KR20140041632A - Composition for improving obesity and fatty liver using an extract of leaves of sasa quelpaertensis or p-coumaric acid - Google Patents

Abstract

The present invention discloses a composition for alleviating obesity and fatty liver using an extract of leaves of Sasa quelpaertensis Nakai or p-coumaric acid separated therefrom. The extract of Sasa quelpaertensis Nakai restrains the weight increase and the accumulation of fat, increases the manifestation content of adiponectin, and activates AMP-activated protein kinase (AMPK) in animal testing. In addition, the extract of Sasa quelpaertensis Nakai decreases the contents of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and lactate dehydrogenase (LDH).

Description

Technical Field [0001] The present invention relates to a composition for improving obesity and fatty liver using an extract of Jeju Syrup or a para-coumaric acid isolated therefrom, and a composition for improving obesity and fatty liver using an extract of Sasa quelpaertensis or p- coumaric acid

The present invention relates to a method for producing Sasa quelpaertensis leaf extract or para-coumaric acid isolated therefrom.

In recent years, the population of obesity has been rapidly increasing due to improvements in living standards due to economic development, lack of exercise due to busy living environment, and excessive intake of nutrition. The proportion of obesity (BMI> 25) in Korea was 11.7% in 1995, 30.7% in 2008, and the proportion of obesity (BMI> 30) increased rapidly from 2.3% in 1998 to 4.1% , 2009).

Obesity is a condition in which energy is excessively accumulated in the body due to unbalanced energy intake and consumption, resulting in abnormally increased adipose tissue (Clinical Obstetrics 28: 675-689, 1998; Clinical Obesity (eds. ) 248-89 (Blackwell Science, Oxford 1998).

The World Health Organization (WHO) estimates that the BMI (body mass index) of the Asian standard is 23 to 25 overweight, 25 to 30 for obesity, and 30 for high obesity.

The causes of obesity include environmental dietary factors such as high fat and high calorie diet, lack of exercise due to busy social environment, and endocrine abnormalities, among which 50 to 70% of obesity is caused by environmental factors And the rest is known to be caused by genetic factors.

Fatty liver refers to a condition in which lipid fears, especially triglycerides, are excessively accumulated in the liver and fat fear is observed in more than half of hepatocytes because normal fat metabolism is not achieved.

Clinically, fat is observed in more than 5% of hepatocytes, or when fat is greater than 5 mg per 100 mg of liver. In recent years, fatty liver patients are rapidly increasing due to high fat, high calorie diet, lack of exercise due to the development of civilization, and the age group is also generally developing from the 10's to 50's and 60's. Fatty liver, if left untreated, can lead to hepatitis, cirrhosis and liver cancer.

The causes of fatty liver include alcohol, diabetes, obesity, malnutrition (long-term low-fat diet, high fat diet, starvation, starvation, vitamin deficiency, excess carbohydrate intake), drug abuse Journal of Medicine 175 (1995.12) pp.785-794).

At present, representative examples of the obesity treatment include Orlistat (Roche), which is the main raw material, and Reductil (Abbott), which is made from sibutramine. However, there is a possibility that nausea, diarrhea, abdominal pain, insomnia, And fibrates such as clofibrate have been used for clinical use in liver diseases, but side effects such as hepatic dysfunction have been reported (Atherosclerosis. 92: 31-40 1992).

As these side effects of synthetic drugs and western medicine show limitations, the value of natural drug products is newly emerging.

The present invention discloses an obesity improving activity and a fatty liver improving activity of Jeju Sori extract.

It is an object of the present invention to provide an obesity improving agent composition and a fatty acid improver composition.

Other objects and specific objects of the present invention will be described below.

The present invention relates to an obesity improving agent composition in one aspect.

As shown in the following examples and experimental examples, the inventors of the present invention found that when the hot water extract of Jeju Syrup Lentil was administered to an experimental animal together with the high fat diet, weight gain was significantly decreased Abdominal fat, and kidney fat.

In addition, the present inventors have found that administration of the extract of Jeju Sori extract increases adiponectin expression, activates AMP-activated protein kinase (p-AMPK) and inhibits the activity of ACC (acetyl-CoA carboxylase) -ACC increase), and in the cell test, it was confirmed that the extract of Jeju Sasa extract activates AMPK and activates CPT-1α (carnitine palmitoyl transferase-1α) (increase of CPT-1α).

The present inventors have also found that p -coumaric acid, a biologically active substance isolated from Jeju Sori extract, inhibits the accumulation of triglycerides in cell experiments and increases AMPK phosphorylation and ACC phosphorylation and activates AMPK Increased the phosphorylation of LKB1 and increased the expression of PPARα, a transcription factor of CPT-1.

AMPK has been shown to be involved in the maintenance of energy homeostasis in the intracellular space [1], [2], [3] and [4] It is known that an enzyme that acts as a sensor is activated when the AMP: ATP ratio is increased by oxidative stress such as glucose deficiency and ROS (J. Biol. Chem. 277: 32571-32577, 2002; J. Appl. Physiol 92: 2475-2482, 2002). When AMPK is activated, ACC is phosphorylated and becomes inactivated. ACC is an enzyme involved in the early stage of fatty acid synthesis and is an enzyme that synthesizes acetyl-CoA with malonyl-CoA (Trends Biochem. Sci. 24: 22-25, 1999, Biochem. Soc. Trans. 31: 162-168, 2003). When ACC is inactivated, it is known that the intracellular concentration of malonyl-CoA is lowered and UCP2, CPT-1α etc. are activated to promote lipid oxidation (J. Biol. Chem. 277: 32571-32577, 2002 , J. Appl. Physiol. 92: 2475-2482, 2002).

Therefore, when the expression level of adiponectin is increased and the AMPK is activated, accumulation of the body fat can be inhibited, consequently, the increase of body weight can be suppressed, and the blood triglyceride and cholesterol concentration can be lowered.

The composition for improving obesity of the present invention is provided on the basis of the results of the experiment described above, and the composition for obesity improvement of the present invention is characterized in that it contains an extract of Jeju Soseki or p -coumaric acid as an active ingredient.

