolating with a less polar solvent; and a step of fractioning using n-hexane-ethyl acetate. The composition is manufactured in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, suppository, and sterilized injection solution.

Description

Composition comprising sauchinone as an active ingredient for preventing or treating insulin resistance}

The present invention relates to the use of Sauchinone or Sauchinone-rich Trichophyll fractions for the prevention and treatment of insulin resistance, insulin resistance related diseases, insulin resistance syndrome.

The research for the present invention was supported in part by the Center for Excellence (ERC) [task unique number: 20100001706 task name: Center for New Drug Development for Metabolic and Inflammatory Diseases].

Insulin resistance is a condition in which liver, fat, and muscle tissues do not respond properly to insulin at normal concentrations, thereby causing abnormalities in the regulation of glucose and nutrient metabolism by insulin. Insulin regulates blood glucose by promoting glucose uptake into the muscle or by inhibiting glucose production in the liver. Insulin resistance refers to a condition in which insulin action is reduced in the absence of insulin (Schulman GI, J. Clin. Invest 106, 171-176, 2000).

As a cause of insulin resistance, abnormal inhibition of insulin signaling process has been suggested. Insulin signaling begins by the activation of insulin receptors with tyrosine kinase activity by phosphorylating tyrosine residues of the insulin receptor substrate (IRS) protein. Subsequent signaling molecules, such as Akt and GSK3-beta, are recruited and activated to interact with each other. They activate the key regulatory enzymes involved in cell growth and metabolism, such as the transport of glucose transporter-4 (GLUT 4), which is essential for glucose transport, to the cell membrane, as well as the synthesis of glycogen into cells. , Promotes protein synthesis, anti-cell death, inhibits lipolysis, promotes growth, gene expression and other various insulin effects. Inactivation of IRS is thought to be a major cause of insulin signaling inhibition, ie insulin resistance (Lee, YH et al., Arch. Pharm. Res. 27, 361 (2004)).

Hyperinsulinemia due to insulin resistance is known as a central etiology of cardiovascular diseases such as type 2 diabetes, obesity, hypertension, hypertriglyceridemia, hypoHDL cholesterol, coronary artery disease and atherosclerosis. In addition, sleep apnea (Punjabi NM et al., Respiratory Physiology & Neurobiology, 136, 167-178, 2003), prostate cancer (Barnard RJ et al., Obesity Reviews, 3, 303-308, 2002), diabetes mellitus (Greenbaum, Diabetes Metab) Res. Rev, 18, 192-200, 2003), affective disorders (Rason N et al., J. Gerontol. A Biol. Sci. Med. Sci. 59, 178-183, 2004), Alzheimer's disease (Watson GS et al., CNS Drugs 17, 27-45, 2003), stroke (Kernan WN et al., Neurology 59, 809-815, 2002), breast cancer (Stoll BA, Int. J. Obes. Relat. Metab. Disord. 26, 747 -753, 2002), inflammation (Perseghin G et al., Int. J. Obes. Relat. Metab. Disord. 27 Suppl. 3, S6-11, 2003), rheumatoid arthritis (Dessen PH et al., J. Rheumatol. 30, 1403-1405, 2003) is also suggested to be due to insulin resistance.

Reaven claims that insulin resistance is the cause of cardiovascular risk factors such as impaired glucose tolerance, hypertension, and dyslipidemia in one person, and is called insulin resistance syndrome (Reaven GM, Diabetes 1988, 37, 1595-1607, 1988). Insulin resistance syndrome also includes melanoma, polycystic ovary syndrome, hyperuricemia, increased plasmainogen activator inhibitor-1 (PAI-1), thrombolytic disorders, dysfunction of vascular endothelium and smooth muscle, and microproteinuria. (Peter P. et al., J. Clinical Pharmacy and Therapeutics, 28, 167-174, 2003).

Methformin and thiazolidinedione (TZD) drugs are known to improve insulin resistance. Metformin is mainly used to inhibit glucose production in the liver through AMPK activation, and TZD drugs are peroxisome. Increasing glucose metabolism in peripheral tissues through the activation of proliferator activated receptor (PPAR) γ is known to be the main action. However, metformin is a risk of lactic acidosis, and the glycozone-based drug, a PPARγ agonist, lowers blood sugar and decreases insulin resistance, but has side effects of accumulating fat by participating in gene expression associated with adipocyte differentiation (Singh S). Et al., JAMA, 298, 1189-1195, 2007). Therefore, not only normalizes the metabolic abnormalities, but also corrects the lipid metabolism and glucose metabolism disturbance at the same time, and further normalizes protein metabolism is required, and the development of an ideal therapeutic agent that does not occur drug resistance even when repeated use of the drug is urgently required. do.