In the present specification, "Jeju bamboo sake berry leaf extract" means a bamboo sake berry leaf used as an extraction target, and water, methanol, ethanol, butanol, Means an extract obtained by using ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof do. The extraction method may be applied by any method such as cold rolling, refluxing, heating, ultrasonic irradiation, etc. in consideration of the degree of extraction of the active ingredient and the degree of preservation. In addition, a polysaccharide degrading enzyme can be used at the time of extraction to change the active substance (an unknown single substance exhibiting activity) or to induce diversification of the active substance. The polysaccharide degrading enzyme may be prepared by combining three or more molecules, Any enzyme capable of decomposing a saccharide may be used. Such enzymes include, for example, cellulase, xylanase, hemicellulase and beta-glucanase, cellobihtydrolase, 1,4-beta- 1,4-β-D-glucosidase and 1,4-β-D-glucanase, amyloglucosidase, A-amylase, pectinase, pentosanase, mannase, pectintransliminase, polygalactonase, pectinesterase, pectinase, ) And / or plant and animal extracts containing these enzymes. Actually, a mixture of PectinexTM (Novo Nordisk, Denmark), Ultraflo LTM (Novo Nordisk, Denmark), Viscozyme LTM (Novo Nordisk, Denmark), CeremixTM (Novo Nordisk, Denmark), CelluclastTM (Novo Nordisk, Commercially available enzymes such as Filterase BR ™ (Gist-Brocades, Netherlands) and Termamyl (Novozymes, Denmark) will be used. The extract may be a concentrated liquid extract or a solid extract, from which the extraction solvent has been removed by lyophilization, vacuum drying, hot air drying, spray drying and the like. In particular, "Jeju Sori extract" means preferably obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent.

In the present specification, the term " active ingredient "alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is not itself active.

In the present specification, "obesity" means an abnormally increased state of fat tissue, whether it is caused by genetic factors or environmental factors, and the obesity (BMI of 25 or more ) And overweight (when BMI is between 23 and 25).

In the present specification, the term "improvement of obesity" is meant to include reduction of body fat and / or reduction of body weight, including prevention of obesity and treatment of obesity.

The composition for improving obesity of the present invention may contain the active ingredient in any amount (effective amount) as long as it exhibits the activity of improving obesity, the formulation, the purpose of compounding, etc. A typical effective amount is based on the total weight of the composition And will be determined within the range of 0.001 wt% to 99.990 wt%. Here, "effective amount" refers to the amount of effective ingredient capable of inducing an obesity-improving effect. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The subject to which the composition of the present invention can be applied (prescription) is preferably a mammal and a person, particularly a human.

In another aspect, the present invention relates to a composition for diet comprising as an active ingredient pomegranate extract or p -cumaric acid.

In another aspect, the present invention relates to a composition for improving hyperlipemia comprising an extract of Jeju Soso or p -cumaric acid as an active ingredient.

As used herein, a "diet " is defined as a condition in which the weight is not obese or overweight, but is desirable or necessary for weight or body fat reduction for cosmetic or health purposes. In general, the composition for diet of the present invention will be manufactured and used for the beauty and health of a normal person.

In the present specification, the term "hyperlipidemia" refers to a disease caused by a high amount of fat in the blood because fat metabolism such as triglycerides and cholesterol is not properly performed.

With respect to the meaning of the Jeju Somen Sauce Extract, the effective amount thereof, and the like in the composition for diet and the composition for improving hyperlipemia of the present invention, the above-mentioned items relating to the obesity-improving composition of the present invention are effective as they are.

In another aspect, the present invention relates to a composition for preventing diseases caused by obesity comprising an extract of Jeju Sauce as an active ingredient.

When obesity persists, obesity can cause hypertension, elevated blood cholesterol, kidney disease, stroke, atherosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes.

Therefore, the term " a disease caused by obesity "can be understood as one of hypertension, elevated blood cholesterol, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea and diabetes.

With respect to the composition for preventing diseases caused by obesity of the present invention, the above-described effects with respect to the meaning of the extract of Jeju Sauce Extract, which is an effective ingredient thereof, and the effective amount thereof and the like, with respect to the composition for obesity-relieving agent of the present invention are still valid.

In another aspect, the present invention relates to a composition for improving liver fat remediation comprising as an active ingredient a bovine Sasa extract or p -coumaric acid.

In the following Experimental Example, it was confirmed that the extract of Jeju Soso is the content of glutamic acid pyruvic acid transaminase (hereinafter "GPT"), glutamic acid oxaloacetate transaminase (hereinafter "GOT") and lactate dehydrogenase (hereinafter "LDH" In addition, in the observation of the tissue sections of the liver tissues of the extracted experimental animals, in the case of the group administered with the high-fat diet and the extract of the low fat diet group, the hepatic tissue morphology was similar to that of the normal diet, whereas only the high fat diet The army showed a marked progression of fatty liver.

Since GPT, GOT and LDH are secreted from the liver into the blood when they are damaged due to liver obesity, alcohol and the like, the activity of these enzymes is used as a general indicator of liver dysfunction. Low means that liver damage is low.

In the case of p - cumaric acid isolated from Jeju Sori extract, SREBP - 1c (concentration - dependent inhibition of activation of SREBP - 1c) was induced in fat accumulation - induced HepG2 cells. The SREBP-1c is known to be involved in the synthesis of fatty acids and triglycerides in the liver (Cell, 89 (3): 331-340, 1997)

These results suggest that the extract of Jeju Sori extract may be useful for the improvement of fatty liver.

As used herein, the term "fatty liver" refers to a disease that causes angina, myocardial infarction, stroke, arteriosclerosis, etc., and refers to a disease in which fat is accumulated in hepatocytes excessively due to liver fat metabolism disorder.

Regarding the fatty liver improvement composition of the present invention, regarding the meaning of the Jeju Somen berry extract, the effective amount thereof, and the like, the above-mentioned effects with respect to the composition for obesity-relieving agent of the present invention are still valid as they are.

On the other hand, when the liver is left untreated, it may progress to hepatitis, liver cirrhosis, liver cancer and the like. Therefore, the composition of the present invention can be used for prevention of hepatitis, hepatopathy or liver cancer caused by fatty liver.

Therefore, in another aspect, the present invention can be grasped as a composition for preventing hepatitis, hepatopathy or liver cancer, which is a disease caused by fatty liver containing the extract of Jeju Soseki as an active ingredient.

In yet another aspect, the present invention relates to an insulin resistance improving composition comprising as an active ingredient a bovine sage extract or p -coumaric acid.

Obesity is one of several causes of insulin resistance. It is known that obesity is associated with insulin resistance, but obesity is closely correlated with insulin resistance. It is generally known that the more obesity is, the more visceral obesity is, the more insulin resistance becomes.

Adipocytokines secrete physiologically active adipocytokines, which play an important role in maintaining metabolic homeostasis. Adipocytokines play an important role in the maintenance of obesity, that is, excessive accumulation of fat. It is understood that excessive or ineffective destruction of balance leads to insulin resistance.

Among the adipocytokines, there are those that promote insulin sensitivity and induce insulin resistance. Representative examples of the former are adiponectin and AMPK (AMP-dependent protein kinase), and the latter representative examples are TNF-a, lL-6, and resistin.

In particular, studies have shown that AMPK activation may be a candidate for improving insulin resistance, since the decrease in AMPK activity is accompanied by insulin resistance and hyperglycemia (J. Biol. Chem., 277 ): 25226-32, 2002; Diabetes. 51 (7): 2074-81,2002; J. Clin. Invest. 108 (8): 1167-74,2001).

The following Examples and Experiments show that the above-described Jeju bacillus Sogarifide extract or p -cumaric acid isolated therefrom, which is an active ingredient of the composition for improving obesity of the present invention, activates AMPK. These results suggest that Jeju Sori extract can be used not only as an obesity - improving agent but also as an insulin resistance improving agent.

In the present specification, the term "insulin resistance" refers to a state in which cells, organs, the human body, etc. require a normal amount of insulin for normal metabolic activities, that is, insulin action- It is accepted in the same sense as non-dependent diabetes or type 2 diabetes. Diabetes mellitus is the result of the breakdown of β-cells in the pancreas. As a result, the insulin-dependent diabetes mellitus (type 1 diabetes) Type 2 diabetes), so insulin resistance, non-insulin dependent diabetes mellitus and type 2 diabetes are accepted in the same sense in the art.

In the insulin resistance improving composition of the present invention, regarding the meaning of the extract of Jeju Soseki, the effective amount thereof and the like, the above-mentioned facts relating to the composition for obesity-improving agent of the present invention are still valid as they are.

(Hereinafter, referred to as "the composition of the present invention"), a composition for preventing obesity, a composition for preventing obesity, a composition for preventing fatty liver, a preventive composition for diseases caused by fatty liver and a composition for improving insulin resistance Can be grasped as a food composition in a specific embodiment.

The food composition of the present invention can be produced as a health supplement food, a special nutrition supplement food, a functional beverage and the like.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

In addition, the food composition of the present invention may further contain a powder derived from any natural substance known to have an activity of improving obesity, hangover activity, etc., in order to enhance flavor or palatability, to enhance its functionality, or to add other functionalities Or extract of the present invention may be selected from the group consisting of a pregelatinized powder or extract, a bean sprouts powder or extract, a shell powder or extract, an oyster powder or extract, an oxalate powder or extract, a jujube or an extract, a cucumber juice or extract, Ginseng powder or extract, licorice powder or extract, garlic powder or extract, powder or extract powder, extract powder, extract powder, extract powder, extract powder, Peanut powder or extract, ginger powder or extract, jujube powder or The present invention relates to a powder or extract of a ginger powder, a powder or extract of green tea, a powder or extract of a green tea, a powder or extract of a ginger, a powder or extract of a ginger, a powder or extract of a ginger, Omega powder or extract, hinoki powder or extract, dirt powder or extract, candy powder or extract, cinnamon powder or extract, decaccinol and the like. The meaning of the extract in the above is as described above in connection with the jeju jeoldae extract. Such an extract may be obtained by mixing the object to be extracted.

In another specific embodiment, the composition of the present invention can be identified as a pharmaceutical composition.

The pharmaceutical composition of the present invention may be in a form suitable for oral use (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or non- Ointments, ointments, solutions, creams, ointments, gels, lotions, patches, and the like).

The term "pharmaceutically acceptable" as used herein means that the application (subject) does not have an adaptive or higher toxicity (sufficiently low toxicity) without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a tableting agent, a stabilizer, an antioxidant, a preservative, water, a saline solution and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, when the pharmaceutical composition of the present invention is prepared by an aqueous suspension, suitable excipients include sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , Sodium alginate, polyvinylpyrrolidone, and the like can be used. As a suitable excipient when prepared by injection, Ringer's solution, isotonic sodium chloride and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

As described above, according to the present invention, an obesity improving agent composition can be provided. Also, according to the present invention, a composition for improving liver fat remedy can be provided. Also, according to the present invention, a composition for improving hyperlipemia, a composition for improving insulin resistance, and the like can be provided. The composition for obesity-relieving agent of the present invention and the like can be commercialized as a functional food or medicine.

FIG. 1 shows the chromatogram of the hydrothermal extract of Jeju Saury and its molecular structure.
FIG. 2 is a graph showing the body weight (body weight) of experimental animals (HFD + SQE) when fed a normal diet (ND), when fed a high fat diet (HFD) and when fed a high fat diet weight) was measured every 5 days for 70 days.
FIG. 3 is a graph showing the results of an experiment in which the animals were fed a normal diet (ND), a high fat diet (HFD), a high fat diet and an oral diet (HFD + SQE) It is the result of confirming the cell size of epididymal adipose tissue.
FIG. 4 is a graph showing the results of an experiment in which animals were fed a normal diet (ND), a high fat diet (HFD), a high fat diet, and an oral diet (HFD + SQE) This is the result of measuring RNA expression of adiponectin in abdominal fat.
FIG. 5 is a graph showing the results of the experiment (HFD) when the normal diets were fed to the experimental animals (ND), the animals fed the high fat diet (HFD) and the animals fed the oral diet (HFD + SQE) The expression level of p-AMPK and p-ACC protein in abdominal fat was measured.
FIG. 6 is a graph showing the results of the experiment (HFD) when the normal diet was fed to the experimental animals (ND), the experimental animals (HFD + SQE) The results of microscopic observation of liver sections.
FIG. 7 shows the results of measurement of protein expression of p-AMPK and p-ACC in differentiated 3T3-L1 adipocytes of the sake brewer's extract (SQE).
FIG. 8 shows the results of measurement of protein expression of p-AMPK and p-ACC in differentiated L6 skeletal muscle cells of Jeju Bacillus subtilis extract (SQE).
FIG. 9 shows the results of measurement of p-AMPK and p-ACC protein expression in HepG2 liver cells induced by localization of Jeju Bacillus subtilis extract (SQE).
FIG. 10 shows the results of measurement of RNA expression of CPT-1a in differentiated 3T3-L1 adipocytes of the spermatogonium extract (SQE).
Fig. 11 shows that p -cumaric acid inhibits the accumulation of triglycerides in 3T3-L1 adipocytes differentiated.
Figure 12 shows that p -cumaric acid increases phosphorylation of LKB1, phosphorylation of AMPK and phosphorylation of ACC in a concentration-dependent manner in 3T3-L1 adipocytes differentiated.
FIG. 13 shows that p -cumaric acid increases phosphorylation of AMPK and ACC in a concentration-dependent manner in L6 skeletal muscle cells differentiated.
Fig. 14 shows the results showing that PPAR [alpha] expression is increased in a concentration-dependent manner in the L6 skeletal muscle cells in which p -cumaric acid has been differentiated.
FIG. 15 shows that p -cumaric acid increases phosphorylation of AMPK and ACC in a concentration-dependent manner in HepG2 cells induced by fat accumulation.
FIG. 16 shows that p -cumaric acid increases the expression of SREBP-1c in a concentration-dependent manner in HepG2 cells induced by fat accumulation.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Preparation of Jeju Sauce Extract and Isolation of Physiologically Active Substances