On the other hand, three hundred seconds (三 白草) is a perennial herb of more than three hundred is known to have antioxidant, anti-aging, detoxification, anti-cancer, anti-diabetic effect. It was reported that three hundred seconds contains flavonoids, alkaloids, amino acids, fatty acids, quinones, essential oils and the like. The essential oils of 300 seconds are methyl-n-nonyl-ketone, alpha-pinene, campene, linalool, safrol, beta- Caryophyllene (β-caryophyllene), humulene (humulene) has been reported, and flavonoids such as hyperin (hyperin), quercetin (quercitin), isoquercitrin (isoquercitrin), etc. Examples of rice crops include emodin, physcion, and sachinone, which are lignan components of the carpanone family. However, it is not well known which component exerts the specific bioactive effect of 300 seconds.

It is an object of the present invention to provide methods and novel compositions useful for the prevention, improvement and treatment of insulin resistance and related diseases, insulin resistance syndrome.

For this purpose, the present invention provides a pharmaceutical composition or a food composition comprising a sacchinone or a three hundred sec extract rich in sacchinone and a method of using the same.

Administration of the sacchinone or sacchinone-rich 300 sec extract of the present invention may be useful for improving insulin resistance and preventing, improving and treating insulin resistance, diseases related to insulin resistance, and insulin resistance syndrome.

1 is a result of observing the effect of Sauchinone (Sau) on serine phosphorylation of IRS1 induced by TNFα in hepatocytes.
Figure 2 is a result of observing the effect of Saucinon (Sau) on tyrosine phosphorylation of IRS1 interfered by TNFα in hepatocytes.
Figure 3 shows the effect of Sauchinone (Sau) on the glucose production of HepG2 cells, hepatocytes. (** means a significant (p <0.01) reduction in glucose production compared to Control.)
FIG. 4 shows the effect of Saucinone (Sau) on glucose utilization in C2C12 cells, which are muscle cell lines, and 3T3-L1 cells, which are adipocyte lines. (** means statistically significant compared to the control group (p <0.01) and ## means statistically significant compared to the insulin treatment group (p <0.01).)
5 is a result of evaluating the hypoglycemic effect of sacchinone in an animal model induced by a high lipid diet. (** means statistically significant compared to the normal diet group (p <0.01), and # and ## are statistically significant compared to the high lipid diet group (p <0.05 and p <0.01) Means.)
FIG. 6 shows the effect of Succinone on weight change in animals fed with a high lipid diet. (** means statistically significant (p <0.01) compared to the normal diet group, and # means statistically significant compared to the high lipid diet group (p <0.05).)

The present inventors found that during the screening of drugs useful for effectively preventing, improving and treating insulin resistance, insulin resistance was improved by administration of sachinone, which is one of the components of 300 seconds.

It is well known that TNF-α interferes with insulin signaling. As a result, TNF-α phosphorylates the serine group of IRS-1 activated by insulin to inhibit the tyrosine phosphorylation of IRS by insulin, thereby inhibiting insulin signaling. The inventors of the present invention found that sacchinone inhibits the phosphorylation of serine of IRS-1 activated by TNF-α (FIG. 1). Insulin binds to insulin receptors present in the cell membrane and delivers insulin signals into cells through tyrosine phosphorylation of IRS, a target of insulin receptors. Tyrosine phosphorylated IRS activates PI3K and transmits signals via Akt and GSK3-beta. The inventors of the present invention inhibited insulin signaling by reducing the phosphorylation of GSK3-beta and IRS1 tyrosine caused by insulin, which is known to interfere with insulin signaling, whereas insulin inhibited by TNFα when treated with sacchinone. It was found that the signal was significantly improved (FIG. 2). These results suggest that sacchinone may be effective in preventing, improving or treating insulin resistance.