Example 1 Preparation of Jeju Sauce Extract Preparation Example 1

6 kg of the berry sake of the present invention was washed with tap water, lyophilized and then pulverized. The pulverized Jeju Sauce was immersed in water and subjected to hot water extraction at 90 ° C for 4 hours. After concentration under reduced pressure, the extract solvent was removed to obtain a powdery Jeju sake extract.

Example 2 Preparation of Jeju Sauce Extract Preparation Example 2

6 kg of the berry sake of the present invention was washed with tap water, lyophilized and then pulverized. The ground Jeju bran was added to water containing 3% by weight of viscozyme ™ (Novo Nordisk, Denmark), 3% celluclast ™ (Novo Nordisk, Denmark) and citric acid (pH 4.5) And the enzyme was inactivated at 100 DEG C for 1 hour. After concentration under reduced pressure, the extract was freeze-dried to remove the solvent to prepare a powdery Jeju bacillus subtilis extract.

Example 3 : Isolation of physiologically active substance

(1) Fraction of Jeju Sori Sago Extract

100 g of the bovine syrup extract was suspended in 1 L of distilled water, and 1 L each of n-hexane, EtOAc and n-BuOH was added thereto. The extract was sequentially extracted three times and concentrated at 40 ° C using a rotary evaporator.

(2) Preparation of standard solutions

Standard solutions for the identification of components in Jeju Sulfur hydrothermal extract were prepared by dissolving chlorogenic acid, p - cumaric acid and tricin (10 mg) in 1 mL of DMSO (dimethylsulfoxide) and diluting with 9 mL of methanol.

(3) Preparation of Test Solution

2.5 mL of DMSO was added to 2.5 mL of methanol and sonication was performed for 10 minutes. The extract was washed with 0.50 mm polytetrafluoroethylene (PTFE) and a syringe filter (Advantec Toyo Roshi Kaisha Ltd., DISMIC ^R- 13 _JP , Japan).

(4) Analysis of compounds

Qualitative analysis of Jeju Sasa hot-water extract were used for HPLC (Waters Corporation, 2695 Alliance system, USA), ^TM Sunfire C ₁₈ column (250 × 4.6 mm ID. 5㎛ ), the column temperature was maintained at 40 ℃. The components of Jeju Syrup fractions were detected by UV absorption in the range of 200 ~ 600 nm using PDA detector and mobile phase was separated by gradient elution method using MeOH and ultrapure water (Table 1). The flow rate was maintained at 1.0 mL / min.

(5) Method for Isolation and Identification of Compound

HPLC (Waters, 2695 Alliance system, USA), Symmetryprep ^TM C ₁₈ column (7.8 300 mm ID, 7 m) was used for the separation of compounds from the hydrothermal extract of Jeju Sulfur. Column temperature was kept at 40 ℃. Detectors were detected at UV absorbance ranging from 200 to 600 nm using a PDA detector. The mobile phase was separated by gradient elution using MeOH and ultrapure water. The flow rate was maintained at 1.8 mL / min and repeated peaks with the same retention time were collected. The nuclear magnetic resonance (NMR) spectrometer used in the structure analysis was 400 MHz FT-NMR (JEOL, JNM-LA, Japan). The solvent used for the measurement was CD ₃ OD and CDCl ₃ from Merck.

(6) Identification of isolated compounds

Three compounds contained in the hydrothermal extract of Jeju Saury were separated by HPLC and used in the method described in J. Agric . Food Chem . , 2007, 55 (25), 10086-10092; J. Appl . Biol . Chem., 2008, 51 (4), 148-154;. .. Arch Pharm Res, 2007, 30 (2), 161-166;... Bull Koeran Chem Soc, compared with 2010, 31 (4), 1088-1090 ) chlorogenic acid, p - coumaric acid, and tricin. In order to confirm this, qualitative analysis was carried out using standard substances of each separated compound. In order to identify the constituent compounds isolated from the hot - water extract, the retention of each compound peak was confirmed by repeating 3 times at 320 nm. As a result, the peak of p - Peaks were detected between 18 and 19 minutes and showed the same retention and absorbance as the standards of the standard. Chlorogenic acid, p - coumaric acid, and tricin were found to contain 0.132, 23.706, and 0.286 mg, respectively, in the extract from Jeju Sori (Fig. 1). The major components of the hydrothermal extract of Jeju Sage were p - cumaric acid. Therefore, the effects of p - coumaric acid, a major component of hot - water extract, on cellular lipid metabolism were investigated in the following experiments.

< Experimental Example > Jeju Sori extract and its isolated p - Experiments to improve metabolic disease activity of cumaric acid

< Experimental Example  1> Experimental study on improvement of metabolic diseases of Jeju Sasa extract

<Experimental Example 1-1> Measurement of body weight and body fat in an animal experiment

Four weeks old C57BL / 6 male mice purchased from Nara Biotech Co., Ltd. were adapted for 1 week while feeding solid feed and water, and mice having similar weight were randomly divided into three groups of 10 mice. (HFD + SQE) and the Jeju bokbunja extract (150 mg / kg of body weight / day) were administered orally while the control group (HFD + SQE) Respectively. The incubation environment was maintained at a temperature of 23 ° C, a humidity of 50 ± 5%, and night and day at a cycle of 12 hours.

In this study, 10% kcal diets (Research Diets, USA) were used for normal diet and 60% kcal diets (USA) for high fat diet.

The effect of Jeju Sori extract on body weight was measured by using an animal scales at every 5 days for 70 days. The results of the experiment were shown in FIG. 2, and the dietary intake was measured every 5 days for 70 days at the same time, The average (g / cage / 5day) of the unit measurements is shown in Table 2. And the total weight gain for 70 days (measured by first weight loss in the final body weight after 70 days) is shown in Table 3.

Dietary intake  division ND HFD HFD + SQE Dietary intake (g / cage / 5day) 26.57 ± 0.40 ^a 21.19 ± 0.28 ^b 21.04 ± 0.28 ^b * There was statistical significance between a, b and c ( p <0.05)

Total weight gain division ND HFD HFD + SQE Weight gain (g) 5.65 ± 0.60 ^a 15.60 ± 0.78 ^c 10.76 ± 0.59 ^b * There was statistical significance between a, b and c ( p <0.05)

FIG. 2 and Table 3 show that the weight gain of HFD + SQE group administered with Jeju Sori extract was significantly lower than that of HFD group. Table 2 shows that the HFD + SQE group had lower dietary intake than the HFD group.

In order to confirm the effect of Jeju Sori extract on the abdominal fat (white fat) and kidney fat weight, after 70 days of experimentation, the animal was anesthetized with ether and then weighed and weighed. The results of the experiment are shown in Table 4 , And HFD + SQE were less than those of the HFD group.

Increase in abdominal fat and kidney fat division ND HFD HFD + SQE Abdominal fat (g) 0.86 ± 0.05 ^a 2.02 ± 0.12 ^c 1.42 ± 0.06 ^b Kidney fat (g) 0.50 0.06 ^a 1.23 + 0.07 ^c 0.97 + 0.06 ^b * There was statistical significance between a, b and c ( p <0.05)

On the other hand, in order to confirm the effect of Jeju Sori extract on the cell size of epididymal adipose tissue, after 70 days of experiment in Experimental Example 1, anesthetized with ether and then extracted, And the results of the experiment are shown in Fig. 3 as the tissue section photographs of 2 mice per group. FIG. 3 shows that the fat tissue size of the HFD + SQE group was similar to that of the ND group, whereas the fat tissue size of the HFD group was significantly increased.

<Experimental Example 1-2> Measurement of adiponectin expression level by RT - PCR

In order to confirm the effect of Jeju Sori extract on the expression of cytokines of epididymal adipose tissue, after 70 days of experiment in Experimental Example 1, the cells were anesthetized with ether and then extracted. -PCR technique, and the experimental results are shown in the graph of FIG.

For total RNA for real-time PCR, 500 μl of Trizol Reaent (Invitrogen, USA) was added, and the cells were homogenized at room temperature for 5-10 minutes. Then, 100 μl of chloroform was added to the tube, centrifuged (12,000 × g, 15 Min). The supernatant was recovered and transferred to a new tube. The same amount of isopropanol was added and the RNA was precipitated by centrifugation (12,000 × g, 15 minutes). The precipitated RNA was washed three times with 75% ethanol, dried, and dissolved in DEPC-treated distilled water. The absorbance at 260 nm was measured using a nanodrop 2000 spectrometer (Nanodrop, USA). RNA samples with a ratio of A260 / A280 nm within the range of 1.8-2.2 were treated with DNase (Wako, Japan) to isolate pure RNA and used in the experiments.

cDNA was synthesized using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). 1 μg total RNA and nuclease-free water were added to the tube in a total volume of 20 μl. The cDNA was synthesized by reacting at 45 ° C for 60 minutes and 95 ° C for 5 minutes, and then adding 30 μl of nuclease-free water to dilute Were used for PCR.

Real time polymerase chain reaction (PCR) was performed using 5 μl of cDNA and 0.4 μl (10 pmol / L / μl) of each primer, 2 μl of nuclease-free water and 6 μl of iQ ™ SYBR® (Bio-rad, USA) using a Peltier thermal cycler (Bio-Rad, USA). The PCR conditions were amplified 49 times at 95 ° C for 20 seconds, 65 ° C for 20 seconds, and 72 ° C for 30 seconds. The fluorescence intensity was measured for each amplification. The melting curve analysis of the primers was performed. Results were analyzed using a Chromo4 Real-Time PCR Detection System v1.10 (Bio-Rad, USA) relative to β-actin. The nucleotide sequences of the primers used in the real-time PCR were: adiponectin, 5'-GAC CTG GCC ACT TTC TCC TC-3 '(SEQ ID NO: 1) and 5'- GTC ATC TTC GGC ATG ACT GG- ); 3 '(SEQ ID NO: 3) and 5'-ACC CAA GAA GGA AGG CTG GA-3' (SEQ ID NO: 4).

FIG. 4 shows that the expression level of adiponectin in the HFD group was significantly lower than that in the ND group, whereas the expression level of adiponectin in the HFD + SQE group was significantly increased compared with the obesity control group.

<Experimental Example 1-3> Western Expression of p- p- AMPK and ACC by measuring the amount blot experiments

In order to confirm the effect of Jeju Sori extract on the abdominal fat (white fat) activation, after 70 days of experiment in Experimental Example 1, the extract was anesthetized with ether, and the protein was separated and measured by Western blotting. The results of the experiment are shown in Fig.

For Western blot analysis, the adipose tissues treated with the sample were washed twice with PBS and then incubated with 1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / The supernatant was obtained by centrifugation (15000 rpm, 4 ° C, 20 minutes) by vortexing for 1 hour using a protein separation reagent containing RIPA lysis buffer (Upstate Biotechnology, USA). Protein concentrations were measured and quantified at 595 nm using a micro-reader using the Bio-rad protein assay kit (Bio-Rad, USA). Protein was separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane (Milipore, USA) for 100 min (120 min). Protein-transferred PVDF membrane was blocked with Tris buffered saline (TTBS) solution containing 0.1% Tween 20 containing 5% skim milk or 5% BSA for 1 hour at room temperature. The primary antibody (p-AMPK, p-ACC, and? -actin). (1: 10,000, Sigma, USA), p-AMPK antibody (1: 2,000, Cell Signaling, USA) and p-ACC antibody RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; overnight. After washing the membrane with 0.1% TTBS, the peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, USA) was diluted to 1: 2,000, 1: 5,000 or 1: 10,000 and reacted for 1 hour TBS-T. Expression of each protein was detected by X-ray film (Ortho CP-G plus; Agfa Gevaert NV, Belgium) after reacting with WEST-ZOL western blot detection system (iNtRON Biotechology, Korea).