Insulin resistance is in a state of inhibiting glucose production and glucose transport ability, so if a substance inhibits glucose production or increases glucose absorption, it is likely to be used as an insulin resistance therapy. The inventors of the present invention, in the hepG2 cells, hepatocytes, the sacchinone treatment group significantly inhibited glucose production compared to the control group (FIG. 3), and significantly increased the glucose absorption capacity that was suppressed in the TNFα treatment group in the adipocyte and myocyte lines. It was revealed that the (Fig. 4). In addition, the inventors of the present invention confirmed that saucionone reduced high-fat diet-induced blood glucose in mice fed with high-fat diet as a model of obesity and diabetes (Fig. 5), and significantly reduced body weight (Fig. 6). .

Based on the above experimental results, the present invention provides the prevention of insulin resistance, insulin resistance-related diseases or insulin resistance syndrome, which comprises a sacchinone or a three hundred-second processed extract fraction rich in sacchinone represented by the formula (1) as an active ingredient, It provides a composition for improvement or treatment or a kit comprising the same. The present invention also provides a method for preventing, ameliorating or treating insulin resistance, insulin resistance related disease or insulin resistance syndrome by administering sacchinone or three hundred seconds processed extract fraction enriched in sacchinone.

Hereinafter, the present invention will be described in detail.

The sacchinone and sacchinone-rich tritical herb extracts of the present invention can be obtained, for example, through a manufacturing process such as the following process, and any method available to those skilled in the art can be used. N-hexane was added to the finely divided three hundred seconds for 1 hour to 7 days, preferably 24 hours to 72 hours, followed by extraction at 10 to 100 ° C. to remove the n-hexane soluble extract, followed by chloroform, dichloromethane, Or reflux extraction, cold leaching, ultrasonic extraction, warm impregnation at room temperature for 10 hours to 100 ° C. for 1 hour to 7 days, preferably 24 hours to 72 hours, by adding a low polar solvent such as ethyl acetate, preferably dichloromethane. Extraction method such as extraction method, supercritical extraction method, preferably, extracting three hundred seconds using ultrasonic extraction method containing a large amount of sautinone [Rf = 0.8, CHCl ₃ -methanol (9: 1)] of the present invention Sambaekcho extract can be prepared. The extract may further comprise a step of fractionating using n-hexane-ethyl acetate. Pure sacchinone can be obtained by recrystallization of the extract fraction rich in sacchinone.

Fractions or sautinones of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art. As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, besylic acid, and the like can be used. .

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

The present invention provides a pharmaceutical composition or food composition for the prevention, improvement, treatment of insulin resistance, insulin resistance-related diseases, or insulin resistance syndrome, which comprises sacchinone or three hundred seconds-rich extract fractions rich in sacchinone. The composition of the present invention preferably contains 0.01 to 99.9%, more preferably 0.1 to 90% of sacchinone or three hundred sec. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

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

Compositions comprising fractions or sacchinones according to the present invention are oral formulations, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, respectively, according to conventional methods. Carriers, excipients and diluents which may be formulated in the form of, and may include in the composition, 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 include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, 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.

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 comprising fractions or sacchinone of the present invention as an active ingredient is administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day It is good. 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 composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Insulin resistance-related diseases that can be prevented, improved, and treated by the composition of the present invention include hyperinsulinemia, type 2 diabetes, obesity, hypertension, hypertriglyceridemia, hypoHDL cholesterol, coronary artery disease, atherosclerosis, and cardiovascular disease. Diseases, sleep apnea, prostate cancer, diabetes mellitus, affective disorders, Alzheimer's disease, stroke, breast cancer, inflammation, rheumatoid arthritis, and the like, but not limited to those recognized by those skilled in the art as insulin resistant diseases All of this is included.

In another aspect, the present invention provides a dietary supplement for the prevention and improvement of insulin resistance, insulin resistance-related diseases, insulin resistance syndrome, including a sacchinone or three hundred s. Foods to which the composition of the present invention may be added include, for example, 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. The amount of the sacchinone or the three hundred sec. Extract fraction rich in the sacchinone in the food or beverage can generally be added to 0.01-50% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is based on 100 ml. It may be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, but can be adjusted according to the purpose.

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 are conventional 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. Sugars 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. .

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice beverages and vegetable drinks. 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 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples, Reference Examples and Formulation Examples. The following examples merely illustrate the invention in detail, but are not intended to limit the scope of the invention.

Example  One. Sauuccinon  And Sauuccinon  Rich white extract processing Fraction  Produce

Succinone and three hundred vinegar-rich extract fractions rich in Succinone were obtained from Br J Pharmacol. The method described in 2003 May; 139 (1): 11-20 was prepared with a slight modification.