FIG. 5 shows that the AMPK activation (p-AMPK) and ACC inactivation (p-ACC) of the HFD group were significantly decreased compared with the ND group, whereas the AMPK activation and the ACC inactivation of the HFD + Compared with the control group.

<Experimental Example 1-4> Measurement of total cholesterol and triglyceride in blood

The effect of Jeju Sori extract on serum total cholesterol and blood triglyceride content was measured by using an assay kit (Asan Pharmaceutical Co., Ltd.) after separating the serum after completion of the experiment for 70 days in Experimental Example 1, As shown in Table 5, the serum total cholesterol and blood triglyceride contents were lower in the HFD + SQE group than in the HFD group.

Total increase in serum total cholesterol (T-CHO) and blood triglyceride (TG) division ND HFD HFD + SQE T-CHO (mg / dL) 119.71 + 4.09 ^a 179.14 ± 3.90 ^c 158.57 ± 2.38 ^b TG (mg / dL) 92.29 ± 4.86 ^a 138.43 + - 9.15 ^b 83.43 + 3.03 ^a * There was statistical significance between a, b and c ( p <0.05)

<Experimental Example 1-5> Experimental Observation of Liver Tissue

The effect of Jeju Sori extract on hepatic tissue injury was confirmed by observing microscopic observation of the tissue section after anesthetizing with ether after 70 days of experiment in Experimental Example 1. The results are shown in Fig. Tissue sections. FIG. 6 shows that the HFD + SQE group is similar to the ND group, and the HFD group has a larger amount of fat globules than the HFD + SQE group or ND group.

<Experimental Example 1-6> Measurement of enzyme activity of liver damage index

The levels of GPT, GOT, and LDH, which are used as indicator enzymes for liver damage, were measured by the use of an assay kit (Asan Pharmaceutical, Korea) after separating the serum after 70 days of experiment in Experimental Example 1, The results are shown in Table 6. Table 6 shows that the HFD + SQE group had lower GPT, GOT and LDH levels than the HFD group, and was similar to the ND group value.

GPT, GOT and LDH activity division ND HFD HFD + SQE GPT (IU / L) 8.57 ± 0.85 ^a 17.29 ± 3.03 ^b 8.29 ± 0.65 ^a GOP (IU / L) 42.29 ± 1.62 ^a 57.43 ± 3.14 ^b 44.00 ± 1.21 ^a LDH (IU / L) 440.86 ± 80.48 ^a 981.86 ± 117.44 ^b 566.57 ± 26.43 ^a * There was statistical significance between a, b and c ( p <0.05)

<Experimental Example 1-7> Experiment of cells in Western Measurement of p- AMPK expression level by blot

In order to confirm the results of the activation of AMPK in animal experiments, 3T3-L1 cells (adipocytes), L6 cells (skeletal muscle cells) and HepG2 cells (hepatocytes) were cultured and the results were confirmed. 7, 8 and 9, respectively.

(AMPK) and its substrate, Acetyl Co-A carboxylase (ACC), on the differentiated adipocytes were investigated. The differentiation of adipocytes Respectively. 3T3-L1 preadipocytes were inoculated into 6 well cell culture plates (1.6 or 2.0 × 10 ⁵ cells / well) using DMEM supplemented with 10% BCS and 1% P / confluent state. The culture medium was further exchanged in the pre-confluent state for 48 hours. In the confluent state (0 day after induction of differentiation), the culture was inoculated into differentiation-inducing medium [10% fetal bovine serum (FBS; Gibco, USA), 1% P / S, 1 μM dexamethasone (DEXA .; Sigma, USA), 0.5 μM 3-isobutyl (DMEM medium containing 5 μg / ml-1-methylxanthine (IBMX; Sigma, USA) and 5 μg / ml insulin (Sigma, USA)] for 2 days. After 2 days, the cells were changed to DMEM medium containing 10% FBS and 1% P / S every 2 days until the experiment. At 8-10 days after induction of differentiation, the medium was removed from the culture vessel, washed twice with DMEM, and cultured for 16 hours in serum-free medium (0.5% BSA + DMEM) / Ml) for 24 hours to confirm the phosphorylation of AMPK and ACC.

To elucidate the effects of Aβ-activated protein kinase (AMPK) and its substrate Acetyl Co-A carboxylase (ACC) on L6 myotube, the differentiation of muscle cells was performed as follows. L6 myoblast was inoculated into a 12-well cell culture plate (1 × 10 ⁵ cells / well) using DMEM supplemented with 10% FBS and 1% P / S and incubated for 48 hours in a pre-confluent state. The culture medium (DMEM supplemented with 10% FBS) was preincubated once more in the pre-confluent state and cultured for another 48 hours. At the confluent state (day 0 differentiation induction), the culture broth was induced to differentiate by changing to differentiation induction medium (DMEM medium supplemented with 2% FBS). The medium was replaced every two days with the same medium until use in the experiment. At 9-10 days after induction of differentiation, the medium was removed from the culture vessel, washed with DMEM medium, and cultured in DMEM medium supplemented with 0.5% BSA for 4 hours. Then, phosphorylation of AMPK and ACC was confirmed by treatment with each concentration (125, 250, 500 and 1,000 / / ml) for 24 hours.

In order to analyze the effect of the extract of Jeju Bacillus subtilis on lipid-induced hepatocytes on the phosphorylation of AMP-activated protein kinase (AMPK) and its substrate Acetyl Co-A carboxylase (ACC), lipid induction of hepatocytes was performed as follows . HepG2 cells were inoculated into 12 well cell culture plates (1 × 10 ⁶ cells / well) using DMEM supplemented with 10% FBS and 1% P / S, cultured for 48 hours, washed twice with DMEM medium The cells were cultured for 16 hours in serum-free medium (0.5% BSA + DMEM) and cultured in serum-free medium (0.5% BSA + DMEM) containing 0.4 mM palmitate for 24 hours to induce fat in HepG2 cells. After the induction of the fat, the medium was removed and the phosphorylation of AMPK and ACC was confirmed by treatment with serum free (0.5% BSA + DMEM) for 24 hours in concentration (62.5, 125, 250, 500 ㎍ / ㎖).