15 liters of 100% n-hexane was added to 1.2 kg (Daewon Pharmaceutical Co., Ltd., Daegu) in the shade for 48 hours, and the extract was discarded. Then, 15 liters of 100% chloroform was added, extracted at room temperature for 72 hours, and filtered with a filter paper. The filtered filtrate was concentrated under reduced pressure with a vacuum concentrator (N-21NS, ELYA) to obtain 80 g of a brown gel extract 300 sec (hereinafter referred to as SBC-1).

Three hundred sec chloroform extract (SBC-1) was loaded onto silica gel column chromatography (70-230 mesh, 700 g), 2 L of n-hexane was poured, and then n-hexane-ethyl acetate (n-hexane- EtOAc [50: 1 (3L), 30: 1 (5L), 15: 1 (5L)]). The n-hexane-ethyl acetate 30: 1 (2L) fraction was concentrated under reduced pressure to obtain 13 g of a light brown small fraction. This fraction was again extracted with n-hexane-ethyl acetate (50: 1 (2 L), 25: 1 (3 L), 9: 1 (2 L), then 1: 1 (1 L)]. Using a gradient solvent gradient, secondary silica gel column chromatography (230-400 mesh, 200 g) was carried out and divided into small fractions again. Fractions 18-25 of the subfractions separated from the secondary silica gel column chromatography were identified as fractions rich in sacchinone [Rf = 0.8, CHCl ₃ -methanol (9: 1)], concentrated and concentrated 5 g of a light brown small fraction, which was more abundant in rice fields, was obtained (SBC-2). The small fraction was recrystallized in ether to give 2.5 g of pure sautinone.

Experimental Example  One: TNF by -α IRS For -1 serine phosphorylation Sauucci  effect

It is known that cytokines such as leptin, interleukin, and tumor necrosis factor (TNF-α) are closely related to insulin resistance. In particular, TNF-α is well known to interfere with insulin signaling. As a mechanism, TNF-α phosphorylates the serine group of IRS-1 activated by insulin, thereby inhibiting tyrosine phosphorylation of IRS by insulin, thereby inhibiting insulin signaling. In this experiment, we evaluated whether serine phosphorylation of IRS-1 induced by TNF-α in hepatocytes was inhibited by pretreatment of sacchinone.

H4IIE (purchased from ATCC) Hepatocytes were plated on plates (1 × 10 ⁶ cells / well) and cultured for 2 to 3 days (80% confluency), then serum depleted for 12 hours, Sauunon (Sau) 30 μM After 1 hour of treatment with and vehicle (DMSO), respectively, 10 μg / ml TNFα was treated for 0.25, 0.5, 1 and 3 hours to induce serine phosphorylation of IRS-1. The serine phosphorylation of IRS-1 after TNFα treatment was observed by Western blot.

Saucinone significantly inhibited serine phosphorylation of IRS-1 by TNF-α in hepatocytes (FIG. 1). These results suggest that sacchinone has the effect of preventing, improving or treating insulin resistance.

Experimental Example  2: TNF by -α GSK3 Beta and IRS1 of Tyrosine  For inhibition of phosphorylation Sauucci  Improvement effect

In order to assess whether insulin signaling inhibited by TNFα is improved by sacchinone, the effect of sacchinone on the inhibition of tyrosine phosphorylation of GSK3beta and IRS1 by TNF-α in hepatocytes was observed.

H4IIE (purchased from ATCC) Hepatocytes were plated on plates (1 × 10 ⁶ cells / well) and cultured for 2 to 3 days (80% confluency), then serum depleted for 12 hours, Sauunon (Sau) 30 μM 1 hour before and vehicle (DMSO), respectively, and treated with 10μg / ml TNFα for 6 hours to inhibit the insulin signal. 10 nM insulin was treated for 10 minutes after TNFα treatment, and changes in insulin signal molecules were observed by Western blot.

Insulin-induced phosphorylation of GSK3beta and IRS1 tyrosine (pGSK3β, pTyr-IRS1) decreases when treated with TNFα to inhibit insulin signals, but significantly inhibits insulin signal suppressed by TNFα when treated with sautinone Improved (FIG. 2). These results suggest that sacchinone has the effect of preventing, improving or treating insulin resistance.

Experimental Example  3: To Sauuccin  Caused by hepatocytes Sugar production  Inhibitory effect

Hepatocytes respond to hormones such as insulin and glucagon to convert glucose into glycogen to lower blood sugar, and to convert glycogen to glucose or to convert non-carbohydrates into glucose to raise blood sugar. Therefore, glycemic control of hepatocytes is an essential condition for glycemic control. Insulin inhibits glucose production in the liver, and insulin resistance is in a state of diminished glucose production. Therefore, if a substance inhibits glycogenogenesis in hepatocytes, it is likely to be used as a therapeutic agent for insulin resistance.

In this experiment, HepG2 cells (purchased from ATCC), a hepatocyte cell line, were used to evaluate the change of intracellular glucose production by sacchinone. Hepatocytes were plated into plates (1 × 10 ⁶ cells / well) and cultured for 2-3 days (80% confluency), then serum depleted for 24 hours and treated with 30 μM of sacchinone or 100 μM of insulin. In order to evaluate the glucose production, after 6 hours of treatment, the medium was changed to glucose-free DMEM medium, and after 3 hours, the medium was collected to measure glucose in the medium (Amplex Red glucose / glucose oxidase assay, Invitrogen).

Compared with the glucose production amount of the control group (DMSO treatment group) (100%), the sugar treatment group produced 39.6 ± 5.6% and the insulin treatment group produced 68.8 ± 1.5% of sugar (FIG. 3). Succinone significantly inhibited glucose production (p <0.01) compared to the control.

Experimental Example  4: To Sauuccin  Caused by muscle and fat cells Sugar transport efficiency  increase

Muscle and adipose tissue, responsible for about 90% of the body's blood glucose homeostasis, absorb blood sugar into cells through a glucose transporter called GLUT4. Therefore, smooth glucose transport from the cytoplasm to the cell membrane by the expression and activity of GLUT4 and insulin stimulation is an essential condition for glycemic control. Insulin resistance is a condition in which sugar mobility is reduced. Thus, if a substance increases the mobility of sugar into cells, it is likely to be used as an insulin resistance therapy.

In this experimental example, the change of intracellular glucose uptake by sacchinone was evaluated using adipocytes obtained by culturing 3T3-L1 preadipocytes, preadipocytes, in a cell incubator for 10 days in the presence of various hormones. Adipocyte precursor which divides the 3T3-L1 adipocyte differentiation from the cells to the plate ^{(1 × 10 6 cell / well} ) in 2-3 days incubation (80% confluency), the depleted serum was for 12 hours, Succinone was treated with 30 μM while incubated in Krebs-Ringer Hepes Buffer at 37 ° C. To evaluate glucose absorption, 0.5 μCi of 2- [3H] deoxyglucose (2-DOG) was added after 1 hour of sacchinone treatment, and the reaction was stopped after 15 minutes to 2 hours, and then absorbed into cells. The radioactivity of the 2-DOG was measured with a liquid scintillation counter.

C2C12 (mouse myoblast cell line) was also treated with 3T3-L1 adipocytes in the same manner to measure glucose uptake.

In the 3T3-L1 cell line, glucose uptake was increased by 2.23 ± 0.17 times in the insulin treated group compared to the control group (DMSO treated group), and 1.02 ± 0.01 times in the insulin and TNFα treated groups. Treatment of sacchinone with insulin and TNFα resulted in a 2.05 ± 0.05 fold increase in glucose uptake, which was significantly increased in the insulin and TNFα treated groups (p <0.01).

In the C2C12 cell line, glucose uptake was increased by 2.77 ± 0.18 times in the insulin-treated group compared with the control group (DMSO-treated group), and the group treated with insulin and TNFα was 1.12 ± 0.08-fold. Treatment of sacchinone with insulin and TNFα resulted in a 2.45 ± 0.05 fold increase in glucose uptake, which was significantly increased in the insulin and TNFα treated groups (p <0.01).

In myocytes and adipocytes, sacchinone increased glucose transport efficiency (2- [3H] deoxyglucose uptake) from the initial time and significantly increased the influx of glucose into the cells for 1-2 hours.

Experimental Example  5: High fat diet  Fasting blood sugar in one mouse and Glucose  For blood sugar by administration Sauuccinon  Effect of dosing

The hypoglycemic effect of sacchinone was evaluated using high-fat diet mice as obesity and diabetes models. Normal and high lipid diets were administered to mice for 11 weeks and oral administration of 10-30 mg / kg five times per week of sautinone for the last 5 weeks. Each group consisted of a total of 10 mice.