For Western blot analysis, the cells treated with the sample were washed twice with PBS and incubated with 1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin 1 μg / ml leupeptin and 10% RIPA lysis (Upstate Biotechnology, USA) for 1 hour, followed by centrifugation (15000 rpm, 4 ° C, 20 minutes) to obtain supernatant. Protein concentrations were measured and quantified at 595 nm using a micro-reader using the Bio-rad protein assay kit (Bio-Rad, USA). Protein was separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane (Milipore, USA) for 100 min (120 min). Protein-transferred PVDF membrane was blocked with Tris buffered saline (TTBS) solution containing 0.1% Tween 20 containing 5% skim milk or 5% BSA for 1 hour at room temperature. The primary antibody (p-AMPK, AMPK, p-ACC, ACC and beta-actin). ACC antibody (1: 1,000, Cell Signaling, USA), ACC antibody (1: 1,000, Cell Signaling, USA) and p-AMPK antibody Cell Signaling, USA) and β-actin antibody clone AC-74 (1: 10,000, Sigma, USA) overnight at 4 ° C. After washing the membrane with 0.1% TTBS, the peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, USA) was diluted to 1: 2,000, 1: 5,000 or 1: 10,000 and reacted for 1 hour TBS-T. Expression of each protein was detected by X-ray film (Ortho CP-G plus; Agfa Gevaert NV, Belgium) after reacting with WEST-ZOL western blot detection system (iNtRON Biotechology, Korea).

Referring to FIGS. 7, 8 and 9, it can be seen that the extract of Jeju Soser is increased in AMPK activity in all three kinds of cells.

<Experimental Example 1-8> Measurement of CPT- 1a expression level by RT - PCR

In particular, the expression of the RNA of the AMPK subadjustor CPT-1a, which is an index for measuring the degree of β-oxidation of fatty acids in 3T3-L1 cells, was confirmed by Jeju Sori extract and the results of the experiment are shown in FIG. Referring to FIG. 10, it can be seen that the extract of Jeju Sauce Extract increased the expression of CPT-1a.

For total RNA for real-time PCR, 500 μl of Trizol Reaent (Invitrogen, USA) was added, and the cells were homogenized at room temperature for 5-10 minutes. Then, 100 μl of chloroform was added to the tube, centrifuged (12,000 × g, 15 Min). The supernatant was recovered and transferred to a new tube. The same amount of isopropanol was added and the RNA was precipitated by centrifugation (12,000 × g, 15 minutes). The precipitated RNA was washed three times with 75% ethanol, dried, and dissolved in DEPC-treated distilled water. The absorbance at 260 nm was measured using a nanodrop 2000 spectrometer (Nanodrop, USA). RNA samples with a ratio of A260 / A280 nm within the range of 1.8-2.2 were treated with DNase (Wako, Japan) to isolate pure RNA and used in the experiments.

cDNA was synthesized using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). 1 μg total RNA and nuclease-free water were added to the tube in a total volume of 20 μl. The cDNA was synthesized by reacting at 45 ° C for 60 minutes and 95 ° C for 5 minutes, followed by addition of 30 μl of nuclease-free water PCR.

Real time polymerase chain reaction (PCR) was performed using 5 μl of cDNA and 0.4 μl (10 pmol / L / μl) of each primer, 2 μl of nuclease-free water and 6 μl of iQ ™ SYBR® (Bio-rad, USA) using a Peltier thermal cycler (Bio-Rad, USA). The PCR conditions were amplified 49 times at 95 ° C for 20 seconds, 65 ° C for 20 seconds, and 72 ° C for 30 seconds. The fluorescence intensity was measured for each amplification. The melting curve analysis of the primers was performed. Results were analyzed using a Chromo4 Real-Time PCR Detection System v1.10 (Bio-Rad, USA) relative to β-actin. The base sequences of the primers used in the real-time PCR were carnitine palmitoyltransferase-1a (CPT-1a), 5'-ACC CTG AGG CAT CTA TTG ACA-3 'and 5'- TGA CAT ACT CCC ACA GAT GGC-3 '(SEQ ID NO: 6); 3 '(SEQ ID NO: 7) and 5'-ACC CAA GAA GGA AGG CTG GA-3' (SEQ ID NO: 8).

< Experimental Example  2> p - Experiments to improve metabolic disease activity of cumaric acid

<Experimental Example 2-1> Inhibitory activity on differentiation of adipocytes in adipocytes

3T3-L1 precursor adipocytes were purchased from the American Type Culture Collection (ATCC). 3T3-L1 precursor adipocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing 1% penicillin / streptomysin (PS) and 10% bovine calf serum (BCS) at 37 ° C and 5% CO ₂ .

Cells were incubated in DMEM medium containing 1% PS, 10% fetal bovine serum (FBS), MDI (0.5 mM isobutylmethylxanthine (IBMX), 1 M dexamethasone, 1 μg / To induce differentiation. After 2 days of induction of differentiation, the medium was replaced with DMEM medium containing 1% PS, 10% FBS and 1 μg / mL insulin. After 2 days, the medium was replaced with DMEM medium containing 1% PS and 10% FBS The medium was changed every 2 days until the experiment.

During induction of adipocyte differentiation ( p - coumaric acid) was treated in each medium at a concentration of 100 uM and the degree of adipocyte differentiation was observed on the 8th day when the differentiation was completed. Specifically, 3T3-L1 precursor adipocytes were washed twice with phosphate-buffered saline (PBS) 8 days after induction of differentiation and fixed with 3.7% formaldehyde for 1 hour. After washing twice with distilled water, 0.6% Oil Red O reagent was applied and stained for 1 hour. After dyeing, Oil Red O was removed, washed twice with primary distilled water, and cells were dried. 4% Nonidet P-40 was added to quantify stained fat droplets. Thereafter, the absorbance at 540 nm was measured, and the results were compared with the absorbance of the cells induced by pure differentiation and the absorbance of the cells treated with the sample.

The results are shown in Fig. 11, it can be seen that the TG content was significantly decreased in the group treated with p -cumaric acid during Day 4 to Day 8. This effect can be assumed to be related to the synthesis and lipolysis of TG after the adipocyte differentiation of p - coumaric acid.

<Experimental Example 2-2> The differentiation 3 T3 - L1 adipocytes in the Western LKB1 p- and p-AMPK expression level measured in the experiment with the blot

To examine the effect of p -coumaric acid on lipid oxidation in differentiated 3T3-L1 adipocytes, the adipocytes differentiated on day 8 were starvated in DMEM medium containing 0.2% BSA and 1% PS for 12 hours, And DMEM medium containing 10% FBS and 1% PS and p - coumaric acid were treated by concentration.

The cultured cells were washed with cold PBS and lysed with 1 mM RIPA (Upstate Biotechnology, Temecula, CA, USA), 1 mM phenylemthyl-sulfonyl fluoride, 1 mM, 1 mM NaF, 1 μg / mL aprotinin, Cells were harvested using 1 μg / mL leupeptin and reacted on ice for 1 hour. Then, cell debris was removed by centrifugation, and proteins were separated. The separated proteins were quantified using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, Calif., USA). The quantified protein was electrophoresed through 10% polyacrylamide gel containing SDS and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline (TBS) containing 5% BSA and 0.1% Tween 20 for 1 hour. Then, the primary anti-body was treated at 4 ° C for at least 16 hours, and the secondary anti-body was treated at room temperature for 1 hour. Target proteins were identified by ECL western blotting detection reagent (Amersham Biosciences).

The results are shown in Fig. Referring to FIG. 12, it was confirmed that p- cumaric acid increased phosphorylation of LKB1, which is known to activate AMPK in differentiated 3T3-L1 cells, and phosphorylation of AMPK and ACC were also increased. These results suggest that p - coumaric acid inhibits lipid accumulation in adipocytes and activates lipid metabolism such as β - oxidation through AMPK signal in mature adipocytes.

<Experiment 2-3> In Western differentiated L6 cells Expression of p- p- AMPK and ACC by measuring the amount blot experiments

Rat derived L6 skeletal muscle cell line was purchased from ATCC. Cells were cultured in DMEM containing 10% FBS and 1% PS and maintained at 37 ° C in 5% CO ₂ . For cell differentiation, the medium was replaced with DMEM medium containing 2% FBS every two days before use in the experiment. The differentiated L6 cells were cultured for 48 hours in serum-free DMEM containing 0.5% bovine serum albumin (BSA) and treated with p-coumaric acid for 48 hours.

The degree of phosphorylation of AMPK and ACC was analyzed by Western blot in the same manner as in <Experimental Example 2-2>.

The results are shown in Fig. 13, phosphorylation of AMPK and ACC in the group treated with p -coumaric acid was increased in a concentration-dependent manner as compared with the control group. In particular, at 50 uM, the highest concentration, AMPK and ACC showed a marked increase in the degree of phosphorylation. This suggests that ACC, which is the downstream of AMPK, is phosphorylated by activating AMPK in p6 - lysates of p - coumaric acid. Phosphorylated ACC is ultimately reported to be involved in fatty acid β-oxidation in mitochondria (FENS let., 223 (2): 217-222, 2003). Therefore, p - coumaric acid may be involved in intracellular β - oxidation by activating AMPK.

<Experimental Example 2-4> In the differentiated L6 cells, Western Measurement of PPARα expression level by blot

The expression level of PPAR? Was analyzed by Western blotting in the same manner as in <Experimental Example 2-3>, and p -cumaric acid was treated for 48 hours according to the concentration in the same manner as in <Experimental Example 2-2>.

The results are shown in FIG. Referring to FIG. 14, PPARa expression in the group treated with p -cumaric acid was increased compared with the expression of PPARa in the control group. Peroxisome proliferator-activated receptors (PPARs) are known to modulate gene expression associated with lipid metabolism and energy homeostasis (Diabetes, 52: 2249-2259, 2003). In particular, PPAR plays an important role in the transcriptional regulation of genes involved in β-oxidation, a process that uses fatty acids to produce energy (Molecular and cellular biology, 20 (5): 1868-1876, 2000). Thus, the increase in PPARα expression, which regulates gene expression associated with β-oxidation, suggests that p -coumaric acid is involved in β-oxidation in differentiated L6 cells.

<Experimental Example 2-5> HepG2 cells were treated with Western Expression of p- p- AMPK and ACC by measuring the amount blot experiments

HepG2 cell line was purchased from ATCC. Cell culture was performed in DMEM medium supplemented with 10% FBS and 1% PS at 37 ° C and 5% CO ₂ . Cells were maintained in subculture at the bottom of the T-75 cell culture flask when cells were 80% full. The induction of lipid accumulation of HepG2 cells was performed by modifying the method of Gomez-Lechon et al. (Chem Biol Interact., 165 (2): 106-116, 2007). HepG2 cells were inoculated into a 6-well cell culture plate (1 × 10 ⁶ cells / well) using DMEM supplemented with 10% FBS and 1% PS . Starvation overnight was performed in DMEM medium containing 0.5% BSA in a state where the cells were about 80% cold. Oleic acid, a free fatty acid, was replaced with media supplemented with 600 uM and p - coumaric acid, and cultured for 24 hours to induce lipid accumulation in HepG2 cells. Cells were treated with oleic acid 600M and p -coumaric acid at various concentrations and cultured for 24 hours. The expression levels of p-AMPK and p-ACC were analyzed by Western blot in the same manner as in <Experimental Example 2-2>.

The results are shown in Fig. Referring to FIG. 15, it can be seen that AMPK and ACC phosphorylation, which are involved in fatty acid oxidation, increase in a concentration-dependent manner.

<Experimental Example 2-5> HepG2 cells were treated with Western Measurement of expression level of SREBP- 1c by blot

In the same manner as in <Experimental Example 2-4>, oleic acid 600M and p -coumaric acid were treated at different concentrations for 24 hours, and SREBP- 1c were analyzed by Western blotting.

The results are shown in Fig. 16, SREBP-1c, which is involved in the synthesis of fatty acids and triglycerides, decreases in a concentration-dependent manner.

Claims (9)

A composition for improving obesity comprising p - coumaric acid as an active ingredient.
The method of claim 1,
Wherein the composition is a food composition.
The method of claim 1,
Wherein the composition is a pharmaceutical composition.
A composition for improving hyperlipemia comprising p - coumaric acid as an active ingredient.
5. The method of claim 4,
Wherein the composition is a food composition.
5. The method of claim 4,
Wherein the composition is a pharmaceutical composition.
A composition for improving fatty liver comprising p - coumaric acid as an active ingredient.
8. The method of claim 7,
Wherein the composition is a food composition.
8. The method of claim 7,
Wherein the composition is a pharmaceutical composition.

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