Basal glucose level (mg / dl) of the mice fasted for 10 hours on the last day of the experiment was measured (fasting blood glucose measurement), and sacchinone was administered. After 1 hour, blood glucose level (BGL) was measured (0 hours), and glucose solution was orally administered in an amount of 2 g / kg, and blood glucose was measured hourly. Glucose concentrations were measured using Accu-Check Active (Roche, Germany).

Fasting blood glucose levels measured after fasting were 115.0 mg / dl in the normal diet group and 194.5 mg / dl in the high-fat diet group, and 167.0 mg / dl in the 10 mg / kg administration group of sacchinone with high-fat diet. In the group administered with 30 mg / kg of sacchinone, 171.0 mg / dl was significantly reduced and fasting blood glucose was significantly decreased (p <0.05).

Succinone also affected the increase in blood glucose due to the administration of glucose solution. As shown in FIG. 5, the blood glucose level induced by lipid diet was significantly decreased after administration of 30 mg / kg of sacchinone compared to before administration of the drug.

Experimental Example  6: High fat diet  For weight change in a mouse Sauucci  effect

The effect of sacchinonone on the weight change of mice fed a high-fat diet as a model of obesity and diabetes was observed. Normal and high-lipid diets were administered to mice for 11 weeks and 10-30 mg / kg of sacchinone five times per week for the last 5 weeks. Each group consisted of a total of 10 mice. The high-fat diet group was significantly higher in weight than the normal diet group, and the weight loss was significantly decreased by administration of sacchinone to the high-fat diet group (Fig. 6).

Reference Example  1: Experimental Animals and Diet

Male C57BL / 6 mice (average weight 25-30 g) used as experimental animals were purchased from Charles River Orient (Seoul. Korea). Before the experiment, animals were adapted from the Seoul National University College of Pharmacy Animal Experimental Research Building with controlled humidity of 55 ± 5%, temperature of 22 ± 2 ℃ and ventilation for more than 1 week. Changed the contrast in time periods. No significant changes were observed in the amount of food and water for the duration of the experiment. The weight and condition of the animals were examined once a week.

Reference Example  2: Sample Preparation

A high-lipid diet for fatty liver induction was purchased from Diet Co., USA, and the desired concentration of sautinone was prepared by dilution in 40% PEG 400.

Reference Example  3: Weston Blot  ( Western blot )

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Mighty Small II SE 250 apparatus according to the Laemmli UK method (1970). Dissolve the fraction of liver sample in sample dilution buffer [63mM Tris (pH.6.8), 10% glycerol, 2% SDS, 0.0013% bromophenol blue, 5% b-mercaptoethanol] and use 7.5%, 9% gel Electrophoresis was performed in buffer (including Tris 15g, glycine 72g, SDS 5g in 1L solution). The electrophoresis gel was transferred to nitrocellulose paper for 1 hour at 190 mAmps in transition buffer [25 mM Tris, 192 mM glycine, 20% v / v methanol (pH.8.3)] using a transfer electrophoresis device. Anti-Ser-phospho-IRS1 (cell signal), anti-IRS1 (santacruz), anti-Tyrosine (cell signal), anti-GSK3β (cell signal), anti-GSK3β (cell signal), anti-β-actin (sigma ) Was reacted as a primary antibody for 1 hour with Horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody and developed using an ECL chemiluminecence system (Amersham, Gaithesberg, MA). The homogeneity of the protein content in the samples was confirmed using anti-β-actin antibodies (Sigma, St. Louis, MO).

Reference Example  4: analysis method

The data presented in the following examples were analyzed using a pharmacological calculation program. The significance of the various experimental groups was analyzed by one-way square deviation analysis (Fisher, RA, Statistical). Methods for Research Workers, Edinburgh : Oliver & Boyd , 1925) Newman-Kells test (Norman GR et al., Biostatistics: The Bare Essentials, 20000) (* p <0.5, ** p <0.01) (1925).

Although the formulation examples of the composition containing the sacchinone of the present invention will be described, the present invention is not intended to be limited thereto but merely to explain in detail.

Formulation example  One. Powder  Produce

Succinone 20 mg / Lactose 100 mg / Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Succinone 10mg / Corn Starch 100mg / Lactose 100mg / Magnesium Stearate 2mg

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

Formulation example  3. Manufacture of capsule

Succinone 10 mg / crystalline cellulose 3 mg / lactose 14.8 mg / magnesium stearate 0.2 mg

It is prepared by mixing the above components according to the conventional capsule preparation method and filling into gelatin capsules.

Formulation example  4. Preparation of injections

Succinone 10mg / Mannitol 180mg / Sterile distilled water for injection 2974mg / Na ₂ HPO ₄ 12H ₂ O 26mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Saucinone 20 mg / isomerized sugar 10 g / mannitol 5 g / purified water

After dissolving by adding each component to purified water according to the conventional method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components and adding purified water to adjust the whole to 100ml and then filled in a brown bottle Prepared by sterilization.

Formulation example  6. Manufacture of dietary supplements

SBC-1 1000 mg /

Vitamin mixture quantity

Vitamin A Acetate 70 μg / Vitamin E 1.0 mg / Vitamin B ₁ 0.13 mg / Vitamin B ₂ 0.15 mg / Vitamin B ₆ 0.5 mg / Vitamin B ₁₂ 0.2 μg / Vitamin C 10 mg / Biotin 10 μg / Nicotinamide 1.7 mg / Folic acid 50 μg / calcium pantothenate 0.5 mg /

Mineral mixture

Ferrous sulfate 1.75 mg / zinc oxide 0.82 mg / magnesium carbonate 25.3 mg / potassium monophosphate 15 mg / dibasic calcium phosphate 55 mg / potassium citrate 90 mg / calcium carbonate 100 mg / 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.

Formulation example  7. Manufacture of health drinks

SBC-2 100 mg / Vitamin C 15 g / Vitamin E (powder) 100 g / Iron lactate 19.75 g / Zinc oxide 3.5 g / Nicotinamide 3.5 g / Vitamin A 0.2 g / Vitamin B ₁ 0.25 g / Vitamin B ₂ 0.3 g Proper amount of water

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (6)

Pharmaceutical composition for the prevention or treatment of insulin resistance, insulin resistance-related diseases or insulin resistance syndrome comprising a sacchinone or three hundred seconds extract fraction rich in sacchinone as an active ingredient. The method of claim 1, wherein the three hundred seconds extract fraction rich in the sacchinone is extracted with a non-polar solvent to remove the non-polar components, the extraction with a low polar solvent, fractionation using n-hexane-ethyl acetate Pharmaceutical composition for the prevention or treatment of insulin resistance, insulin resistance-related diseases or insulin resistance syndrome, characterized in that it is prepared by a method comprising a. According to claim 1 or 2, wherein the insulin resistance-related diseases are hyperinsulinemia, type 2 diabetes, obesity, hypertension, hypertriglyceridemia, hypoHDL cholesterol, coronary artery disease, atherosclerosis, cardiovascular disease, Insulin resistance, insulin resistance related disease, or insulin resistance syndrome, characterized in that selected from the group consisting of sleep apnea, prostate cancer, first diabetes, affective disorder, Alzheimer's disease, stroke, breast cancer, inflammation, rheumatoid arthritis Pharmaceutical composition for the prophylaxis or treatment of. A dietary supplement for the prevention or improvement of insulin resistance, insulin resistance-related diseases or insulin resistance syndrome, which contains sacchinone or three hundred seconds extract fraction rich in sacchinone as an active ingredient. The method of claim 4, wherein the three hundred seconds extract fraction rich in the sacchinone is extracted with a non-polar solvent to remove the non-polar components, extracting with a low polar solvent, fractionating using n-hexane-ethyl acetate Functional food for the prevention or improvement of insulin resistance, insulin resistance-related diseases or insulin resistance syndrome, characterized in that it is prepared by a method comprising a. According to claim 4 or 5, wherein the insulin resistance-related diseases are hyperinsulinemia, type 2 diabetes, obesity, hypertension, hypertriglyceridemia, hypoHDL cholesterol, coronary artery disease, atherosclerosis, cardiovascular disease, Insulin resistance, insulin resistance related disease or insulin resistance syndrome, characterized in that it is selected from the group consisting of sleep apnea, prostate cancer, first diabetes, affective disorder, Alzheimer's disease, stroke, breast cancer, inflammation, rheumatoid arthritis Functional foods for the prevention or improvement of

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